1. Our main objective was to understand how interact with
ILCs and TH17 cells to maintain gut homeostasis, and the
role of IL-10 in this crosstalk. Here, we used a mouse
model conditionally deficient for expression of IL-10 by
Tregs. These animals developed, similar to IL-10
knockout mice.6
This points to the significant contribution
of Treg IL-10 to colon homeostasis. The absence of
pathology in the small intestine is in line with the known
role of microbiota in colitis.
Out findings suggest that ILC3s and TH17 cells increase
in concert with colitis, when Tregs fail to produce IL-10.
The innate nature of ILC3s strongly implies that their
response precedes that of the TH17 cells, which belong
to the adaptive immune compartment. Interestingly, the
reverse trend was seen in the small intestine. The exact
sequence of events and manner of communication
between ILC3s and TH17 cells remains to be
investigated.
Colitis was associated with splenomegaly and systemic
expansion of pro-inflammatory monocytes at the expense
of regulatory monocyte populations. It remains to be
determined if this shift is a direct consequence of IL-10
deficiency or gut inflammation.
The gut mucosa is continuously exposed to microbial and
non-microbial antigenic stimuli, which demands a
stringent balance between tolerance and protective
immune response. The cross talk between Tregs, ILC3s,
and TH17 cells is central role in this regulation.
Lisa Levoir*, Abdulrahman Saadalla M.D.1,
Khashayarsha Khazaie, Ph.D.1
*Macalester College 1
Department of Immunology, Mayo Graduate School
Mayo Clinic, Rochester, MN
Discussion & Conclusions
1. Vignali, Dario AA, Lauren W. Collison, and Creg J. Workman. "How regulatory
T cells work." Nature Reviews Immunology 8, no. 7 (2008): 523-532.
2. Dennis, Kristen L. et al. "Adenomatous polyps are driven by microbe-instigated
focal inflammation and are controlled by IL-10–producing T cells." Cancer
research 73, no. 19 (2013): 5905-5913.
3. Dennis, Kristen L. et al. "T-cell expression of IL-10 is essential for tumor
immune surveillance in the small intestine." Cancer immunology research (2015)
4. Laidlaw, Brian J., Weiguo Cui, Robert A. Amezquita, Simon M. Gray, Tianxia
Guan, Yisi Lu, Yasushi Kobayashi et al. "Production of IL-10 by CD4+ regulatory
T cells during the resolution of infection promotes the maturation of memory
CD8+ T cells." Nature immunology (2015).
5. Blatner, Nichole R. et al. "Expression of RORγt marks a pathogenic regulatory
T cell subset in human colon cancer." Science translational medicine 4, no. 164
(2012): 164ra159-164ra159.
6. Kühn, Ralf, Jürgen Löhler, Donna Rennick, Klaus Rajewsky, and Werner
Müller. "Interleukin-10-deficient mice develop chronic enterocolitis." Cell 75, no. 2
(1993): 263-274.
Acknowledgements: I would like to thank the entire Khazaie lab for their support
and teaching this summer as well as Mayo Graduate School and the Immunology
Department for welcoming undergraduate students.
References
The function of regulatory T cells (Tregs) is to regulate
immune responses to various self and non-self antigens. At
mucosal interfaces as in the gut, the commensal microbiome
provides ample stimuli that promote the regulatory T cell
maintenance and response.1
Tregs regulate and coordinate
the innate and adaptive arms of the immune system through
cytokines, (such as IL-10 and TGFß) and co-inhibitory
signaling. In the gut, Tregs represent the major cellular source
of IL-10, which is a known immune suppressor cytokine.
Specifically, in the colon, the vast majority of IL-10 is produced
by Tregs.2
We and others have recently highlighted the
essential role of IL10 in the establishment of T cells Th1
immunity.3
Following this, we concluded that immune
modulation by Tregs is necessary for antitumor immunity.4
Our lab has shown that in colorectal cancer, Tregs switch to
an inflammatory phenotype marked by the expression of the
transcription factor RORγt.5
Although RORγt+ Tregs are more
suppressive, they secrete IL-17 which promotes cancer
through excess Th-17 inflammation. It remain to be shown if
IL-10 plays a role in this switch.
We employed a conditional knockout of IL10 in Tregs using a
Cre-Lox knockout system. We profiled changes in both
adaptive and innate cells deregulated in consequence of IL10
deficiency causing inflammation and impacting immunity. In
this project, we studied the crosstalk between Tregs, Th17
cells, innate lymphoid cells and its impact on mucosal
immunity.
Introduction Figure 2: IL10+
Tregs suppress RORγt
IL-10 Regulation of Gut Innate Immunity
Frequencies and proliferation status of RORγt expressing
Tregs and conventional CD4+ T-cells in the MLN of Foxp3cre IL-
10 fl/fl
as compared to WT mice . A) Increases in frequencies and
proliferation in MLN but not spleen (n= 6-8) B) Representative
FACS dot plot showing KI67 expression (n=4) C) Distribution of
CD4+ cells expressing proliferation marker KI-67 among CD4+
cells that are Foxp3+/- or RORγt+/- . p<0.05* p<0.005**
Figure 1: IL-10+
Tregs prevent colitis
Foxp3cre
IL-10fl/fl WT
Foxp3cre
IL-10fl/fl
WT
smallbowelcolon
spleen
MLNMLN
spleen
Gated on : CD4 Ki-67+
WT Foxp3cre
IL-10fl/fl
Foxp3cre IL-10fl/fl WT
A
B
C
H&E stained histology sections of colon and small
intestine of Foxp3 Cre IL-10 fl/fl
vs. healthy WT mice.
A) Elongated crypts in colon but no crypt/villus
elongation in the small intestine of representative
Foxp3 Cre IL-10 fl/fl
mouse B) Plots of crypt lengths
in mm of Foxp3 Cre IL-10 fl/fl
vs. healthy WT in the
colon and small bowel n=2,2 p<.005=***
Foxp3 Cre IL-10 fl/fl
WT
100x100x
colon
WTFoxp3 Cre IL-10 fl/fl
200x200x
smallbowel
BA
Increased frequencies of ILC3s ( )in intestine and colon of
Foxp3cre IL-10fl/fl
vs. WT mice. (A) Small intestine. (B) Colon.
Gated: CD45+ Lin- CD127hi
Figure 3: Treg IL-10 controls ILC3s
A
B
Flow plots of gut lamina propria CD4 Th17 cells. Gated on
CD45+ cells.
Foxp3cre
IL-10fl/fl
WT
smallbowel
WTFoxp3cre
IL-10fl/fl
colon
Figure 4: Th17 cells increase in colitis
Increased frequencies of inflammatory monocytes ( ) in
spleen of Foxp3cre IL-10fl/fl
vs. WT mice. A) Representative FACS
dot plots, gated on CD11b+ splenocytes B) Expansion of
proinflammatory monocytes and resident monocytes in the
spleen (n=3,2).
Figure 5: IL-10+
Tregs suppress monocytes
Foxp3cre
IL-10fl/fl
WT
**
A
B