Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation

Abstract

Effector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the corresponding type of inflammatory response. Regulatory T (Treg) cells integrate environmental cues to suppress particular types of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative regulatory circuits operating in immune effector cells.

Figure 1. IL-10 induces robust STAT3 phosphorylation in Treg cells

(A) Flow cytometric analysis of pSTAT3 (Y705) amounts after treatment of splenic CD4⁺Foxp3^- T cells, CD4⁺Foxp3⁺ Treg cells and CD8 T cells with recombinant IL-6 (16 ng/ml) or IL-10 (100 ng/ml) for 15 mins. Analysis of pSTAT3 levels by either flow cytometry (B) or western blotting (C) after stimulation of indicated cells with titrating doses (3, 10 and 15 ng/ml for IL-6; 50, 150 and 300 ng/ml for IL-10) of recombinant IL-6 or IL-10. (D) Immunoblot detection of pSTAT3 and STAT3 in Treg cells isolated from IL-10R or IL-6R deficient and sufficient mice. Data are representative of four independent experiments.

Figure 2. Treg cell-specific IL-10R deletion results in severe intestinal inflammation

(A, B) Spleen and lymph node cellularity in Foxp3^CreIl10ra^fl/wt and Foxp3^CreIl10ra^fl/fl mice (** p<0.001; * p<0.01). (C, D) Frequencies of activated T cells (E, F) Absolute numbers of CD4⁺Foxp3⁺ T cells and (G) body weights of Foxp3^CreIl10ra^fl/fl mice (n=7). (H) IBD scores derived from evaluation of colon and cecum from 14-16 week-old Foxp3^CreIl10ra^fl/wt and Foxp3^CreIl10ra^fl/fl mice (n=4/group). (I) Representative H&E stained sections of large bowel at the level of the ileocecal-colic junction from Foxp3^CreIl10ra^fl/wt and Foxp3^CreIl10ra^fl/fl mice. Note: Mucosa (M) with expanded irregular, hyperplastic crypts, glandular loss and fibrosis with multifocal transmural inflammation (arrow). Lumen (L). Original magnification, 5x (n=7).

Figure 3. IL-10R ablation does not affect Foxp3 expression and Treg cell lineage stability

(A) Flow cytometric analysis of Foxp3 and YFP expression on splenic CD4⁺ T cells in Foxp3^CreIl10ra^fl/flR26Y and control littermate mice. (B) Expression of Foxp3 in flow cytometry-sorted CD4⁺YFP⁺ T cells from indicated mice. (C) Quantitative PCR (qPCR) analysis of Foxp3 and IL-10Rα expression in flow cytometry-sorted CD4⁺YFP⁺ T cells. The grey bar represents CD4⁺YFP^- T cells. The results represent the mean+/-SD of relative expression values for the indicated genes relative to hypoxanthine-guanine phosphoribosyl transferase. Data are representative of three independent experiments.

Figure 4. Increased Treg cell activation and selective dysregulation of Th17 cell responses in Foxp3^CreIl10ra^fl/fl mice

(A) Flow cytometric analysis of Treg cell activation markers and putative effector molecules on splenic CD4⁺Foxp3⁺ T cells in Foxp3^CreIl10ra^fl/fl and control littermate mice. (B, C) Flow cytometric analysis of cytokine production by splenic CD4⁺Foxp3^- T cells in Foxp3^CreIl10ra^fl/wt and Foxp3^CreIl10ra^fl/fl mice after PMA and Ionomycin treatment for 4-6 hours (** p<0.001; * p<0.01).

Figure 5. IL-10R-deficient Treg cells efficiently control in vitro T cell proliferation but fail to suppress Th17 cell differentiation and colitis

(A) IL-10R-deficient and -sufficient Treg cell-mediated suppression of effector T cell (T_Eff) proliferative responses in vitro. (B) Frequencies of Th1, Th2 and Th17 skewed cells among the transferred T cell population isolated from the Foxp3^- mice. (C) Frequencies of splenic Treg cells, CD4⁺IL-17⁺Foxp3^- T cells, CD4⁺IFN-γ⁺ Foxp3^- T cells, and CD4⁺IL-4⁺Foxp3^- T cells within the indicated donor-derived population 5-6 weeks after co-transfer of either Foxp3^CreIl10ra^fl/wt or Foxp3^CreIl10ra^fl/fl Treg cells with Foxp3^- CD4⁺ T cells into Rag2^-/- recipients. (D) Representative H&E stained sections of colon collected from recipient mice 5-6 week after adoptive T cell transfer (original magnification, 10x). Note: Mucosa (M), submucosal edema (E), dilated lymphatics (*) and lumen (L).

Figure 6. IL-10R signaling is required for IL-10 expression by Treg cells

(A) qPCR analysis of STAT3-bound chromatin isolated from wild type Treg cells using primers corresponding to the indicated genes. House-keeping gene Gmpr was used as a specificity control while IgG-immunoprecipitated chromatin was used as a negative control. (B) qPCR analysis of relative expression of indicated genes in Treg cells isolated from either Foxp3^CreIl10ra^fl/wt or Foxp3^CreIl10ra^fl/fl mice. The results represent the mean+/-SD of relative expression values for the indicated genes relative to hypoxanthineguanine phosphoribosyl transferase.