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 1Department of Immunology, Mayo Graduate SchoolMayo 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 RORt.5 Although RORt+ 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 RORt

IL-10 Regulation of Gut Innate Immunity

Frequencies and proliferation status of RORt 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 RORt+/- . p<0.05* p<0.005**

Figure 1: IL-10+ Tregs prevent colitis

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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=***

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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

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Flow plots of gut lamina propria CD4 Th17 cells. Gated on CD45+ cells.

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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

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