THE

JOURNAL OF
EXPERIMENTAL
MEDICINE
SELECTED ARTICLES MARCH 2015 www.jem.org

DENDRITIC CELLS
AND MACROPHAGES
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 Welcome
D e n d r i t i c C e l l s a n d M a c ro p h a ge s
The Journal of Experimental Medicine now prints topic-specific mini collections to showcase a handful
of our recent publications. In this installment, we highlight papers focusing on dendritic cells and
macrophages in health and disease.
Immunotherapy has proven successful in many types of cancer treatment, but has also been
associated with dangerous inflammatory responses in some patients. Our collection begins with
an Insight by Merghoub and Wolchok discussing the findings of Mirsoian et al. who show that
lethal inflammation in response to anti-CD40 and IL-2 immunotherapy is triggered by increased
inflammatory macrophages in the accumulated visceral fat of aged mice. Similarly, young obese mice succumbed to treatment
while older mice on diets were protected. The study highlights factors to be considered with use of immunotherapeutics.
In the gut, the immune system must balance recognition of infectious pathogens with minimal responses to commensal
bacteria. An Insight by Giorgio Trinchieri describes these immune challenges and the findings from Longman et al. showing that
in both mice following Citrobacter rodentium infection and in patients with colitis, CX3CR1+ mononuclear phagocytes (MNPs)
are potent producers of IL-23 and IL-1b. The production of these cytokines by MNPs efficiently induces IL-22 production by
group 3 innate lymphoid cells to promote barrier integrity and mucosal protection.
Lambrecht and Guilliams examine genetic fate mapping of myeloid cells that distinguishes macrophages derived from
embryonic precursors vs. circulating monocytes. They highlight the contribution to macrophage biology from Molowi et al.
reporting that cardiac macrophages are initially of embryonic origin, but are progressively replaced by monocyte-derived cells as
mice age. They speculate about how manipulation of different macrophage populations could be used in therapeutics.
Myelin destruction in multiple sclerosis (MS) is mediated by inflammatory macrophages, but the origin of these cells has been
unclear. An Insight from Michael Heneka discusses findings from Yamasaki et al., who use a mouse model of MS to distinguish
tissue-resident microglial cells from infiltrating monocytes. Using double chemokine reporter mice, the authors find that
monocyte-derived macrophages initiate myelin destruction, mainly in the nodes of Ranvier, while microglia-derived macrophages are
involved with clearing debris.
An article by Woodruff et al. relies on imaging techniques and examines aspects of flu infection relevant to vaccination.The
authors describe how, during immunization, resident lymph node dendritic cells can rapidly relocate to sites of viral influenza
antigen, driving early activation of T cells, and contributing to germinal center formation and B cell memory to establish an
appropriate immune response.
Together these studies characterize the many roles and functions of DCs and macrophages. We hope you enjoy this
complimentary copy of our dendritic cell and macrophage collection. We invite you to explore additional collections at
www.jem.org and to follow JEM on Facebook, Google+, and Twitter.

Selected Articles March 2015

Immunotherapy and the belly of the beast

Taha Merghoub and Jedd D. Wolchok

Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy
regimens in aged mice
Annie Mirsoian, Myriam N. Bouchlaka, Gail D. Sckisel, Mingyi Chen, Chien-Chun Steven Pai, Emanuel Maverakis,
Richard G. Spencer, Kenneth W. Fishbein, Sana Siddiqui, Arta M. Monjazeb, Bronwen Martin, Stuart Maudsley,
Charles Hesdorffer, Luigi Ferrucci, Dan L. Longo, Bruce R. Blazar, Robert H. Wiltrout, Dennis D. Taub,
and William J. Murphy

Critical role for CX3CR1+ mononuclear phagocytes in intestinal homeostasis

Giorgio Trinchieri
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CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22
Randy S. Longman, Gretchen E. Diehl,1 Daniel A. Victorio, Jun R. Huh,1 Carolina Galan, Emily R. Miraldi,
Arun Swaminath, Richard Bonneau, Ellen J. Scherl, and Dan R. Littman

Monocytes find a new place to dwell in the niche of heartbreak hotel

Bart Lambrecht and Martin Guilliams

Progressive replacement of embryo-derived cardiac macrophages with age

Kaaweh Molawi, Yochai Wolf, Prashanth K. Kandalla, Jeremy Favret, Nora Hagemeyer, Kathrin Frenzel,
Alexander R. Pinto, Kay Klapproth, Sandrine Henri, Bernard Malissen, Hans-Reimer Rodewald, Nadia A. Rosenthal,
Marc Bajenoff, Marco Prinz, Steffen Jung, and Michael H. Sieweke

Macrophages derived from infiltrating monocytes mediate autoimmune myelin destruction

Michael T. Heneka

Differential roles of microglia and monocytes in the inflamed central nervous system
Ryo Yamasaki, Haiyan Lu, Oleg Butovsky, Nobuhiko Ohno, Anna M. Rietsch, Ron Cialic, Pauline M. Wu,
Camille E. Doykan, Jessica Lin, Anne C. Cotleur, Grahame Kidd, Musab M. Zorlu, Nathan Sun, Weiwei Hu,
LiPing Liu, Jar-Chi Lee, Sarah E. Taylor, Lindsey Uehlein, Debra Dixon, Jinyu Gu, Crina M. Floruta, Min Zhu,
Israel F. Charo, Howard L. Weiner, and Richard M. Ransohoff

Trans-nodal migration of resident dendritic cells into medullary interfollicular regions initiates
immunity to influenza vaccine
Matthew C. Woodruff, Balthasar A. Heesters, Caroline N. Herndon, Joanna R. Groom,Paul G. Thomas,
Andrew D. Luster, Shannon J. Turley, and Michael C. Carroll
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INSIGHTS

Immunotherapy and the belly of the beast

Immunotherapy has emerged as an effective means to restore immune recognition of cancer.
Numerous forms of immunotherapy are currently being explored clinically. The most common are
vaccines (dendritic cell, viral, and whole tumor cell based), adoptive T cell therapy and immune
checkpoint blockade. The recent successes in melanoma, renal cancer, and lung cancer have gener-
ated renewed optimism for treatment of multiple cancer types that were believed not amenable to
immune-based therapies. However, immune-related events such as colitis, dermatitis, and, less
frequently, endocrinopathy and pneumonitis have been reported and can be a challenge in the
clinical use of these approaches. Cytokine release syndrome has been reported in the case of CAR Insight from Taha Merghoub (left)
(chimeric antigen receptor) T cell therapy and treatment with agonist antibodies. In this issue, and Jedd Wolchok (right)
Mirsoian et al. provide evidence that adiposity in aged mice induces a lethal cytokine storm follow-
ing systemic administration of stimulatory immunotherapy consisting of anti-CD40 agonist antibody with IL-2.
This is an elegant follow up to a previous study by the same authors in which they showed that systemic immunotherapy adminis-
tration in aged mice resulted in the induction of a rapid and lethal cytokine storm. In the current paper, they find that it is the fat that
accumulates during aging, and mainly the visceral fat, that is associated with toxicity. This results in increased levels of proinflammatory
M1 macrophages within the peritoneal cavity and visceral adi-
pose tissues, leading to heightened production of TNF. In or-
der to confirm that it is indeed the fat that is associated with
lethal effects, the authors repeated their studies in young mice
with genetic (ob/ob) or diet-induced obesity (DIO). While
young obese mice also displayed severe toxicity to the therapy,
it was much more severe in older mice suggesting that age is
also a significant factor. Reciprocally, calorie-restricted aged
mice had lower visceral body fat content and reduced cytokine
levels, with increased survival following immunotherapy.
Obese mice were also protected by macrophage depletion or
TNF blockade. These data demonstrate the need to consider
age and body fat content as variables in preclinical assessment
of therapeutics and when modeling diseases such as cancer.
Since many cancer patients fall into the elderly category,
this study brings up a critical point that aged mice respond dif-
ferently to immunotherapy than younger mice. This also
highlights an important issue when considering potential tox-
icities associated with any therapy, as most animal studies are
performed in young, healthy mice. Another interesting point
is the potential impact of body mass index and adipose accu-
mulation in predicting side effects when investigating and uti-
lizing immunotherapies. This is a very important and impactful
The Murphy group previously showed that immunotherapy with anti-CD40 finding for the field, and further investigations in tumor bear-
and IL-2 leads to a productive immune response in young mice (A) but ing mice with commonly used therapies are warranted. If the
lethal cytokine storm in older mice (B). They now find that the visceral fat findings are confirmed in other therapeutic settings, the inclu-
that accumulates in aged mice (or young obese mice) is the primary trigger
sion of supportive measures, such as TNF blockade, should
of inflammation after immunotherapy, resulting in increased inflammatory
M1 macrophages and toxic cytokine storm (B). Immunotherapy-induced
continue to be considered for early management of immune-
lethality was prevented by blocking TNF or depleting macrophages related toxicities to improve clinical outcomes.
(B). Whether the success of immunotherapy in the treatment of tumor- Mirsoian, A., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/
bearing mice will also be impaired by obesity remains to be determined. jem.20140116.

Taha Merghoub and Jedd D. Wolchok, Memorial Sloan Kettering Cancer Center: merghout@mskcc.org and wolchokj@mskcc.org

2 INSIGHTS | The Journal of Experimental Medicine

 Article

Adiposity induces lethal cytokine storm

after systemic administration of stimulatory
immunotherapy regimens in aged mice
Annie Mirsoian,1* Myriam N. Bouchlaka,1* Gail D. Sckisel,1 Mingyi Chen,2
Chien-Chun Steven Pai,1 Emanuel Maverakis,1 Richard G. Spencer,5
Kenneth W. Fishbein,5 Sana Siddiqui,5 Arta M. Monjazeb,3
Bronwen Martin,5 Stuart Maudsley,5 Charles Hesdorffer,5 Luigi Ferrucci,5
Dan L. Longo,5 Bruce R. Blazar,6 Robert H. Wiltrout,7 Dennis D. Taub,4,8**
and William J. Murphy4**
1Department of Dermatology, 2Department of Pathology and Laboratory Medicine, 3Department of Radiation Oncology,
and 4Department of Dermatology and Internal Medicine, University of California, Davis, Sacramento, CA 95817
5National Institute on Aging-Intramural Research Program, National Institutes of Health, Biomedical Research Center,

Baltimore, MD 21224
6Division of Blood and Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455
7National Cancer Institute, Frederick, MD 21702
8Hematology and Immunology Translational Research Center, VA Medical Center, Washington, DC 20422

Aging is a contributing factor in cancer occurrence. We recently demonstrated that sys-

temic immunotherapy (IT) administration in aged, but not young, mice resulted in induction
of rapid and lethal cytokine storm. We found that aging was accompanied by increases in
visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice.
Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are
not well defined. We determine the effects of adiposity on systemic IT tolerance in aged
compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory
cytokine levels and organ pathologies are comparable to those in aged ad libitum mice
after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2
macrophages within the peritoneal and visceral adipose tissues and higher percentages of
TNF+ macrophages in response to CD40/IL-2 as compared with young lean mice. Macro-
phage depletion or TNF blockade in conjunction with CD40/IL-2 prevented cytokine
storms in young obese mice and protected from lethality. Calorie-restricted aged mice
contain less visceral fat and displayed reduced cytokine levels, protection from organ
pathology, and protection from lethality upon CD40/IL-2 administration. Our data dem-
onstrate that adiposity is a critical factor in the age-associated pathological responses to
systemic anti-cancer IT.

CORRESPONDENCE
William J. Murphy:
wmjmurphy@ucdavis.edu
Abbreviations used: AL, ad
libitum; ALT, alanine amino
transferase; CR, calorie restricted;
DIO, diet-induced obese; HD,
high dose; IT, immunotherapy;
LD, low dose; MRI, magnetic
resonance imaging.
The use of immunotherapy (IT) in cancer has
recently resulted in impressive responses.Yet, the
usages of IT regimens, especially those involving
cytokine therapies, have also resulted in the in
duction of severe systemic toxicities often not pre
viously characterized in their preclinical animal
studies.Because such toxicities often require inten
sive care management of patients, the causes for
*A. Mirsoian and M.N. Bouchlaka contributed equally to
this paper.
**D.D. Taub and W.J. Murphy contributed equally to
this paper.
the discrepancies in observations between clin
ical and preclinical toxicity merit further study.
Cancer has been considered a disease of aging,
with persons over 65 accounting for over 60%
of newly diagnosed malignancies (Balducci and
Ershler, 2005). Consequences of aging include
gradual decreases in immunological function,
thymic involution leading to decreased naive
T cell output, restriction within T cell repertoire
2014 Mirsoian et al. This article is distributed under the terms of an Attribution
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the publication date (see http://www.rupress.org/terms). After six months it is
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J. Exp. Med. 2014 Vol. 211 No. 12 23732383 2373
www.jem.org/cgi/doi/10.1084/jem.20140116
diversity, increases in genomic instability, and increases in cel
lular senescence (Campisi, 2013). However, despite the ma
jority of patients being of this demographic, the overwhelming
majority of preclinical studies are performed in young inbred
mice.We recently demonstrated that treatment of young mice
with a combination IT regimen consisting of agonistic mono
clonal antibody to CD40 (CD40) given in conjunction with
IL-2 resulted in synergistic anti-tumor effects and was well
tolerated (Murphy et al., 2003).Yet, administration of the same
regimen in an aged cohort resulted in 100% lethality within
two days of treatment (Bouchlaka et al., 2013). Importantly, the
mortality in the aged was perpetuated by an aberrant cytokine
storm mirroring a systemic inflammatory response syndrome
(SIRS), which resulted in multi-organ damage (Bouchlaka et al.,
2013). These systemic toxicities were closely representative of
those previously noted clinically with FDA-approved immu
nostimulatory IT therapeutics such as high-dose IL-2 and
IFN- (Lee and Margolin, 2011).
Aging is associated with a gradual decline in immunolog
ical function but is also accompanied by the coincidence of
a systemic, chronic, and low-grade proinflammatory response
termed inflammaging that leads to increased systemic cyto
kine levels, namely IL-1, IL-6, and TNF (Franceschi, 2007).
Emerging data suggests that inflammaging may be influenced
by age-associated changes in body mass composition such as
decreased lean muscle mass, increased adiposity, and increases
in overall body mass index (BMI; Franceschi et al., 2000).
Whether adiposity plays a role in the generation of systemic
toxicities or whether it is primarily an age-associated occur
rence remains unknown.
Like aging, hallmark to the obese microenvironment is
the development of a low-grade chronic meta-inflammatory
state. Excess adiposity is associated with increased infiltration
of macrophages and proinflammatory mediators such as den
dritic cells, NK cells, and T cells into fat depots that ultimately
results in environmental remodeling. Consequences include
increased circulating levels of C-reactive protein (CRP), IL-6,
leptin, and TNF (Ronti et al., 2006;Vzquez-Vela et al., 2008).
Importantly, the immunomodulatory effects of increased adi
pose tissue extends through the ability for both adipocytes and
the stromal vascular fraction to express a variety of immunologi
cally important surface receptors, such as the leptin receptor,
IL-6 receptor, and both p55 and p75 TNF receptors (Ailhaud,
2000). Systemic consequences of increased adiposity are also
seen at distance sites through premature induction of aging-
associated changes such as thymic involution, which leads to
the restriction of naive T cell output and ultimately restriction
of the T cell repertoire (Yang et al., 2009).
Collectively, the field of IT has made great progress in the
last decade toward the development of therapeutic candidates
against cancer.Yet, these candidates have been met in the clinic
with the induction of severe, often limiting, systemic toxici
ties that hinder their usage. Additionally, given the rise of
obesity within society, as well as the estimation that cancer is
a disease primarily of the aged, using lean and young models
may not accurately reflect patient outcomes. Therefore, this
2374
study seeks to establish the consequences of increased adipose
tissue accumulation throughout aging upon IT-induced tox
icities. Here, we find that adiposity results in a skewing toward
increased levels of proinflammatory M1 macrophages within
the peritoneal cavity and visceral adipose tissues that ultimately
result in heightened production of proinflammatory cytokines,
namely TNF, during strong immune stimulation with IT.
Importantly, aged ad libitum (AL)fed mice and young obese
mice succumb to TNF-mediated pathological responses culmi
nating in rapid lethality, which are prevented through caloric
restriction in the aged or through either macrophage depletion
or TNF blockade.
RESULTS
IT administration in the aged results in rapid lethal cytokine
storm and is associated with increased accumulation
of visceral adipose tissue
The combination IT regimen consisting of CD40 and IL-2
(CD40/IL-2) has previously shown synergistic anti-tumor
effects resulting in the regression of metastatic tumors in
young (<6 mo) mouse models (Murphy et al., 2003). Re
cently, we demonstrated that administration of the therapeu
tic dose (high dose [HD]) of CD40/IL-2 into aged mice
(15 mo) resulted in a heightened cytokine storm that prompted
multi-organ damage and resulted in 100% lethality by day 2
of treatment (Bouchlaka et al., 2013). To address whether the
increased lethality was specific to this therapeutic dosage in
the aged, we administered CD40/IL-2 at a lower dose (LD),
less than half, into aged AL-fed mice. Consistent with our
previous findings, treatment of aged mice with LD IT re
sulted in 100% mortality by day 2 of treatment (Fig. 1 A) and
demonstrated increased serum levels of proinflammatory cy
tokines TNF, IL-6, and IFN-, indicative of a cytokine storm
(Fig. 1 B). In addition, to ensure that the lethality observed in
the aged mice was not IT regimenspecific, we administered
an IT combination of CpG and IL-2 (CpG/IL-2) into young
WT and aged AL mice. Confirming our previous findings,
aged mice treated with CpG/IL-2 therapy incurred a similar
cytokine storm that culminated in rapid lethality by day 3
(Fig. 1, CE), in contrast to young mice which exhibited lower
levels of cytokines and demonstrated complete treatment tol
erance (Fig. 1, CE).
Normal aging is inherently associated with changes in body
mass compositionspecifically with decreases in muscle mass
and increases in intra-abdominal visceral adiposity (Harris,
2002). Equally, within our animal studies an observable differ
ence between aged and young mice is noted in overall size, in
particular increased waist circumference (Fig. 1 F). On average,
aged mice weigh twice as much as young (Fig. 1 G).To deter
mine whether the difference in size is due to increases in fat
deposits, we sought to quantify differences in body mass and
the presence of visceral adiposity. Young (<6 mo) and aged
(15 mo) C57BL/6 mice were analyzed through magnetic
resonance imaging (MRI) to quantify the presence of visceral
adiposity. Aged mice exhibited markedly increased body mass
indices and total visceral fat volumes in comparison with young
Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.

mice (Fig. 1, G and I).These results indicate that aged mice are
not only heavier and larger in size but that these size differences
are attributed to increases in adipose tissue accumulation.
Adipose tissue is widely published to be a highly active
metabolic organ with immune modulatory capabilities. To
address whether in our aged AL-fed mice, who were allowed
to freely access food, the increased adiposity noted was con
tributory toward the development of IT-induced toxicities in
the aged, we aimed to determine the effects of IT administra
tion into an aged calorie-restricted (CR) cohort. CR mice are
placed on a food restriction diet beginning at 14 wk of age
and maintained throughout life. To ensure that age-matched
CR aged mice contained a decreased accumulation of body
fat in comparison with aged AL mice, we quantified total body
weight and visceral fat through MRI (Fig. 1, H and I). CR
mice weighed significantly less than AL aged mice and exhib
ited a decreased presence of visceral adiposity to levels that
JEM Vol. 211, No. 12
Ar ticle
Figure 1. Aged AL mice show increased
proinflammatory cytokines, rapid lethality
to LD IT, and display increased fat volume
ratios in comparison to aged CR mice.
(A) Ad libitumfed C57BL/6 mice were treated
with LD CD40/IL-2 or rIgG/PBS and survival
was monitored, n = 58 mice/group. (B) Serum
cytokines TNF, IL-6, and IFN- were measured
day 2 by CBA after the start of CD40/IL-2 or
rIgG/PBS therapy in aged AL mice, n = 35
mice/group. (C) Young AL (3 mo) and aged AL
(18 mo) mice were treated with CpG/IL-2 IT
and survival was monitored, n = 5 mice/group.
(D and E) Serum cytokine levels of TNF (D) and
IL-6 (E) were measured by CBA on day 2 after
the start of therapy, n = 5 mice/group.
(F) Representative image of aged and young
C57BL/6 mice showing size and waist circum-
ference. (G) Total body weight (left graph) and
visceral fat weight (right graph), was mea-
sured for young (2 mo) and aged AL mice
(20 mo), n = 5 mice/group. (H) Total body
weight (left graph) or visceral fat (right graph)
was measured in naive 15-mo-old C57BL/6
mice on an AL or CR diet, n = 34 mice/group.
(I) Visceral fat in young (4 mo), middle-aged
(9 mo), and aged (15 mo) mice on either an AL
(left) or CR (right) diet was evaluated by MRI.
WS is indicative of water suppression, where
fat is bright. FS is indicative of fat suppression,
fat is dark/black. Data in all panels are repre-
sentative of one of at least five experiments
with similar results. MRI scans are representa-
tive of one of at least three mice imaged per
group with similar results. Survival analysis
was plotted according to the Kaplan-Meier
method, and statistical differences were deter-
mined with the log-rank test. Bar graph (mean
value SEM) statistics were performed using
either one-way or two-way ANOVA with
Bonferroni post-hoc tests. ***, P < 0.001; **,
P < 0.01; *, P < 0.05.
were similar to young mice (Fig. 1, GI). MRI scans of young
(4 mo), middle-aged (9 mo), and aged (15 mo) mice that were
either AL-fed or CR throughout life showed significant dif
ferences in adipose accumulation over time (Fig. 1 I). We found
that with increasing age, AL-fed mice have a proportional in
crease in body fat content, whereas mice placed on a CR diet
lack increased fat accumulation regardless of age. Aged CR
mice show no significant difference in fat volume ratios to
their middle-age and young counterparts, and remain similar
to young AL mice (Fig. 1 I).
Caloric restriction in aged mice results in decreased
accumulation of adiposity and protection
from IT-induced toxicity
To examine the effect of adiposity and aging on the develop
ment of systemic toxicities after IT, aged AL, aged CR, and
young AL mice were administered IT or a control therapy
2375
Figure 2. Calorie restriction in the aged confers protection by decreasing cytokine levels and IT-associated organ pathology, thereby
allowing increased survival during IT. (AC) Aged AL and age-matched aged CR mice (18 mo) were treated with HD CD40/IL-2 or rIgG/PBS; at day 2
of IT, mice were sacrificed for histological analysis of livers (A) and intestines (C). Serum was collected on day 2 and assessed for ALT (B) levels, n = 3 mice/
group. Representative H&E images of liver and intestines (gut) for aged AL and aged CR groups. Bars, 200 m. Asterisks represent steatosis and arrows
denote areas of severe immune infiltration and/or necrosis. (DF) Serum was analyzed day 2 after treatment for TNF (D), IL-6 (E), and IFN- (F). (G) Aged
(18 mo) C57BL/6 mice on AL or CR diet were treated with either LD or HD of CD40/IL-2 and survival was monitored. Control groups received rIgG/PBS.
All data panels are representative of one of four independent experiments with similar results for all panels. Survival analysis was plotted according to
the Kaplan-Meier method and statistical differences were determined by using the log-rank test. Bar graph (mean value SEM) statistics were performed
using one-way ANOVA with Bonferroni post-hoc test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01.

(rIgG/PBS). IT resulted in increased lymphocytic infiltrates
to the liver of aged CR and aged AL mice, in contrast to age-
matched control-treated mice (Fig. 2, AC). To assess whether
IT resulted in organ damage, livers and intestines were col
lected on day 2 of treatment, the day of mortality in aged AL
mice, and assessed for pathological changes. Importantly, livers
and intestines from aged CR mice demonstrated significantly
less inflammation and less necrosis in comparison with aged
AL mice (Fig. 2, A and C). Serum analysis of alanine amino
transferase (ALT) enzyme levels, a correlate of liver damage,
showed a significant reduction in ALT levels in aged CR mice
in comparison with aged AL mice (Fig. 2 B).Additionally, serum
proinflammatory cytokine analysis of TNF (Fig. 2 D), IL-6
(Fig. 2 E), and IFN- (Fig. 2 F) resulted in all cytokines being
significantly decreased in aged CR mice in comparison with
the aged AL mice. These decreases in cytokine values were to
levels that were either lower-than or as-low-as those in young
AL mice that received IT (Fig. 2, DF). To determine if these
lower cytokine values during IT therapy would confer protec
tion against IT-induced mortality, aged AL mice and aged CR
mice were administered either LD IT or HD IT and monitored
for mortality. Independent of treatment dose, aged AL mice all
succumb to mortality on day 2 of treatment (Fig. 2 G). How
ever, 100% of aged CR mice placed on the HD amount of
IT demonstrated complete protection from CD40/IL-2
mediated lethality (Fig. 2 G). Collectively, these data suggest
that increased body fat plays a critical role in the induction of
systemic toxicities by IT. Increased adiposity played a critical
role in the age-associated cytokine storm and subsequent organ
pathologies after IT and calorie restriction diminished the de
velopment of such toxicities conferring a protective effect that
allowed for increased survival in the aged.
Increased adipose tissue accumulation through obesity, in the
absence of age, results in IT-induced lethal cytokine storm
To further dissect the role of adipose tissue in the IT-associated
inflammatory responses seen in aged mice, we next sought to
determine the impact of adiposity, in the absence of age, as a
possible cofactor.Young ob/ob mice (B6.Cg-Lepob/ob) are leptin-
deficient and therefore start exhibiting obesity by 3 wk of age.
In comparison to age-matched young WT (2 mo) control mice
placed on an AL diet (young AL), ob/ob mice have significantly
higher body masses and weigh, on average, 2.5 greater
(Fig. 3 A). MRI analysis of body fat content demonstrated

2376 Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.


that young ob/ob mice have higher total body fat content that
is similar in accumulation to the aged mice, being deposited
largely in the intra-abdominal cavity, in comparison with young
AL mice (Fig. 3 B).
To determine if adiposity could solely induce toxic con
sequences in aged AL, young ob/ob and age-matched young
AL mice were treated with HD IT and at day 2 of treatment
analyzed for the presence of immune-mediated organ patholog
ical changes within the liver and intestines (Fig. 3, C and D).
IT resulted in mild inflammatory infiltrates in young AL mice
but resulted in moderate to severe inflammation in the ob/ob
mice, which was comparable to the aged within the intestines
(Fig. 3 D).Yet, analysis of IT-induced liver pathology was hin
dered by the presence of extensive steatosis in ob/ob mice, re
sulting in a paradoxical interference in determining the intensity
of immune-mediated inflammatory liver damage (Fig. 3 C).
IT resulted in both the intestines and liver displaying the most
immune-mediated inflammatory damage within the ob/ob that
JEM Vol. 211, No. 12
Ar ticle
Figure 3. Increased adiposity indepen-
dently of age results in systemic release of
proinflammatory cytokines and organ pa-
thology after IT in young ob/ob (obese)
and aged AL mice. (A) Body weights of naive
young (2 mo) WT and young ob/ob C57BL/6
mice, n = 6 mice/group. Representative pic-
tures of young-AL WT mice and obese ob/ob
naive mice are shown. (B) Total fat content in
naive young WT (2 mo), young ob/ob (2 mo),
and aged (18 mo) C57BL/6 mice was visualized
by MRI. Fat is bright. (C and D) Young AL (2 mo),
young ob/ob (2 mo), and aged AL (18 mo) mice
were treated with HD CD40/IL-2 and liver
(C) and gut (D) were stained with H&E and
scored for histopathology on day 2. Bars,
200 m. Arrows indicate areas of steatosis
and/or immune cell infiltration and asterisks
indicate patchy necrosis and/or fibrosis.
(EG) Young AL (2 mo), young ob/ob (2 mo),
aged AL (18 mo), and aged CR (18 mo) were
treated with LD CD40/IL-2 and serum was
collected day 2 of treatment and analyzed by
CBA for levels of: TNF (E), IL-6 (F), and IFN-
(G), n = 3 mice/group. (H and I) Young AL (2 mo),
young ob/ob (2 mo), aged AL (18 mo), and
aged CR (18 mo) were treated with LD CD40/
IL-2 (H) or CpG/IL-2 (I) or rIgG/PBS control and
survival was monitored. (J) Serum levels of TNF
were measured on day 2 of CpG/IL-2 therapy
by CBA. Data in all panels are representative of
one of at least three experiments with similar
results. MRI scans are representative of at
least three mice imaged per group with similar
results. Survival data were plotted using the
Kaplan-Meier method and statistical differ-
ences were determined with the log-rank test.
Bar graph (mean value SEM) statistics per-
formed using Students t test or one-way
ANOVA with Bonferroni post-hoc tests. ***,
P < 0.001; **, P < 0.01; *, P < 0.05.
was comparable to the aged AL mice and significantly greater
than the young AL (Fig. 3, C and D).
Because HD IT resulted in significant changes within the
obese ob/ob mice, we next administered the lower dose (LD)
of IT to young ob/ob, young age-matched AL mice, aged AL,
and aged CR mice. Administration of LD IT still resulted in
significant increases in TNF and IL-6 proinflammatory cyto
kine levels within the young ob/ob that were not significantly
different to the aged AL mice (Fig. 3, E and F). Both ob/ob and
aged AL were significantly greater than both the young AL
and aged CR mice, where CR in the aged led to significant
down-regulation in both TNF and IL-6 cytokine production
(Fig. 3, E and F). Although the young ob/ob and the aged AL
mice displayed increased levels of IFN-, these levels were not
significant among the groups (Fig. 3 G). Consistent with the
increase in proinflammatory cytokines at day 2 of IT treatment,
100% of young ob/ob mice succumbed to IT-induced lethality
within 4 days of treatment in contrast to 100% survival in the
2377
Figure 4. IT toxicity is TNF-dependent through M1 macrophage accumulation. (AD) Young (2 mo) WT and young (2 mo) ob/ob mice were
treated with either control rIgG/PBS or LD CD40/IL-2. Visceral adipose tissue and peritoneal lavages were collected on day 2 and assessed for the pres-
ence of macrophages identified as CD45+CD19F4/80+CD11b+. Ratio of M1/M2 macrophages (A), and total numbers (B) and percentages (C) of TNF-
producing macrophages. Macrophages identified as M1 macrophages were characterized as CD206; M2 macrophages were gated as CD206+. (EH)
Young AL (2 mo) and young ob/ob (2 mo) were treated with either control or macrophage depleting clodronate liposomes before and during LD CD40/IL-2
and survival was monitored; n = 5 mice/group. (F and G) On day 2, visceral adipose tissues and peritoneal lavages were collected and assessed for the
percentages TNF+ macrophages (F and G) as characterized in experiments in AD and serum TNF levels (H), n = 4 mice/group. (IL) Young AL (2 mo) and
young ob/ob (2 mo) mice were treated with either control hIgG/PBS or subcutaneous injection of 1.5 mg/0.1 ml etanercept before and during LD CD40/
IL-2 and survival was monitored (I), n = 5 mice/group. (J and K) On day 2, visceral adipose tissue and peritoneal lavages were collected and assessed by
flow cytometry for the percentages of TNF+ macrophages (J and K) and serum TNF levels (L); n = 4 mice/group. Data in all panels are representative of one
of at least three experiments with similar results. Survival data were plotted using the Kaplan-Meier method and statistical differences were determined
with the log-rank test. Bar graph (mean value SEM) statistics performed using one-way ANOVA with Bonferroni post-hoc tests. **, P < 0.01; *, P < 0.05.

young WT cohort (Fig. 3 H). 100% of aged AL mice yielded
to lethality from the IT by day 2 of treatment, whereas 100%
of aged CR mice survived the entire regimen (Fig. 3 H).
The pathological and lethal response observed within the
young ob/ob mice was not CD40/IL-2 IT administration
specific. Young AL and age-matched young ob/ob mice were
administered CpG in combination with HD IL-2 and, like
wise, resulted in lethality of 75% of young ob/ob mice by day 5
of treatment but resulted in complete tolerance by young
AL mice (Fig. 3 I). CpG/IL-2 therapy led to significantly in
creased levels of serum proinflammatory cytokine TNF in an
analogous trend as CD40/IL-2 administration on day 2 of
therapy (Fig. 3 J).
This data further supports that increased adiposity plays a
crucial role in the induction of systemic toxicities after IT.
These data are suggestive that not only does an aging environ
ment result in lethal toxicities but toxicity may also be depen
dent on the increase in fat accumulation as the young ob/ob
mice succumb to cytokine storm induction and mortality after
IT, similar to that observed in the aged mice.
IT toxicity is TNF- and M1 macrophagedependent within
the peritoneum and visceral adipose tissues
A hallmark of obesity is the increasing infiltration of macro
phages into adipose tissues (ATMs) through the secretion of
MCP-1 by adipocytes. Phenotypically, these macrophages are

2378 Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.


predominately classically activated M1 cells that also express
proinflammatory mediators IL-1, TNF, and IL-6 (Lumeng
et al., 2007; Fujisaka et al., 2009). As such, we next sought to
determine if increases in M1 macrophages within the perito
neal cavity and visceral adipose tissues could account for the
pathological increases in systemic TNF during IT administra
tion.Young AL and age-matched young ob/ob mice were ad
ministered LD CD40/IL-2. At day 2 of treatment, visceral
adipose tissues and peritoneal lavages were phenotypically as
sessed by flow cytometry for the presence of M1 (CD206)
macrophages, M2 (CD206+) macrophages, and percentages
of TNF-expressing macrophages. Consistently, young ob/ob
mice demonstrated significant increases in M1 macrophage
proportions during IT administration, whereas young AL mice
were able to maintain consistent M1-to-M2 ratios (Fig. 4 A).
Additionally, both the visceral adipose tissues and peritoneal
lavages of ob/ob mice experienced significant increases in per
centages of TNF-expressing macrophages as opposed to young
AL mice (Fig. 4, BD). Significant changes were not observed
in the spleen of either young ob/ob or AL mice (unpublished
data).This data, consistent with obesity literature, suggests that
increases in adipose tissue accumulation within the visceral
cavity may impact regulation of M1 and M2 macrophage ra
tios that ultimately may affect TNF responses to immune stim
ulation with immunotherapeutic regimens.
Given the increases in M1 macrophages within the obese
mice and dysregulation observed with IT, young ob/ob and
young AL mice were treated with LD CD40/IL-2 in conjunc
tion with either liposomal clodronate for macrophage deple
tion (Fig. 4, EH) or etanercept (Enbrel) for TNF blockade
JEM Vol. 211, No. 12
Ar ticle
Figure 5. DIO succumb to cytokine storm,
organ pathology, and lethality similar to aged
AL mice after IT. Young C57BL/6 mice were
placed on a 60% fat diet starting at 3 wk of age
to induce obesity (young DIO). Lean age-matched
controls (young lean) were fed a 10% fat diet
beginning at 3 wk of age. Young mice were ana-
lyzed at 5 mo of age, and aged AL mice were
analyzed at 18 mo of age, n = 6 mice/group. Av-
erage body weight (A), representative pictures (B),
and total fat content as measured by MRI (C) are
shown for each group. (DF) Young lean (5 mo),
young DIO (5 mo), and aged AL (18 mo) mice
were treated with HD CD40/IL-2 or rIgG/PBS.
Serum was collected day 2 after the start of
treatment and analyzed by CBA for levels of TNF
(C), IL-6 (D), and IFN- (E) and survival was ana-
lyzed (F). Data in AC are representative of one of
at least three experiments with similar results and
data in DG are representative of one of at least
four experiments with similar results. Survival
data were plotted using the Kaplan-Meier method
and statistical differences were determined with
the log-rank test. Bar graph (mean value SEM)
statistics performed using one-way ANOVA with
Bonferroni post-hoc tests. ****, P < 0.0001;
***, P < 0.001; **, P < 0.01; *, P < 0.05.
(Fig. 4, IL). Both macrophage depletion and co-treatment
with etanercept during LD CD40/IL-2 resulted in complete
protection of young ob/ob mice from lethality (Fig. 4, E and I).
As expected, treatment with liposomal clodronate led to signif
icantly decreased levels of TNF-producing macrophages within
both the visceral fat and peritoneal lavages (Fig. 4, F and G),
which significantly reduced systemic circulating serum levels
of proinflammatory cytokine TNF (Fig. 4 H). Additionally,
co-administration of LD CD40/IL-2 with etanercept did
not reduce the percentages of TNF-expressing macrophages
within the visceral fat or peritoneal cavity (Fig. 4, J and K) but
mediated complete protection through an overall reduction
in systemic TNF levels (Fig. 4 L).
Together, these data indicate that proinflammatory M1
macrophages are the cellular mediator in the induction of IT
toxicity, particularly through increased presence within the peri
toneal and visceral adipose tissues. Importantly, these effects are
TNF-dependent, as blocking TNF levels either through etan
ercept administration or macrophage depletion resulted in com
plete protection of young ob/ob mice from lethality.
Diet-induced obese (DIO) mice succumb to cytokine
storm, organ pathology, and lethality similar to ob/ob
and aged AL mice after IT
ob/ob mice are congenic for the spontaneous mutation in the
OB gene, resulting in leptin hormone deficiency. To extend
our findings, we generated young DIO mice. Mice were placed
on a purified 60% fat diet to induce obesity beginning at 3 wk
of age and were determined to be obese when weighing >40 g
(DIO). Age-matched counterparts were placed on a matching
2379
purified diet that was 10% fat and served as lean controls. In
creases in body mass were validated in DIO mice before IT
administration (Fig. 5, AC). DIO mice displayed increases in
overall body weight and size (Fig. 5, A and B). MRI analysis
of body fat content showed increased total body fat accumu
lation, largely as increased visceral fat, in young DIO mice that
was similar to the aged AL mice (Fig. 5 C).
Confirming our previous findings, DIO mice treated with
HD CD40/IL-2 displayed increased serum TNF, IL-6, and
IFN- levels compared with age-matched young mice on a
10% fat diet after IT administration (Fig. 5, DF).The increases
in proinflammatory cytokines in DIO mice were lower, yet not
significantly so, than aged mice after IT (Fig. 4, CE). Young
DIO mice started succumbing to IT lethality at day 3 of treat
ment (>60%), and <40% were still alive by day 4 (Fig. 5 G).
Together, these results suggest that adipose tissue is a criti
cal component to the development of systemic cytokine storm
and lethality after IT.Yet, due to the observation that treatment
of DIO mice did not result in 100% of mice succumbing to
lethality, aging itself may also likely contribute to the overall
heightened inflammatory responses incurred by IT.
DISCUSSION
After initiation of strong immune stimulation with IT, we
show that both young obese and aged AL mice demonstrate
increased levels of adiposity, which led to aberrant increases in
both TNF and IL-6 levels that ultimately resulted in multi-
organ damage. Similar to aging, obesity has been shown to be
immunomodulatory, where the cross talk between adipocytes
and macrophages is thought to lead toward the development
and perpetuation of a meta-inflammatory state through con
tinual NF-B activation, which is currently hypothesized to
be responsible for the induction of metabolic diseases. A study
conducted by Spaulding et al. (1997) examined the effects of
age and calorie restriction on the production of TNF and IL-6
in resting mice and demonstrated that serum levels of both
cytokines were significantly higher in aged mice in compari
son to young, and yet aged mice subjected to long-term calo
rie restriction resulted in TNF and IL-6 serum levels that
were comparable to the young mice.These findings lend sup
port to the protective effects seen within our studies in the
aged CR cohort administered IT.
Importantly, the data presented here demonstrate the need
for using age and body fat content as variables in preclinical
assessment of therapeutics and modeling diseases, such as can
cer. However, the vast majority of inbred mouse studies are
housed in specific pathogen-free colonies where normal aging
may result in higher body fat content and yet less immune
challenge from pathogens making them more susceptible to
proinflammatory responses and toxicities after immune thera
pies. It will therefore be important that future studies ascer
tain responses in mice that have been exposed to pathogens
and immune challenges as they age.
Furthermore, our data demonstrate that an obese phenotype
resulted in lethal consequences that were ameliorated through
calorie restriction in the aged. Also, differences in lethality
2380
were observed between young obese and aged AL mice, where
aged mice die by day 2 but young obese mice die at day 4 of
treatment. Therefore, although our data strongly demonstrates
that adiposity plays a central role in the induction of toxicities,
aging is also a likely key factor. Therefore, our findings high
light the importance in considering both the status of aging
and obesity of patients receiving systemic administration of stim
ulatory IT regimens. It will be important to conduct studies
dissociating the consequences of aging upon increased cytokine
responsiveness, possibly through either investigating epigene
tic modifications induced by the meta-inflammatory and/or
inflammaging states associated with increased adipose accumu
lation or whether aging is accompanied by changes in expres
sion levels of various receptors that allow for a quicker response.
The development of IT regimens continues on a positive
trajectory with increasing successes within a variety of can
cers. Active IT has taken a greater comprehensive form focus
ing on activating humoral and cell-mediated immunity, and
these strategies have included antigen-specific vaccines (i.e.,
MART-1 vaccines), DC-based vaccines (i.e., Sipuleucel-T),
inhibitors of checkpoint blockade (i.e., CTLA-4 and PD-1),
and systemic stimulatory regimens through cytokine based
therapies and application of agonistic antibodies (i.e., CD40,
IFN-, and IL-2). Our data presented herein demonstrates
that both young obese and aged AL mice succumb to rapid
lethality during systemic administration of stimulatory thera
pies. Our data strongly suggests that excess adiposity may be a
prognostic factor in determining patient outcome and treat
ment tolerance. However, additional studies need to be con
ducted examining the effects of adiposity on the development
of adverse reactions within other classes of IT regimens, such
as checkpoint inhibitors.
Clinically, we hypothesize that taking into account patient
BMI, adipose accumulation, and age may better modulate treat
ment type choices as well as the inclusion of supportive thera
peutics such as TNF blockade in conjunction with anti-cancer
therapies that, overall, may increase patient survival and lead
to greater anti-cancer responses. Follow-up investigations in
tumor-bearing models merit further study.
MATERIALS AND METHODS
Animals and diets. Female young (25 mo old), middle-aged (912 mo old),
and aged (1522 mo old) C57BL/6 mice were purchased from Charles River
or from the animal production area at the National Cancer Institute. Young
female B6.Cg-Lepob/ob and young age-matched female WT control C57BL/6
mice were purchased from The Jackson Laboratory at 812 wk of age.
DIO obese and control lean mice were generated from WT C57BL/6
mice purchased at 3 wk of age from Charles River or from the animal produc
tion area at the National Cancer Institute. Mice were placed on an open-source
purified diet consisting of either 60% high-fat (D12492) or 10% low-fat
(D12450J) constitution (Research Diets, Inc.). Age-matched mice were placed
on either a high-fat or low-fat diet beginning at 3 wk of age for 1620 wk
and weighed weekly. Mice were deemed obese upon reaching a minimum of
40 g of weight.
For studies comparing aged AL and CR dietary effects, both female aged
CR and age-matched female AL-littermates (1522 mo old) were purchased
from the CR rodent colony at the National Institutes on Aging, National In
stitutes of Health (NIH). In brief, mice were either raised through free access,
AL, feeding on the NIH31 regular chow, or placed on a limited daily feeding
Obesity induces immunostimulatory therapy toxicity | Mirsoian et al.

Table 1. Grading of acute liver damage
Global assessment
0: None
I: Minimal/indeterminate Portal inflammatory infiltrate is in a minority of the portal triads, which is minimal
II: Mild Inflammatory infiltrate in 50% of the triads, which is generally mild, and confined within the portal spaces
III: Moderate Inflammatory infiltrate, expanding most or all of the triads, and associated with mild interface and lobular activity
IV: Severe As above for moderate, with spillover into periportal areas and moderate to severe perivenular inflammation that
extends into the hepatic parenchyma and is associated with perivenular hepatocyte necrosis
schedule with the NIH31-fortified chow. CR is initiated at 14 wk of age at
10% restriction, increased to 25% restriction at 15 wk, and to 40% restriction
at 16 wk of age where it is maintained throughout the life of the animal. In
formation on survival curves, food intake curves, and body weights of the
NIA strains has been previously published (Turturro et al., 1999).
All experimental mice were housed in the Animal Facilities at the Uni
versity of California, Davis under specific pathogen-free (SPF) conditions.
For the comparison in fat volume ratios, young, middle-aged, and aged AL
and CR mice were ordered and sent to the National Institute on Aging for
MRI analysis and also housed under SPF conditions. All of the animal proto
cols were approved by the Institutional Animal Care and Use Committees of
both institutions. Body weights were also measured for several mice at differ
ent ages and on dietary regimens. Mice were euthanized by CO2 asphyxia
tion and the visceral fat pads (mesenteric, gonadal, and renal) were collected
and weighed together for each mouse to determine the visceral fat weight.
Reagents. The agonistic antimouse CD40 antibody (clone FGK115B3)
was generated via ascites production in our laboratory as previously described
(Murphy et al., 2003; Bouchlaka et al., 2013). The endotoxin level of the
CD40 was <1 endotoxin unit/mg of antibody as determined by a quantita
tive chromogenic limulus amebocyte lysate kit (QCL-1000; Bio Whittaker).
Recombinant human IL-2 (rhIL-2; TECIN Teceleukin) was provided by the
National Cancer Institute (NCI). Murine CpG ODN 1826 was purchased
from InvivoGen. All were injected i.p.
Schedule of CD40/IL-2 and CpG/IL-2 IT treatments. Mice were
treated with agonist CD40 antibody and rhIL2 as previously described
(Murphy et al., 2003; Bouchlaka et al., 2013). CD40 or CpG was adminis
tered daily for a total of 5 consecutive days (days: 0, 1, 2, 3, and 4) and IL-2
was administered twice a day for a total of 4 d (days: 1, 4, 8, and 12). Control
mice received rat IgG (rIgG; Jackson ImmunoResearch Laboratories, Inc.)
and PBS (Cellgro). Survival was monitored daily. An HD or LD of CD40/
IL-2, or HD CpG/IL-2, was administered as follows on the same scheduling
pattern as previously described (Bouchlaka et al., 2013): HD CD40/IL-2,
mice received 80 g of agonist CD40 and 106 IU of IL-2 in 0.2 ml PBS i.p.;
control mice received 80 g rIgG in PBS; LD CD40/IL-2, mice received
40 g of agonist CD40 and 4 105 IU of IL-2 in 0.2 ml PBS i.p; control serum samples and standards were incubated for 1 h at 25C with a bead
mice received 40 g rIgG in PBS; CpG/HD IL-2, mice received 100 g CpG
ODN 1826 (InvivoGen) and 106 IU of IL-2 in 0.2 ml PBS i.p.
Macrophage depletion and TNF blockade with etanercept (Enbrel).
In some experiments, mice were in vivo depleted of macrophages using lipo
somal clodronate or control-loaded liposomes (Encapsula NanoScience) be
fore and during IT administration, as previously described (Bouchlaka et al.,
2013). For experiments where mice were euthanized by day 2 of treatment,
0.2 ml per dose was injected i.p. of either clodronate or control liposomes on
days 2 and 0. For all survival experiments, clodronate or control liposomes
were administered on days 2, 0, 2, and 4.
TNF blockade was preformed through in vivo subcutaneous injection
of 1.5 mg/0.1 ml etanercept (Enbrel) on days 1 and 1 for experiments where
mice were euthanized on day 2 of IT therapy. For all survival experiments,
etanercept was administered on days 1, 1, 3, 7, and 9.
JEM Vol. 211, No. 12
Ar ticle
Criteria
No portal inflammation
Murine macrophage studies. On day 2 of treatment, murine macrophages
were isolated from either the peritoneal cavity or visceral adipose tissue. For
peritoneal lavages, 4 ml of cold PBS was injected into the peritoneal cavity
of each mouse and then aspirated. Lavages were spun down at 1,200 rpm for
5 min and the resulting pellet was resuspended in RF10c media and plated
for flow cytometry staining.
The stromal vascular fraction of visceral adipose tissues was collected day
2 of treatment. Adipose tissues from the visceral cavity (gonadal) were col
lected and then incubated for 60 min in a digestion solution consisting of
2 mg/ml type IV collagenase (Sigma-Aldrich) dissolved fresh in a 2% BSA in
PBS solution. After incubation samples were centrifuged at 1,200 rpm for
5 min for layer separation of the stromal vascular fraction from adipocytes.
The resulting stromal vascular fraction was then removed, resuspended into
a single cell suspension, counted, and plated for flow cytometry staining.
Flow cytometry analysis of macrophages. Single cell suspensions from
the peritoneal lavages and visceral adipose tissues were plated in RF10c media
with the addition of GolgiPlug (containing Brefeldin A) and GolgiStop (con
taining Monensin) Protein Transport Inhibitors (BD) according to manufac
turers protocols. Plates were incubated for 9 h at 37C and 5% CO2.
After incubation, cells were washed with PBS and stained for flow analy
sis. Single cell suspensions were labeled with Fc block (purified antimouse
CD16/32; BD) for 10 min, followed by labeling with antibodies for 20 min
at 4C. Cells were washed twice with a staining buffer consisting of 5% FBS
(Gemini Bio-products) in DPBS (Corning). For intracellular staining, the
Cytofix/Cytoperm kit (BD) was used per manufacturers protocols. After
staining, cells were analyzed using a custom configured Fortessa cytometer,
and by using FACSDiva software (BD). Data were analyzed using FlowJo
software vX (Tree Star). Antibodies used to distinguish macrophages were:
PB-conjugated antimouse CD45, BV711-conjugated antimouse CD11b,
BV605- and PE-conjugated antimouse CD206, BV785-conjugated anti
mouse CD19, APC-conjugated antimouse F4/80, and PE-Cy7conjugated
antimouse TNF (BioLegend).
Serum cytokine bead array. Serum levels of TNF, IFN-, and IL-6 were
quantified by multiplex measurement using the Cytometric Bead Array
(CBA; BD) kit as described previously (Bouchlaka et al., 2013). In brief,
mixture containing bound antibodies to TNF, IFN-, and IL-6 according to
manufacturers instructions. Samples and standards were resuspended in wash
buffer and analyzed by flow cytometry. Data were acquired on a custom-
configured Fortessa cell analyzer using FACSDiva software (BD) and analyzed
using FlowJo software (Tree Star). Each sample was analyzed in triplicate.
Upon analysis of raw data, the mean fluorescent intensities (MFIs) of each
bead cluster were quantified.Where indicated in the figures, protein concen
trations were extrapolated relative to a standard curve created by serial dilu
tion of the mouse positive control cytokine.
Colorimetric liver enzyme assay. Serum ALT was quantified using the
ID Labs ALT Enzymatic Assay kit (ID Labs Biotechnology Inc.) as previously
described (Bouchlaka et al., 2013). Colorimetric determination of ALT levels
was performed by reading the absorbance of each well at 340 nm on a plate
reader (VERSAmax turntable plate reader).The concentration of ALT (U/liter)
2381
Table 2. Grading of acute intestinal damage
Global assessment Criteria
0: None No inflammation
I: Minimal/indeterminate Scattered infrequent inflammatory infiltrate in a minority of crypts, which is minimal
II: Mild Inflammatory infiltrate in 50% of the crypts, which is generally mild, and confined within the mucosa epithelium with
mild architectural distortion
III: Moderate Inflammatory infiltrate involving most of the crypts, and associated with architectural distortion (villous blunting and
focal mucosal atrophy/erosion) and luminal inflammatory exudate
IV: Severe Extensive inflammatory infiltrate (large lymphoid aggregates) with expanding into the lamina/muscular propria, and
associated with prominent mucosal ulceration/denuding, muscular wall necrosis, and/or perforation

in each sample was then directly determined from the change in absorbance
within 5 min time. Dilutions of the Pyruvate Control, included in the kit,
were used to construct a standard curve to calibrate the assay. Every serum
sample was assayed in triplicate.
Histopathology and grading/scoring. Liver and whole intestines were
collected on day 2 of IT, flushed, and fixed in 10% paraformaldehyde, embed
ded in paraffin, cut into 5-m sections, and stained with hematoxylin and
eosin (H&E). All tissues were prepared and stained at Histology Consultation
Services, Inc. (Everson, WA). Images were captured with a microscope (BX4;
Olympus) equipped with a Q-color3 camera and 10 numerical aperture
objective lens. Magnification for each captured image is specified in the figure
legends. Grading of histopathological inflammation was performed using a
scale from 0 to 4 in a blind fashion by a board-certified pathologist (M. Chen)
at the UC Davis Medical Centers Department of Pathology and Laboratory
Medicine. Specifically, the grading score for liver and gastrointestinal tract
followed our previously published grading method (Bouchlaka et al., 2013),
summarized in Tables 1 and 2.
normal body temperature using warm air. Mice were inserted into the scan
ner in a head-first prone orientation. Spin-echo T1-weighted images were
obtained through the mouse body (neck-to-base of the tail) using a 72-mm
linear volume coil. Scan sequence parameters were the following: TR 1,000 ms,
TE 15 ms, and 2 averages. The field of view was 7.7 3.85 2.0 cm, with a
matrix size of 256 128 40. The corresponding voxel size was 0.3 0.3
0.5 mm. Images were acquired using ParaVision 5.0 software. After acquisi
tion, images were transferred into Invenon Research Workplace 4.0 software
(Siemens Preclinical) allowing fat to be displayed with high intensity (white).
Statistical analysis. Statistical analyses were performed using Prism soft
ware (GraphPad Software Inc.). Data were expressed as mean SEM. For
analysis of three or more groups, a nonparametric ANOVA test was per
formed with a Bonferroni post-hoc test. Analysis of differences between two
normally distributed test groups was performed using the Students t test.
Non-parametric groups were analyzed with the Mann-Whitney test.Welchs
correction was applied to datasets with significant differences in variance be
fore Students t test.

We thank Monja Metcalf and Weihong Ma for technical help.

MRI. To quantify abdominal fat, MR images were acquired in collaboration
with the National Institute on Aging. Images were acquired using a Biospec 7
Tesla 30-cm MRI scanner (Bruker Biospin) with a 72-mm diameter transmit-
receive birdcage coil. In each experiment, two mice were inserted into the
scanner, side by side, in feet-first prone orientation immediately after sacrifice.
During scanning, the mice were cooled with a stream of cold air from a vortex
tube (Exair, Inc.) to minimize decomposition. Two different pulse sequences
were used: a heavily T1-weighted fast spin echo (RARE) sequence yielding
bright-fat (water-suppressed [WS]) images and a fat-suppressed proton density
weighted fast gradient echo (FLASH) sequence yielding fat-suppressed (FS)
images. 3D datasets were acquired in axial orientation with a field of view of
72 36 80 mm (left-right anterior-posterior head-foot) and matrix size
256 128 128.The voxel size (spatial resolution) was 281 281 625 m.
The spectral bandwidth in both sequences was 100 kHz (391 Hz/pixel) and
two averages were acquired for improved signal-to-noise ratio. For the RARE
sequence, the RARE factor (i.e., number of spin echoes per shot or number
of k-space lines per segment) was 8, the repetition time (TR) was 125 ms, the
actual echo time (TE) was 11.4 ms, and the effective echo time (TEeff) was
46.3 ms. Each RARE scan took 8 min and 32 s to acquire. For the FLASH
sequence, the repetition time was 25 ms and the echo time was 3.2 ms. The
excitation pulse had a flip angle of 30. Each FLASH scan took 13 min 39 s to
acquire. Image processing was performed in ImageJ 1.45 (NIH). The statisti
cal analyses were performed using one-way ANOVA and the Newman-Keuls
post-hoc test. P < 0.05 was considered to be significant.
To visualize fat distribution and content imaging of young ob/ob, DIO,
and lean age-matched controls were conducted in collaboration with the
Center for Molecular and Genomic Imaging (CMGI) Facility at the Univer
sity of California, Davis. Mice were anesthetized before imaging using 2%
isoflurane in an induction chamber and then maintained on 12% isoflurane
throughout imaging via a nose cone fitted for anesthetic inhalation. Mice
were imaged using a BioSpec 70/30 7T (Bruker Biospin) horizontal bore
system designed specifically for small animal imaging. Animals were kept at
This work has been supported by NIH grants CA0905572 and AG034874 and
by the Intramural Research Program of the National Institute on Aging, NIH.
The authors declare no competing financial interests.
Author contributions: A. Mirsoian, M.N. Bouchlaka, D.D. Taub, and W.J. Murphy
designed the research; A. Mirsoian, M.N. Bouchlaka, G.D. Sckisel, M. Chen, C.C.S. Pai,
A.M. Monjazeb, and D.D. Taub performed the research; A. Mirsoian, M.N. Bouchlaka,
D.D. Taub, and W.J. Murphy analyzed the data; M. Chen analyzed and scored all
histology data; E. Maverakis provided etanercept (Enbrel) for TNF blockade studies;
R.G. Spencer, K.W. Fishbein, and S. Siddiqui performed and analyzed MRI studies;
B. Martin, C. Hesdorffer, L. Ferrucci, and S. Maudsley provided aged mice and assisted
with data interpretation; A. Mirsoian, M.N. Bouchlaka, D.D. Taub, and W.J. Murphy
wrote the paper; and G.D. Sckisel, D.L. Longo, B.R. Blazar, and R.H. Wiltrout helped with
the editing of the paper. All authors read and approved the manuscript.
Submitted: 18 January 2014
Accepted: 6 October 2014
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2383
INSIGHTS

Cr itical role for CX 3 CR1 + mononuclear phagocytes in

intestinal homeostasis
A key challenge at the intestinal barrier is to minimize responses to commensal bacteria, which can lead to
inflammatory bowel disease (IBD) in genetically predisposed individuals, but retain the ability to recognize
and control the growth of infectious pathogens. Group 3 innate lymphoid cells (ILC3) help maintain intes-
tinal homeostasis by producing the cytokine IL-22, which promotes mucosal healing and maintains barrier
integrity. Microbial signals trigger production of IL-23 and IL-1b, which stimulate ILC3s to produce IL-22,
leading to the induction of antibacterial peptides and epithelial cell regeneration.
But the identity of the cell type producing IL-23 in response to microbial signals is unclear and has
Insight from been the subject of much debate; resident mononuclear phagocytes, inflammatory monocytes, and
Giorgio Trinchieri
conventional dendritic cells have all been implicated. In this issue, Longman et al. provide compelling
evidence, both in mouse following Clostridium rodentium infection and in patients with colitis, that
CX3CR1+ mononuclear phagocytes (MNPs) are the most potent producers of IL-23 and IL-1b and are very efficient in
inducing IL-22 production by ILC3.
The authors demonstrated the importance of microbial stimulation in IL-22 induction
in patients with surgical diversion of the fecal streamIL-22 production by ILC3 was lower in
the sites unexposed to the gut microbiota compared with exposed sites. Microbial TLR4 and
TLR9 agonists were particularly efficient at inducing IL-23 and IL-1b production by CX3CR1+
MNPs. The authors also discovered that a gene significantly associated with both ulcerative
colitis and Crohns disease in GWAS, TNF-like ligand 1A (TL1A or TNFSF15), is overexpressed
in mouse CX3CR1+ MNPs and synergizes with IL-23 and IL-1b to induce IL-22 production
in both human and mouse ILC3.
The study does not completely exclude the role of other cell types in the production of IL-23 and
induction of IL-22 production but in the conditions studied (mouse infection with C. rodentium and
human colitis), CX3CR1+ MNPs were crucial in mediating this mucosal protective loop. In basal
conditions or in other types of inflammatory or preneoplastic conditions, and under stimulation by
different microbial TLR agonists (e.g., flagellin), it is quite possible that other cell types, including
conventional dendritic cells, may play an important role, as suggested by published studies. However, Confocal immunofluorescence
the study by Longman et al. greatly contributes to our understanding of the mechanisms of human image of mouse colon shows
IBD and reassures us that when mouse data are carefully combined with experimental human studies juxtaposition (white arrows)
and GWAS, they are powerful in providing mechanistic evidence that explains human pathology. of CX3CR1+ MNPs (green) and
Longman, R.S., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140678. RORgt+ ILC3 (red).

Giorgio Trinchieri, National Cancer Institute, National Institutes of Health: trinchig@mail.nih.gov

2 INSIGHTS | The Journal of Experimental Medicine

 Article

CX3CR1+ mononuclear phagocytes support

colitis-associated innate lymphoid cell
production of IL-22
Randy S. Longman,1,3 Gretchen E. Diehl,1 Daniel A. Victorio,1,3
Jun R. Huh,1 Carolina Galan,1 Emily R. Miraldi,1,5,6 Arun Swaminath,4
Richard Bonneau,5,6 Ellen J. Scherl,3,4 and Dan R. Littman1,2
1The Kimmel Center for Biology and Medicine of the Skirball Institute and 2Howard Hughes Medical Institute, New York
University School of Medicine, New York, NY 10016
3The Jill Roberts Center for IBD, Department of Medicine, Weill-Cornell Medical College, New York, NY 10021
4Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032
5Center for Genomics and Systems Biology, Department of Biology; and 6Courant Institute of Mathematical Sciences,

Computer Science Department, New York University, New York, NY10003

Interleukin (IL)-22producing group 3 innate lymphoid cells (ILC3) promote mucosal heal-
ing and maintain barrier integrity, but how microbial signals are integrated to regulate
mucosal protection offered by these cells remains unclear. Here, we show that in vivo
depletion of CX3CR1+ mononuclear phagocytes (MNPs) resulted in more severe colitis and
death after infection with Citrobacter rodentium. This phenotype was rescued by exogenous
IL-22, which was endogenously produced by ILC3 in close spatial proximity to CX3CR1+
MNPs that were dependent on MyD88 signaling. CX3CR1+ MNPs from both mouse and
human tissue produced more IL-23 and IL-1 than conventional CD103+ dendritic cells
(cDCs) and were more efficient than cDCs in supporting IL-22 production in ILC3 in vitro
and in vivo. Further, colonic ILC3 from patients with mild to moderate ulcerative colitis or
Crohns disease had increased IL-22 production. IBD-associated SNP gene set analysis
revealed enrichment for genes selectively expressed in human intestinal MNPs. The product
of one of these, TL1A, potently enhanced IL-23 and IL-1-induced production of IL-22
and GM-CSF by ILC3. Collectively, these results reveal a critical role for CX3CR1+ mono-
nuclear phagocytes in integrating microbial signals to regulate colonic ILC3 function in IBD.

CORRESPONDENCE
Randy S. Longman:
ral2006@med.cornell.edu
OR
Dan R. Littman:
dan.littman@med.nyu.edu
Abbreviations used: CD,
Crohns disease; DT, diphtheria
toxin; DTR, DT receptor; IBD,
inflammatory bowel disease;
ILC3, group 3 innate lymphoid
cell; MAMP, microbe-associated
molecular pattern; MNP,
mononuclear phagocyte;
PAMP, pathogen-associated
molecular pattern; UC, ulcer-
ative colitis.
Inflammatory bowel disease (IBD) has been de-
fined as a dysregulated cellular immune response
to environmental triggers in genetically predis-
posed individuals. Although the initial discovery
linking single-nucleotide polymorphisms in the
IL23R locus with susceptibility to IBD (Duerr
et al., 2006) was consistent with a role for IL-23
responsive T cells, more recent evidence supports
the importance of IL-23responsive innate lym-
phoid cells (ILC) in maintaining epithelial ho-
meostasis (Sonnenberg and Artis, 2012). These
RORt-dependent ILCs (now named group 3
ILCs, or ILC3 (Spits et al., 2013)) were initially
R.S. Longman and G.E. Diehl contributed equally
to this paper.
J.R. Huhs present address is University of Massachusetts
Medical School, Worcester, MA 01605.
A. Swaminaths present address is Lennox Hill Hospital,
New York, NY 10075.
characterized in mouse models of colitis as pre-
dominant producers of IL-22 (Satoh-Takayama
et al., 2008), an IL-10 family member that sig-
nals via STAT3 to regulate mucosal healing, a
critical clinical endpoint in IBD (Pickert et al.,
2009; Hanash et al., 2012). In light of their ro-
bust production of IL-22 and close proximity
to the intestinal epithelial layer (Cella et al., 2009),
ILC3 have been proposed to play an important
role in mucosal healing and maintenance of
barrier integrity, and understanding how they
are induced to produce IL-22 has great poten-
tial for therapeutic benefit.
2014 Longman et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (AttributionNoncommercial
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).

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J. Exp. Med. 2014 Vol. 211 No. 8 1571-1583 1571
www.jem.org/cgi/doi/10.1084/jem.20140678
 Mononuclear phagocytes (MNPs) are sentinels of the intes-
tinal lamina propria, capable of responding to microbial prod-
ucts, and play a crucial role in orchestrating intestinal lymphocyte
homeostasis. MNPs can be subdivided based on their expression
of CD103 or CX3CR1, and each group has been ascribed criti-
cal functions in maintaining intestinal homeostasis (Bogunovic
et al., 2009; Merad et al., 2013). CD103+ cells, which can be
further subdivided based on the expression of CD11b, differen-
tiate from a common DC precursor and are thought to be
the conventional, migratory myeloid DCs (Varol et al., 2010).
CD103+ CD11b DCs require Irf8, Id2, and Batf3 for their de-
velopment and are thought to play a critical role in cross-priming
virus- and tumor-specific CTLs (Hildner et al., 2008; Merad
et al., 2013). Loss of these cells, however, does not alter intestinal
T cell homeostasis or lead to spontaneous inflammation (Edelson
et al., 2010). CD103+CD11b+ DCs, in contrast, require Notch2
signaling, produce IL-23 in response to flagellin-induced TLR5
activation, resulting in IL-22 production by ILC3, and have ad-
ditionally been proposed to support Th17 polarization (Lewis
et al., 2011; Kinnebrew et al., 2012). These cells can produce
retinoic acid, which promotes the expression of the gut-homing
receptor CCR9 and synergizes with TGF to induce regulatory
T cells (Sun et al., 2007). One recent study suggests that Notch2-
dependent CD103+ CD11b+ DCs regulate protection from
C. rodentiuminduced colitis (Satpathy et al., 2013). However,
specific depletion of CD103+ CD11b+ intestinal DCs revealed
that these cells are not the MNP subset required for protection
against C. rodentium or IL-22 production (Welty et al., 2013).
In contrast to CD103+ cDCs, CX3CR1+ MNPs differenti-
ate from monocyte precursors (Varol et al., 2010). Although
these cells were previously thought to be tissue-resident and to
promote local Treg differentiation (Hadis et al., 2011), recent data
from our group showed that they can up-regulate CCR7 and
migrate to secondary lymphoid organs, suggesting a broader role
in orchestrating immunity (Diehl et al., 2013). Notably, we ob-
served that interaction with microbiota limits the migration of
these cells to mesenteric LNs (MLNs; Diehl et al., 2013), and an
increase in CX3CR1+ cells has been described in the lamina
propria during mouse (Zigmond et al., 2012) and human colitis
(Kamada et al., 2008). A recent study reported that fractalkine
receptor (CX3CR1) expression supports innate celldependent
clearance of C. rodentium infection (Manta et al., 2013), but a
functional role for CX3CR1+ MNPs in regulating colitis-
associated ILC3 remains unclear.To evaluate this question, we
employed novel mouse models to enable selective depletion of
CX3CR1+ MNPs in vivo. Our results reveal a critical role for
CX3CR1+ MNPs from both mouse and human tissue in sup-
porting IL-22 induction in ILC3 in vitro and in vivo. Moreover,
we identify the ability of TL1A produced by MNPs to potently
enhance IL-23 and IL-1induced production of IL-22 and
GM-CSF by ILC3.
RESULTS
CX3CR1+ cells protect against C. rodentiuminduced colitis
To investigate the role of the expanded population of CX3CR1+
cells in the intestinal lamina propria during colitis, we generated
1572
a mouse with the diphtheria toxin receptor (DTR) cDNA in-
serted into the Cx3cr1 locus (Diehl et al., 2013). Analysis of
colonic lamina propria mononuclear cells (LPMCs) after infec-
tion of DT-treated mice revealed a reduction in the percentage
of CD11c+ MHCII+ LPMCs (Fig. 1 A), which reflected a pref
erential loss of the CX3CR1+ CD11b+ CD14+ fraction of MNPs
(Fig. 1 B; Tamoutounour et al., 2012), as well as CX3CR1+
monocytes in Cx3cr1DTR/+ mice compared with control mice.
CD103+ CD11b+ cDCs were not depleted (Fig. 1 C).To induce
colitis, mice were infected with C. rodentium, a mouse model
for infectious colitis (Zheng et al., 2008; Sonnenberg et al., 2011;
Qiu et al., 2012). DT-treated infected Cx3cr1DTR/+ mice, but
not uninfected or control infected mice, lost more weight
(Fig. 1 D), displayed more severe intestinal pathology (Fig. 1 E),
and ultimately succumbed to infection (Fig. 1 F). Infected
Cx3cr1DTR/+ mice also had increased bacterial burden in the
spleen, consistent with the loss of barrier integrity (Fig. 1 G).
To examine potential involvement of signaling pathways
for receptors of pathogen- or microbe-associated molecular
patterns (PAMPs or MAMPs) in mediating this phenotype, mice
with a conditional deletion of MyD88 in CD11c-expressing
MNPs (CD11c-Cre/Myd88fl/fl) were infected with C. roden-
tium. Infection of CD11c-Cre/Myd88fl/fl mice, but not litter-
mate controls, was lethal by 15 d after infection (Fig. 2 A),
implicating PAMP/MAMP signaling as having a critical role
in barrier protection mediated by CD11c-expressing MNPs.
The C. rodentium colitis model depends on IL-22 for protec-
tion (Zheng et al., 2008). Thus, to test if exogenous IL-22
could rescue the susceptibility phenotype described above,
CD11c-Cre/Myd88fl/fl (Fig. 2 A) and DT-treated CX3CR1-
DTR (Fig. 2 B) mice were hydrodynamically injected with a
plasmid encoding IL-22 (Qiu et al., 2012). The exogenous
IL-22 rescued both lines of mice from colitis-induced death.
Colonic CX3CR1+ MNPs regulate ILC3 production of IL-22
High-dose infection with C. rodentium is controlled by ILC3,
which represents the large majority of LPMCs producing IL-22
(Sonnenberg et al., 2011). At day 7 after infection, both the
percentage and absolute number of IL-22+ colonic, lineage,
CD90hi, and RORt+ ILCs (Fig. S1 shows gating strategy)
from mice depleted for CX3CR1+ cells were reduced in com-
parison to ILCs from mice with intact CX3CR1+ cells (Fig. 2,
CE). Depletion of CX3CR1+ cells did not affect the abso-
lute number of ILC3 (Fig. 2 F). Although T cells can also con-
tribute to IL-22 production in low-dose C. rodentium infection
(Basu et al., 2012), no statistically significant difference in the
total IL-22+ (or IL-17+) T cells was noted in mice depleted
for CX3CR1+ cells (Fig. 2 G). To assess the ability of colonic
CX3CR1+ MNPs to interact with ILC3 within the colonic tis-
sue, Cx3cr1GFP/+ mice were used to visualize CX3CR1+ MNPs
in situ ( Jung et al., 2000). Consistent with the ability of these
cells to regulate ILC3 function, confocal microscopy revealed
the spatial proximity of RORt+ ILC3 cells with CX3CR1+
MNPs in the colonic lamina propria (Fig. 2 H).
To evaluate the ability of intestinal CX3CR1+ cells and
cDCs to support ILC3 activation, CX3CR1+ (GFP+) cells and
CX3CR1+ MNPs regulate ILC3 | Longman et al.

CD103+ CD11b+ DCs were sorted from the lamina propria
of Cx3cr1GFP/+ mice (Fig. 3 A) and co-cultured with intestinal
ILCs. TLR-stimulated CX3CR1+ cells were markedly more
efficient than CD103+ CD11b+ DCs in supporting IL-22 pro-
duction (Fig. 3, B and C). CX3CR1+ cells in the LP include
Ly6Chi monocytes and Ly6Clo MHCIIhi MNPs (Fig. 3 D;
Zigmond et al., 2012; Diehl et al., 2013).To evaluate the role of
these distinct CX3CR1+ populations in supporting ILC3 func-
tion, we sorted CX3CR1+ monocytes and CX3CR1+ MNPs
and co-cultured them with ILCs in the presence of TLR stim-
uli.TLR-stimulated MNPs were much more potent inducers of
IL-22 than monocytes (Fig. 3, E and F). In an effort to confirm
the functional potential of MNPs compared with monocytes
in vivo, we selectively ablated CD11c-expressing CX3CR1+
MNPs. CD11c-cre mice were bred to mice engineered to
JEM Vol. 211, No. 8
Ar ticle
Figure 1. Intestinal CX3CR1+ cells pro-
tect mice from C. rodentium-induced
colitis. (A and B) Depletion efficiency in the
Cx3cr1DTR/GFP mice was assessed by flow
cytometry of small intestinal lamina propria
cells Cx3crDTR/GFP or littermate control mice
after administration of DT to both groups
daily for 2 d. (A) Surface staining for CD11b
versus CX3CR1-GFP. (B) MHCII+ CD11c+ cells
were assessed for expression of CX3CR1,
CD103, and CD14. Results are representative
of five independent experiments with a mini-
mum of three animals per group. (C) Total
number of MHCII+ CD11c+CD103+ (left) and
MHCII+ CD11c+CX3CR1+ cells (right) per intes-
tine as determined by flow cytometry analy-
sis. n.s., P > 0.05; **, P 0.01. Two-tailed
Students t test. Error bars represent the SEM.
Results are representative of five independent
experiments with a minimum of 3 animals per
group. (D) Weight of DT-treated littermate WT
(control) mice or Cx3cr1DTR/+ mice following
infection with C. rodentium (n = 7 mice/
group). DT was administered at days 2, 1,
and 0 and every other day after infection.
Data are representative of two independent
experiments. (E) Representative colonic histol-
ogy from littermate control mice or Cx3cr1DTR/+
mice (analyzed in D) infected with C. rodentium
at day 7 after infection. <, areas of lympho-
cyte infiltration; *, areas of epithelial erosion.
Bar, 100 m. (F) Survival curves of DT-treated
Cx3cr1DTR/+ and littermate control mice in-
fected with C. rodentium or uninfected (n = 7
mice/group). Data are representative of three
independent experiments. Animals were
treated with DT as in D. (G) Bacterial CFUs of
spleens from littermate WT (control, n = 5)
mice or Cx3cr1DTR/+ mice (n = 5) infected with
C. rodentium and treated with DT as above.
*, P 0.05. Two-tailed Students t test. Error
bars represent the SEM. One of two represen-
tative experiments is shown.
express DTR only upon cre-mediated deletion of a LoxP-Stop
cassette inserted into the Cx3cr1 locus (Diehl et al., 2013). In-
jection of DT resulted in a selective loss of Ly6Clo MHCIIhi
MNPs, which express both intermediate and high levels
of CX3CR1-GFP, and spared the Ly6Chi monocytes, which
express intermediate levels of CX3CR1 (Fig. 4 A; Diehl
et al., 2013). Similar to results with CX3CR1DTR/+ mice, in
which both monocytes and MNPs were ablated, depletion of
CX3CR1+ CD11c+ cells led to reduction in colitis-induced
ILC3 production of IL-22 (Fig. 4 B).
Because both IL-23 and IL-1 regulate ILC3, we wished
to determine the contribution of these cytokines to the ob-
served regulation of IL-22 production by MNPs. LPS and CpG
stimulation induced markedly increased IL-23 and IL-1 pro-
duction by CX3CR1+ MNPs compared with CD103+ CD11b+
1573

DCs in vitro (Fig. 4, C and D). To evaluate the role of MNP-
derived IL-23 and IL-1, co-cultures were performed with in-
testinal CD11c+ cells derived from WT or Il23p19/ mice and
ILCs from WT or Il1r/ mice. IL-23deficient MNPs and
IL1R-deficient ILCs yielded significantly reduced production
of IL-22 (Fig. 4 E), consistent with the importance of MNP-
derived IL-1 and IL-23 in supporting IL-22 production.
These data reveal a mechanistic role for IL-23 and IL-1 pro-
duced by colonic MNPs in supporting colitis-associated ILC3
secretion of IL-22.
Human intestinal ILC3 production of IL-22
is regulated by microbial stimulation of MNPs
To evaluate the regulation of intestinal ILC3 in humans with
IBD, we prepared LPMCs from descending colon biopsies of
patients with endoscopically mild to moderate Crohns disease
1574
Figure 2. CX3CR1+ cells support colonic ILC3 pro-
duction of IL-22. (A) Survival curves of C. rodentium
infected Myd88f/f littermate controls (n = 10, open circle)
as compared with CD11c-cre/Myd88f/f mice without
(n = 13, filled circle) or with (n = 5, open triangle) exog-
enous hydrodynamic delivery of an IL-22producing
plasmid. Results are a composite of two independent
experiments. (B) Survival curves of DT-treated Cx3cr1DTR/+
mice infected with C. rodentium after hydrodynamic
delivery of a plasmid expressing IL-22 (n = 8) or control
vector (n = 9). DT was administered at days 2, 1, and 0
and every other day after infection. Results are a com-
posite of three independent experiments. (CE) Percent-
age (C and D) and total number (E) of colonic Lin
CD90.2+ ILCs producing IL-22 from DT-treated Cx-
3cr1DTR/+ (n = 10) and littermate control mice (n = 9) 7 d
after C. rodentium infection and from uninfected mice
(NT; n = 3). Results are a composite of two independent
experiments. DT was administered at days 2, 1, and 0
and every other day after infection. Intracellular IL-22 was
assayed by flow cytometry after 4-h culture. A represen-
tative flow cytometry plot from each group is shown
in C. **, P 0.01. One way ANOVA with Bonferronis
correction. Error bars represent the SEM. (F) Total
number of ILC3 per colon in Cx3cr1DTR/+ (n = 9) or control
(n = 8) mice administered DT. Error bars represent the
SEM. Results are one of three representative experiments.
(G) Total number of IL-17+ or IL-22+ colonic CD4+ T cells
from DT-treated Cx3cr1DTR/+ (n = 10) and littermate con-
trol mice (n = 9) 7 d after C. rodentium infection and
from uninfected mice (n = 3). Intracellular IL-22 and IL-17
was assayed by flow cytometry after 4-h culture.
*, P 0.05. One way ANOVA with Bonferronis correction.
Error bars represent the SEM. Results are a composite of
two independent experiments. (H) Confocal immuno-
fluorescence of colonic samples from Cx3Cr1GFP/+ mice
stained for CD3 and RORt. Bar, 10 m. White arrows
indicate sites of MNP and ILC3 juxtaposition.
(CD, n = 8) or ulcerative colitis (UC, n = 6;Table S1) as well as
age-matched non-IBD control patients undergoing routine
screening colonoscopy (n = 8). Analysis of intracellular cytokine
production revealed significantly increased IL-22 production
in the CD3 fraction of colonic LPMCs from sites of colonic
inflammation in both CD and UC compared with the non-IBD
controls (Fig. 5 A and Fig. S2). In contrast, IL-22 production
by T cells was not significantly different between the groups.
Further characterization of the nonT cells producing IL-22
revealed that a large fraction of these cells expressed c-Kit and
CD56, markers of ILCs (Cella et al., 2009; Fig. 5 B). Consis-
tent with their being ILC3, these cells were lineage negative
(Lin; Fig. S3 A); expressed RORt (Fig. 5 C); were CD45int
CD127+ (Fig. 5 D); expressed CD161, NKp44, and CCR6
(Fig. 5 E), which are phenotypic surface markers of ILC3; and
produced IL-22 in response to IL-23 stimulation (Fig. 5 F). As
CX3CR1+ MNPs regulate ILC3 | Longman et al.

Myd88 deficiency abrogated ILC3 production of IL-22, we
hypothesized that signals from the microbiota could induce
ILC3 to produce IL-22. To investigate this in human tissue, we
evaluated three patients who had a surgical diversion of the
fecal stream (i.e., a diverting ostomy) as part of their therapy
for IBD. Endoscopic biopsies were taken from a site proximal
to the diversion (afferent limb), where the mucosa was exposed
to intestinal microbiota in the fecal stream, and from mucosa
distal to the diversion (efferent limb), that was unexposed to
the fecal stream. ILCs were present at both mucosal locations,
but not in PBMCs from the same donor (Fig. S3 B). In all
three donors, ILCs from tissue exposed to bacteria in the fecal
stream produced substantially more IL-22 compared with
ILCs from unexposed tissue (postdiversion; Fig. 5 G).
JEM Vol. 211, No. 8
Ar ticle
Figure 3. TLR-stimulated CX3CR1+ MNPs
are stronger inducers of ILC3 production
of IL-22 than CD103+ CD11b+ DCs and
monocytes. (AC) CD103 or CX3CR1+ MHCII+
CD11c+ CD11b+ cells were isolated from the
lamina propria of CX3CR1GFP/+ mice (sort strat-
egy shown in A and co-cultured with Lin
RORt-GFP+ ILCs with or without the indi-
cated bacterial TLR ligands or IL-23. IL-22 was
assessed by intracellular staining of CD90.2+
ILCs after 18 h. A representative flow cytom-
etry plot is shown in B. (C) Percent IL-22+ ILCs
is shown from seven independent experi-
ments. **, P 0.01; *, P 0.05. One way
ANOVA with Bonferronis correction. Error
bars represent SEM. (DF) Ly6C+ MHCIIlo
(monocytes) and Ly6C MHCIIhi (MNPs) were
isolated from CX3CR1+ CD11b+ lamina propria
cells (sort strategy is shown in D and co-
cultured with intestinal ILCs with LPS or IL-23
as indicated. Intracellular cytokine staining for
IL-22 is shown after 18 h (E). Supernatants
were harvested after 18 h and IL-22 produc-
tion quantitated by ELISA (F). Results are rep-
resentative of two independent experiments
performed in triplicate. ***, P 0.001. One-
way ANOVA with Bonferroni correction. Error
bars represent the SEM.
Parallel subpopulations of CD11c+ MNPs present in the
mouse intestine similarly exist in the human intestine (Fig. 6 A;
Merad et al., 2013). To evaluate whether these distinct sub-
populations of MNPs from human intestinal tissue functioned
similarly, we examined the phenotypic properties of CD103+
DCs and CD14+ MNPs (which express CX3CR1; Kamada
et al., 2008) within the CD11c+ MHCII+ fraction of LPMCs
(Fig. 6 B). In contrast to CD103+ DCs, CD14+ MNPs ex-
pressed CD64 as well as higher levels of CD86. Consistent with
the phenotypic characterization of these subsets, transcriptional
analysis of these populations by RNA-seq revealed higher
levels of CLEC9A, XCR1, and CD207 expression in the
CD103+ cells, whereas MERTK, STAB1, and CX3CR1 were
higher in the CD14+ cells (Fig. 6 C). We tested the potential
1575
Figure 4. CX3CR1+ MNP-derived IL-23 and IL-1 activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1STOP-DTR/GFP
mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX3CR1hi MNPs. (bottom) Expression of Ly6C and MHCII on
CX3CR1hi and CX3CR1int populations. (B) Expression of IL-22 in Lin CD90.2+ colonic ILCs from Cx3cr1STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1STOP-DTR/+
(Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days 2, 1, and 0 and every other day postinfection. One representative
intracellular cytokine flow cytometry plot is shown on the left and a composite graph (n = 6/group) on the right. *, P 0.05, two-tailed Students t test.
Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures (Fig. 3, B and C) were
harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX3CR1+ MNPs or
CD103+ CD11b+ DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1 by ELISA. Results are the mean of
two independent experiments performed in duplicate and the SEM is shown. *, P 0.05; **, P 0.01. (E) Lin CD90.2hi ILCs from WT or Il1r/ mice were
co-cultured with sorted intestinal MNPs from WT or Il23p19/ mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after
18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P 0.05; ***, P 0.001. One-way ANOVA with Bonferroni
correction. Error bars represent the SEM.

of these subsets to induce IL-22 production by co-culturing
TLR-stimulated CD14+ MNPs and CD103+ DCs from human
intestinal resections with intestinal ILCs. Intracellular cyto-
kine staining at 18 h revealed that the CD14+ MNP were more
effective than the CD103+ DCs at stimulating IL-22 production
by ILCs (Fig. 6 D). Neither cell population induced signifi-
cant IL-17 or IFN- production by ILCs (Fig. 6, D and E).
Consistent with the importance of IL-23 and IL-1 in
the mouse co-culture experiments, a higher level of IL23A
expression was observed by RNA-seq in CD14+ MNPs com-
pared with CD103+ DCs (Fig. 6 C). Stimulation of these human
MNP subsets with LPS or flagellin revealed increased IL-23p19
mRNA and IL-1 protein produced by the CD14+ cells com-
pared with the CD103+ cells (Fig. 6 F). In accord with this
finding, in co-cultures of human intestinal ILC3 and CD14+
MNPs, IL-23 and IL-1 antibody blockade blunted IL-22
production (Fig. 6 G).These data reveal a mechanistic role for
the expanded population of CD14+ MNPs (Kamada et al.,
2008) in supporting colitis-associated IL-22 production by
ILC3, through their secretion of IL-23 and IL-1.
CX3CR1+ MNP-derived TL1A synergizes
with IL-23 and IL-1 to induce IL-22
We hypothesized that MNP-derived factors in addition to
IL-23 and IL-1 would contribute to the regulation of ILC3
function. The RNA-seq data from CD14+ human MNPs re-
vealed a significant enrichment for genes associated with IBD in
GWAS studies, suggesting that IBD-associated pathways are im
portant in these cells (Fig. 7 A and Table S2). Notably, we iden
tified TNF-like ligand 1A (TL1A, also designated TNFSF15)
as a significant contributor to the IBD GWAS-derived gene
set enrichment. Evaluation of Tnfsf15 transcript in sorted
mouse colonic APC subsets by qPCR confirmed higher ex-
pression in CX3CR1+ MNPs (Fig. 7 B). DR3/TNFRSF25,

1576 CX3CR1+ MNPs regulate ILC3 | Longman et al.

 Ar ticle

Figure 5. Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from de-
scending colon biopsies from patients with endoscopically mild to moderate Crohns disease (n = 8, gray) or ulcerative colitis (n = 6, black; Table S1),
as well as age-matched non-IBD control patients undergoing routine screening colonoscopy (n = 8, white), were stimulated ex vivo with PMA/ionomycin
and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3+ or CD3 fraction expressing IL-17 or IL-22 is
indicated. *, P 0.05, two-tailed Students t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3
IL-22+ (black lines) and CD3 IL-22 (gray) LPMCs. (C) Expression of RORt by c-Kit+CD56+ LPMCs. Lin cells (CD14/CD19/CD3/CD11b/CD11c/TCR;
Fig. S3 A) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORt. Lin CD56+ c-Kit+ ILC3 (black line) were compared
with Lin CD56+ c-Kit NK cells (gray) for RORt expression. (D) Surface staining of Lin c-Kit+ ILCs for the indicated markers (black line) compared with
isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with con-
trol media (dotted line). Cells shown were gated on Lin CD56+ c-Kit+. The isotype control is in gray. (F) CD11c+ MHCII+ human colonic APCs were elec-
tronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed
(prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry.
(left) Result from one representative donor. (right) Percentage of IL-22+ ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients.
**, P 0.01, two-tailed Students t test. Black bars represent the geometric mean.

the receptor for TL1A, is expressed both on T cells and ILC
subsets, with the highest expression on ILC2 and ILC3 (Meylan
et al., 2014). Ex vivo stimulation with mouse or human re-
combinant TL1A significantly enhanced the ability of IL-23
and IL-1 to induce IL-22 by both mouse (Fig. 7 C and Fig. S4)
and human (Fig. 7 D) intestinal ILC3, respectively. TL1A and
IL-1 also cooperated in inducing GM-CSF production by
mouse ILC3 (Fig. 7 E). To assess the specificity of TL1A for
DR3/TNFRSF25 on ILCs and its functional role in enhancing
CX3CR1+ support of ILC3 activation, DR3 expression was
specifically knocked down using siRNA nucleofection of sorted
mouse intestinal ILC3. Efficiency of knock-down was con-
firmed by surface staining for DR3, comparing with a scram-
bled control siRNA (Fig. 7 F). Compared with the scramble
control, cells targeted with siRNA for Tnfrsf25 had signifi-
cantly reduced enhancement of IL-22 production upon treat-
ment with recombinant TL1A (Fig. 7, G and H).The targeted
ILCs also produced reduced amounts of IL-22 upon co-culture
with LPS-stimulated CX3CR1+ MNPs. These data reveal a
potent ability of TL1A to enhance IL-22 production via DR3/
TNFRSF25 on ILC3 in both mouse and human.
DISCUSSION
Mononuclear phagocytes are spatially and functionally poised
to integrate microbial signals from the luminal microbiota
(Niess et al., 2005; Varol et al., 2010). Although MNPs were
previously thought to remain in the tissue, we recently showed
that CX3CR1+ MNPs can migrate to draining lymph nodes
and initiate immune responses under conditions of dysbiosis
(Diehl et al., 2013). In the context of inflammation, however,
these CX3CR1+ MNPs expand within the lamina propria dur-
ing chemical (Zigmond et al., 2012) or infectious colitis and
in IBD patients (Kamada et al., 2008), and their function has
remained obscure. Conventional DCs (cDCs), rather than the
MNPs, have been postulated to regulate intestinal Th17 cell
differentiation in response to microbiota (Denning et al., 2011;

JEM Vol. 211, No. 8 1577

Figure 6. Human ILC3 production of IL-22 is supported by IL-23 and IL-1 produced by TLR-stimulated CD14+ and CX3CR1+ MNPs.
(AC) HLA-DR+ CD11c+ cells from intestinal resection tissue were sorted into CD103+ DCs and CD14+ MNPs subpopulations and transcriptional profiles
were assessed by RNA-seq. (A) Sorting strategy. (B) Each subset was examined for expression of the indicated cell surface markers. Isotype controls are
shown in gray. One of three donors is shown. (C) Heatmap of relative expression of relevant MNP-related genes. Values represent the mean of two inde-
pendent donors, and an asterisk denotes individual genes differentially expressed at an FDR = 0.01. (D and E) Induction of IL-22 in human ILCs in co-
culture with CD14+ MNPs or CD103+ DCs in the presence of media alone, LPS, or flagellin, as indicated. c-Kit+ cells were examined for intracellular IL-22
production after 18-h culture. Data are representative of five independent experiments. (F) CD14+ MNPs or CD103+ DCs sorted from human intestine (as
in A) were stimulated with the indicated TLR ligands for 18 h and qPCR or cytometric bead array analysis were used to quantitate IL-23p19 and IL-1,
respectively. Results are averaged from three independent donors, and technical replicates were performed in duplicate or triplicate, respectively. *, P 0.05.
N.D., not detected. Two-tailed Students t test. Error bars represent the SEM. (G) Sorted human intestinal CD14+ MNPs and ILCs were left unstimulated or
were co-cultured in the presence of LPS with or without neutralizing antibodies against IL-1 and IL-23. IL-22 ELISA was performed after 18h. Results
are averaged from two independent donors performed in duplicate. *, P 0.05. Two-tailed Students t test. Error bars represent the SEM.

Lewis et al., 2011; Persson et al., 2013; Schlitzer et al., 2013;
Goto et al., 2014), but there has been conflicting evidence as
to which myeloid cell populations regulate IL-22 produc-
tion by ILC3 cells (Kinnebrew et al., 2012; Manta et al., 2013;
Satpathy et al., 2013).
Our data reveal that colonic CX3CR1+ MNPs regulate
ILC3 production of IL-22 and likely play a critical role in pro-
moting mucosal healing during colitis. The requirement for
CX3CR1+ MNPs in regulating colitis-induced ILC3 shown
here appears to contrast with recent work suggesting that
Notch2-dependent CD103+ CD11b+ cDCs are required for
the control of C. rodentiuminduced colitis (Satpathy et al., 2013).
These disparate results may reflect a coordinated contribution
of multiple myeloid subsets in the immune response to C. ro-
dentium (Schreiber et al., 2013), and our data do not exclude a
contribution of other myeloid subsets. Alternatively, Notch2
may affect cellular subpopulations in addition to CD103+
CD11b+ cDCs. This latter hypothesis is supported by the re-
port that an alternate selective depletion strategy for CD103+
CD11b+ cDCs, by expression of DT under the human Lan-
gerin promoter (Welty et al., 2013), did not result in increased
susceptibility to C. rodentium. Some of the conflicting data
may additionally reflect differential roles of inflammatory mono-
cytes and MNPs in colitis. Inflammatory monocytes may ex-
acerbate pathology, as antibody-mediated depletion of Ly6Chi
monocytes during DSS treatment improved pathology in DSS

1578 CX3CR1+ MNPs regulate ILC3 | Longman et al.

 Ar ticle

Figure 7. CX3CR1+ MNP-derived TL1A synergizes with IL-23 and IL-1 to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis
was used to determine whether the indicated disease-related SNP were differentially expressed between CD14+ MNPs and CD103+ DCs. Significance was
estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two
independent donors. (B) B cells (CD3 CD19+), CX3CR1+ MNPs, CD103+ DCs, and Ly6C+ monocytes were sorted from the intestinal lamina propria of
Cx3cr1GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by Ct normalized to GAPDH expression. Data are from
two biological replicates performed with two technical replicates. *, P 0.05. Two-tailed Students t test. (CE) Sorted intestinal ILCs from mice (C and E)
or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added
to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments.
(FH) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by
flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured
with media alone (-) or IL-23 and TL1A or co-cultured with CX3CR1+ MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production
was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative
of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin.
*, P 0.05; **, P 0.01. Two-tailed Students t test. Error bars represent SEM.

colitis (Zigmond et al., 2012). Selective depletion of such
monocytes may hence explain the intermediate results seen
in the CCR2-deficient mice exposed to C. rodentium (Satpathy
et al., 2013). In addition to supporting ILC3, MNPs may also
directly promote epithelial barrier repair (Wynn et al., 2013).
Although our data suggest a critical role for CX3CR1+ MNPs
in regulating production of IL-22 by ILC3 during acute coli-
tis, it will be important to examine the role for these myeloid
subsets during chronic colitis and their impact on another major
clinical endpoint in IBDtumorigenesis (Huber et al., 2012;
Kirchberger et al., 2013).
Our results also highlight a beneficial role for microbial sig-
nals in promoting intestinal homeostasis and driving ILC3 pro-
duction of IL-22 through TLR/MyD88-dependent induction
of IL-23 and IL-1. At steady state, commensal-dependent sig-
naling may negatively regulate ILC production of IL-22 via in-
testinal epithelial cell production of IL-25 (Sawa et al., 2011),
but we and others (Satoh-Takayama et al., 2008) find that
microbes support colitis-associated ILC production of IL-22,
which promotes mucosal healing. Although intravenous de-
livery of flagellin can induce CD103+ CD11b+ cDCs to produce
IL-23 that regulates ILC3 in the small intestine (Kinnebrew
et al., 2012), we and others (Kamada et al., 2008) found that
in vivo colonic inflammation and other bacterial-derived signals
induced more robust production of IL-23 and IL-1 by MNPs,
suggesting that the type of stimulation and/or colonic inflam-
mation may confer specificity. Similar to our results in mice,
we found marked reductions in IL-22 production by intestinal

JEM Vol. 211, No. 8 1579

ILC3 from human tissue distal to a surgical diversion of the
fecal stream.These findings may offer clinically relevant mech-
anistic insight into diverted IBD patients with persistent in-
flammatory disease or even non-IBD patients with de novo
mucosal inflammation after diversion (e.g., diversion colitis;
Harig et al., 1989). Although some evidence supports a role
for luminal replacement of short chain fatty acids in diversion
colitis (Harig et al., 1989;Vernia et al., 1995), further work is
required to determine if particular metabolites or intestinal
microbes may regulate ILC3 function to promote healing.
In light of their robust production of IL-22 and close
proximity to the intestinal epithelial layer (Cella et al., 2009),
ILC3 have been proposed to play an important role in muco-
sal healing and maintenance of barrier integrity, but their char-
acterization in IBD patients has been limited. ILCs producing
IL-17 or IFN- (classified as ILC1) have also been described in
mouse models of colitis (Buonocore et al., 2010; Klose et al.,
2013) and human IBD (Geremia et al., 2011; Bernink et al.,
2013), supporting a potentially important role for ILCs in IBD
pathogenesis. Here, we observed a specific increase in IL-22
production by ILCs from colonic biopsies of patients with mild
to moderate CD and UC. Despite characteristically distinct
phenotypes, both CD and UC share genetic susceptibility risk
alleles within the IL-23 pathway (Duerr et al., 2006), which
may underlie a common role for ILCs during colonic inflam-
mation in both diseases. In contrast to previous studies, we did
not observe any significant IL-17 production by CD3 cells
from patients with CD (Geremia et al., 2011) or a reduction
in ILC3 in CD patients (Bernink et al., 2013). This difference
may reflect clinical differences in the patient cohorts and
samplescolonoscopic biopsies in our cohort were taken from
ambulatory patients with mild to moderate colonic CD and UC,
in contrast to surgical resection of ileal tissue from medically
refractory patients in the other studies. As such, mild to mod-
erate colitis may be the ideal human model to evaluate ILC3-
producing IL-22 promoting mucosal healing. The association
of clinical phenotype with ILC phenotype and the potential
plasticity of these ILCs in colitis remain to be evaluated.
Consistent with our results in mouse models, CX3CR1+
MNPs from human intestinal tissue were more potent than
CD103+ DCs in supporting human intestinal ILC3 produc-
tion of IL-22. In addition to the increased production of IL-23
and IL-1 by the CX3CR1+ subset described by us and oth-
ers (Kamada et al., 2008), RNA seq analysis of these subsets
revealed enrichment for transcripts from genes having IBD-
associated SNPs, notably TL1A/TNFSF15. Polymorphisms
in TNFSF15, the gene that encodes TL1A, are strongly asso-
ciated with IBD, and expression of both TL1A and its recep-
tors DR3 and DcDR3 is elevated in IBD patients (Kugathasan
et al., 2008). TL1A has been shown to enhance Th17 differen-
tiation and effector function (Pappu et al., 2008; Kamada et al.,
2010); promote Treg expansion and ameliorate allergic asthma
(Schreiber et al., 2010); and promote IL-13 production by
ILC2s (Meylan et al., 2014). Interestingly, microbial stimula-
tion and Fc-receptor engagement induces TL1A/TNFSF15
in monocytes and monocyte-derived mononuclear phagocytes
1580
(Shih et al., 2009).The data shown here reveal a potent ability
of TL1A to enhance ILC3 production of IL-22 and suggest
that the increased expression of TL1A by colonic CX3CR1+
MNPs enables their selective ability to potently support ILC3.
Furthermore, the ability of TL1A to induce GM-CSF pro-
duction by ILC3 is consistent with recent data suggesting
that, even at steady state, CSF1R-dependent MNPs support
ILC3 production of GM-CSF that, in turn, regulates oral tol-
erance (Mortha et al., 2014). Collectively, these data support a
broader role for colonic CX3CR1+ MNPs in regulating mu-
cosal homeostasis.
Mucosal ILC3 play a crucial role in barrier homeostasis,
mucosal healing, and oral tolerance. CX3CR1+ MNPs are aptly
poised to integrate microbial signals to regulate ILC3 and
may serve as important targets for therapeutic manipulation.
Further understanding of the contributions of discrete commen-
sal bacterial species and of the mechanistic interactions be-
tween CX3CR1+ MNPs and ILC3 may provide novel strategies
to promote intestinal healing. In light of the specific and po-
tent regulation of ILC3 by colonic CX3CR1+ MNPs during
colitis, diagnostics that correlate susceptible genotypes (Hueber
et al., 2012) with clinical disease immunophenotype will pro-
vide insight into therapeutic strategies to manipulate colonic
MNPs in the clinical management of IBD.
MATERIALS AND METHODS
Antibodies and flow cytometry. Staining of human cells was performed
with c-Kit-e450 (104D2), CD56-PECy5.5 (CMSSB), HLA-DR-PE (LN3),
CD11c-FITC (3.9), CD3-e780 (UCHT1), CD19-PerCP5.5 (HIB19), CD103
PECy7 (B-Ly7), CD14 PE (61D3), CD86-PE (IT2.2), CD123 PE (6H6),
CD11b PE (CBRM1/5), and RORt (AFKJS-9) obtained from eBiosci-
ence; CD161-FITC, CD83-PE, CD11c PE (555392), CD127 FITC (M21),
CCR6-biotin (11A9) were purchased from BD; BDCA-1-APC were ob-
tained from Miltenyi Biotec; CD45-APC (4505) and CD64 (6404) were ob-
tained from Invitrogen; NKp44-APC (p44-8.1) was purchased from R&D
Systems; and BDCA-3 APC (M80) was purchased from BioLegend. Intra
cellular human cytokine staining was performed with IL-22 PE (IC7821P;
R&D Systems), IL-17A-FITC (eBio64CAP17; eBioscience), or IFN- (45.
B3; eBioscience). Staining of mouse cells was performed with CD90.2-e450
(532.1), CD3e (145-2C11), NKp46-e710 (29A1.4), RORt-PE (B2D),
CD11c-PECy7 (N418), MHCII A700 (M5/114.15.2), CD103 APC (2E7),
CD14 PerCP5.5 (Sa2-8), CD11b e780 (M1/70), Ly6C PerCP5.5 (AL-21),
and F4/80 PE (BM8) were from eBioscience. DR3-PE (4C12) was obtained
from BioLegend. Intracellular mouse cytokine staining was performed with
IL-22 APC (IL22JOP), IFN- PECy7 (XMG1.2), IL17A FITC (eBio17B7),
all from eBioscience. Intracellular cytokine staining was performed according
to the manufacturers protocol (Cytofix/Cytoperm buffer set; BD). Intra
nuclear staining for RORt was performed according to manufacturers protocol
(Intracellular Fixation and Permeabilization kit; eBioscience). Flow cytome-
try and analysis were performed with a LSR II (BD) and FlowJo software
(Tree Star). Dead cells were excluded using the Live/Dead fixable aqua dead
cell stain kit (Invitrogen).
Mice. C57BL/6, Myd88flox, CD11c-cre (Caton et al., 2007), and Il1r/ mice
were purchased from The Jackson Laboratory. Myd88-deficient mice were
obtained from S. Akira (Osaka University, Osaka, Japan; Adachi et al., 1998)
and Il23p19/ mice were obtained from D. Cua (Merck Research Labora-
tories, Palo Alto, CA; Cua et al., 2003). RORc(t)GFP/+ (Eberl et al., 2004),
Cx3cr1GFP/+ (Jung et al., 2000) and inducible CX3CR1-DTR mice (Diehl et al.,
2013), were previously described. The latter mice were subsequently bred to
a germline-expressing cre mouse to excise the transcriptional stop sequence
CX3CR1+ MNPs regulate ILC3 | Longman et al.

and produce the Cx3cr1DTR/+ mice. All mice were kept in specific pathogen
free (SPF) conditions at the animal facility of the Skirball Institute. Mouse
experiments were performed with at least three mice per group and multiple
experiments were combined to assess statistically significant differences as
noted. Littermates of the same genotype were randomly assigned to experi-
mental groups. Animals were used between 8 and 16 wk of age. Males and
females were used in approximately equal ratios. All animal experiments were
performed in accordance with approved protocols for the NYU Institutional
Animal Care and Usage Committee.
Preparation of LPMCs. Endoscopic biopsies were obtained under IRB-
approved protocols at Weill Cornell Medical College (1103011578 and Co-
lumbia University Medical Center (AAAE5448) including patients >18 yr of
age and able to give informed consent. IBD sample was defined based on en-
doscopic inflammation with history of ulcerative colitis or Crohns disease.
Endoscopic score (mild, moderate, or severe) was based on Mayo Endoscopic
subscore or SES-CD (1, 2, 3, respectively) at the site of biopsy (Pineton de
Chambrun et al., 2010). All endoscopic biopsies were taken from the sigmoid
or descending colon to reduce sampling variation. Study sample sizes for
human biopsies were based on preliminary data and powered to achieve sta-
tistically significant differences in the production of IL-22 or IL-17. Surgical
resections were obtained under an IRB-approved protocol at New York Uni-
versity Langone Medical Center. Mouse and human intestines were washed
in PBS and 1 mM DTT twice with 30 mM EDTA, and then digested in col-
lagenase 8 (Sigma-Aldrich) and DNase-containing media with 10% fetal bo-
vine serum. Digested material was passed through a cell strainer and separated
on a discontinuous 40%/80% Percoll gradient. LPMCs were cultured ex vivo
in the presence of GolgiPlug (BD) for 4 h or stimulated with phorbol my-
ristate acetate (PMA; 20 ng/ml) and ionomycin (1 g/ml) or IL-23 (40 ng/ml;
eBioscience) in the presence of GolgiPlug (BD) for 4 h before staining.
Intestinal ILC cultures. Surgical resections were obtained from the NYU
Biorepository (Rachel Brody). Lin c-Kit+ CD45int ILCs were sorted and
cultured in tissue culture media (RPMI 1640; Invitrogen) supplemented with
10% (vol/vol) heat-inactivated FBS (HyClone), 50 U penicillin-streptomycin
(Invitrogen), 2 mM glutamine, and 50 M -mercaptoethanol), supplemented
with IL-7 (50 ng/ml; PeproTech) and IL-2 (1,000 U/ml; PeproTech) for 810 d
before stimulation. Lin, CD90.2+, RORt-GFP mouse ILC3 were sorted
from LPMCs and resuspended in RPMI-based tissue culture media for stim-
ulation directly ex vivo. Human and mouse ILCs were stimulated with human
or mouse IL-23 (eBioscience; 40 ng/ml), IL-1 (eBioscience; 10 ng/ml), or
TL1A (R&D Systems; 200 ng/ml) as indicated, respectively. After 18 h, super-
natants were harvested for IL-22 ELISA (eBioscience) and Golgi Plug (BD)
was added to cells for 4 h for subsequent intracellular cytokine staining.
Co-culture assay. ILCs and APC populations were sorted on a FACSAria
and co-cultured with 5 103 and 2.5 103 cells, respectively, in 96-well
round-bottom plates in tissue culture media.TLR stimulation was performed
with 1 g/ml LPS (Escherichia coli; Sigma-Aldrich), 1 M CpG 1668 (mouse),
1 M CpG 2216 (human), or 1 g/ml flagellin (Salmonella Typhi; InvivoGen).
Cultures were incubated for 18 h. Supernatants were harvested for ELISA
and remaining cells were incubated with Golgi Plug (BD) for 4 h and sub
sequently analyzed by flow cytometry.
siRNA transfection. Sorted intestinal ILCs were cultured overnight in IL-7
(20 ng/ml) and SCF (20 ng/ml). After 24 h, 4 105 ILCs were transfected
using AMAXA T cell nucleofection protocol. 300 pmol of Tnfrsf25 siRNA
pool or scramble control (Thermo Fisher Scientific) was used per transfec-
tion. Cells were rested overnight and harvested at 24 h for experimental use.
Knockdown efficiency was assessed at 24 h by DR3 surface staining.
Colitis models. C. rodentium DBS100 (ATCC 51459; American Type Culture
Collection) was harvested at log phase growth and 1010 CFU were delivered by
gavage in PBS. 200 ng of DT was administered i.p. as indicated for depletion.
Plasmid DNA expressing IL-22 or control plasmid (Qiu et al., 2012) were
JEM Vol. 211, No. 8
Ar ticle
delivered i.v. at 5 g DNA/mouse diluted in TransIT-EE Hydrodynamic Deliv-
ery Solution (Mirus) at 0.1 ml/g body weight. Immune cell functional analysis
and histology were performed at day 7 after exposure. Spleens were harvested on
day 21, homogenized, and plated at serial dilutions to determine CFU/spleen.
RNA-seq processing and gene set enrichment analysis. Sequence reads
were mapped to the human genome (version hg19) by Tophat (version 2.0.6),
using Bowtie2 (version 2.0.2) and Samtools (version 0.1.18). Reads are depos-
ited at Bioproject PRJNA219394. Reads mapped per transcript served as input
to DESeq (version 1.12.0), an R package that calculates differential gene ex-
pression. To improve detection of APC lineage-dependent gene-expression
changes and overcome donor-dependent variability, we used the following
strategy: independently for each donor, differential gene expression, comparing
CD103+ to CD14+ human myeloid cells, was estimated using the negative bi-
nomial distribution (nbinomTest), and then results from biological replicates
were combined using Fishers method. Several gene set enrichment techniques
(e.g., hypergeometric test and area under precision-recall curve) were used to
test whether specific gene sets (Table S2) were significantly differentially regu-
lated between the two lineages.The GWAS gene sets are derived from disease-
associated SNPs from the NHGRI GWAS Catalog. False-discovery rates
(FDRs) were calculated using the Benjamini-Hochberg procedure. The en-
richment analyses were implemented in Matlab R2013a (8.1.0.604).
qPCR. RNA from primary intestinal APCs stimulated as indicated was pre-
pared with TRIzol (Invitrogen). RNA was reverse transcribed into cDNA
(SuperScript III; Invitrogen) and QPCR was performed with a Roche Light-
Cycler with SYBR Green Supermix (Bio-Rad Laboratories), 20 pmol forward
and reverse primers, and 0.1 g of cDNA from 5-TGTTCCCCATATC-
CAGTGTGG-3 and 5-CTGGAGGCTGCGAAGGATTT-3 for human
IL23p19, 5-ATGCTTCGGGCCATAACAGA-3 and 5-TGAAGGC-
CATCCCTAGGTCA-3 for mouse TL1A; 5-ACCACAGTCCATG
CCATCAC-3 and 5-TCCACCACCCTGTTGCTGTA-3 for human
GAPDH; and 5-AATGTGTCCGTCGTGGATCT-3 and 5-CATCGA
AGGTGGAAGAGTGG-3 for mouse GAPDH. The thermocycling pro-
gram was 40 cycles at 95C for 15 s, 60C for 30 s, and 72C for 30 s, with
an initial cycle of 95C for 2 min. Relative levels of target gene were deter-
mined by using the delta Ct value compared with delta Ct (GAPDH).
Immunofluorescence. Intestinal tissue was Swiss-rolled before fixing for 4 h
in 4% paraformaldehyde. Tissue was incubated overnight in 30% sucrose be-
fore freezing in OCT. Tissue was cut into 5-M sections. Tissue was blocked
in PBS-XG (0.1% Triton X-100, 10% goat serum) before incubating over-
night in primary antibody in PBS-XG. Tissue was washed and then incu-
bated with secondary antibody for 1 h before DAPI staining. The following
primary antibodies are from eBioscience: antihuman/mouse RORt (clone
AFKJS-9) and antimouse CD3e (clone 145-2C11). Secondary antibodies
are from Jackson ImmunoResearch Laboratories (Cy3-AffiniPure goat antirat
IgG and goat antiArmenian hamster). Tissue was imaged using an LSM 710
confocal (Carl Zeiss) and images were processed using ImageJ.
Online supplemental material. Fig. S1 shows the gating strategy for co-
lonic ILC3. Fig. S2 shows increased production of IL-22 in ILCs from pa-
tients with IBD. Fig. S3 shows Lin gating and surface phenotype for human
intestinal ILC3. Fig. S4 shows that TL1A enhances IL-23 and IL-1 induc-
tion of IL-22 by intestinal ILC3s. Online supplemental material is available at
http://www.jem.org/cgi/content/full/jem.20140678/DC1.
We thank Rachel Brody (NYU Biorepository), Fatiha Chabouni, Priyanka Patel, Ryan
Warren and members of The Roberts Center for IBD for help with sample collection
as well as the patients that participated in this study. We would like to thank
Michael Cammer for assistance with microscopy.
This work was supported by the American Gastroenterological Association
Research Foundation (R.S. Longman), National Institutes of Health K08 DK099381
(R.S. Longman), American Cancer Society (G.E. Diehl), NIH T32 CA009161 (G.E. Diehl),
K99DK091508 (J.R. Huh), and the Howard Hughes Medical Institute (D.R. Littman).
The authors declare no competing financial interests.
1581
Author contributions: R.S. Longman and G.E. Diehl designed and performed the
experiments. R.S. Longman, G.E. Diehl and D.R. Littman planned experiments and
wrote the manuscript with input from the co-authors. J.R. Huh, C. Galan, and
D.A. Victorio helped plan and perform experiments. E.R. Miraldi and R. Bonneau
performed data analysis. A. Swaminath and E.J. Scherl helped with sample collection
and experimental design.
Submitted: 9 April 2014
Accepted: 18 June 2014
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1583
INSIGHTS

Monocytes find a new place to dwell in the niche of

heartbreak hotel
Monocytes and macrophages belong to the mononuclear phagocyte system (MPS). The con-

Lambrecht Photo courtesy of

cept of the MPS is based on the idea that circulating monocytes represent the immediate
precursors of tissue macrophages. Recent genetic fate mapping of myeloid cells caused a con-
ceptual frameshift, as it was found that many macrophages are derived from embryonic pre-
cursors that seed the tissues before birth and then self-maintain without any significant

www.vib.be
contribution from circulating monocytes. In this new view, monocytes merely represent an
emergency squad that can be rapidly recruited to sites of inflammation to provide a transient
Insight from Bart Lambrecht (left)
supplement of inflammatory macrophages to the local tissue-resident macrophage pool of and Martin Guilliams (right)
embryonic origin.
In this issue, Molawi et al. challenge this new concept by reporting that cardiac macrophages are initially of embryonic
origin but are progressively replaced by monocyte-derived cells. These newcomer macrophages lack the capacity to self-renew
and are slowly but constantly replaced by circulating monocytes. In this regard, the heart resembles the skin and intestine.
There, macrophages are also mainly derived from circulating monocytes. Because the skin and the intestine are barrier sites with
an important influence of the microbiome, a continuous low-grade inflammation could explain continuous recruitment of
circulating monocytes. Molawi et al. report that cardiac macrophages of embryonic origin gradually lose the capacity to self-
renew with age and hypothesize that this allows circulating monocytes to occupy the heart niche and join the cardiac macrophage
pool. Why embryonic macrophages would lose their self-renewal capacity in the heart but not in the liver, the lung, or the
spleen remains to be determined. Another unexplored hypothesis is that continuous microtrauma induced by cardiac contraction
is the driver of monocyte influx to the sterile heart.
Tissue-resident macrophages are astonishingly diverse in terms of gene expression and are adapted to perform specific functions
in their tissue of residence. The unique functions of cardiac macrophages remain largely unknown. Molawi et al. describe
four subsets of cardiac macrophages based on the expression of CX3CR1 and MHC class II (see figure). Embryonic macrophages
preferentially give rise to MHC class IIlow cells, while most monocyte-derived macrophages express high levels of MHC class II.
This implies a different capacity to
interact with lymphocytes and distinct
functional adaptations for embryonic
macrophages. Macrophages play an im-
portant role in wound healing, and many
cardiovascular diseases are associated with
poor or exaggerated tissue repair. It will
therefore be interesting to investigate
whether embryonic macrophages are
better at tissue repair compared with
monocyte-derived macrophages, and
whether the presence of the embryonic
pool explains the superior regeneration
capacity of the heart at a young age.
Manipulation of macrophage self-renewal
might yield important therapeutic appli-
Cardiac macrophages derived from embryonic progenitors gradually lose their capacity to cations to increase macrophage popula-
self-renew and are continually replaced in adulthood by macrophages derived from circulating tions involved in tissue homeostasis and
monocytes, even in the absence of inflammation. repair, while avoiding the recruitment of
macrophages that fuel inflammation.
With these exciting prospects and new concepts, macrophage biology is going through a revival period, and this study
certainly contributes to that. Given the breadth of tissue-specific macrophage heterogeneity, both in terms of functional specialization
and cellular origin, we have many years ahead until we fully understand the cell that initiated immunology research more than
a century ago.
Molawi, K., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20140639.

Bart Lambrecht and Martin Guilliams, VIB Inflammation Research Center, UGent: bart.lambrecht@irc.vib-Ugent.be and martin.guilliams@irc.ugent.be
 Brief Definitive Report

Progressive replacement of embryo-derived

cardiac macrophages with age
Kaaweh Molawi,1,2,3,4 Yochai Wolf,5 Prashanth K. Kandalla,1,2,3
Jeremy Favret,1,2,3 Nora Hagemeyer,6 Kathrin Frenzel,6 Alexander R. Pinto,8
Kay Klapproth,9 Sandrine Henri,1,2,3 Bernard Malissen,1,2,3
Hans-Reimer Rodewald,9 Nadia A. Rosenthal,8,10 Marc Bajenoff,1,2,3
Marco Prinz,6,7 Steffen Jung,5 and Michael H. Sieweke1,2,3,4
1Centre dImmunologie de Marseille-Luminy (CIML), Aix-Marseille Universit, UM2, 13288 Marseille, France
2Institut National de la Sant et de la Recherche Mdicale (INSERM), U1104, 13288 Marseille, France
3Centre National de la Recherche Scientifique (CNRS), UMR7280, 13288 Marseille, France
4Max-Delbrck-Centrum fr Molekulare Medizin (MDC), Robert-Rssle-Strasse 10, 13125 Berlin, Germany
5Department of Immunology, The Weizmann Institute of Science, 7610001 Rehovot, Israel
6Institute of Neuropathology and 7BIOSS Centre for Biological Signaling Studies, University of Freiburg, 79106 Freiburg, Germany
8Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton 3800, Victoria, Australia
9Division of Cellular Immunology, German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany
10National Heart and Lung Institute, Imperial College London, London SW7 2AZ, England, UK

Cardiac macrophages (cM) are critical for early postnatal heart regeneration and fibrotic
repair in the adult heart, but their origins and cellular dynamics during postnatal develop-
ment have not been well characterized. Tissue macrophages can be derived from embryonic
progenitors or from monocytes during inflammation. We report that within the first weeks
after birth, the embryo-derived population of resident CX3CR1+ cM diversifies into
MHCII+ and MHCII cells. Genetic fate mapping demonstrated that cM derived from
CX3CR1+ embryonic progenitors persisted into adulthood but the initially high contribution
to resident cM declined after birth. Consistent with this, the early significant prolifera-
tion rate of resident cM decreased with age upon diversification into subpopulations.
Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative
replacement of all cM populations. Furthermore, parabiotic mice and BM chimeras of
nonirradiated recipient mice revealed a slow but significant donor contribution to cM.
Together, our observations indicate that in the heart, embryo-derived cM show declining
self-renewal with age and are progressively substituted by monocyte-derived macrophages,
even in the absence of inflammation.

CORRESPONDENCE
Michael H. Sieweke:
sieweke@ciml.univ-mrs.fr
Abbreviations used: cM,
cardiac macrophages; HSC,
hematopoietic stem cell;
YS, yolk sac.
Cardiovascular disease represents a leading
cause of death in the developed world, and
many pathologies result from insufficient repair
of cardiac injury. Mammalian wound healing
mechanisms in the adult heart involve scar for-
mation, but for a short time window after birth
the neonatal heart maintains full regeneration
capacity of cardiac tissue (Porrello et al., 2011),
a process which requires macrophages (Aurora
et al., 2014). Studies of cardiac repair after myo-
cardial infarction in the adult have highlighted
the critical role of infiltrating monocytes and
monocyte-derived macrophages for the healing
K. Molawi and Y. Wolf contributed equally to this paper.
Steffen Jung and M.H. Sieweke contributed equally to this paper.
process (Nahrendorf et al., 2007; Swirski et al.,
2009). The focus of these studies has been on
monocyte-derived cells, consistent with the tra-
ditional view that macrophages are part of the
mononuclear phagocyte system and monocyte-
derived (van Furth and Cohn, 1968; Geissmann
et al., 2010). Recent evidence, however, sug-
gests that monocyte contribution to macro-
phages only represents an emergency pathway,
as many tissue macrophage populations get
seeded in the developing embryo and can self-
maintain without major monocyte contribution
2014 Molawi et al. This article is distributed under the terms of an Attribution
NoncommercialShare AlikeNo Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
it is available under a Creative Commons License (AttributionNoncommercial
Share Alike 3.0 Unported license, as described at http://creativecommons.org/
licenses/by-nc-sa/3.0/).

The Rockefeller University Press $30.00

J. Exp. Med. 2014 Vol. 211 No. 11 2151-2158 2151
www.jem.org/cgi/doi/10.1084/jem.20140639
Figure 1. cM develop into 4 subpopulations after birth. (A and B) Cytometry analysis of cM from adult CX3CR1GFP/+ mice. (A) FACS profiles of
cM populations, pregated on living single CD11b+ cells with low CD11c and Ly6C expression. (B) Mean of total and subpopulations of cM in absolute
numbers (left) or as percentage of total (right) as determined by bead-normalized flow cytometry. Error bars represent SEM (n = 4). (C and D) Cytometry
analysis (C) and mean percentage (D) of cM subpopulations from CX3CR1GFP/+ mice of indicated age. Error bars represent SEM (n = 34). (E) MHCII and
MHCII+ cM from adult CX3CR1cre:R26-yfp mice (black line) were analyzed for YFP expression and compared with Cre littermate controls (gray area;
n = 34). Data in all panels are representative of at least two independent experiments.

(Chorro et al., 2009; Ginhoux et al., 2010; Hoeffel et al.,
2012; Schulz et al., 2012; Hashimoto et al., 2013;Yona et al.,
2013). Macrophages can massively expand in the tissue by
local proliferation in response to challenge (Jenkins et al.,
2011; Hashimoto et al., 2013; Sieweke and Allen, 2013) and
can extensively self-renew without loss of differentiated func-
tion in culture upon inactivation of MafB and cMaf tran-
scription factors (Aziz et al., 2009). This has led to the
proposition that tissue macrophages may have a long-term
self-renewal capacity akin to that of stem cells (Sieweke and
Allen, 2013).
Examples of tissue macrophages that are independent
from monocytes are microglia in the brain and epidermal
Langerhans cells (Ajami et al., 2007; Chorro et al., 2009;
Ginhoux et al., 2010; Hoeffel et al., 2012). In contrast, intesti-
nal or dermal macrophages have a high turnover rate and are
constantly replaced from Ly6C+ blood monocytes (Zigmond
et al., 2012; Tamoutounour et al., 2013). Thus, origin and
turnover of tissue macrophages are highly tissue specific and
need to be assessed individually for different organ systems
(Sieweke and Allen, 2013).
Resident macrophages can be found in all tissues, where
they fulfill a variety of tissue-specific functions contributing
to homeostasis, development, and regeneration (Davies et al.,
2013), making them prominent candidates for therapeutic in-
tervention.This holds in particular for the heart, where tissue-
resident cardiac macrophages (cM) have been described as
CX3CR1+ cells that can be found throughout the myocar-
dium (Pinto et al., 2012). A recent study identified several
cM populations, including short-lived monocyte-derived
cells that resemble monocyte-derived DCs as well as tissue-
resident cM, which were suggested to be primarily of embry-
onic origin and to self-maintain under homeostatic conditions
(Epelman et al., 2014).

2152 Replacement of embryonic cardiac macrophages with age | Molawi et al.


Here, we examined the origin and turnover of tissue-
resident cM during postnatal development. Using genetic
lineage tracing, parabiotic mice, and unconditioned BM chi-
meras, we demonstrate that embryo-derived cM are gradu-
ally replaced by monocyte-derived macrophages with age and
in the absence of inflammation or injury. This replacement is
mirrored by dynamic changes in the resident cM population
composition and decreasing cM self-renewal over time.
RESULTS AND DISCUSSION
We focused on the resident cM population and applied a
rigorous flow cytometry gating strategy to exclude potential
infiltrating leukocytes (Fig. S1, AC). Tissue-resident macro-
phages were identified as positive for the core macrophage
signature markers CD14, CD64, and MerTK (Gautier et al.,
2012; Tamoutounour et al., 2013) and the classical macro-
phage marker F4/80 (Fig. 1 A). The chemokine receptor
CX3CR1 is widely expressed in the mononuclear phagocyte
system (Jung et al., 2000) and cM have been reported to be
CX3CR1-positive (Pinto et al., 2012). Therefore, we refined
our analysis of the global cM population by testing CX3CR1
expression in CX3CR1GFP/+ knock-in mice (Jung et al.,
2000). Additionally, we included MHCII in our analysis,
which can be differentially expressed on tissue macrophages
JEM Vol. 211, No. 11
Br ief Definitive Repor t
Figure 2. Decreased contribution of embryo-
derived macrophages to cM with age.
(A) CX3CR1creER:R26-yfp embryos were treated with
TAM on E9 or E13 and analyzed on the day of delivery
(P0) or 6 wk after delivery (P42). (B) Percentage of
microglia-normalized YFP+ cM and representative
cytometry plots pregated on total cM at P0 and
P42 after E13 labeling. Bars show median (n = 913).
***, P 0.005, Mann-Whitney test. Data were pooled
from two independent experiments. (C) Representa-
tive cytometry plot showing YFP+ within total cM
at P42 and percentage of microglia-normalized YFP+
cM at P0 and P42 after E9 labeling. Bars show
median (n = 7) *, P 0.05. Data were pooled from
three independent experiments. (D) Representative
cytometry plots showing YFP+ cells within MHCII
and MHCII+ cM at P0 and P42 after E13 labeling
(n = 913). (E) Quantification of YFP+ cells within
MHCII and MHCII+ cM at P42 after E13 labeling.
Bars show the median (n = 913). ***, P 0.005,
Mann-Whitney test. Data were pooled from two
independent experiments. (F) Adult TAM-treated
CX3CR1creER:R26-yfp mice were analyzed for YFP+
cM and microglia at 1 and 4 wk after treatment.
Percentage of microglia-normalized YFP+ cM is
shown as median with extreme samples as error
bars (n = 3). Representative cytometry plots
show YFP+ within total cM. Unless otherwise
indicated data in all panels are representative of
two independent experiments.
(Tamoutounour et al., 2013). We found that in 8-wk-old
mice, the majority of cM was CX3CR1+ (80%) and
MHCII+ (70%), allowing the delineation of four distinct
cM subpopulations (Fig. 1, A and B). Additional populations
of CD11c+MHCII+ or Ly6C+ cells, referred to as cM
by others (Epelman et al., 2014), were excluded from our
analysis (Fig. S1 B), as data from other tissues suggest that
these are monocytes and monocyte-derived dendritic cells
(Tamoutounour et al., 2013). Consistent with this, these cells
have been shown to be short-lived and monocyte-derived
(Epelman et al., 2014).
To investigate the development of the four cM popula-
tions, we analyzed CX3CR1 and MHCII expression in cM
from newborn to 30-wk-old mice (Fig. 1, C and D). We ob-
served that almost all embryo-derived cM present at birth
were CX3CR1+MHCII. This homogenous cM compart-
ment diversified with age into four subpopulations with a pro-
gressive increase of MHCII+ cM and a decrease of CX3CR1+
cM. Importantly, genetic fate mapping analysis using
CX3CR1cre mice crossed to R26-yfp reporter mice (CX3CR1cre:
R26-yfp; Yona et al., 2013) revealed that all adult cM sub-
populations must have developed from a CX3CR1+ stage
(Fig. 1 E). These results suggested two not mutually exclusive
possibilities for cM subpopulation development: persistence
2153
Figure 3. Decreased proliferation rate of embryo-derived cM with age. (A) cM subpopulations of adult CX3CR1GFP/+ mice were analyzed for
BrdU incorporation and expression of Ki67 by flow cytometry four hours after BrdU injection (i.p.). Bars show median (n = 1213). *, P 0.05; **, P 0.01;
***, P 0.005; Wilcoxon test. (B and C) Total (B) or CX3CR1+MHCII (C) cM of CX3CR1GFP/+ mice of indicated age were analyzed for BrdU incorporation
and Ki67 expression by flow cytometry 4 h after BrdU injection (i.p.). Bars show median (n = 415). ***, P 0.005, Mann-Whitney test. Data in all panels
were pooled from 23 independent experiments.

and diversification of embryo-derived cM as suggested else-
where (Epelman et al., 2014), or replacement of embryo-
derived cM by adult monocyte-derived macrophages.
To address these alternatives, we first analyzed the persis-
tence of embryo-derived cM by genetic lineage tracing using
CX3CR1-driven tamoxifen (TAM)-inducible Cre recom-
binase (CreERT2) and R26-yfp reporter mice (CX3CR1creER:
R26-yfp; Fig. 2 A; Yona et al., 2013). Tamoxifen-induced
pulse labeling at E9 permits us to identify cells derived from
yolk sac (YS) CX3CR1+ macrophages before the onset of
definitive hematopoiesis. Labeling at E13 cannot distinguish
YS- and hematopoietic stem cell (HSC)derived macro-
phages, but should increase cM labeling because the heart
is colonized by CX3CR1+ macrophages at this time (Epelman
et al., 2014). cM labeling was normalized to YFP+ mi-
croglia, which are YS-derived, remain CX3CR1+ throughout
development, self-maintain without contribution of HSC-
derived cells (Ginhoux et al., 2010; Kierdorf et al., 2013),
and therefore represent an internal control for maximal
CX3CR1 labeling efficiency. E13 labeling revealed that rela-
tive cM labeling declined from 35% in newborn mice
to 18% in 6-wk-old mice (Fig. 2 B), indicating a loss of
embryo-derived cM with age. E9 labeling showed that
embryo-derived cM were at least partially of YS origin and
similarly declined from 14% in neonates to 4% in 6-wk-
old mice (Fig. 2 C). Interestingly, all embryo-derived YFP+
cM were MHCII at birth but had partially differentiated
into MHCII+ cM after 6 wk (Fig. 2 D). Although this
demonstrated that both populations can develop from cells of
embryonic origin, the relative contribution of embryo-derived
YFP+ cM to MHCII+ cM was significantly lower than to
MHCII cM, (Fig. 2 E).
We further assessed the turnover of CX3CR1+ cM in
the adult heart by pulse labeling adult CX3CR1creER:R26-yfp
(Fig. 2 F). 1 wk after treatment, labeling in cM corresponded
to 60% of microglia labeling but drastically decreased to
20% after 4 wk. By comparison, microglia labeling was
nearly complete after 1 wk and remained unchanged there-
after (Goldmann et al., 2013). Collectively, our lineage tracing
experiments argue that a significant proportion of macro-
phages established in early embryonic development is still
present at birth but is gradually lost with age.
The relatively fast turnover of labeled tissue-resident cM
suggested that local self-renewal was insufficient to maintain
the resident cM pool. We therefore analyzed the prolifera-
tive activity of cM subpopulations in adult mice by BrdU
incorporation after a 4-h pulse labeling period and by Ki67
staining (Fig. 3 A). The overall number of proliferating mac-
rophages in adult hearts was low, particularly in MHCII+
populations, but the proliferative rate of CX3CR1+MHCII
cM significantly exceeded that of all other subpopulations.
Interestingly, this population was also the only cM subset
present at birth but declined with age at the expense of the other
cM populations (Fig. 1, C and D). We therefore analyzed the
evolution of cM proliferation with age. Both BrdU incor-
poration and Ki67 staining showed a high proliferative rate
in newborn CX3CR1+MHCII cM, which gradually de-
creased by 810-fold both in total cM (Fig. 3 B) and in the
CX3CR1+MHCII cM population (Fig. 3 C). Together,
these data suggest that both a successive loss of the cell popu-
lation with the highest proliferative rate (CX3CR1+MHCII
cM) and a strong decrease of the proliferation rate collectively
result in progressively decreasing self-renewal of the tissue-
resident cM pool.

2154 Replacement of embryonic cardiac macrophages with age | Molawi et al.

 Br ief Definitive Repor t

Figure 4. Monocytes contribute to four cM subpopulations in adult mice. (A) WT mice were lethally irradiated and reconstituted with BM from
Ubow:CX3CR1GFP/+ mice. cM were analyzed for contribution of dTomato+ cells at indicated time points after reconstitution and graft-derived cells were
analyzed for CX3CR1 and MHCII expression. Data are presented as mean percentage SEM (n = 37) and derived from two independent experiments.
(B) Mixed BM chimeras were generated by reconstituting lethally irradiated WT mice (CD45.1/CD45.2) with BM from CCR2/:CX3CR1GFP/+ (CD45.2) and
WT CX3CR1GFP/+ (CD45.1) mice. cM, circulating CD11b+CD115+ monocytes (Ly6C+ and Ly6C) and B220+ B cells were analyzed for WT (CD45.1) and
CCR2/ (CD45.2) contribution. Host- and graft-derived cM were analyzed for the ratio of MHCII+/MHCII cM. Bars show median (n = 4). *, P 0.05,
Mann-Whitney test. (C) Parabiosis was established between adult CCR2/ (CD45.2) and WT (CD45.1) mice. After 10 wk, CCR2/ mice were analyzed for
contribution of CD45.1+ non-host cells to cM (total, MHCII+, and MHCII), circulating monocytes (Ly6C+ and Ly6C), and B cells. For CD45.1 host and
CD45.1+ non-host cM, the ratio of MHCII+/MHCII cM was calculated and compared. Bars show median (n = 4). **, P 0.01, Mann-Whitney test.
(D) BM chimeras were generated by transfer of LT-HSC isolated from panYFP mice into nonirradiated Rag2/c/KitW/Wv mice. cM (total, MHCII+, and
MHCII) and neutrophils (Gr1hi and SSChi) were analyzed for contribution of grafted YFP+ cells 8 wk after transplantation. Bars show median (n = 6).
**, P 0.01, Mann-Whitney test. Data in all panels are representative of two independent experiments.

We next addressed the question of how the age-dependent
loss of embryo-derived macrophages is compensated. To test
the ability of monocytes to contribute to cM populations,
we generated BM chimeras using WT hosts and BM from
transgenic mice expressing dTomato from a ubiquitously ex-
pressed human Ubiquitin-C promoter (Ubow mice; Ghigo
et al., 2013) and GFP from the CX3CR1 locus that makes it
possible to monitor grafted cells in all four cM populations.
Graft-derived dTomato+ cM contributed to all four cM
populations from 4 wk after transplantation and almost com-
pletely replaced host cM populations within 8 wk (Fig. 4 A).
However, a small radio-resistant population (10%) persisted
for 36 wk without monocyte contribution. To further analyze
whether cM replacement was monocyte-dependent, we
generated mixed chimeras with BM from WT (CD45.1)
and CCR2/ (CD45.2) CX3CR1GFP/+ mice in CD45.1/
CD45.2 double-positive WT hosts (Fig. 4 B). CCR2/ mice
have reduced numbers of Ly6C+ monocytes in the blood
(Serbina and Pamer, 2006) and can therefore be used to ana-
lyze the contribution of circulating monocytes and CCR2-
dependent progenitors to tissue macrophage populations (Yona
et al., 2013; Tamoutounour et al., 2013). Blood analysis indeed

JEM Vol. 211, No. 11 2155

confirmed that the vast majority of monocytes but not B cells
were of WT origin. Graft-derived cM were almost com-
pletely derived from WT cells (Fig. 4 B). Collectively these
two experiments established that HSC-derived CCR2-
dependent cells, most likely Ly6C+ monocytes, have the capac-
ity to differentiate into all cM subsets. Residual host-derived
cM included both MHCII+ and MHCII cM but showed
a lower proportion of MHCII+ cells than grafted monocyte-
derived cM (Fig. 4 B).
To examine monocyte contribution to cM populations
under homeostatic conditions, we generated parabiotic cou-
ples of WT (CD45.1) and CCR2/ (CD45.2) mice and
analyzed non-host contributions after 10 wk of parabiosis in
CCR2/ partners (Fig. 4 C). Circulating B cells were equally
distributed between the partner mice. In the CCR2-deficient
host Ly6C+ monocytes had reached 70% non-host chime-
rism. About 16% of total cM in CCR2-deficient mice were
of non-host origin. Given a 70% chimerism in Ly6C+ mono-
cytes, the likely cells of origin, and the possibility that de-
pending on lifetime and exchange rate cM may not be fully
equilibrated after 10 wk, these observations indicated a signif-
icant monocyte contribution to tissue-resident cM. Again,
non-host contribution to MHCII+ cM (30%) was signifi-
cantly higher compared with MHCII cM (5%) as high-
lighted by MHCII+/MHCII cM ratios for non-host and
host-derived cells (Fig. 4 C).
As a complimentary experimental approach that does not
depend on myeloablative conditioning, we used Rag2/c/
KitW/Wv mice, which are universal HSC recipients that accept
grafts without prior irradiation (Waskow et al., 2009), and
generated BM chimeras by transplantation of HSC from
panYFP mice (Luche et al., 2013). More than 90% of neutrophils
were YFP+ after 8 wk, demonstrating efficient reconstitution
(Fig. 4 D). Analysis of cM revealed 7% YFP+ cells in the
total population. Again, donor cells showed a higher contribu-
tion to MHCII+ (8% YFP+) than to MHCII cM (4%
YFP+) (Fig. 4 D). In the same mice, no donor contribution
to microglia cells was observed (K. Klapproth and H.R.
Rodewald, personal communication). Together, these experi-
ments clearly demonstrated a slow but significant replacement
of tissue-resident cM by infiltrating monocyte-derived mac-
rophages, which preferentially contributed to MHCII+ cM.
In the present study, we have analyzed homeostasis, origin,
and turnover of resident macrophages in the heart during
postnatal development. We have shown that the cM com-
partment is undergoing dynamic changes with age. Embryo-
derived macrophages present in newborn mice are all CX3CR1+
MHCII and at least partially YS-derived. After birth, the
cM compartment diversifies into four subpopulations
defined by MHCII and CX3CR1 expression. Our results
show that this diversification is due both to differentiation
of persisting embryo-derived CX3CR1+MHCII cM and
infiltrating monocyte-derived macrophages, which both can
contribute to all four cM subpopulations. However, mono-
cytes preferentially give rise to MHCII+ cM, whereas embryo-
derived or radio-resistant cM had a higher proportion of
2156
MHCII cells, explaining the relative increase of MHCII+ cM
and the reduction of the CX3CR1+MHCII cM subpopu-
lation with age.The dynamic change of the cM subpopulation
distribution therefore appears to reflect the gradual replace-
ment of embryo-derived cM by monocyte-derived macro
phages. The increasing contribution of monocyte-derived
macrophages to the cM pool with age may be explained by
the decreasing self-renewal of embryo-derived cM. Although
CX3CR1+MHCII cM that contain the majority of em-
bryo-derived macrophages have a higher proliferative rate than
the other subpopulations, overall cM self-renewal is decreas-
ing with age and is insufficient to sustain the cM pool with-
out input from infiltrating monocytes. It will be interesting to
determine whether the loss of self-renewal capacity in cM is
permanent or might be reactivated. The observed mechanism
of cM homeostasis differs from other macrophage popula-
tions such as microglia, Langerhans cells, or alveolar macro-
phages, which in the absence of tissue damage or inflammation
appear to self-maintain in tissues without replacement from
circulating cells. It is also distinct from the high turnover rate of
dermal tissue macrophages or intestinal lamina propria macro-
phages that are constantly replenished from circulating mono-
cytes (Zigmond et al., 2012; Tamoutounour et al., 2013). In
contrast to what was proposed elsewhere (Epelman et al., 2014),
our results also show that inflammation or injury are not neces-
sarily required for replacement of embryo-derived cardiac
tissue macrophages by monocytes. It will be interesting to
determine whether dynamic age-dependent changes in resi-
dent tissue macrophage populations also occur in other tissues
and whether they may be important for the differences in re-
pair mechanisms and regenerative capacity observed between
young and old individuals.
MATERIALS AND METHODS
Mice. CD45.1 and CD45.2 C57BL/6 mice were obtained from Charles
River. All transgenic mice used in this study have a C57BL/6 background
(>7 backcrosses). CX3CR1GFP/+ mice (Jung et al., 2000), CCR2/, Rosa-26-
yfp, CX3CR1cre (JAX Stock No. 25524 B6.C-Cx3cr1<tm1.1(cre)Jung>/J), and
CX3CR1creER mice (JAX Stock No. 20940 B6J.129-Cx3cr1<tm1.1(cre/
ERT2)Jung>/J; Yona et al., 2013), Ubow mice (Ghigo et al., 2013), Rag2/c/
KitW/Wv mice (Waskow et al., 2009), and panYFP mice (Luche et al., 2013)
were described elsewhere. Experiments were performed on 610-wk-old
mice, if not stated otherwise. Experiments involving CX3CR1cre and
CX3CR1creER mice included littermate controls. For other experiments
C57BL/6 mice served as controls. All mouse experiments were performed
under specific pathogen-free conditions. Animals were handled according
to protocols approved by the Weizmann Institute Animal Care Committee
(Israel), the Federal Ministry for Nature, Environment and Consumers Pro-
tection of the state of Baden-Wrttemberg (Germany) or the animal ethics
committee of Marseille (France) and were performed in accordance to the
respective international, national, and institutional regulations.
Parabiosis. 9-wk-old C57BL/6 CD45.1 mice were sutured together with
9-wk-old B6 CCR2/ CD45.2 and subsequently kept under Bactrim for
10 wk before analysis.
BM chimeras. 710-wk-old host animals were lethally irradiated and were
reconstituted with donor BM by i.v. injection of minimum 106 BM cells.
Donor BM was isolated from femurs and tibias of donor mice, filtered through
a 70-m mesh and resuspended in PBS for i.v. injection. Rag2/c/KitW/Wv
Replacement of embryonic cardiac macrophages with age | Molawi et al.

mice were reconstituted with long-term (LT)HSC from panYFP BM
without prior conditioning as previously described (Waskow et al., 2009).
LT-HSCs were defined as lineage-negative (B220, CD3e, CD4,
CD8a, CD11b, CD19, Gr1, Ter119, NK1.1), Kit+, Sca1+, CD48, Fondation pour la Recherche Mdicale (DEQ. 20071210559 and DEQ.
and CD150+.
Tamoxifen treatment. Tamoxifen (TAM; Sigma-Aldrich) was dissolved in
100% ethanol to get a 1 g/ml solution and was 10-fold diluted in corn oil
(Sigma-Aldrich) to get the 100 mg/ml final solution for oral or i.p. adminis-
tration. To induce Cre-mediated gene recombination in 5- to 7-wk-old
CX3CR1creER mice, 5 mg TAM was administrated orally for 5 consecutive
days (25 mg total). For Cre induction in the embryo, 200 l of 20 mg/ml
TAM and 10 mg/ml Progesterone dissolved in corn oil was injected i.p. into
pregnant females at 9 or 13 days post coitum (dpc).
BrdU pulsing. BrdU was purchased from Sigma-Aldrich. Mice were in-
jected with 0.1 mg BrdU/g body weight. Newborn mice were injected sub-
cutaneously, older mice intraperitoneally. cM isolated from BrdU injected
mice were analyzed 4 h after injection for BrdU incorporation using the
BrdU Flow kit (BD).
Flow cytometry. Cells were acquired on FACSCanto, LSRII, and LSRFor
tessa systems (BD) and analyzed with FlowJo software (Tree Star). The fol-
lowing antibodies were used for staining cells: anti-CD11b (clone M1/70;
eBioscience), anti-CD11c (clone N418; eBioscience), anti-CD14 (clone
Sa2-8; eBioscience), anti-F4/80 (clone BM8; eBioscience), anti-MHCII
(clone M5/114.15.2; eBioscience), anti-CD45.1 (clone A20; eBioscience),
anti-CD45.2 (clone 104; eBioscience), anti-B220 (clone RA3-6B2; eBiosci-
ence), anti-CD115 (clone AFS98; eBioscience), anti-CD117 (clone 2B8;
eBioscience), anti-Sca1 (clone D7; eBioscience), anti-CD48 (clone JM48-1;
eBioscience), anti-Gr1 (clone RB6-8C5; BioLegend), anti-CD64 (clone
X54-5/7.1; BioLegend), anti-Ly6G (clone 1A8; BioLegend), anti-CD150
(clone TC15-12F12.2; BioLegend), anti-Ly6C (clone AL21; BD), anti-
Ki67 (clone B56; BD), anti-CD4 (clone H129.19; BD), anti-CD8 (clone
536.7; BD), anti-CD19 (clone ID3; BD), Ter119 (clone Ter119; BD),
NK1.1 (clone PK136; BD), anti-F4/80, anti-MerTK (clone BAF591; R&D
Systems), anti-BrdU (clone MoBU-1; Life Technologies). Before staining,
cells were preincubated with Fc receptor blocking antibody (clone 2.4 G2;
BD). LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Life Technologies)
was used to stain dead cells. Absolute cell numbers were determined by
using Flow-Count Fluorosperes (Beckman Coulter) according to the manu
facturers instructions.
Preparation of tissue samples. Peripheral blood leukocytes were isolated
by density separation using Lympholite-M solution (Cedarlane) according to
manufacturers instructions. For preparing single cell suspension from heart
and brain, the mice were perfused with PBS before collecting the tissue. For
cM, the heart was macerated and incubated with 1 mg/ml collagenase-2
(Worthington Biochemical Corporation) and 0.15 mg/ml DNase1 (Sigma-
Aldrich) at 37C for 30 min during constant agitation. Resulting cell suspen-
sion was filtered through a 70-m mesh and erythrocytes were removed by
ACK lysis. For microglia isolation a gradient of 70, 37, 30% Percoll was used.
The brain tissue was homogenized and incubated with HBSS solution con-
taining 2% BSA (Sigma-Aldrich), 1 mg/ml collagenase d (Roche), and
0.15 mg/ml DNase1, filtered through a 70-m mesh, and resuspended in
70% Percoll, before density centrifugation (800 g for 30 min at 20C with
low acceleration and no brake).
Online supplemental material. Fig. S1 depicts the full gating strategy and
population characterization of cM. Online supplemental material is avail-
able at http://www.jem.org/cgi/content/full/jem.20140639/DC1.
We thank Dr. Sandrine Sarrazin for critical reading of the manuscript and help
with statistical analysis, L. Razafindramanana for animal handling, and M. Barad,
A. Zouine, and S. Bigot for cytometry support.
JEM Vol. 211, No. 11
Br ief Definitive Repor t
K. Molawi was supported by an HFSP long-term fellowship. The work
was supported by grants to M.H. Sieweke from Aviesan ITMO IHP exploratory
Project A12194AS and CNRS PICS program No. 5730. M.H. Sieweke is a
20110421320) and INSERM-Helmholtz group leader. The work was further
supported by the Israel Science Foundation (ISF), the Deutsche Forschungs
gemeinschaft (DFG) Research Unit (FOR) 1336 (S. Jung and M. Prinz), and
DFG-SFB 938-project L (H.-R. Rodewald and K. Klapproth).
The authors declare no competing financial interests.
Submitted: 6 April 2014
Accepted: 19 August 2014
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Replacement of embryonic cardiac macrophages with age | Molawi et al.
INSIGHTS

M acrophages der ived from infiltrating monocytes mediate

<doi>10.84/jem2nsight1<do>ajem.218insght</ad>uMicelT.Hnka</>AF1UiverstyofBn</AF1>crmihael.nk@ub-ode</cr>haInsigt</doce> piNws</doct>

autoimmune myelin destruction

Macrophages mediate myelin destruction in multiple sclerosis (MS), but the origin of these cells (whether de-
< ID >j e m .2 1 8i n s gh t f j p e < / ID>

rived from tissue-resident microglial cells or infiltrating monocytes) has been widely debated. Now, Yamasaki
and colleagues distinguish these cells in a mouse model of MS and show that monocyte-derived macrophages
(MDMs) mediate myelin destruction, whereas microglia-derived macrophages (MiDMs) clear up the debris.
Previous attempts to decipher the nature and role of cells involved in autoimmune demyelination have
proven challenging. Although ontogenetically distinct, it has not been possible to distinguish macrophages
derived from tissue-resident or -infiltrating cells based on morphological features (by light microscopy) or
surface phenotype. Previous attempts to address this problem include parabiosis and bone marrow trans-
Insight from
plantation after irradiation, both strategies with substantial technical problems and limitations. Michael Heneka
Yamasaki et al. studied double chemokine receptor (CCR2-RFP+; < I D > j em . 2 1 8i ns g h t f p < / I D >

CX3CR1-GFP+) mice in the experimental autoimmune encephalo-

Nodes of Ranvier represent a prime
site of attack for MDMs at the onset
of EAE. This 3D reconstruction of SBF-
SEM images shows a monocyte-derived
macrophage encircling the node of
Ranvier, as shown by the two primary
processes (white and black arrows).
myelitis (EAE) mouse model of MS. Inflammatory lesions were filled with both MDMs and
MiDMs. Confocal immunohistochemistry, serial block-face scanning electron microscopy
(SBF-SEM), and subsequent 3D reconstruction revealed that myelin destruction was initiated
by MDMs, often at the nodes of Ranvier, whereas MiDMs were not detected at this site.
Disruption of MDM infiltration by CCR2 deficiency completely abolished the presence of
macrophages at the nodes of Ranvier. Gene expression profiling of both cell types at disease
onset revealed substantial differences, which correlated well with the observations obtained
by SBF-SEM. MDMs expressed genes attributable to effector functions, including those
involved in phagocytosis and cell clearance. In contrast, MiMD gene expression patterns at
disease onset were characteristic of a repressed metabolic state.
This paper sets a new standard for further studies in the field. For the first time,
MDMs and MiDMs have been clearly differentiated and their morphological relation-
ship to axoglial structures has been analyzed. The finding that MDMs rather than
MiDMs initiate myelin destruction at disease onset should enable this cell population to
be targeted more effectively in future. The next stage is to verify these findings in human
tissue. Future research should also assess further time points over the entire disease
course, in particular to exclude that MiDMs do not join MDMs at the node of Ranvier
at later stages of disease. A precise distinction between local and infiltrating cell popula-
tions may also contribute to a better understanding of pathogenesis in other CNS disor-
ders such as stroke and brain trauma and will hopefully lead to the development of new
therapeutic strategies.
Yamasaki, R., et al. 2014. J. Exp. Med. http://dx.doi.org/10.1084/jem.20132477.

Michael T. Heneka, University of Bonn: michael.heneka@ukb.uni-bonn.de

 Article

Differential roles of microglia and monocytes

in the inflamed central nervous system
Ryo Yamasaki,1 Haiyan Lu,1 Oleg Butovsky,5 Nobuhiko Ohno,2
Anna M. Rietsch,1 Ron Cialic,5 Pauline M. Wu,2 Camille E. Doykan,2
Jessica Lin,1,6 Anne C. Cotleur,1 Grahame Kidd,2 Musab M. Zorlu,1,7
Nathan Sun,8 Weiwei Hu,2,9 LiPing Liu,1 Jar-Chi Lee,3 Sarah E. Taylor,10
Lindsey Uehlein,1,6 Debra Dixon,1,11 Jinyu Gu,1 Crina M. Floruta,1,12 Min Zhu,1
Israel F. Charo,13 Howard L. Weiner,5 and Richard M. Ransohoff 1,4,11
1Neuroinflammation Research Center and 2Department of Neurosciences, Lerner Research Institute; 3Department
of Quantitative Health Sciences; and 4Mellen Center for Multiple Sclerosis Treatment and Research, Neurological Institute,
Cleveland Clinic, Cleveland, OH 44106
5Center for Neurological Diseases, Brigham and Womens Hospital, Harvard Institutes of Medicine, Boston, MA 02115
6Ohio State University College of Medicine, Columbus, OH 43210
7Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
8Vanderbilt University, Nashville, TN 37235
9Department of Pharmacology, School of Basic Medical Sciences, Zhejiang University, Hangzhou, 310058 Zhejiang, China
10Case Western Reserve University, School of Medicine, Cleveland, OH 44106
11Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44106
12Baylor University, Waco, TX 77030
13Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, CA 94158

In the human disorder multiple sclerosis (MS) and in the model experimental autoimmune
encephalomyelitis (EAE), macrophages predominate in demyelinated areas and their num-
bers correlate to tissue damage. Macrophages may be derived from infiltrating monocytes
or resident microglia, yet are indistinguishable by light microscopy and surface phenotype.
It is axiomatic that T cellmediated macrophage activation is critical for inflammatory
demyelination in EAE, yet the precise details by which tissue injury takes place remain
poorly understood. In the present study, we addressed the cellular basis of autoimmune
demyelination by discriminating microglial versus monocyte origins of effector macro-
phages. Using serial block-face scanning electron microscopy (SBF-SEM), we show that
monocyte-derived macrophages associate with nodes of Ranvier and initiate demyelination,
whereas microglia appear to clear debris. Gene expression profiles confirm that monocyte-
derived macrophages are highly phagocytic and inflammatory, whereas those arising from
microglia demonstrate an unexpected signature of globally suppressed cellular metabolism
at disease onset. Distinguishing tissue-resident macrophages from infiltrating monocytes
will point toward new strategies to treat disease and promote repair in diverse inflamma-
tory pathologies in varied organs.

CORRESPONDENCE
Richard M. Ransohoff:
ransohr@ccf.org
Abbreviations used: CNS,
central nervous system; EAE,
experimental autoimmune
encephalomyelitis; MDM,
monocyte-derived macrophage;
MiDM, microglia-derived mac-
rophage; MS, multiple sclerosis;
SBF-SEM, serial block-face
scanning electron microscopy.
Blood-derived monocytes and resident microglia
can both give rise to macrophages in the cen-
tral nervous system (CNS). In tissue sections,
macrophages derived from these two distinct
precursors are indistinguishable at the light
microscopic level both morphologically and by
surface markers. Using flow cytometry, microglia-
and monocyte-derived macrophages can be
isolated separately from CNS tissue lysates and
expression profiling suggests distinct functional
R. Yamasaki, H. Lu, and O. Butovsky contributed equally to
this paper.
capacities (Gautier et al., 2012; Chiu et al., 2013;
Butovsky et al., 2014).
Microglia and monocytes are ontogeneti-
cally distinct: microglia derive from yolk-sac pro-
genitors during embryogenesis (Ginhoux et al.,
2010; Schulz et al., 2012), whereas monocytes
continuously differentiate throughout postnatal
life from bone marrow hematopoietic stem cells
2014 Yamasaki et al. This article is distributed under the terms of an Attribution
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J. Exp. Med. 2014 Vol. 211 No. 8 1533-1549 1533
www.jem.org/cgi/doi/10.1084/jem.20132477
(HSCs), which require the transcription factor Myb. Microg-
lial precursors are Myb independent, and microglia self-renew
independently of bone marrow HSCs (Gomez Perdiguero
et al., 2013). Distinct developmental origin and renewal mech-
anisms imply that monocyte-derived macrophages (MDMs)
and microglia-derived macrophages (MiDMs) might exert dif-
ferent functions in pathological processes. Microglia represent
one instance of tissue-resident macrophages, which reside in all
organs. Studying the CNS as compared with other organs
may carry advantages for distinguishing tissue-resident my-
eloid cells from infiltrating monocytes during disease, as there
is virtually no background trafficking of monocytes in the
CNS parenchyma of healthy animals.
In EAE, which models inflammatory aspects of MS
(Williams et al., 1994; Ransohoff, 2012), macrophages dominate
the inflammatory infiltrates and their numbers correlate to
EAE severity (Huitinga et al., 1990, 1993; Ajami et al., 2011).
However the cellular mechanisms by which macrophages
promote disease progression are uncertain. Whether MiDMs
or MDMs are functionally distinct and whether the two cell
types differentially initiate demyelination or promote repair
(Steinman et al., 2002) also remains elusive (Bauer et al., 1995).
In MS autopsy tissues, prominent macrophage accumulation
correlates with active demyelination (Ferguson et al., 1997;
Trapp et al., 1998). Based on kinetics of cell accumulation and
differential marker expression, its estimated that 3050% of
activated macrophages in active MS lesions derive from mi-
croglia (Brck et al., 1995; Trebst et al., 2001). Therefore, dif-
ferential functions of MDMs and MiDMs are relevant for
human demyelinating disease.
To date, no research techniques have permitted distinction
between monocytes and microglia in CNS tissue without ir-
radiation chimerism or parabiosis, techniques that confound
interpretation or impose practical limitations (Ajami et al.,
2007, 2011; Ransohoff, 2007). When F4/80+ macrophages
were isolated from CNS and analyzed by flow cytometry
using cells from double-heterozygous Ccr2rfp::Cx3cr1gfp mice
with EAE, GFP was expressed by CD45dim/Ly6C microglia,
whereas RFP was restricted to CD45high/Ly6C+ monocytes
(Saederup et al., 2010; Mizutani et al., 2012). These findings
suggested an approach to clarifying distinct roles of MDMs
and MiDMs in EAE based on differential expression of GFP
and RFP reporters. Here, we use that strategy to extend pre-
vious findings and address the hypothesis that MDMs and
MiDMs exert different functions in neuroinflammation. We
detected detailed ultrastructural characterization of MDMs
and MiDMs at EAE onset.
Unexpectedly, this approach provided insight into the cel-
lular basis for autoimmune demyelination, which has remained
obscure despite >80 yr of study in the EAE model. Here we
provide evidence that MDMs initiate demyelination, often at
nodes of Ranvier. In contrast, phagocytic microglia appear rela-
tively inert at disease onset. Results from expression profiling
provided insight into mechanisms and signaling pathways un-
derlying the disparate effector properties of MDMs and MiDMs
in EAE.The distinct functions of tissue-resident myeloid cells
as compared with infiltrating macrophages broadly underlie
disease pathogenesis in manifold circumstances and also hold
promise for innovative treatment strategies.
RESULTS
In the CNS of mice of EAE, MDMs and MiDMs
exhibit different accumulation kinetics
The histological strategy in this study is shown in Table 1. At
onset of EAE, two pools of CD11b+ mononuclear phagocytic
cells (putative red MDMs and green MiDMs) predominated
in spinal cord (Fig. 1 A), indicating that fluorochrome mark-
ers could be distinguished at this time point. Using cells iso-
lated from Ccr2rfp/+::Cx3cr1gfp/+ spinal cords at disease onset,
flow cytometry demonstrated distinct expression of RFP and

Table 1. Histology analysis strategy

Method Purpose Finding
Confocal analysis of 0.2 mm optical To distinguish MDMs (RFP+)from MDMs and MiDMs can be distinguished by cell volume and primary
sections (n = 104 cells). MiDMs (GFP+). processes.
SBF-SEM inspection in 0.2 mm sections To detect MDMs and MiDMs in SBF- Using criteria detected in the previous step, it is possible to distinguish
from 14 lesions, 7 mice at EAE onset. SEM using cell volume and process MDMs and MiDMs in SBF-SEM images.
criteria.
SBF-SEM inspection of ultrastructure To detect ultrastructural characteristics MDMs and MiDMs show characteristic ultrastructural differences
of MDMs and MiDMs. of MDMs and MiDMs. regarding their mitochondria, nuclei, osmiophilic granules and
microvilli.
Quantification of relation of MDMs To determine relationship of MDMs Most (55/75; 73%) axoglial units are contacted in limited fashion by
(n = 169) and MiDMs (n = 86) to and MiDMs to axoglial units and MDMs and MiDMs. If one cell type is present (20/75 cells), its nearly
axoglial units (n = 29 intact axons, characterize presence of myelin debris. always (18/20 segments) MDMs.
46 demyelinated axons).
Reconstruction of 3D shape of four To detect relationship of MDMs with In all, 49 MDMs interacting with axoglial units in absence of nearby
representative MDMs at axoglial axoglial units at EAE onset. MiDMs, 2-3 MDMs were attached to each (n = 18) axoglial unit.
units. MDMs have close relationship with nodes of Ranvier (7 MDMs/75
axoglial units). 3D reconstructions showed four representative MDMs
at axoglial units: show one carrying out active demyelination, three at
nodes of Ranvier.
1534 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
 Ar ticle

GFP by F4/80+/CD45high MDMs and F4/80+/CD45dim
MiDMs, respectively (Fig. 1 B). Enumeration of cells recovered
from cell sorting using F4/80+RFP+ as MDMs gate and F4/
80+GFP+ as MiDMs gate indicated that MDMs and MiDMs
showed equal numbers at disease onset when explosive MDM
accumulation occurred. MiDM expansion began at peak
(Fig. 1 B). At recovery, MiDMs were found near preonset
numbers as MDM frequency continued to decline, which is
compatible with previous studies (Ajami et al., 2011). There-
fore, there were unequal numbers of MiDMs/MDMs before
and after disease onset (Fig. 1 B). Morphological analyses and
definitions of relations between myeloid cells and axoglial
units were conducted at disease onset so that equal numbers
of MDMs and MiDMs could be assayed and early events in
the demyelinating disorder could be explored.
Morphological features distinguish
MDMs and MiDMs at EAE onset
Immunofluorescence staining for RFP and GFP in spinal cord
at EAE onset showed that red MDMs exhibited elongated or
spindle shape, whereas green MiDMs showed a process-bearing
morphology (Fig. 1 C). Quantification in 3D reconstructions
from 0.2-m confocal z-stack images showed that MiDMs
exhibited much larger size than MDMs along with multiple
primary processes, which were sparse in MDMs (Fig. 1 D).
Several 3D shape parameters also discriminated between MDMs

Figure 1. MDMs and MiDMs exhibit different time courses of accumulation in the CNS of mice with EAE and morphological characteristics
can distinguish them. (A) Immunohistochemistry shows expression of CD11b for red RFP+ MDMs and green GFP+ MiDMs in the spinal cords of
Cx3cr1gfp/+::Ccr2rfp/+ mice at EAE onset. Bars: 25 m. We studied 6 mice at EAE onset from 3 EAE inductions. In each EAE induction, 810 mice were used and
2 mice were selected from each induction. (B) Flow cytometric analysis of CCR2-RFP+ and CX3CR1-GFP+ populations in cells gated for F4/80 expression
(top); CD45 expression of F4/80+RFP+ MDMs and F4/80+GFP+ MiDMs populations (middle); and MDMs and MiDMs numbers at EAE onset, peak, and recovery
(bottom). We studied 3 mice for naive groups; 12 for onset; 15 for peak; 13 for recovery from 5 EAE inductions. For each induction, 810 mice were used
and 23 mice were selected at each time point (onset, peak, and recovery) for analysis. (C) Confocal microscopy assessment of myeloid cell morphology in
lumbar spinal cord from mice at EAE onset. We studied 54 MDMs and 51 MiDMs of 5 EAE onset mice from 3 EAE inductions for (CE); 2 sections/mouse;
46 cells/section; 812 cells/mouse. In each EAE induction, 810 mice were induced and 12 EAE onset mice were selected from each experiment. Bars,
25 m. (D) Cell volumes of 500 m3; surface areas of 1,000 m2; primary process numbers 3 or 5; solidity3D of 0.4; and Formfactor3D of 0.3 discrimi-
nate between MDMs and MiDMs. (E) Model plot of cell volume against primary process number to distinguish MDMs (red symbols and pink area) from
MiDMs (green symbols and green area).

JEM Vol. 211, No. 8 1535

and MiDMs (Fig. 1 D). We observed scant overlap of several
values between MDMs and MiDMs (Fig. 1 D), and entirely
nonoverlapping distributions for cell volume and primary
processes (Fig. 1 E).
MDMs and MiDMs exhibit differentiating
ultrastructural characteristics at EAE onset
We used confocal microscopy in 0.2-m optical sections to
correlate structural features of MDMs and MiDMs with RFP
or GFP fluorescence, as a bridge to characterizing cells in 0.2 m
SBF-SEM images (Table 1). Using this approach (Fig. 1 E),
MDMs and MiDMs were identified by estimating volume
and counting primary processes. Volume estimations came
from multiplying the midcell area by the number of sections in
which the cell was identified. In electromagnetic (EM) images,
quantitative analysis also demonstrated differentiating ultrastruc-
tural characteristics for mitochondria, nuclei, cytoplasmic os-
miophilic granules and microvilli (unpublished data). MDMs
had shorter, thicker mitochondria than MiDMs (unpublished
data). Total mitochondrial numbers and volumes were equal
in MDMs and MiDMs (unpublished data). MDMs had bi-
lobulated or irregular nuclei, whereas MiDMs had round nu-
clei (unpublished data). MDMs, but not MiDMs, frequently
contained osmiophilic granules and microvilli (unpublished
data). Collectively, these ultrastructural features provided con-
firmatory ultrastructural characteristics to distinguish MDMs
from MiDMs.
MDMs initiated demyelination at EAE onset
Results from confocal and EM analysis yielded a secure basis
for examining the relationships of MDMs and MiDMs to ax-
oglial units at EAE onset (n = 7 mice; 14 lesions) using serial
block-face scanning electron microscopy (SBF-SEM), as pre-
sented diagrammatically (Table 1). We quantified contacts
made by MDMs (n = 169) and MiDMs (n = 86) with axoglial
units (n = 75; Fig. 2), and observed that most (55/75; 73%) of
all segments (both intact and demyelinated) contacted both
MDMs and MiDMs (Fig. 2). Where only one myeloid cell
type was present (20/75; 27%), nearly all axoglial units made
contacts to MDMs (Fig. 2). In particular, 8/29 intact and 10/46

Figure 2. SBF-SEM shows MDMs initiating demyelination at EAE onset. Quantitation of MiDMs and MDMs interacting with axoglial units in SBF-
SEM images of CNS at EAE onset. Intact (69%) and demyelinated (76%) segments interacted with MDMs and MiDMs. Red and pink, MDMs; green and
light green, MiDMs; yellow, both MDMs and MiDMs. We studied 29 intact axon segments, 46 demyelinated axon segments, 86 MiDMs, and 169 MDMs in
14 lesions of 7 EAE onset mice from 3 EAE inductions as follows: 810 mice were immunized at each experiment and 23 onset mice were selected from
each induction.

1536 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.


demyelinated axoglial units were contacted solely by MDMs.
We found 23 MDMs attached to each of the 18/20 (90%)
axoglial units where only MDMs were present (Fig. 2). More
than half of all analyzed MDM and MiDM cells (n = 255
total) contained myelin debris, regardless of whether axon
segments were intact or demyelinated (Fig. 2). Of the MDMs
found in sole contact with axoglial units, virtually all (>90%)
MDMs contained myelin when found in sole contact with a
demyelinated axon (Fig. 2). These findings motivated evalua-
tion of relationships of MDMs to axoglial units by 3D recon-
struction of SBF-SEM image stacks.
MDMs frequently exhibited morphological characteristics
suggesting an involvement in active demyelination. Reconstruc-
tion of one representative image stack shows MDMs with
large intracellular myelin inclusions tightly encircling a partially
demyelinated axon (Fig. 3 A). The myelin peeled away from
the axon remained in continuity with a large myelin inclusion
JEM Vol. 211, No. 8
Ar ticle
inside the MDMs (Fig. 3 A). Remaining myelin was undergoing
vesicular breakdown (Fig. 3 A). In contrast, a nearby MiDM
encompassed a large fragment of myelin debris (Fig. 3 B) and
contacted the nearby MDMs (Fig. 3 B), but made minimal
connection to the axoglial unit (Fig. 3 B). In our SBF-SEM
data, only MDMs seemed to be implicated in active damage
to myelin. These observations suggested that MDMs initiated
demyelination at the onset of EAE.
MDMs surrounded apposed and invaded
nodes of Ranvier at EAE onset
We analyzed axoglial units to examine the nature of contacts
with myeloid cells. Unexpectedly, 7/75 (9%) of axoglial units
demonstrated MDMs attached to nodes of Ranvier. In each
case, the contact between MDMs and node appeared to be
pathogenic. One representative monocyte surrounded a node
of Ranvier with two microvilli interposed between myelin
Figure 3. SBF-SEM shows example of
MDM-initiating demyelination at EAE
onset. (A) Representative MDMs encircles the
axoglial unit. A myelin ovoid within an intra-
cellular phagolysosome shows physical conti-
nuity with myelin remaining attached to an
axoglial unit which is undergoing active de-
myelination. In serial images, disrupted myelin
shows continuity from outside to inside the
MDM. (B) Rotated view from B demonstrating
MDM-extensive attachment to axoglial unit
and MiDM nearby with limited attachment to
axon. A, axon; m, myelin; c, cytosol; n, nucleus.
Red, MDM cytosol; green, MiDM cytosol;
yellow: nuclei; blue, myelin and myelin debris;
gray, axoplasm; red line, MDM plasma mem-
brane. We studied 14 lesions from 7 EAE on-
set mice from 3 EAE inductions as follows:
810 mice were immunized at each experi-
ment and 23 EAE onset mice were selected
from each induction. Bar, 2 m.
1537
and axolemma near the paranode complex (Fig. 4 A). The ax-
oglial unit appeared otherwise healthy and no myelin debris
was found in the MDM cytosol. This observation suggested
that initial MDMaxoglial contacts might occur at nodes of
Ranvier. Further, we detected an intratubal (Stoll et al., 1989)
MDMs with myelin debris interposed between compact my-
elin and axolemma near a node of Ranvier (Fig. 4 B). Addi-
tionally we identified an MDM apposed to a node of Ranvier
and actively phagocytizing myelin (Fig. 4 C). At this node,
paranode loops were disrupted and surrounded by MDM cy-
tosol (Fig. 4 C), indicating likely involvement in damaging my-
elin near the node. No MiDMs contacted nodes of Ranvier.
Nodal pathology without demyelination
at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice
We interpreted our ultrastructural findings to indicate that
MDMs recognized altered nodal structure and initiated demy-
elination at EAE onset. CCR2 is essential for monocyte recruit-
ment to CNS tissues during immune-mediated inflammation
(Fife et al., 2000; Izikson et al., 2000; Savarin et al., 2010). To
1538
address the role of MDMs in demyelination at EAE onset, we
investigated clinical characteristics in relation to node pathology
and demyelination in Ccr2rfp/rfp::Cx3cr1gfp/+ mice in which
MDMs were virtually absent from inflamed EAE tissues and
replaced in large part by neutrophils (Saederup et al., 2010).We
observed equivalent magnitude of weight loss in Ccr2rfp/+::
Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and
onset stages of EAE, showing that CCR2 deficiency did not
affect systemic inflammation in this model (Fig. 5 A). There was
a moderate delay in disease onset (Fig. 5 B) and slight reduction
in EAE onset severity (Fig. 5 A) in Ccr2rfp/rfp::Cx3cr1gfp/+ mice.
SBF-SEM was used to evaluate nodal pathology, myeloid
cell relations to axoglial units and demyelination at and before
EAE onset. In three distinct tissues from individual Ccr2rfp/+
::Cx3cr1gfp/+ mice with EAE preonset, we found five MDMs
attached to disrupted nodes of Ranvier. In an equivalent sam-
ple of EAE tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ mice, only
one MDM was found in contact with a node of Ranvier, despite
the presence of disrupted nodes in proximity to neutrophils.
One representative MDM from Ccr2rfp/+::Cx3cr1gfp/+ tissue
Figure 4. MDMs surrounded, apposed,
and invaded nodes of Ranvier at EAE
onset. (A) SBF-SEM images and 3D reconstruc-
tion of SBF-SEM images of MDMs with a node
of Ranvier. White and black arrow: microvil-
lus. (B) SBF-SEM images and 3D construction
of intratubal MDMs with demyelinated axon
and node of Ranvier. (C) SBF-SEM images and
3D reconstruction of an MDM with intracel-
lular myelin debris apposed to a node of Ran-
vier. Red, MDM cytosol; yellow, nucleus; blue,
myelin; gray, axoplasm. M, myelin; c, cytosol.
red line, MDM plasma membrane. We studied
14 lesions from 7 EAE onset mice collected as
follows: 810 mice were immunized at each
induction and 23 EAE onset mice were
selected from each immunization. Bar, 2 m.
Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
 Ar ticle

Figure 5. Nodal pathology without demyelination at EAE onset in Ccr2rfp/rfp::Cx3cr1gfp/+ mice. (A) Magnitude of weight loss in Ccr2rfp/+::Cx3cr1gfp/+ and
Ccr2rfp/rfp::Cx3cr1gfp/+ mice at preonset and onset stages of EAE. Clinical score in Ccr2rfp/+::Cx3cr1gfp/+ and Ccr2rfp/rfp::Cx3cr1gfp/+ mice at EAE onset stage. (B) Days
at disease preonset and onset stages of EAE. We studied 28 Ccr2rfp/+::Cx3cr1gfp/+ mice and 26 Ccr2rfp/rfp::Cx3cr1gfp/+ mice for A and B. Data were collected from
12 EAE inductions in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 EAE inductions in Ccr2rfp/rfp::Cx3cr1gfp/+ mice as follows: 810 mice were immunized at each induction and
13 EAE recovery mice were selected from each immunization. **, P < 0.01; ***, P < 0.001. (C) SBF-SEM imaging of MDMs with nodes of Ranvier phagocytosis in
Ccr2rfp/+::Cx3cr1gfp/+ mice at EAE preonset. Pink, MDM cytosol; red arrow, myelin inclusion of MDM connecting to the node of Ranvier. We studied 3 tissues from 3
Ccr2rfp/+::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 810 mice were immunized at each experiment and one EAE preonset mouse was selected
from each induction. Bar, 2 m. (D) SBF-SEM of disrupted nodes (black arrows) in preonset spinal cord tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice. Bar, 2 m. (E) SBF-
SEM of neutrophil is with myelin phagocytosis from internode at preonset stage of Ccr2rfp/rfp::Cx3cr1gfp/+ mouse. Blue, neutrophil cytosol. For DE, we studied
three tissues from three Ccr2rfp/rfp::Cx3cr1gfp/+ EAE mice at preonset stage from 3 EAE inductions: 810 mice were immunized at each experiment and one EAE
preonset mouse was selected from each induction. Bar: 2 m. (F) Histochemical staining and with aurohalophosphate complexes (black gold staining) and quanti-
fication of demyelinated area of Ccr2rfp/+::Cx3cr1gfp/+ mice and Ccr2rfp/+::Cx3cr1gfp/+ mice. We studied 5 naive Ccr2rfp/+::Cx3cr1gfp/+ mice, 5 naive Ccr2rfp/rfp
::Cx3cr1gfp/+ mice, 5 onset Ccr2rfp/+::Cx3cr1gfp/+ mice, and 5 onset Ccr2rfp/rfp::Cx3cr1gfp/+ mice from 3 EAE inductions as follows: 810 mice were immunized at each
experiment and 12 onset mice were selected from each induction. **, P < 0.01. Bar: 250 m.

JEM Vol. 211, No. 8 1539

Figure 6. Inflammatory signature in MiDMs versus MDMs in the CNS of Cx3cr1gfp/+::Ccr2rfp/+ mice with EAE. (A) Quantitative nCounter expres-
sion profiling of 179 inflammation related genes was performed in CNS-derived GFP+ microglia and RFP+ recruited monocytes from naive and EAE mice
at onset, peak and recovery stages. Each row of the heat map represents an individual gene and each column an individual group from pool of 5 mice at
each time point. The relative abundance of transcripts is indicated by a color (red, higher; green, median; blue, low). For AH, we studied five mice in each
time point (onset, peak, recovery) from 3 EAE inductions; 810 mice were immunized in each induction. (B) Heat map of differentially expressed microglia

1540 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.


having concave nucleus (Fig. 5 C, left) had multiple intracellu-
lar myelin inclusions, one of which (Fig. 5 C, left middle) was
physically connected to a myelin sheath (Fig. 5 C, right mid-
dle) at a paranode (Fig. 5 C, right), indicating active ongoing
demyelination at a node of Ranvier. By distinct contrast, EAE
onset tissues of Ccr2rfp/rfp::Cx3cr1gfp/+ mice were characterized
by nodal pathology often without cellular infiltrates (Fig. 5 D).
In one instance, we detected a neutrophil abstracting myelin
from the myelin internode (Fig. 5, left and right) despite a
nearby disrupted node (Fig. 5, left) in tissues from a Ccr2rfp/rfp::
Cx3cr1gfp/+ mouse. Importantly, there was no evidence for
neutrophil recognition of disrupted nodes of Ranvier. We in-
terpreted these observations to suggest that MDMs specifically
recognized nodal components to initiate demyelination, and that
absence of MDMs at disrupted nodes of Ccr2rfp/rfp::Cx3cr1gfp/+
mice with EAE was caused by the virtual absence of infiltrat-
ing monocytes (Saederup et al., 2010).
To quantify the outcome of these ultrastructural differences,
we monitored demyelination using histochemical staining with
aurohalophosphate complexes at disease onset in Ccr2rfp/rfp::
Cx3cr1gfp/+ and Ccr2rfp/+::Cx3cr1gfp/+ mice. Demyelination was
significantly reduced at EAE onset in CCR2-deficient mice
(Fig. 5 F), indicating the importance of MDM recognition of
disrupted nodes for efficient inflammatory demyelination. Fur-
thermore, as nodal pathology was equivalent in Ccr2rfp/rfp::
Cx3cr1gfp/+ (Fig. 5 D) and Ccr2rfp/+::Cx3cr1gfp/+ mice at the
preonset stage of EAE, the results suggested that inflammatory
nodal disruption could be reversible if MDMs were prevented
from initiating demyelination at those sites.
Expression profiling demonstrates differential
MiDMs and MDMs gene expression
across the time course of an EAE attack
We reasoned that different phenotypes (Fig. 1) and effector
properties (Figs. 24) of MDMs and MiDMs should be re-
flected in distinct gene expression profiles in the dynamic CNS
microenvironment during EAE. To address this hypothesis,
nCounter digital multiplexed gene expression analysis (Kulkarni,
2011) was performed using directly ex vivo naive microglia
and splenic F4/80+ macrophages (here termed monocytes and
considered similar to microglia by expression profiling; Gautier
et al., 2012), as well as flow-sorted MiDMs or MDMs across the
time course of an EAE attack. Microglia and MiDMs clus-
tered together during unsupervised hierarchical clustering, as
did monocytes and MDMs (Fig. 6, A and B). In both MiDMs
and MDMs, naive and recovery-stage expression profiles were
more alike than were onset and peak-stage profiles (Fig. 6,
A and B) suggesting a return to homeostasis at EAE recovery.
and monocyte genes. (C) Enriched monocyte genes as compared with resident microglia. Bars represent fold changes of gene expression across naive and
all disease stages versus resident microglia. (D) Enriched microglia genes as compared with recruited monocytes. Bars represent fold changes of gene
expression across naive and all disease stages versus recruited monocytes. (EH) K-means clustering of inflammation genes in resident microglia and
recruited monocytes. K-means clustering was used to generate 5 disease stagerelated clusters in MiDMs. Heat map (E) and expression profile (F) of in-
flammation genes in MiDMs are shown by generated clusters. MDM expression matrix overlaid on microglial based clusters shows (G) heat map and
(H) expression profile.
JEM Vol. 211, No. 8
Ar ticle
We noted a subset of genes that were expressed in microglia
and highly regulated in MiDMs during EAE, but not ex-
pressed at all in monocytes or MDMs (Fig. 6, A and B). Con-
versely, a subset of MDM-enriched genes were dynamically
regulated in monocytes and MDMs but not in microglia
(Fig. 6, A and B). MDM-enriched genes were sharply up-
regulated from naive monocytes to onset and peak-stage
MDMs (Fig. 6 B), descending toward naive levels during
recovery (Fig. 6 B). In contrast, MiDM-enriched genes
were strongly expressed in naive cells, almost uniformly si-
lenced at onset, and began a return toward naive levels at peak
and recovery (Fig. 6 B). Comparing MDM-enriched genes
with MiDM-enriched genes showed that MDMs were
more likely to express effector functions, including secreted
factors and surface molecules (18/28; 64.3% of MDM-
enriched genes encoded effector functions; Fig. 6 C: and
Table S1, purple genes). In contrast, only 18/48 (37.5%) of
MiDM-enriched genes encoded effector functions (Fig. 6 D,
Table S1, purple genes). These observations indicated that
MiDMs and MDMs exhibited markedly distinct expression
profiles during EAE.
Differential expression of macrophage
effector functions by MiDMs and MDMs
Our ultrastructural analysis of myeloid cells in EAE focused
on myeloid cell relationships to tissue elements. Expression
profiling also addressed the cytokine and growth factor output
of MiDMs and MDMs, potentially providing insight into disease
pathogenesis.We used k-means clustering to discriminate five
distinct patterns of MiDM gene expression during the course
of EAE (Fig. 6, E and F). The red, blue and green groups in-
creased in MiDMs at onset, peak, and recovery, respectively.
Red group genes involved several surface molecules. Green
group genes, up-regulated at onset and transiently further
up-regulated at peak, were comprised mainly of complement-
system elements (C3aR1; C4a, C1qa, C1qa, C3, and Cfb);
mononuclear cellspecific chemokines (CCl2, 3, 4, 5, 7, and
CXCL9); proliferation related genes ( fos, jun, myc, and CSF1);
and acute inflammationrelated genes (IL1a, IL1b, TNF,
CEBP, STAT1). Cell growthrelated genes expressed at this
time point correlated to reported patterns of microglial pro-
liferation during EAE (Ajami et al., 2011). Blue group genes
up-regulated at recovery included heterogeneous cytokines
(IFN-, IFN-, TGFB3, IL2, IL3, IL4, IL12, IL12,
PDGFA, CSF2, and CXCL2). Both yellow group and golden
group genes were strongly expressed in naive microglia, re-
duced drastically at onset, and either returned to preEAE lev-
els during recovery (yellow) or failed to do so (golden).These
1541
genes included a large spectrum of intracellular signaling com-
ponents from the MAP-kinase pathways, as well as TGF and
receptor, both of which are implicated in the nave microglial
phenotype (Butovsky et al., 2014).
These five gene groups were also analyzed for MDM ex-
pression patterns during EAE (Fig. 6, G and H). None of the
gene groups showed coordinate regulation patterns in MDMs,
as were observed in MiDMs (Fig. 6 H). This observation un-
derscored disparate responses of MiDMs and MDMs to the
inflammatory CNS microenvironment of EAE, despite their
being present in close proximity (Fig. 1 A).
Expression patterns at EAE onset
in relation to MiDM and MDM function
To determine whether gene expression patterns could be in-
formative for understanding the relationships of cells to ax-
oglial elements in tissues at EAE onset, we interrogated naive
versus onset MDM and MiDM gene expression related to
cellular functions (Fig. 7). MiDMs showed highly significant
up-regulation of functions associated with cell movement, che-
moattraction, and migration (Fig. 7 B). In the Ingenuity IPA
database, the terms cell movement, chemoattraction, and mi-
gration indicated production of chemokines such as CCL2,
CCL3, CCL4, CCL5, and CCL7, which are up-regulated at
onset and further increased at peak (Fig. 6, E and F, green
group and genes). In other respects, MiDMs exhibited a re-
pressed metabolic and activation phenotype by comparison
to naive microglia (Fig. 7 B) including proliferation, RNA
metabolism, cytoskeletal organization, microtubule dynamics,
extension of processes, phagocytosis and generation of reac-
tive oxygen species.
MDMs showed up-regulation of functions associated to
macrophages, including phagocytosis, calcium signaling, pro-
duction of prostanoids, adhesion, autophagy, and cell clearance
(Fig. 7 B).This pathway analysis corresponded well to effector
properties displayed by MDMs in our SBF-SEM analysis
(Figs. 24). No functions were reported to be down-regulated
in MDMs at EAE onset as compared with naive monocytes.
A comprehensive listing (Table S1) of all genes regulated
by at least twofold in MiDMs or MDMs as compared with
expression levels in naive mice affirmed and extended these
interpretations. At EAE onset when SBF-SEM analyses were
performed, MiDMs predominantly suppressed the distinctive
gene expression pattern which correlates to their unique phe-
notype (Chiu et al., 2013), reflected by the observation that
MiDMs down-regulated far more genes than were up-regulated
(Table S1). In contrast, MDMs up-regulated far more genes
than did MiDMs and up-regulated more transcripts than were
down-regulated. Additionally, the extent of gene up-regulation
in MDMs exceeded that seen in MiDMs.
MiDM and MDM gene expression kinetics reflected
return toward homeostasis in recovery stage
These expression profiles showed consonant changes for the
vast majority of genes analyzed: if a gene was up-regulated at
any time point, then its expression level showed an increase at
1542
other time points as well. However, a substantial minority of
genes both for MDMs and MiDMs showed some dissonant
time points, at which a previously down-regulated gene might
show up-regulation (unpublished data). We show this subset of
recovered genes in Fig. 8. In virtually every case (Fig. 8, AD),
these dissonant compensatory changes took place during recov-
ery and almost always showed an increase in a gene that had
been down-regulated during onset and peak. Both MiDMs
(Fig. 8 B) and MDMs (Fig. 8 D) demonstrated this pattern of
gene-expression kinetics.
Convergent and divergent responses to upstream
regulatory signaling by MiDMs and MDMs
Translation of observations made using expression profiles can
be enabled through identification of upstream regulators. We
used Ingenuity IPA software to identify putative upstream reg-
ulators of the gene expression alterations demonstrated by
MiDMs and MDMs at disease onset. Putative regulatory ele-
ments were then grouped in signaling modules and subjected
to pathway analysis. Cell motility pathways were clearly differ-
ent in MiDMs and MDMs (unpublished data). Core elements
such as RhoA (Xu et al., 2009) were regulated divergently and
associated signaling components were predicted to be enhanced
in MDMs but depressed in MiDMs, consistent with our pheno-
typic characterization using SBF-SEM. Both HIF-1 (Fig. 9 A)
and TNF pathways (not depicted) were also differentially reg-
ulated in MiDMs and MDMs. By contrast, type I IFN pathway
(Fig. 9 B) was regulated virtually identically in MiDMs and
MDMs. Collectively, these data suggest that HIF-1 and TNF
signaling may partly drive pathogenic properties of MDMs.
Additionally, these data indicated that the separate ontogeny
of microglia and monocytes will lead, probably by epigenetic
influences, to divergent responses to some but not all environ-
mental stimuli, with phenotypic consequences according to
the CNS microenvironment.
DISCUSSION
In this study, we developed a novel strategy to discriminate
MDMs from MiDMs. We used SBF-SEM to address the de-
tailed relationships of MiDM and MDM to axoglial units in
the spinal cords of mice at EAE onset and expression profiling
to examine potential mechanisms. Selection of the EAE disease
model ensured that both recruited monocytes and resident mi-
croglia were exposed to the same intensely inflammatory en-
vironment to increase the likelihood that ambient conditions
could activate these two myeloid cell types toward a convergent
inflammatory phenotype. Instead, we found strikingly diver-
gent relationships of MDMs and MiDMs to axoglial units, by
quantitative and qualitative ultrastructural analysis. Results
from expression profiling supported this interpretation by show-
ing that MiDM metabolism was severely down-regulated,
whereas expression profiles of MDMs reflected the activated
phagocytic phenotype observed through SBF-SEM.
Several salient new observations emerged from these ex-
periments. First, we showed that MDMs initiate demyelination
at EAE onset, as MDMs were the overwhelmingly dominant
Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.
 Ar ticle

cells found in isolation attached to axoglial units and demon-
strated destructive interactions with myelinated axons in 3D
reconstructions. Second, MDMs were unexpectedly observed
at nodes of Ranvier in 9% of axoglial units and showed remark-
ably invasive behavior, including extension of microvilli (Fig. 4 A)
or localization of cell soma (Fig. 4 B) between axolemma and
myelin sheath. Our observed frequency of MDMnodal inter-
action represents a minimum estimate as MDMs found at hemi-
nodes adjacent to a demyelinated segment (Fig. 3 B) were not
scored. Comparison of Ccr2rfp/rfp::Cx3cr1gfp/+ and Ccr2rfp/+::
Cx3cr1gfp/+ mice at and before EAE onset emphasized the
importance of MDMs for this mechanism of demyelination. In
particular, neutrophils in inflamed CNS of Ccr2rfp/rfp::Cx3cr1gfp/+
mice did not recognize disrupted nodes. These observations
are clinically pertinent: our detection of MDMs at nodes of
Ranvier is consistent with recent reports of nodal pathology
in clinical demyelinated tissues (Fu et al., 2011; Desmazires
et al., 2012).The present observations extend this concept and
provide a cellular basis for nodal pathology at the earliest stages
of demyelination. Given the presence of potential phagocytic
signals at nodes (antibodies to paranodal proteins such as contac-
tin and neurofascins; Meinl et al., 2011); complement-derived

Figure 7. Affected functions in MiDMs and MDMs at EAE onset. nCounter inflammatory gene expression data were uploaded to IPA. Genes with
fold change (EAE onset vs. Naive) 1.5 or 1.5 were included in downstream effects analysis. (A) MDMs up-regulated functions, sorted by activation
z-score. (B) MiDMs up-regulated (left) and down-regulated (right) functions, sorted by activation z score. The bias term indicates imbalanced numbers of
up- and down-regulated genes associated with a distal function requiring significance at the P < 0.01 level. We studied pooled samples from 5 mice in
each time point (onset, peak, recovery) from 3 EAE inductions; 810 mice were immunized in each induction.

JEM Vol. 211, No. 8 1543

Figure 8. Restoration of affected inflammatory genes in resident microglia and recruited monocytes at recovery stage. For each gene, fold
change of all different disease stages versus naive state were calculated. Genes that contained at least one fold change >2 or < 0.5 and average fold
change for peak and onset >2 or <0.5 were presented. (A) Microglial up-regulated; (B) Microglial down-regulated; (C) monocyte up-regulated; (D) monocyte
down-regulated. We studied pooled samples from five mice in each time point (onset, peak, recovery) from three EAE inductions; 810 mice were immu-
nized in each induction. FC, fold change.

1544 Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.

 Ar ticle

Figure 9. Function networks in MiDMs and MDMs. nCounter inflammatory gene expression data were uploaded to IPA. Genes with fold change
(EAE onset vs. Naive) 1.5 or 1.5 were included in upstream regulators analysis. Predicted upstream regulators were manually curated to form func-
tional clusters. Clusters were uploaded to IPA using Z scores as reference value for each gene. Networks were generated for each cluster consisting of
uploaded genes and additional predicted molecules. (A) Typical example of functions with dissimilar activation pattern in MiDMs and MDMs: HIF1A.
(B) Function with similar activation pattern in MiDMs and MDMs: Type I IFN. Red object denotes positive (>2) z score and green object denotes negative

JEM Vol. 211, No. 8 1545

opsonins (Nauta et al., 2004); and stress-induced eat-me sig-
nals (Hochreiter-Hufford and Ravichandran, 2013), it may be
feasible to identify a direct molecular pathway for initiating de-
myelination in this model. Third, we characterized a molecu-
lar signature for resident microglia at EAE onset. Grouping of
regulated genes into functional categories demonstrated a
remarkable down-regulation of microglial metabolism at the
nuclear, cytoplasmic and cytoskeletal levels.
In our initial experiments we found that the presence of
myelin debris at the peak of EAE did not discriminate MDMs
from MiDMs. We considered that SBF-SEM would exhibit
advantages for spatial resolution (Denk and Horstmann, 2004)
required for characterizing relationships of myeloid cells to
axoglial units during the inflammatory demyelinating process
at EAE onset.To take advantage of this technique we developed
methods based on cell volume and process number (Fig. 1 E),
to distinguish MDMs from MiDMs in 0.2-m confocal optical
sections, and translated this approach directly to SBF-SEM
image sets at 0.2-m intervals.We also noted differential nuclear
morphology, mitochondrial shape, and osmiophilic granule
content between MDMs and MiDMs. These characteristics of
MDMs and MiDMs may not be universally present in other
pathological circumstances but demonstrate an approach
to ultrastructural distinction of myeloid cell populations in
tissue sections.
Gene expression profiling across the time course of EAE
yielded intriguing kinetics as analyzed by k-means clustering.
Five patterns were observed. Red group genes (increased at
onset) comprised the smallest number and involved several sur-
face molecules: CCR1, CCR7, CXCR2, and CD40. Of these,
CCR7 and CD40 have been reported on activated microglia,
including those observed in MS tissue sections (Kiviskk et al.,
2004; Serafini et al., 2006). GAPDH was up-regulated in
MiDMs at onset. Although often regarded as a housekeeping
gene, GAPDH is found in complexes that limit the translation
of inflammatory gene transcripts in activated mouse macro-
phages (Mukhopadhyay et al., 2009; Arif et al., 2012). As pre-
viously reported (Chiu et al., 2013), MiDM gene expression
during the course of EAE did not correspond to the M1/M2
pattern of peripheral macrophage responses to infection or tis-
sue injury. Microglial morphological transformation can be
relatively uniform regardless of the inflammatory process that
provokes it. Despite this apparent uniformity, gene expression
by morphologically identical microglia can differ drastically
contingent on context (Perry et al., 2007).
Unsupervised hierarchical clustering provided insight into
gene expression patterns of MDMs and MiDMs. Naive and
recovery patterns were similar for both cell types. At disease
onset, microglia showed drastic down-regulation of the expres-
sion profile observed in cells from healthy brain. Brisk mi-
croglial proliferation (Ajami et al., 2011) may have accelerated
(<2) z-score. Orange object denotes predicted activation of the network object. Blue object denotes predicted inhibition of the network object. Predicted
relationships (connecting lines): orange, leads to activation; blue, leads to inhibition; yellow, finding inconsistent with state of downstream molecule;
gray, effect not predicted.
1546
a gradual return toward a homeostatic expression profile, as
suggested by MiDM up-regulation of fos, jun, myc, and CSF1
(Wei et al., 2010) at disease onset. By striking contrast, MDMs
up-regulated a large suite of inflammation-associated genes at
EAE onset, with subsequent regression to the expression phe-
notype of circulating monocytes.
Blood monocytes and resident microglia were exposed to
the same inflammatory environment. However, their preEAE
states were extremely distinct, with monocytes being gener-
ated from a bone marrow progenitor within weeks of entry
into CNS, whereas microglia originated during early embryo-
genesis and had inhabited a serum-free unique environment
from midgestation. In a recent study, we characterized resident
microglia by profiling mRNA, miRNA, and protein in com-
parison with infiltrated brain macrophages, nonmicroglial resi-
dent brain cells, and peripheral macrophages (Butovsky et al.,
2014). The detailed profiling after separating cells via CD45dim
status showed distinct mRNA, miRNA, and protein expres-
sion by microglia as compared with infiltrating monocytes or
neuroepithelial brain cells (Butovsky et al., 2014). The study
described transcription factors and miRNAs characteristic of
microglia in healthy brain but not in peripheral monocytes.
These findings partially explain a divergent response of these
two cell types to the same stimuli (Butovsky et al., 2014).
The strength of the study is that the dual reporter system
is sufficient to accurately distinguish monocyte versus mi-
croglial cells and thus to address the general concept that
monocytes and microglia can exert differential functions in
a CNS disease process. At the onset of EAE, the time point
at which our imaging studies were focused, we are able to
make an unequivocal distinction between resident microglia
(CX3CR1gfp) and infiltrating monocytes (CCR2rfp). Two em-
pirical observations underline this discrimination: microglia
are uniformly CX3CR1+ from early embryonic time points
through adulthood (Cardona et al., 2006; Ginhoux et al., 2010;
Schulz et al., 2012), and CCR2+Ly6C+ cells constitute the vast
majority of infiltrating monocytes at EAE onset (Saederup
et al., 2010; Mizutani et al., 2012).
There were unavoidable limitations of our research; spe-
cifically, to address how monocytes and microglia respond to
a shared microenvironment, we focused on a single, patho-
genically relevant time point: onset of EAE. For this reason, it
was beyond the scope of our study to decipher the phenotypic
fate of infiltrated monocytes. In peripheral models of inflam-
mation, Ly6Chi/CCR2rfp monocytes down-regulate the re-
porter over time and show phenotypic evolution. Furthermore,
our conclusions should not be generalized beyond the present
disease paradigm: in other models, such as spinal cord contusion,
the inflammatory infiltrate includes Ly6Clow/CX3CR1gfp
monocytes, which are highly pathogenic (Donnelly et al.,
2011). Our findings carry biological and medical significance
Distinguishing microglia and monocytes in EAE CNS | Yamasaki et al.

by demonstrating and characterizing differential responses of
infiltrating monocytes and resident microglia in a relevant dis-
ease model at a prespecified time point, at which point patho-
genic events are taking place.Therefore, we focused our analysis
on the day of EAE onset rather than subsequent events to
challenge our overall hypothesis that infiltrating monocytes
versus resident microglia respond very differently to acute in-
flammatory stimuli.
Activated myeloid cells are the proximate effectors of a
bewildering array of acute and chronic disorders (Wynn et al.,
2013). The technical and conceptual approach taken in this
study may be applicable to other tissues and disease processes.
In many pathological conditions, tissues harbor a mixed pop-
ulation of activated resident and recruited monocytes. The
therapeutic strategy will differ conclusively based on the spe-
cific effector properties of each cell type and the stage of dis-
ease. In particular, if monocytes are pathogenic, then their
trafficking should be blocked using a peripherally active agent.
The optimal application of agents that regulate leukocyte mi-
gration and intracellular signaling will be promoted by de-
tailed examination of each individual myeloid population.
MATERIALS AND METHODS
Mice. C57BL/6 mice were obtained from the National Cancer Institute.
Ccr2rfp/+::Cx3cr1gfp/+ mice were generated by crossbreeding Ccr2rfp/rfp::C57BL/6
mice (Saederup et al., 2010) with Cx3cr1gfp/gfp::C57BL/6 mice ( Jung et al.,
2000). Ccr2rfp/rfp::Cx3cr1gfp/gfp mice were generated by breeding Ccr2rfp/+
::Cx3cr1gfp/+ mice. Ccr2rfp/rfp::Cx3cr1gfp/+ mice were generated by crossbreed-
ing Ccr2rfp/rfp::C57BL/6 mice with Ccr2rfp/rfp::Cx3cr1gfp/gfp mice. Animal ex-
periments were performed according to the protocols approved by the
Institutional Animal Care and Use Committee at the Cleveland Clinic fol-
lowing the National Institutes of Health guidelines for animal care.
EAE induction and clinical evaluation. EAE was induced in Ccr2rfp/+
::Cx3cr1gfp/+ mice and Ccr2rfp/rfp::Cx3cr1gfp/+ mice of 2428 wk of age using
myelin-oligodendrocyte-glycoprotein peptide 3555 (MOG) as previously
described (Huang et al., 2006). All mice were weighed and graded daily for
clinical stages as previously reported (Saederup et al., 2010).We defined clinical
stage of EAE as follows: pre-onset was the day sudden weight loss for 810%
occurred; onset was the day EAE signs appeared; peak was the second day score
didnt increase after sustained daily worsening; and recovery was the second
day score didnt decrease after a period of sustained daily improvement.
To address our research questions, we integrated flow cytometry, immuno
histochemistry with quantitative morphometry, cell sorting for expression
profiling, and serial block-face scanning electronic microscopy. In all, we per-
formed 12 EAE immunizations in Ccr2rfp/+::Cx3cr1gfp/+ mice and 19 immu-
nizations in Ccr2rfp/rfp::Cx3cr1gfp/+ mice for this project, with 810 mice in
each immunization. We selected EAE mice at onset, peak or recovery de-
pending on the specific studies underway at that time, with the majority of
mice coming from the onset stage of EAE. Each experiment incorporated
samples from at least three separate immunizations. Details of mouse numbers
and how they were selected for each experiment were included in the figure
legends as requested.
Cell isolation and flow cytometry. Brains and spinal cords were removed
and homogenized. Mononuclear cells were separated with a 30%/70% Per-
coll (GE Healthcare) gradient as previously reported (Pino and Cardona,
2011). Single-cell suspensions from CNS were stained with antiF4/80-APC
(BM8; eBioscience) and antiCD45-PerCP (30-F11; BioLegend). Cells were
either analyzed on a LSR-II (BD) or sorted on a FACSAria II (BD) running
Diva6. Data were analyzed with FlowJo software (Tree Star).
JEM Vol. 211, No. 8
Ar ticle
Histological and immunohistochemical analysis. Spinal columns were
removed after mice were perfused with 4% paraformaldehyde (PFA). For im-
munofluorescence assay, free floating sections of the lumbar spinal cord were
prepared as previously described (Huang et al., 2006). For immunofluores-
cence assay, sections were blocked with 10% normal serum for 2 h and stained
with primary antibodies at 4C for 2448 h. After washing with PBS-T (PBS
with 0.1% Triton X-100; Sigma-Aldrich) three times, the sections were incu-
bated with secondary antibodies at room temperature for 2 h and mounted
in ProLong Gold antifade reagent (Invitrogen). Antibodies used include rat
anti-CD11b (BD), mouse anti-GFP (Abcam), rabbit anti-RFP (Abcam), Alexa
Fluor 488 goat antimouse IgG (Invitrogen), Alexa Fluor 594 goat antirabbit
IgG (Invitrogen), and Alexa Fluor 647 goat antirat IgG (Invitrogen). Nuclei
were labeled by DAPI. Images were collected by confocal laser-scanning mi-
croscope (SP5; Leica).
Quantitative 3D morphology. Quantitative 3D morphology of MDMs
and MiDMs was analyzed in confocal images from spinal cord of mice at
EAE onset. Free floating sections of the lumbar spinal cord were stained with
RFP for MDMs, GFP for MiDMs, and DAPI for nuclei. Stack images were
taken at 0.2-m step size along the z-direction with a 63 objective (numer-
ical aperture [NA] = 1.4) and zoom factor 2. A square (1,024 1,024 pixels)
corresponding to 123 123 m2 was used for the analysis. Cells were 3D re-
constructed by ImageJ software and all analyses were performed using ImageJ
with 3D Convex Hull plugin. The parameters analyzed include voxel (volu-
metric pixel), convex voxel, volume, convex volume, surface, and convex sur-
face area. Other calculated parameters were: Solidity3D = volume/convex
volume; Convexity3D = convex surface area/surface area; Formfactor3D =
3 36 volume 2 surface area 3 . The number of primary processes was esti-
mated visually. We included 5 mice, 54 MDMs; 51 MiDMs in this assay with
2 sections/mouse, 46 cells/section and 812 cells/mouse. Those mice came
from three EAE inductions.
SBF-SEM. Spinal cords were removed after mice were perfusion-fixed
using 4% PFA with 1% glutaraldehyde. Lumbar spinal cord sections were
made on a vibratome (Leica). Sections were stained with 0.4% OsO4, uranyl
acetate and lead aspartate, then embedded in epon resin (Electronic Micros-
copy Sciences). SBF-SEM images were acquired using a Sigma VP SEM
(Carl Zeiss) with 3View (Gatan). Serial image stacks of images at 100-nm
steps were obtained by sectioning 48 48 20 m3 tissue blocks (length
width depth) at a resolution of 8192 8192 pixels. Image stacks were pro-
cessed for 3D reconstruction by TrakEM2 in FIJI software (National Institutes
of Health). Alternating sections from the same stacked images were chosen to
make stacks for 3D reconstructions which matched the 0.2-m step size used
for acquiring confocal stacked images. In SBF-SEM images, we discriminated
MDMs and MiDMs using the volume/primary processes model (Fig. 1 E)
generated from analyzing confocal images. Quantifications of myeloid-cell
spatial relationships to axoglial units, including myelin incorporation, were
done in SBF-SEM images.
Quantification of nuclei and mitochondria. Characterizations of nu-
clear shapes were conducted in SBF-SEM images. Nuclei were categorized
as follows: round, round shape and smooth surface with ratio of length/
width 1.5; elongated, elongated or oval shape with length/width >1.5, and
may have small indentations; Bilobulated: two connected lobes with single
intervening large indentation; Irregular: complicated shape with corrugated
surface, and may have multiple and variable sizable indentations. Blinded ob-
servers (n = 3) scoring the nuclear morphology from SBF-SEM images in-
cluded a research student, a research fellow and a neuroscientist. Observers
were trained on the same nuclear examples in each category and practiced
using 20 nuclei comprising all shapes before scoring the nuclei. Kappa test
showed good pairwise agreement rates among observers (>0.8) and the data
from the neuroscientist are used. Quantifications were done in 3 individual
mice from 3 EAE inductions including 2835 cells from two separate lesions
from each mouse in the assay.
1547
 Mitochondria of MDMs and MiDMs at EAE onset were reconstructed
from SBF-SEM images to 3D images using TrakEM2. 5 MDM and 5 MiDM
cells from 3 separate mice at EAE onset (total 10 cells) were included in the
assay.Those mice came from 3 EAE inductions. Mitochondria were quantified
for length, cross-sectional area, volume and ratio of length/cross-sectional
area using Fiji software.
Quantification of demyelination. Black-gold staining was performed ac-
cording to a protocol described previously (Liu et al., 2010). In brief, 5 free
floating lumbar spinal cord sections were stained in 0.2% black-gold solution
at 65C water bath for 10 min. After staining with black-gold, sections were
pictured by 3-CCD video camera interfaced with an Image-Pro Plus Analy-
sis System (Version 4.1.0.0; MediaCybernetics) and analyzed with ImageJ
software. Demyelinated areas are those void of black-gold staining. Mean
percentage of demyelinated areas in white matter were calculated. We in-
cluded 5 mice from 3 EAE inductions in this assay.
Statistical analysis of cellular elements. Statistical analyses were per-
formed using SAS (SAS Institute Inc.), PRISM (GraphPad Software) and
SPSS 17.5 (SPSS Inc.). Flow cytometry data were analyzed by two-way
ANOVA test and Wilcoxon matched-pairs signed rank test. Nuclear shape
quantifications were compared by paired Students t tests and logistic regres-
sion. Mitochondrial quantifications were compared by Mann-Whitney U
test and linear mixed model. Quantitative relationships of myeloid cells to
axoglial units were compared using logistic regression with generalized esti-
mating equations (GEE). Clinical characteristics of EAE mice were analyzed
using two-way ANOVA test with Bonferroni post test. Percentage of demye-
lination was compared by Students t test. Data were shown as mean SEM
or median (the first quartilethe third quartile) and P < 0.05 was considered
statistically significant.
Gene expression. Mononuclear cells were prepared from brains and spinal
cords as described previously. Cells were sorted on a BD FACSAria II by gat-
ing on F4/80+GFP+ for MiDMs and F4/80+RFP+ for MDMs. RNA was
isolated from FACS-sorted cells mixed from three mice from six EAE induc-
tions per data point in TRIzol Reagent (Ambion) according to manufactur-
ers protocol. RNA samples were analyzed by nCounter gene expression
analysis and quantified with the nCounter Digital Analyzer (NanoString
Technologies). Expressions of 179 genes were analyzed using nCounter GX
Mouse Inflammation kit.
Nanostring data normalization. Normalization was conducted with
nSolver Analysis Software1.1. Data were normalized using positive and nega-
tive controls and housekeeping genes probes. Background level was calcu-
lated for each sample as mean of negative control probes + (x2 SD). Calculated
background was subtracted from each gene expression value. In cases where
the calculated value was <1, values were set to 1.
Hierarchical and k-means clustering analysis. Hierarchical cluster analy
sis was performed using Pearson correlation for distance measure algorithm
to identify samples with similar patterns of gene expression. MiDM samples
expression data were used in k-means clustering using Pearson correlation
for distance measures (Multi Experiment Viewer v. 4.8).
IPA (Ingenuity) analysis. Data were analyzed using IPA (Ingenuity Sys-
tems). Differentially expressed genes (EAE onset MiDMs versus naive mi-
croglia and EAE onset MDMs versus naive splenic monocytes) were used in
downstream effects and upstream regulators analyses. Uploaded dataset for
analysis were filtered using cutoff definition of 1.5-fold change. Level of con-
fidence for analysis was set to high-predicted and experimentally observed.
Terms used in IPA analyses. The p-value is a measure of the likelihood
that the association between a set of genes in the uploaded dataset and a re-
lated function or upstream regulator is due to random association.The smaller
the p-value, the less likely it is that the association is random and the more
significant the association. In general, P < 0.05 indicate a statistically significant,
1548
nonrandom association. The p value of overlap is calculated by the Fishers
Exact Test.
The activation z-score is a value calculated by the IPA z-score algorithm.
The z-score predicts the direction of change for a function or the activation
state of the upstream regulator using the uploaded gene expression pattern
(upstream to the function and downstream to an upstream regulator). An ab-
solute z-score of 2 is considered significant. A function is increased/upstream
regulator is activated if the z-score is 2. A function is decreased/upstream
regulator is inhibited if the z-score -2.
The bias term is the product of the dataset bias and the bias of target
molecules involved in a particular function annotation or upstream regulator
activity. A biased dataset is one where there is more up- than down-regulated
genes or vice versa. The dataset bias is constant for any given analysis and the
function/upstream regulator bias is unique for each upstream regulator/function.
When the absolute value of this term is 0.25 or higher, then that function/up-
stream regulators prediction is considered to be biased and the Fishers exact
p-value must be 0.01 or lower for the analysis to be considered significant.
We thank Dr. Bruce D. Trapp for invaluable suggestions. We thank Flow core
in Cleveland Clinic Foundation for the flow cytometry experiments. We thank
Aishwarya Yenepalli for help with quantification.
This research was supported by grants from the US National Institutes of
Health, the Charles A. Dana Foundation, the National Multiple Sclerosis Society, and
the Williams Family Fund for MS Research, as well as a Postdoctoral Fellowship
from National Multiple Sclerosis Society (to N. Ohno).
The authors have no competing financial interests.
Submitted: 28 November 2013
Accepted: 9 June 2014
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 Article

Trans-nodal migration of resident dendritic

cells into medullary interfollicular regions
initiates immunity to influenza vaccine
Matthew C. Woodruff,1,4 Balthasar A. Heesters,4,5 Caroline N. Herndon,4
Joanna R. Groom,6 Paul G. Thomas,7 Andrew D. Luster,1,8 Shannon J. Turley,1,3,9
and Michael C. Carroll1,2,4
1Graduate Program in Immunology, 2Department of Pediatrics, and 3Department of Microbiology and Immunobiology,
Harvard Medical School, Boston, MA 02115
4The Program in Cellular and Molecular Medicine, Childrens Hospital Boston, Boston, MA 02115
5Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX Utrecht, Netherlands
6Department of Medical Biology, The Walter and Eliza Hall Institute of Medical Research, University of Melbourne,

Parkville, Victoria 3052, Australia

7Department of Immunology, St. Jude Childrens Research Hospital, Memphis, TN 38105
8Division of Rheumatology, Allergy, and Immunology, Center for Immunology and Inflammatory Diseases,

Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129

9Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02215

Dendritic cells (DCs) are well established as potent antigen-presenting cells critical to
adaptive immunity. In vaccination approaches, appropriately stimulating lymph node
resident DCs (LNDCs) is highly relevant to effective immunization. Although LNDCs have
been implicated in immune response, their ability to directly drive effective immunity to
lymph-borne antigen remains unclear. Using an inactive influenza vaccine model and whole
node imaging approaches, we observed surprising responsiveness of LNDC populations to
vaccine arrival resulting in a transnodal repositioning into specific antigen collection sites
within minutes after immunization. Once there, LNDCs acquired viral antigen and initiated
activation of viral specific CD4+ T cells, resulting in germinal center formation and B cell
memory in the absence of skin migratory DCs. Together, these results demonstrate an
unexpected stimulatory role for LNDCs where they are capable of rapidly locating viral
antigen, driving early activation of T cell populations, and independently establishing
functional immune response.

CORRESPONDENCE
Michael C. Carroll:
michael.carroll@
childrens.harvard.edu
Abbreviations used: ALN,
auricular LN; cDC, conven-
tional DC; cIFR, cortical IFR;
IFR, interfollicular region;
LNDC, LN-resident DC; mDC,
migratory DC; mIFR, medullary
IFR; MP-IVM, multiphoton
intravital microscopy; MPM,
multiphoton microscopy; PLN,
popliteal LN; vG, van Gogh.
Since early descriptions of DCs as primary stim-
ulators of adaptive immunity (Steinman, 1991),
their role in establishing and regulating immune
responses has been central to diverse immuno-
logical fields such as transplantation (Larsen et al.,
1990; Hill et al., 2011), autoimmunity (Llanos
et al., 2011), infectious disease (Poudrier et al.,
2012), and vaccinology (Arnason and Avigan,
2012). As critical mediators of antigen presenta-
tion, significant effort has been spent describing
activation of conventional DCs (cDCs) in pe-
ripheral tissue (Moodycliffe et al., 1994; Austyn,
1996; Rescigno et al., 1997) and characterization
of their subsequent migration to secondary lym-
phoid organs (Itano et al., 2003; Randolph et al.,
2005; Alvarez et al., 2008; Braun et al., 2011; Tal
et al., 2011). Once in peripheral LNs, migratory
DC (mDC) populations from the injection site
present antigen to cognate T and B cells and
stimulate adaptive immunity (Qi et al., 2006).
The activation and maturation of mDCs is
thought to follow a three-stage process. First, im-
mature DCs encounter antigen in the periph-
ery, leading to up-regulation of MHC class II and
co-stimulatory molecules with a concomitant
reduction in phagocytic capacity (Rescigno
et al., 1997). Second, antigen-loaded DCs ac-
quire migratory capacity through the expression
of matrix metalloproteases (Yen et al., 2008), mi-
gratory adhesion molecules (Acton et al., 2012),
and rapid actin treadmilling to enter and migrate
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J. Exp. Med. 2014 Vol. 211 No. 8 1611-1621 1611
www.jem.org/cgi/doi/10.1084/jem.20132327
along lymphatic vessels (Lmmermann et al., 2008). Finally, LN-
bound mDCs cross the subcapsular sinus floor into the paracorti-
cal region and interact with cognate T cells and LN-resident
DCs (LNDCs) within the draining LN (Allan et al., 2006; Braun
et al., 2011) to establish protective downstream immunity.
After antigen capture in peripheral tissues, the activation
and migration of mDCs into draining LNs is delayed for up
to 1824 h to allow for transcriptional and translational mod-
ification and a crawling migration sometimes representing dis-
tances of thousands of cell body lengths of the mDC. In the
case of vaccination, however, arrival of injected antigen is rapid,
with detectable antigen arriving in the draining LN via the
afferent lymphatics within minutes (Roozendaal et al., 2009;
Gonzalez et al., 2010).This timing discrepancy between antigen
arrival in the LN and the migration of DCs from the periph-
ery leaves open a potential window whereby targeting a vaccine
to a nondegradative, immunostimulatory compartment within
the LN could have important humoral immune ramifications.
Several studies have focused on the drainage of lymph-borne
antigen from the afferent lymph into the subcapsular sinus of
the draining LN (Szakal et al., 1983; Carrasco and Batista, 2007;
Junt et al., 2007; Phan et al., 2007; Roozendaal et al., 2009;
Gonzalez et al., 2010). A current view is that subcapsular sinus
macrophages rapidly capture antigen from the lymph and par-
ticipate in its active transport to the B cell follicle. Less well
described is the downstream filtration of the lymph within the
medulla by medullary sinus-lining macrophages (Gray and
Cyster, 2012) and LNDCs (Gonzalez et al., 2010). Historically,
DCs residing in the LN (LNDCs) have been described as rela-
tively sessile at steady-state, (Steinman et al., 1997; Lindquist et al.,
2004) and insufficient to drive effective immunity after direct
antigen acquisition (Itano et al., 2003; Allenspach et al., 2008).
However, the recent observation of direct viral capture in the me-
dulla by the LNDC population suggested they may have a more
active role in the establishment of downstream immune response
in the case of influenza vaccination (Gonzalez et al., 2010).
To extend our understanding of the role of LNDCs in es-
tablishing immune response to influenza vaccination, resident
DCs were characterized at a whole-LN level. Unexpectedly,
a major trans-nodal repositioning of LNDCs from the T cell
cortex to the afferent medulla was observed within minutes
of viral antigen arrival from the afferent lymphatics, areas re-
cently shown to be important in vaccine efficacy (Liu et al.,
2014). This migration leads to rapid viral acquisition by
LNDCs and stimulation of viral-specific naive CD4+ T cells.
Furthermore, total elimination of skin mDCs had a negligible
effect on the generation of a protective humoral response in
mice vaccinated with UV-inactive virus. Collectively, the re-
sults suggest a model in which LNDCs are fully competent in
establishing robust, long-term viral immunity, even in the ab-
sence of mDCs from the injection site.
RESULTS
Activation of LNDCs after influenza vaccination
To characterize LNDC response after vaccination, CD11c-
eYFP C57BL/6 mice (Lindquist et al., 2004; Hickman et al.,
1612
2008; Sung et al., 2012; Kastenmller et al., 2013) were im-
munized s.c. in the footpad with UV-inactivated influenza A
virus strain PR8 (UV-PR8). DCs were tracked by multipho-
ton intravital microscopy (MP-IVM) of the popliteal LN (PLN)
by surgically exposing the node in live, anesthetized mice
(Gonzalez et al., 2010). Continuous imaging for 40 min after
UV-PR8 vaccination revealed an influx of LNDCs proxi-
mal to the collagen capsule (<150 m; Fig. 1 a and Video 1).
Quantitation of cellular trafficking over this period identified
a three- to fourfold increase in the number of YFP+ DCs
within this region (3.23 0.24; P < 0.001), suggesting a rapid
repositioning to the periphery of the PLN.
In addition to increased cell number within the LN pe-
riphery, LNDCs exhibited extensive morphological changes
over the 40-min imaging period (Fig. 1 a, inset). Measured in
bulk, DCs within these regions increased in surface area by
almost 50% after vaccination and experienced a concomitant
increase in volume and decrease in spherical index, a measure
of the spherical nature of an object (Fig. 1 c and not depicted).
Importantly, the fluorescence intensity of individual DCs did
not change over this time period, indicating that the observed
phenotypic changes were not caused by changes in YFP ex-
pression. Together, these data suggest an unexpected accumu-
lation and activation of resident DCs in the PLN periphery
immediately after vaccination.
Repositioning of LNDCs to the medulla
To address the origin of the accumulating DCs, in vivo cellu-
lar tracking was applied to the live imaging model. By track-
ing individual DCs, it was observed that rather than infiltrating
from an outside source, the cells originated from the interior
of the PLN (>150 m from the collagen capsule), which is
beyond the imaging depth threshold for our imaging system
(Fig. 1 b and Video 1). Pretreatment of mice with CD62L
blocking antibody or local administration of Pertussis toxin,
two approaches which limit influx of leukocytes from the vas-
culature or lymphatics, had negligible effects on DC accumu-
lation (not depicted).These results provide additional evidence
that the activated DCs originated within the PLN before vac-
cination and confirmed their identity as LNDCs.
To assess the overall movement of LNDCs after vaccina-
tion, 50-m serial cryosections of PLNs were imaged by mul-
tiphoton microscopy (MPM) and serially reconstructed for
analysis of whole PLNs. Similar approaches were reported by
Grigorova et al. (2010) using confocal microscopy for partial
PLN imaging. By in vivo labeling medullary macrophages
through preinjection of an -F4/80 mAb into the footpad of
CD11c-eYFP reporter mice, the medulla could be outlined and
shown to include relatively sparse populations of LNDCs in
resting naive LNs (Fig. 1 d). In agreement with the live imag-
ing data, injection of UV-PR8 into the footpad of CD11c-
eYFP mice stimulated a visible shift of the LNDC population
into the medullary compartment by 60 min after vaccination
(Fig. 1 d). Flow cytometric analysis of single cell suspensions
of PLN indicated no appreciable increase of LNDCs at these
time points. This global repositioning of the LNDCs can be
LNDCs initiate humoral immunity to flu vaccination | Woodruff et al.
 Ar ticle

Figure 1. LNDCs migrate to the medulla after influenza vaccination. (a) MP-IVM of DC arrival in the PLN periphery after UV-PR8 vaccination.
Snapshots were taken at 0 and 36 min after injection and are representative of three independent surgeries (three mice) from different imaging sessions.
(inset) High magnification of two individual LNDCs. (b, left) Real-time tracking of LNDCs from panel a. LNDC tracks are highlighted (white) and final desti-
nations marked (closed circles). (right) Vector representation of complete tracks. Green and red vectors represent LNDCs with migration paths toward or
away from the PLN capsule, respectively. Data are representative of three independent surgeries (three mice) from different imaging sessions. (c) Quanti-
tation of bulk DCs from live imaging in panel a. DC spherical index (red) and surface area (black) are displayed. ANOVA (black), P < 0.005; ANOVA (red),
P < 0.005. (d) Fluorescent reconstructions of in vivolabeled CD11c-eYFP reporter PLNs. PLNs were collected at 60 min after PBS or UV-PR8 vaccination.
Data are representative of three reconstructed PLNs. B, follicles; M, medulla; MM, medullary macrophage; T, T cell cortex. (e) Quantitation of the percent-
age of LNDCs inside or outside of the medulla as in d (n = 3 PLNs). *, P < 0.05. Mean SEM.

quantified through a shift in the ratio of medulla-occupying
versus total LNDCs (Fig. 1 e). Migration data were further
verified through MP-IVM, through which the rapid arrival
of LNDCs into the medulla could be observed (Video 2).
Surprisingly, this change in both LNDC morphology and lo-
calization could not be identified after the injection of tradi-
tional adjuvants such as alum and MF59 despite extensive
inflammation of the PLN, suggesting that this response may
be specific toward viral antigen or endosomal TLR signaling.
Capture of viral antigen by LNDCs
within interfollicular regions (IFRs)
Recent studies have identified interactions of DCs and T cells
outside of the T cell area within IFRs in the stimulation of
memory CD8+ T cell responses (Hickman et al., 2008; Len
et al., 2012; Sung et al., 2012). In these studies, central mem-
ory T cells were generated after viral infection with the pur-
pose of tracking those cells in the LN after secondary challenge.
Although there is debate on the resting location within the
LN, it is clear that after secondary challenge, central memory
CD8+ T cells are rapidly recruited to the IFRs where they
undergo activation by antigen-loaded APCs. Furthermore, a
study by Hickman et al. (2008) suggested a primary role for
these sites in priming CD8 T cell immunity.
We hypothesized that these sites may serve as destinations
for migrating LNDCs. To characterize the architectural iden-
tity of the IFRs, PLNs were in vivo labeled with antibodies
against the lymphatic endothelium (a-Lyve-1) and subcapsu-
lar sinus macrophages (a-CD169) and then optically cleared
for MP imaging. Projections of whole nodes after optical
clearing (Ertrk et al., 2012) identified long extensions of the
medulla that protruded extensively between B cell follicles
and merged with the subcapsular sinus (Video 3). This obser-
vation identified two different types of IFR structure: cortical
IFRs (cIFRs), which represent chemokine boundaries be-
tween the paracortex and B cell follicles, and medullary IFRs
(mIFRs), which represent the most afferent connection points
between the medulla and the subcapsular sinus (Fig. 2 a). In-
terestingly, these regions have recently been targeted by Liu
et al. (2014) in vaccination attempts, resulting in greatly en-
hanced T cell priming. Confocal imaging of mIFRs in PBS or
UV-PR8vaccinated PLNs identified YFP+ clusters similar to
those identified in reconstruction analysis, identifying these
sites as destinations for migrating LNDCs (Fig. 2 b). Histologi-
cal staining confirmed that both CD11bhi and CD8a+ LNDC

JEM Vol. 211, No. 8 1613

Figure 2. LNDCs infiltrate mIFRs and acquire viral antigen. (a) Schematic diagram of the architecture of a PLN. mIFRs and cIFRs are highlighted in red and
blue, respectively. (b) Confocal imaging of mIFRs in CD11c-eYFP PLNs 40 min after vaccination with PBS or UV-PR8. Blue arrowheads: CD8a+ DCs; red arrowheads:
CD11bhi DCs. Images are representative of three independent trials; three mice/trial. B, follicles; M, medulla; T, T cell cortex. (c) Reconstructions of in vivolabeled
C57BL/6 PLNs vaccinated with A633UV-PR8 and collected at 0.5 or 6 h after injection. White arrowheads: IFRs. SS, subcapsular sinus. Images are representative
of three independent experiments; two mice per time point per experiment. (d) MPM of in vivolabeled CD11c-eYFP PLNs 60 min after PBS or A633UV-PR8
vaccination. Dashed lines: IFRs. Images are representative of four independent experiments; two mice per experiment. (e) LNDC capture of A488UV-PR8 by
flow cytometry 60 min after injection. (left) Cells displayed are pregated to be CD11chi (n = 4 PLNs). Mean SEM. (f) MPM of in vivolabeled CD11c-eYFP
PLNs 6 h after A633UV-PR8 vaccination. White arrowhead: UV-PR8 patch on the LNDC. Image is representative of four independent experiments; two
mice per experiment.

populations had accumulated in mIFRs within 60 min of
vaccination, suggesting a multisubset responsiveness to inacti-
vated influenza (Fig. 2 b).
Afferent lymph flowing through the medullary sinus is
constitutively filtered by sinus-lining macrophages (Gray and
Cyster, 2012), and this process predicts a natural gradient of
viral antigen after vaccination. Thus, it is proposed that the
highest antigen concentrations would reside at the tips of
these mIFRs (Video 4). To test this possibility, mice were in-
jected s.c. with UV-PR8 labeled with Alexa Fluor 633. Fluor
escent PLN reconstructions at various time points identified
accumulation of viral antigen within mIFRs over 6 h after
vaccination (Fig. 2 c). In contrast, lower levels of virus were
retained in the subcapsular sinus and the deeper medullary
compartments connecting with the efferent lymphatics. It
was hypothesized that these antigen-rich regions might serve
as a destination for migrating LNDCs. High-resolution imag-
ing of IFRs bearing dense deposits of viral antigen showed
large numbers of infiltrating LNDCs within 60 min of viral
arrival at the node. Analysis of LNDCs by flow cytometry
confirmed viral capture by both CD11bhi and CD8a+ LNDCs
within 30 min after injection (Fig. 2 e), which was verified by
MPM identifying viral patches on the surface of LNDCs at
later time points (Fig. 2 f, inset).
LNDCs present viral antigen to CD4+ T cells near IFRs
Previous work by Itano et al. (2003) interrogated the role of
mDCs versus LNDCs in CD4+ T cell stimulation. In that study,
soluble peptide designed for presentation on MHC II was in-
jected s.c., and downstream T cell responses were tracked in
the draining LN. The authors observed two waves of peptide
presentation, one at 6 h, which resulted in only transitory ac-
tivation, and one at 18 h, which stimulated a more robust, long-
term T cell response. The authors concluded that although
resident DCs can activate T cells, the resident cells induce
only a limited response with mDCs from the injection site
required for robust immunity. To determine whether LNDCs
participate in activation of viral-specific CD4+ T cells within
this study, CD11c-eYFP mice were adoptively transferred with
labeled, ova-specific CD4+ OT-II T cells 24 h before vaccina-
tion with a UV-inactive recombinant strain of PR8 (UV-PR8-
OTII), which was engineered to express the OT-II epitope
(Thomas et al., 2006).
As predicted, vaccination with UV-PR8-OTII stimulated
activation of cognate T cells within 6 h, as indicated by CD69
up-regulation (Fig. 3 a). Although individual DCT cell con-
tacts could be observed as early as 6 h, extensive interaction
between LNDCs and CD4+ OT-II T cells was observed near
IFRs by 12 h after vaccination (Fig. 3 b). Additionally, small

1614 LNDCs initiate humoral immunity to flu vaccination | Woodruff et al.

 Ar ticle

clusters of viral-specific T cells could be identified surround-

ing LNDCs in these regions. To quantitate clustering, labeled,
naive OT-II T cells were transferred into CD11c-eYFP re-
cipients, which were subsequently vaccinated with UV-PR8-
OTII 12 h before LN harvesting.The LNs were serially imaged
for reconstruction, and single plane images were captured from
each reconstruction, representing 400 m of vaccinated ver-
sus control LNs. Using histocytometric analysis (Gerner et al.,
2012), OT-II T cells were identified within each plane, and
each T cell was assessed for the number of T cell neighbors
present in the image within one T cell diameter.The resulting
measure of T cell clustering revealed significant increases in
the mean number of T cell neighbors and increases in the
absolute number of T cells with multiple neighbors within
vaccinated versus nonvaccinated LNs (Fig. 3 c). Significantly,
heat mapping the number of T cell neighbors onto the ori
ginal images identified the border between the paracortex
and IFRs as the most densely populated by clustered T cells
(Fig. 3 d). Analysis of T cell migration at 24 h after immunization
identified discreet clusters at the extremes of the paracortex,
with specific colocalization of T cell clusters at the mIFR
cortical junctions (Fig. 3, e and f).

Clustering of viral-specific CD4 T cells is chemokine dependent

Previously mentioned studies describing central memory
CD8 T cell responses within cIFRs have suggested the che-
mokine CXCR3 as a critical mediator of cellular retention in
these regions. The ligands for CXCR3, CXCL9 and CXCL10,
are produced by both hematopoietic and stromal cells in re-
sponse to viral challenge and are critical to CD8 T cell re-
cruitment to these sites (Sung et al., 2012; Kastenmller et al.,
2013). Additionally, Groom et al. (2012) have recently de-
scribed the up-regulation of CXCR3 as an early first step in
the generation of robust Th1 CD4 T cell responses. Thus, we
hypothesized that the clustering of CD4+ OT-II T cells with
LNDCs and virus in the IFR after immunization with UV-
PR8-OTII could be CXCR3 dependent.To examine whether
CD4+ OT-II T cells become activated and differentiate to
CXCR3+ T cells, LNs were harvested from immunized mice,
and CD4+ T cells were analyzed by flow cytometry for ex-
pression of cell surface markers of activation. Notably, an in-
crease in CD69 expression on CD4+ OT-II T cells was observed,
followed by a corresponding increase in CD44 (Fig. 4 a and
not depicted). By gating on newly activated CD69+ cells, in-
creases in CXCR3 expression could be identified as early as
12 h after vaccination and continued to increase over several
days (Fig. 4, a and b).
As recent reporting has shown that activated DCs arriv-
ing from the periphery express CXCL10 and that direct
injection of LPS/Poly I:C can induce CXCL10 expression
within 12 h in the draining LN (Groom et al., 2012), it was
predicted that LNDCs might express this chemokine after im-
munization with UV-PR8-OTII. Thus, the release of CXCL10
by activated LNDCs within the IFR could explain the extensive
clustering of CD4+ T cells observed within the antigen-rich
IFRs. To test this possibility, CXCL9/10 reporter mice (REX3;
Figure 3. Cognate CD4+ T cells relocate to mIFRs after vaccina-
tion. (a) C57BL/6 mice received naive OT-II T cells and were vaccinated
with UV-PR8-OTII. OT-II T cell activation in PLNs was analyzed by flow
cytometry (n = 4 mice). *, P < 0.05; ***, P < 0.001. Mean SEM. (b) CD11c-
eYFP mice received labeled naive OT-II T cells and were vaccinated
with UV-PR8-OTII. MPM of a PLN 12 h after vaccination. White arrowheads:
LNDC/OT-II contacts. Images are representative of three PLNs; two
independent trials. (c) OT-II neighbor analysis of PLNs as shown in b.
Each symbol type represents an individual PLN, with four XY planes
shown. **, P < 0.005. Horizontal bars indicate mean. (d) Heat map of
OT-II neighbor counts as shown in c. OT-II cells were identified from
images (left) and displayed with color indicative of the number of
corresponding OT-II neighbors (right). B, follicles; M, medulla; T,
T cell cortex. (e) CD11c-eYFP recipients received labeled naive OT-II T cells
and were vaccinated with UV-PR8-OTII. Fluorescent reconstruction of
a PLN 24 h after vaccination is shown. Per, PLN periphery. Image is rep-
resentative of three independent trials; two mice/trial. (f) C57BL/6 mice
received OT-II T cells and were vaccinated with UV-PR8-OTII. PLNs were
collected at 24 h after vaccination, optically cleared, and imaged. Data are
represented with medulla isosurfacing to aid visual interpretation. White
arrowheads: mIFR/T cell cluster colocalization. Image is representative
of two trials; two mice/trial.

JEM Vol. 211, No. 8 1615


Figure 4. CXCR3-dependent clustering of viral-specific CD4+
T cells in mIFRs. (a) Flow cytometry of OT-II T cell activation after UV-
PR8-OTII vaccination at the indicated time points (n = 4). (b) Expression
of CD69 (black) and CXCR3 (red) by OT-II T cells at the indicated time
points as in panel a. CD69+ OT-II cells were gated for CXCR3 expression
analysis. Symbols represent individual mice. ANOVA (CD69), P < 0.0005;
ANOVA (CXCR3), P < 0.005 (n = 4). Mean SEM. (c) CXCL10 expression
in vaccinated REX3 mice or unvaccinated REX3 controls by flow cytom-
etry. (left) Displayed cells gated on CD11chi cDCs, followed by CD11bhi or
CD8a+ as indicated. (right) Percentage of CXCL10-positive DCs by subset
after vaccination (n = 4). ***, P < 0.001. Horizontal bars indicate mean.
(d) MP imaging of REX3 PLN 24 h after UV-PR8 vaccination. Dashed
line: medulla (M). B, follicles; T, T cell cortex. Images are representative
of four PLNs from two independent trials. (e) Confocal imaging of REX3
PLN 24 h after UV-PR8 vaccination. Arrowheads: CXCL10-positive DCs.
Images are representative of four PLNs; two independent trials. (f) C57BL/6
recipients received differentially labeled WT or CXCR3-deficient naive
OT-II T cells. Recipients were vaccinated with UV-PR8-OTII, and PLNs
1616
Groom et al., 2012) were vaccinated with UV-PR8, and LNs
were harvested after 24 h for analysis by flow cytometry and
immunohistochemistry. Flow analysis identified the expres-
sion of CXCL10 by both CD11bhi and CD8a+ LNDCs after
vaccination (Fig. 4 c), although the CD11bhi population ap-
peared more robustly responsive. Fluorescent reconstructions
of vaccinated REX3 PLNs identified high CXCL10 expres-
sion on dendritic-looking cells within mIFRs after vaccina-
tion, suggesting LNDC expression (Fig. 4 d). Confocal analysis
positively identified these cells as DCs through CD11c ex-
pression and confirmed clustering of CXCL10-positive DCs
within IFRs by 24 h after vaccination (Fig. 4 e). The timing
of this event corresponded with both LNDC migration and
formation of T cell clusters.
To confirm the importance of CXCL10 in attracting viral-
specific T cells to the IFRs, mice were adoptively transferred
with differentially labeled CXCR3+/+ and CXCR3/ OT-II
T cells and subsequently immunized with UV-PR8-OTII virus.
Characterization of the vaccinated recipients revealed a sig-
nificant defect in the clustering of CXCR3/ OT-II cells at
24 h after vaccination, confirming the importance of CXCR3
in T cell migration (Fig. 4 f). Additionally, flow cytometric
analysis of CXCR3/ OT-II T cells displayed a profound
deficiency in both CD69 activation and CD40L expression
(Fig. 4, g and h). These results replicate the study by Groom
et al. (2012), which described early activation defects, fol-
lowed by a deficient Th1 response in the absence of CXCR3.
Together, these data suggest a model whereby LNDC activa-
tion of CD4+ T cells at early time points leads to expression of
CXCR3 and contributes to the efficient localization of cog-
nate cells into mIFRs, resulting in efficient early activation.
LNDC-dependent T cell activation
Although the activation of T cells within 6 h of vaccina-
tion suggested a role for LNDCs in early activation of CD4+
T cell responses to UV-PR8, it remained unclear whether
skin-resident mDCs were required to establish an enduring hu-
moral response. Using a system developed by Itano et al. (2003),
the contribution of the mDC population could be effectively
removed through resection of the injection site within 30 min
after immunization (Kissenpfennig et al., 2005). By adminis-
tering UV-PR8 intradermally in the ear and removing the ear
shortly after administration, the relative contribution of mDC
and LNDC populations could be evaluated in the auricular
LN (ALN).
were collected at 24 h. Quantitation of clustering efficiency of WT or
CXCR3-deficient OT-II T cells is shown (n = 5). (g and h) Adoptive trans-
fers were performed as in f. Recipients were vaccinated, and PLN
suspensions were collected 60 h after vaccination for flow analysis.
Individual T cell populations were compared with the same population
in unvaccinated contralateral controls. CD40L (g) and CD69 (h) acquisi-
tion is expressed as fold change (FC) in vaccinated versus unvaccinated
population controls (n = 5). **, P < 0.005; ***, P < 0.001.
LNDCs initiate humoral immunity to flu vaccination | Woodruff et al.
 Ar ticle

Using this approach, C57BL/6 mice were vaccinated with

PBS or UV-PR8 intradermally, with half of the vaccinated
group undergoing ear removal (here referred to as the van Gogh
[vG] group) within 30 min after vaccine administration. Using
REX3 reporter mice, expression of CXCL10 by both CD11bhi
and CD8a+ in WT or vG-vaccinated animals was assessed 24 h
after injection/resection. Interestingly, although CXCL10 ex-
pression was still robust in both LNDC populations, suggesting
normal responsiveness to vaccination despite a lack of mDCs
in the vG group (Fig. 5 a), there was a small decrease in expres-
sion by the CD8a+ subset, perhaps relating to its known reliance
on incoming mDCs from the periphery (Allan et al., 2006).
By transferring CFSE-labeled naive OT-II T cells into B6
recipients and using the vG vaccination system, potential de-
ficiencies in T cell proliferation and activation could be assessed
in the absence of mDCs from the injection site. Surprisingly,
vG group OT-II T cells proliferated similarly to the WT group
(Fig. 5 b) and could be tracked through seven divisions in 60 h.
Tracking T cell activation by division status, cells could be
observed acquiring both CXCR3 and CD40L as they prolif-
erated in both the WT and vG groups (Fig. 5, c and d). Inter-
estingly, although CD40L expression appeared identical in
both groups, there was a slight delay in CXCR3 acquisition Figure 5. mDC-independent LNDC/CD4+ T cell activation. Ear re-
between the WT and vG group. These findings may also sug- section in the vG model 30 min after vaccination. (a) Percentage of CD8a+
gest collaboration with mDCs in reinforcing Th1 immunity or CD11bhi cDCs in the ALN 24 h after ear vaccination. PBS, WT, and vG
development as they arrive from the periphery. groups are displayed. ANOVA (light gray), P < 0.0001; ANOVA (dark gray),
P < 0.0001 (n = 4). *, P < 0.05; ***, P < 0.001. (b) CFSE-labeled OT-II T cells
LNDC-dependent B cell memory were transferred into C57BL/6 recipients. Recipients were vaccinated in
As LNDC and T cell activation appeared to be intact despite the ear and separated into WT and vG groups. ALNs were isolated at 60 h
the absence of mDCs in the vG model, it was hypothesized after injection and analyzed by flow cytometry. (left) Percentage of OT-II
T cells by division number as assessed by CFSE dilution. ANOVA, P > 0.1.
that LNDCs may be sufficient to drive downstream protective
(right) Representative plot of CFSE dilution in WT and vG groups (n = 4
humoral immunity despite the absence of mDCs. To test this mice/group). (c and d) Adoptive transfers were established as in c. OT-II
hypothesis, the vG vaccination model was used in C57BL/6 T cell expression (MFI) of CXCR3 (c) and CD40L (d) was assessed by T cell
mice alongside PBS-vaccinated controls. ALNs were collected division to track acquisition over time. (c) Two-way ANOVA, P < 0.0001
at day 7 after vaccination, and germinal center staining appeared overall, P < 0.05 between groups. (d) Two-way ANOVA, P < 0.0001 overall,
normal in both WT and vG groups, confirming effective T cell P > 0.1 between groups (n = 4 mice/group). Mean SEM.
help despite the absence of mDCs (Fig. 6 a). Antibody titers
collected at day 10 after vaccination showed normal increases
in IgM, IgG1, and IgG2b in vG versus WT vaccination groups in the vG group, providing additional evidence for LNDC/
in comparison with unvaccinated controls, suggesting normal mDC collaboration in a vaccination setting. Altogether, these
class switching and germinal center development (Fig. 6 b). data demonstrate that LNDCs are capable of stimulating
To test the functionality of this antibody response, vacci- CD4+ Th1-dependent protective humoral responses to influ-
nated, or unvaccinated controls were challenged intratrache- enza vaccination.
ally with a lethal dose of live PR8 on day 21 after vaccination.
Morbidity was monitored for 9 d after infection, at which point DISCUSSION
the unvaccinated control group was sacrificed as a result of An earlier study identifying capture of lymph-borne inactive
excessive weight loss. Neither WT nor vG groups experienced influenza virus by LNDCs raised the question of the relative
significant weight loss over the course of infection, indicating importance of this population in overall humoral immunity
protective immunity had been generated with or without mDC (Gonzalez et al., 2010). To gain a broader understanding of
arrival from the injection site (Fig. 6, c and d). Finally, antibody LNDCs in response to vaccination with inactive influenza,
titers were assessed at day 9 and compared with both unvacci- a whole-node imaging approach was developed, and mice
nated and uninfected controls. As expected, antibody titers bearing a fluorescent reporter for DCs (CD11c+) were vacci-
showed extensive class switching in both WT and vG groups, nated with UV-PR8. After s.c. vaccination, viral antigen was
and protective IgG2b responses were identical between groups. observed within the draining LN within minutes, as expected.
It is worth noting that although not required for protection, Surprisingly, however, the virus accumulated over the first 6 h
IgG1 antibody titers were slightly, but insignificantly decreased within specialized sites identified as mIFRs.

JEM Vol. 211, No. 8 1617


Figure 6. mDC-independent protection from influenza. (a) Confocal
imaging of ALN follicles at day 10 after UV-PR8 vaccination in WT and vG
mice. Dashed line: B cell follicle. Images are representative of two inde-
pendent trials; five mice/group. (b) ELISA analysis of PR8-specific serum
antibodies at day 10 after UV-PR8 vaccination. Relative titers of PR8-
specific IgM, IgG1, and IgG2b are shown. Symbols represent individual
animals. (c) Unvaccinated or vaccinated WT or vG groups were challenged
with LD80 PR8 21 d after vaccination. Morbidity (left) and mortality
(right) are displayed as percentage of body weight or percent survival,
respectively. Red dashed line: morbidity/euthanasia experimental cutoff.
Six mice/group. (d) ELISA analysis of PR8-specific serum antibodies at day
9 after PR8 challenge as in c, compared with naive C57BL/6 littermates.
Relative titers of PR8-specific IgM, IgG1, and IgG2b are shown. Symbols
represent individual animals. *, P < 0.05; ***, P < 0.001. Mean SEM.
Using fluorescent reconstructions of full PLNs isolated at
early time points after vaccination, we observed a major repo-
sitioning of LNDCs from the T cell cortex to the mIFR where
they acquired viral antigen and became activated. This was
unexpected because LNDCs are reported to be relatively ses-
sile at steady-state.The capture of virus by the resident DC was
biologically relevant because they became positive for the che-
mokine CXCL10 and formed clusters with viral-specific CD4+
1618
T cells that became activated based on expression of CD69,
CD44, and CXCR3. Thus, three lines of evidence are presented
that support a role for LNDCs in promoting a humoral re-
sponse to inactive PR8 independent of mDCs: (1) activation
of viral-specific CD4+ T cells before expected arrival of skin
mDCs, (2) activation of viral-specific CD4+ T cells within
PLNs in the absence of mDCs, and (3) a normal humoral mem-
ory response despite surgical removal of the injection site
within 30 min of vaccination. Notably, although these find-
ings clearly demonstrate sufficiency of the resident DC popu-
lation in establishing humoral immunity, they do not address
the requirement of mDCs under conditions where lymph-
borne antigen is not readily available.
Although several groups have previously described resi-
dent DC populations in skin-draining LNs (Lindquist et al.,
2004), few have directly assessed their potential in presenting
antigen acquired directly from the lymph. In one study, Itano
et al. (2003) describe a two-step process of antigen presentation
where resident DCs in the LN were capable of acquisition and
presentation of soluble peptides, but skin-resident mDCs were
required for effective immunity. In another study, Allenspach
et al. (2008) found that in the case of soluble peptide adminis-
tered with CFA, resident DCs were similarly deficient in T cell
presentation. Our findings are not inconsistent with these
results, as the robust activation/migration of resident DCs
cannot be identified after traditional adjuvant administration
(alum or MF59). The profound differences in these responses
also raise interesting questions about a potential gap between
traditional vaccination approaches and natural response to viral
antigen. As medullary macrophages rapidly degrade protein
antigen as a part of normal lymphatic filtration, it is important
to understand how to maximize antigen exposure to efficient
antigen presenters, especially when antigen concentration is
highest immediately after vaccination. Data from this study
suggest that LNDCs are capable of effectively using antigen
when appropriately stimulated and could represent an effi-
cient target for vaccination approaches.
An unexpected observation was the rapid kinetics of LNDC
repositioning from the paracortical region to the mIFR in re-
sponse to localization of viral antigen. Although large deposits
of LNDCs were observed in viral-loaded IFRs within hours
of vaccination, individual LNDC morphological changes
could be observed within 12 min of vaccination. The highly
directional migration pattern of LNDCs to the sites of viral
accumulation suggests a response to chemotactic mediators
released from a prestored source after innate sensing of viral
antigen, although this source remains yet unclear.
A recent study has identified CXCR3 and its ligands
CXCL9 and CXCL10 as required for migration of CD8
memory T cells into IFRs, where they make contact with
both antigen and APCs (Sung et al., 2012). Although the dis-
tinction between mIFRs and cIFRs was not made in these
studies, it is clear that this chemoattractant is important in
various conditions of immune response. In our study, formation
of clusters of viral-specific CD4+ T cells with LNDCs was
significantly reduced in CXCR3/ OT-II T cells. Because
LNDCs initiate humoral immunity to flu vaccination | Woodruff et al.

CXCL10 expression is not restricted to LNDCs, this pathway
might provide an efficient mechanism for collaboration be-
tween LNDCs and incoming mDCs, whereby early activa-
tion of naive CD4 T cells, and concomitant expression of
CXCR3, allows activated T cells to more easily find mDCs
from the injection site, which also express CXCL10. Addi-
tionally, it is possible that mDCs might be able to locate hot
spots of immune activation within the LN through their own
expression of CXCR3.
In summary, using novel whole-node imaging approaches,
we observed a multistep system of immune activation whereby
viral antigen, LNDCs, and cognate naive CD4+ T cells rapidly
assemble in highly organized environments within the drain-
ing LNs known to be relevant to vaccine design (Liu et al.,
2014). In addition to identifying an unexpected repositioning
of LNDCs to specialized sites within the draining LN, these
findings clarify the importance of CXCR3 in establishing
immunostimulatory microenvironments in three dimensions
and establish LNDCs as biologically relevant cell populations,
sufficient to drive humoral memory response to an influenza
vaccine in the absence of mDCs.
MATERIALS AND METHODS
Animals. Mice used in this study were bred and housed in standard conditions
and used between 6 and 12 wk of age. All experimental protocols were approved
through Harvard Universitys Institutional Animal Care and Use Committee.
In vivo labeling. In vivo labeling of the PLN subcapsular sinus and medulla
was achieved through injection of Alexa Fluorconjugated (A488, A568,
A633) antibody targeting stromal (aLYVE-1) or macrophage populations
(a-CD169, a-SIGNR1, a-F4/80) s.c. in the footpad 4 h before PLN imaging
or tissue harvest.
PLN live imaging. PLN live imaging was performed through surgical ex-
posure of the PLN in anesthetized mice and MPM. Temperature was moni-
tored using a digital thermometer embedded in the imaging chamber created
around the PLN and controlled using a closed-circuit water circulation sys-
tem (Lindquist et al., 2004; Gonzalez et al., 2010).
Three-dimensional reconstruction. Collected organs were fixed in 4%
PFA, embedded in OCT, and serially cryosectioned (50 m). PLN sections
were serially imaged by MPM and assembled using Imaris software (Bitplane)
to obtain three-dimensional reconstructions. Final analysis was performed
using Volocity image analysis software (PerkinElmer).
Whole-organ imaging. LNs were dissected and fixed overnight in 4%
paraformaldehyde. The sample was then incubated in increasing concentra-
tions of tetrahydrofuran followed by dichloromethane. Finally, the PLN was
immersed in BABB (benzyl alcohol/benzyl benzoate, mix 1:2) for final clear-
ing and imaging (Lindquist et al., 2004; Ertrk et al., 2012).
Viral propagation/labeling. Influenza virus was grown in live chicken
eggs, purified over sucrose gradients (Szretter et al., 2006; Gonzalez et al.,
2010), and labeled using standard Alexa Fluorlabeling protocol (Invitrogen).
1020-l s.c. injections were given in the footpad at 106 PFU.
T cell isolation. LNs of OT-II mice were processed using Liberase DH
(Sigma-Aldrich) digestion, and T cells were isolated through MACS negative
selection (CD11c, CD11b, NK1.1, B220, GR-1, CD69, CD8). T cells were
labeled in 10 m CMTMR for imaging or 5 M CFSE for imaging and flow
JEM Vol. 211, No. 8
Ar ticle
cytometric identification. 1 or 5 106 cells were adoptively transferred into
naive recipients for flow cytometric or imaging experiments, respectively.
Three-dimensional image analysis. Images were processed and
analyzed using Volocity imaging software. Histocytometric analysis of
reconstructions was performed with CellProfiler (Carpenter et al., 2006;
Grigorova et al., 2010), and CellProfiler Analyst (Hickman et al., 2008;
Jones et al., 2008; Len et al., 2012). Analysis of reconstructions was per-
formed with individual XY imaging planes, which were representative of
at least 400 m of reconstructed LNs.
T cell clustering. CXCR3/ and WT OT-II cells were isolated as above,
differentially labeled, and adoptively transferred into C57BL/6 recipients.
PLNs were collected, processed, and serially imaged 24 h after vaccination
with UV-PR8-OTII. T cell clusters were identified using a blinded approach,
and the percentage of each population within these clusters was measured.
Ear resections. Ear removal was performed 30 min after injection of 10 l
PBS or UV-PR8 s.c. between the ear dermal layers. ELISA analysis of serum
was performed through immobilization of UV-PR8 on the plate, addition of
serum, and probing for specific binding of IgM, IgG1, or IgG2b.
Statistics. All statistics/graphical representations of collected data were as-
sembled through Prism (GraphPad Software). Mean and SEM are displayed
where applicable. Results from Students t tests or Tukeys post tests are indi-
cated by asterisks in the figures (*, P < 0.05; **, P < 0.005; ***, P < 0.001).
One- or two-way ANOVA testing is indicated in the legends.
Online supplemental material. Video 1 shows LNDC migration in re-
sponse to influenza vaccination. Video 2 shows that LNDCs infiltrate the
medulla after UV-PR8 vaccination. Video 3 shows reconstruction of a PLN.
Video 4 shows lymph flow through the PLN. Online supplemental material
is available at http://www.jem.org/cgi/content/full/jem.20132327/DC1.
We thank J. Campbell for providing CCR7-deficient mice, N. Anandasabapathy and
S. Jones for critical discussion and review of this manuscript, and H. Leung, E.
Carroll, S. Lavoie, and M. Ma for technical support.
This work was supported by the National Institutes of Health (NIH)National
Institute of Allergy and Infectious Diseases (grants 1 P01 AI078897, R37 AI054636,
and R01 AI039246 to M.C. Carroll; R01CA069212 to A.D. Luster; RO1 DK074500 and
PO1 AI045757 to S.J. Turley; and AI107625 to P.G. Thomas), NIH T32 Training Grant
in Transplantation (T32 AI007498 to M.C. Woodruff), the American Lung Association
(grant RT-224269-N to C.N. Herndon), and the National Health and Medical
Research Council, Australia (fellowship 516791 to J.R. Groom).
The authors declare no competing financial interests.
Author contributions: M.C. Woodruff, B.A. Heesters, and C.N. Herndon performed all
experiments and analysis described in the text. P.G. Thomas, J.R. Groom, and A.D. Luster
developed critical reagents for the completion of the study. M.C. Woodruff, S.J. Turley,
and M.C. Carroll designed this study and developed the manuscript for publication.
Submitted: 6 November 2013
Accepted: 19 June 2014
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