Supplementary Figure Legends
Figure 1(a) IL23R-/-Rag-/-and Rag-/- mice were intraperitoneally injected with 5x105 CD45Rbhigh CD4+ NK1.1-Foxp3YFP- T cells and at the end of week 5 colons were harvested. 1 cm proximal colon was cultured for 48h ex vivo, and supernatants were used for detection of IL-22, IL-17A and IFN-γ.
Figure 2 Distribution and characterization of IL-23R GFP+ innate lymphoid cells (a) Following organs from untreated IL23R+/-Rag-/- mice was examined for presence of IL-23R GFP+ innate lymphoid cells via flow cytometry after surface staining: Spleen, mesenteric (MLN), peripheral lymph node (PLN) colon and small intestine (SI ) lamina propria lymphocytes. (b-c) Control IL23R+/-Rag-/- and IL23R-/-Rag-/- mice colon lamina propria lymphocytes of untreated mice were surface stained for indicated markers and analyzed by flow cytometry. (d) Absolute number colon Lamina propria cells before and 2 days after anti-CD40 injection.Het: IL-23R-/+Rag-/-, Homo: IL-23R-/-Rag-/-(e) Measurement of intestinal epithelial barrier integrity after anti-CD40 colitis induction on day 4.
Figure 3 IL23R+ ILC kinetics remains comparable in IL23R-/-Rag-/- and IL23R-/+Rag-/-mice colon LP during anti CD40 induced colitis. Representative plots of IL-23R+ ILCs over time after colitis induction (a) and quatification of % cells. Het: IL23R-/+Rag-/-, Homo: IL23R-/-Rag-/-.
Figure 4 mRNA profile of colon lamina propria lymphocytes (a) Sorting strategy for colonLP lymphocytes isolated at day 5 of colitis from IL23R+/-Rag-/- or IL23R-/-Rag-/- mice (b) real time-qPCR results for the expression of indicated genes (c) Colon lamina propria cells isolated from IL23R+/-Rag-/- mice at 7 of anti-CD40 injection. IL-22 expressing Thy1.2+ cells were gated and stained for Rorγt.
Figure 5 IL-22 causes increased colon pathology upon anti-CD40 injection. Pictures of colons at day 7 of colitis taken from IL23R+/-Rag-/- mice that received IL-22 neutralizing or isotype antibody (left) or IL23R-/-Rag-/- mice that received IL-22 expressing or mock plasmid (right).Ceca (Ce) and colons harvested from experimental groups as indicated. Grossly, colitis was mild in both groups and characterized by turgid and thickened bowel walls (arrowheads) with poorly formed, large and sticky fecal pellets (arrows).
Figure 6(a) RegIIIγ plasmid is functional. Hydrodynamic delivery of RegIIIγ plasmid but not empty vector protects IL23R+/-Rag-/-mice from bacterial translocation/burden after Citrobacter rodentium infection. (b) IL-22 over expression alone without anti-CD40 injection does not cause colitis. IL-22 plasmid recipient IL23R-/-Rag-/- mice have normal colon with average colitis scores “0”.Original magnification 20X.
Figure 7Chemokine and MMP mRNA expression of indicated genes in the colon LP of IL23R-/-Rag-/- or IL23R-/+Rag-/- mice on day 0, 2 and 7 of anti-CD40 induced colitis.
Figure 8Model explaining the role of IL-23R and IL-22 during anti CD40 induced colitis.
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