Immune system characteristics in mice

with normal immunological experience

Steve Jameson
Center for Immunology
Dept. Lab. Med. & Path.
University of Minnesota

http://speakingofresearch.com

Generating and working with dirty mice

Overall question: How would a more broad, more physiological infectious history alter
the immune system and immunological responses in inbred mice?

Approach included analysis of wild-caught and pet store purchased mice (dirty mice), as well as cohousing pet store animals with inbred strains, so that the latter acquire pet store mouse microbes.

Wild and pet store have been exposed to many pathogens (only some of which we define) and
commensal microbes. Some pathogens/commensals are acquired by co-housed mice, but we dont try
to control this. These are essentially the opposite of gnotobiotic mice we dont know the infectious
history of the mice but may be seen as a more authentic reflection of human microbial experience.

Logistical problems abound. Dirty mice are known to carry SPF-excluded pathogens (and perhaps
others that are equally dangerous) so an effective barrier with SPF colonies was required. We use
BSL3 housing, despite the fact that none of the defined pathogens involved are above BSL2 status.

The approach of deliberate sequential infection is valuable but the choice of which
pathogens/commensals to use is difficult to justify. Weve started using microbes that frequently arise
as chronic infections pet store animals lab strains or naturally isolated strains as well as others that
are of interest and/or easily-spread between mice (e.g. MCMV)

CD8 T cell memory subsets in adult human and C57BL/6 mouse blood
C57BL/6 mouse
Naive

Tcm

Adult Human

Human cord blood

Tcm

Naive
Gated on CD8+
T cells

Tem

Tem

TEMRA
Gated on
non-naive CD8+
T cells

Beura et al. 2016

Nature

Abundance of T cells in human but not mouse non-lymphoid sites

B6 cervix

CD8
DAPI

Adult human cervix

Beura et al. 2016

Nature

Why might mouse and human

immune systems differ at baseline
Divergence in adaptive
& innate immune
system evolution over
last 65-75 million
years.
Animal lifespan & age
at analysis.
Environment
(immunological
experience).

The production and use of germfree and specific-pathogen-free (SPF) laboratory

animals continues to gain momentum in most countries, despite the fact
that some members of the older school of medical research workers consider
that the results of tests carried out with SPF animals may be misleading when
extrapolated to man, who cannot be regarded as "pathogen-free."
J.S. Paterson and R. Cook (1971)
From Utilization of diets sterilized by irradiation for germfree and specific-pathogenfree animals in Defining the Laboratory Animal.
National Academy of Sciences, Washington, DC

Mice used for immunology research are typically

maintained in barrier facilities, under specific pathogen
free (SPF) conditions.
Helps normalize immune status prior to study
but artificially limits normal, physiological
exposure to microbes.
Humans, on the other hand --

If SPF mice are too clean, what do

we see in dirty mice?

CD8 T cell memory subsets in mice from different environments

Naive
Tcm

Tem

% CD8+ T cell

80

***
*** **
***
*** **

60

SPF
Feral mice
Pet store mice

40
20
0

44lo 62Lhi

Naive

44hi 62Lhi

44hi 62Llo

Tcm

Tem

Beura et al. 2016

Nature

Adult pet store mice

Adult B6 mice

Wait
Co-housed for >60d under ABSL3 conditions to avoid SPF colony contamination

Co-housed mice become infected by some SPF microbes.

Petstore
(n=15)

Laboratory
(n=4)

Cohoused
(n=13)

Viruses
Rotavirus (EDIM)
Mouse Hepatitis Virus
Murine norovirus
Mouse parvovirus NS1
Mouse parvovirus type 1
Mouse parvovirus type 2
Minute virus of mice
Theiler's murine encephalomyelitis virus
Sendai virus
Ectromelia virus
Lymphocytic Choriomeningitis virus
Mouse adenovirus 1 and 2
Mouse cytomegalo virus
Polyoma virus
Pneumonia virus of mouse
Reovirus

0
93.3
60
53.3
40
46.7
46.7
60
66.7
0
6.7
0
0
6.7
53.3
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
61.5
38.5
0
7.7
0
0
38.5
23.1
0
7.7
0
0
0
0
0

Bacteria
Cillia-Associated Respiratory Bacillus
Mycoplasma pulmonis
Clostridium piliforme

0
73.3
26.7

0
0
0

0
30.8
0

Parasites/Protozoa/Fungi
Enchephalitozoon cuniculi
Pinworm
Mites

40
100
100

0
0
0

0
100
100

Beura et al. 2016

Nature

Impact of co-housing SPF B6 with pet store mice

Not cohoused

CD8+ T cells

18 10

24

11

CD62L

80

D27 post-cohousing D52 post-cohousing

76

59

CD44
Mixing with the wrong mice can be fatal

Beura et al. 2016

Nature

Co-housing leads to appearance of T cells in NLT

Beura et al. 2016

Nature

Pet store and SPF mice have similar -diversity in

intestinal microbiota
Group

Shannon

ACE

Co-housed

4.03 0.35

548 141

Pet store

3.98 0.57

672 260

SPF

3.93 0.25

494 125

ANOVA P-value

0.905

0.196

Alex Khoruts/Mike Sadowsky

Unpublished

but differ in community composition. The microbiota of cohoused mice becomes more similar to that of pet store mice

Cohoused
Pet store
SPF

Beta diversity (ANOSIM) P < 0.001; group clustering (AMOVA) P < 0.001

Alex Khoruts/Mike Sadowsky

Unpublished

Animal welfare and protecting SPF animals

A major issue is avoiding spread of pathogens to our standard SPF colonies. Since we dont have
personnel investigators who work only with dirty mice, containment was essential. We used a BSL3
facility for all dirty mouse studies.

Co-housing of cohorts of inbred mice leads to some mortality -- ~20% in the case of C57BL/6 mice,
and initial pilot experiments indicate higher percentages for BALB/c strains.

Since SPF-designated pathogens are the most likely cause of inbred mouse death, we use necropsy
data and an iterative process of discerning what transmitted SPF pathogens most clearly associate
with higher mortality. For example, Mycoplasma pulmonis may account for much of the inbred animal
death, based on characteristics of the pathology. We can avoid this pathogen altogether by choice of
pet store mice used for co-housing.

Recent advances, such as use of contaminated bedding in place of co-housing, has led to some
reduction in mortality without an apparent compromise in immune education but presumably only
some pathogens/commensals are efficiently spread by fomites,

Sequential infection models if they recapitulate the dirty mouse phenotype in terms of basal immune
status and immune reactivity may help avoid this issue all together and could potentially be achieved
using BSL2 designated pathogens. Currently, though these studies are still conducted at BSL3.

Comparing human/mouse PBMC gene expression profiles

Comparing human/mouse PBMC gene expression profiles

Beura et al. 2016

Nature

Alternative approach to testing impact of immunological experience

From Reese et al. 2016

Cell Host and Microbe

Alternative approach to testing impact of immunological experience

Petstore/SPF

Co-housed/SPF

Alignment of co-infected versus mock infected PBMC

vs indicated group.

Adult/Neonatal human

From Reese et al. 2016

Cell Host and Microbe

Concluding remarks

The potential for dirty mice in research has captured some attention. We have one current R01 and
several new R01 and P01 awards in preparation in-house at the University of Minnesota, and have
been approached by PIs at other institutions as well as small and large drug companies to collaborate
on conducting various assays using these animals (topics including cancer therapy, allergic asthma,
autoimmune diseases, etc.)

Currently our recommended SOP is to use the BSL3 containment approach, but some groups at other
institutions are exploring other options. BSL3 containment is not required for any of the known
pathogens carried by these mice (and pet store mice have to pass DOA inspections) but potential
contamination of SPF colonies would be catastrophic for many institutions.

Jameson/Hogquist
Lab

Sara Hamilton
Lalit Beura

Other Collaborators
UMN: Marc Jenkins.
Harvard: Nick Haining, Kevin Bi
Support:
NIH awards to D.M. and S.C.J.
UMN BSL3 waiver award

Masopust/Vezys
Lab