Leonid Yurkovetskiy

Aly Azeem Khan

Mike Burrows
Camilla H F Hansen

NIH
JDRF

Dion Antonopoulos, Argonne

Betty Theriault, Uchicago
Tatyana Golovkina, UChicago

How we can or should use the animal models to

study sexual dimorphism of diseases?
What can we learn from these models?

Alexander V. Chervonsky
The University of Chicago

When sex is important?

Infectious diseases
a. Sensitivity to disease
b. Adverse effects of vaccination
c. Vaccination efficacy
Non-infectious diseases (autoimmunity, cancer)

KleinandFlanagan

Glycoprotein-DAdjuvant Vaccine to Prevent Genital Herpes

LawrenceR.Stanberry,M.D.,Ph.D.,SpotswoodL.Spruance,M.D.,Anthony
L.Cunningham,M.D.,DavidI.Bernstein,M.D.,AdrianMindel,M.D.,
StephenSacks,M.D.,StephenTyring,M.D.,Ph.D.,FredY.Aoki,M.D.,
Moncef Slaoui,Ph.D.,MartineDenis,Ph.D.,PierreVandepapeliere,M.D.,
andGaryDubin,M.D.,fortheGlaxoSmithKlineHerpesVaccineEfficacy
StudyGroup*
NEnglJMed2002;347:16521661November21,2002

In 2 trials the vaccine was 73% and 74% efficient in seropositive

females, but not in males.

Sources of sexual dimorphism of diseases

Genes encoded by sex
chromosomes
Mitochondrial genes
Lack of androgens as opposed to
the presence of estrogens

Rubtsova etal.,JCI,2015

physical and
chemical
insults

biological
insults

CSHpress,2013

Confounding factors in studying microbiota-host interactions

a) the enormous complexity of the microbiota;
b) involvement of microbial communities rather than
isolated lineages in interactions with the host;
c) sensitivity of the microbiota to various environmental
insults including dietary, chemical and biological;
d) the multiplicity of host microbial sensors;
e) the difficulties in applying classical Kochs postulates
to commensal microbes;
f) the difficulties in cultivation of commensal microbes

Wheelotella sp.

Reyniers germfreesystem1
massproductionIsolator

Microbes can be both inducers and negative regulators

of autoimmunity
Direct evidence for influence of microbiota on
autoimmunity came from the studies of Germ-Free
animals

A. Monogenic diseases are insensitive to commensal regulation

IPEX, APECED, AID-deficiency
B. Some diseases develop independently of commensals

Type 1 diabetes in mice and rats

C. Some diseases develop independently of commensals, but

commensals amplify the disease
Models of RA in collagen immunized rats; RA in mice;
SLE (MRL.lpr)
D.

Some diseases require commensals

EAE (tcr transgenic model); Ankylosing enthesopathy in B10.BR mice;

RA in IL1R-antagonist KO
Sialitis in NOD mice

Diabetes

Sialitis

Even within one host organism, commensals affect

different autoimmune manifestations differently

Incidence of T1D in different NOD colonies

Culmative incidence of diabetes (%)
100
90

% incidence

80
70
60

Male

50

Female

40
30
20
10
0
1

Center

Pozzilli et al, 1994

Yurkovetskiy etal.,Immunity2013
Institution
SPF Facilities
U of Chicago
Yale 2008
Taconic 2009*
Harvard
T1DR** Jax
2002
2003
2004
2005
2006
2007
2008
2009
Germ-Free
Facilities
Taconic 2008
U of Chicago 2012
Gnotobiotic
Facilities
Taconic ASF 2008

Female
(F)

Male
(M)

Ratio F/M

60%
90%
80%
50-60%
--75%
85%
90%
80%
65%
100%
90%
90%

25%
70%
50%
10-15%
--50%
60%
45%
40%
35%
65%
85%
65%

2.4
1.3
1.6
4.4
--1.5
1.4
2.0
2.0
1.9
1.5
1.1
1.4

100%
95%

85%
84%

1.2
1.1

ND

75%

*CumulativeincidenceofdiabetesinNODmiceat
30weeksofage.
T1DR T1DrepositoryatTheJacksonLaboratory

regulatory
step 1

A
hormonesmicrobes

autoimmunity

microbialcontrol

B
hormonesmicrobes

autoimmunity

C
hormonesmicrobes

autoimmunity

effector
step 2

Linear hypothesis A predicts that males and females

should have different microbiota composition

A
hormonesmicrobes

microbialcontrol

autoimmunity

How do we address the issue of male/female microbiota differences?

Every experiment has some trade-offs.
1. Single-housed animals to be analyzed. Reason: coprophagy
removes individual differences and can bias the results.
Lot more expensive and possible influence of loneliness-induced stress.
2. Progeny of the same parents to be analyzed. Reason: reduction of
cage effects influence of the input microbiota.
Small litter size may affect the statistics of microbial analysis.
3. Colonization of GF mice. Reason: standardization of the input
microbiota.
Expensive and not yet widely-available.
4. Repetition of individual experiments. Reason: removal of the
misleading results and avoidance of the result over-interpretation.
The only problem is the historically high price tag. However, since the
sequencing prices are low, this is a poor excuse.

principal component analysis of microbiota composition in

females (F), males (M), and castrated males (C).

principal component analysis of microbiota composition in

gnotobiotic females (F), males (M) associated with
SPF microbiota after birth

Male and female microbiota are different

A
hormonesmicrobes

microbialcontrol

Is there a male microbiota signature?

autoimmunity

Experiment 1

Experiment 2

Experiment 4

Experiment 3

Proportion of seqquences
belonging to a listed family, %

Phylum Bacteroidetes Actinobacteria Firmicutes Proteobacteria Firmicutes Firmicutes Bacteroidetes Firmicutes Bacteroidetes
Family Porphyromonadceae Kineosporiaceae
Veillonellaceae Enterobacteriaceae
LactobacillaceaeCytophagaceae
Peptococcaceae
Bacteroidaceae
Peptostreptococcaceae
Kineosporiaceae

80

p<0.004
60

0.12

p<0.016

Veillonellaceae

p<0.05

0.8

Enterobacteriaceae
0.4

p<0.04

Peptococcaceae
0.6

p<0.04

p>0.57
0.6

0.08

40

0.3

0.4

0.4

0.2

0.2

M F

Prepubescent

0.00

Postpubescent

0.0

50

p<0.03
p<0.005

40

30 p>0.34

20

10

p<0.0003
q=0.02

p<0.0008
q=0.05

3 p<0.001

q=0.05

0.2

0.04

20

Lactobacillaceae

0.1

0.0

0.0

FF

M
M

FF

M
M

F
F

M
M

conclusions
Input microbiota affects gender differences in postpubescent animals
Hormones affect gender differences
Gender bias in NOD T1D does not depend on a
specific microbial lineage

It is also possible that the expansion of specific microbial

lineages is irrelevant to the gender bias of the disease

VSL3 mix rich in Lactobacillacae

SECS (Similar to E.Coli and Shigella) proteobacterium

Incidence of T1D, %

SFB-monocolonized GF mice show gender bias

100

GF F (n=81)
SFB F (n=10)
GF M (n=25)
SFB M (n=18)

75
50
25
0

10

20

Wks

30

Thus, not ALL bacteria can influence the gender bias

of T1D
Bacteria of different families can affect the gender bias

Linear hypothesis predicts that microbes can affect the

levels of hormones that reduce autoimmunity.
It also infers that male microbiota should affect disease
development in females.
B
hormonesmicrobes

autoimmunity

male
GFNOD
ASFNOD

DiabetesIncidence,%

100
80
60
40
20
0

10

20

30

Wks

Wen etal,Nature,2008Markle etal.,Science 2013

micewithnoprotectivemicrobiota
micewithprotectivemicrobiota

Since
a. protective bacteria protect at a wide range of
androgen concentration in blood, and
b. ASF that elicits reasonable levels of androgens
does not protect males from T1D,
it is likely that linear hypothesis B is also incomplete

Male microbiota does not protect females

single mom

traditional
family

T1D incidence, %

Male microbiota does not protect females

100

traditional family
single mom

80
60
40
20
0

10

20

Age (weeks)

30

20 mice/group

Thus, not ALL bacteria can influence the gender bias

of T1D
Bacteria of different families can affect the gender bias
Importantly, protective bacteria only worked in male mice,
indicating that they require male hormones to protect

A dual-signal model, in which both

androgens and microbes work in
concert to reduce T1D in males.
effector
steps

amplification
regulatory loop

hormones

microbes

Block of
autoimmunity

Sialitis in NOD mice is

a. Micobiota-dependent
b. Sexually dimorphic
a. Hormone-dependent

Sexual dimorphism of sialitis is more than hormones and microbiota

Restricting the studies of mutants to one sex

can lead to incorrect results and to breaking off
the ramifications of interesting projects

What do we learn about autoimmunity by

studying sexual dimorphism?

M
GF
SPF

Gene network (1 log fold cutoff)

Data from T1DR at Jax and from literature

Suggest that IFN-gamma is involved
Gene Knockout Female Male Ratio F/M
IFN
70% 65%
1.1
IFN R
50% 50%
1
STAT 4
45% 40%
1.1
STAT 6
90% 55%
1.6
IL-4
70% 45%
1.6
IL-10
40% 15%
2.7
Caspase-1
95% 75%
1.3
Controls
95% 40%
2.4

IFN-gamma production in the regional lymph nodes

is androgen-dependent

IFN (ng/mg of tissue)

3
P=0.001

Control Castrated

Pancreatic lymph nodes in SPF males have more

IFN producing CD4+ and CD8+ T cells.
CD4
15

P=0.025

P=0.02

% IF N

10

2
1

CD4

CD8

% IF N

% IFN+ cells

CD8

Protection by IFN-gamma is counterintuitive

How can it work?

1. Excessive IFN-gamma production may work as a

T cell-intrinsic regulator

Macrophages from female and male mice elicit very

different levels of IFN-gamma from TCR-transgenic T cells
(8.3)

IFN (pg/ml)

3000

2000

1000

M Gender
SECS

M
WT
-

F
WT
-

M
M
IFNKO WT
+

F
WT
+

M
IFNKO
+

In the same assay, MF from males induced more cell death in

8.3 TCR transgenic T cells than female MF. The effect was
reduced by anti-IFNg antibodies.

Female MF

9.6

PI

Male MF

17.6

Male MF+anti-IFNg

11.5

Sexual dimorpism of T1D in mice is dependent on

microbiota.
Central cell type involved is likely a macrophage
Gene expression analysis suggests a role for
IFN-gamma in male protection
This is supported by genetic data and by T cell
activation experiments
IFN-gamma is likely not the only mechanism