Interleukin-23 restrains regulatory T cell activity to drive T cell-dependent colitis

Abstract

Interleukin-23 (IL-23) is an inflammatory cytokine that plays a key role in the pathogenesis of several autoimmune and inflammatory diseases. It orchestrates innate and T cell-mediated inflammatory pathways and can promote T helper 17 (Th17) cell responses. Utilizing a T cell transfer model, we showed that IL-23-dependent colitis did not require IL-17 secretion by T cells. Furthermore, IL-23-independent intestinal inflammation could develop if immunosuppressive pathways were reduced. The frequency of naive T cell-derived Foxp3+ cells in the colon increased in the absence of IL-23, indicating a role for IL-23 in controlling regulatory T cell induction. Foxp3-deficient T cells induced colitis when transferred into recipients lacking IL-23p19, showing that IL-23 was not essential for intestinal inflammation in the absence of Foxp3. Taken together, our data indicate that overriding immunosuppressive pathways is an important function of IL-23 in the intestine and could influence not only Th17 cell activity but also other types of immune responses.

Figure 1

T Cell-Derived IL-17 Is Not Essential for Colitis (A) Transfer of Il17a^−/− CD4⁺CD45RB^hi T cells into Rag1^−/− mice. Left: colitis scores for recipients transferred with wild-type or IL-17-deficient CD4⁺CD45RB^hi T cells. Each point represents an individual mouse. Data are representative of four independent experiments; graph shows pooled data from two independent experiments. Center and right: Percentage of IL-17⁺ (center) or IFNγ⁺ (right) cells among CD4⁺ cells isolated from the colonic lamina propria from the mice analyzed left. (B) Characterization of Th17 and Th1 cell responses in the absence of IL-23. Amounts of IFN-γ (left) and IL-17 (center) in colon homogenates of Rag1^−/− or Il23a^−/−Rag1^−/− mice transferred with wild-type naive T cells. Right: Amounts of RORγt mRNA in colon homogenate. Values are normalized to CD3γ expression. Data show mean + SEM of between five and ten mice from two independent experiments. ^∗, p < 0.05; ^∗∗∗, p < 0.001.

Figure 2

Reduction of Regulatory Pathways Increases Colitis in Il23a^−/−Rag1^−/− Mice Transferred with Naive T Cells (A) Blockade of the IL-10 pathway after transfer of CD4⁺CD45RB^hi T cells into Il23a^−/−Rag1^−/− mice. Left: Colitis scores for control untreated and anti-IL-10R-treated recipients. Each point represents an individual mouse. Right: Concentration of proinflammatory cytokines (mean + SEM) in colon homogenates from these mice. (B) Blockade of TGF-β after transfer of CD4⁺CD45RB^hi T cells into Il23a^−/−Rag1^−/− mice. Left: Colitis scores for control untreated and anti-TGF-β treated recipients. Each point represents an individual mouse. Right: Representative microphotographs of colonic sections from either control untreated recipients (score 3) or recipients treated with blocking TGF-β antibody (score 6). The scale bars represent 200 μm. Data are pooled from two independent experiments. ^∗, p < 0.05; ^∗∗, p < 0.01.

Figure 3

Absence of TGF-β Signaling in T Cells Increases Intestinal Inflammation in Il23a^−/−Rag1^−/− Mice (A) Colitis score of Il23a^−/−Rag1^−/− mice transferred with wild-type (WT) or dnTGFβRII (DN) naive T cells. Each point represents an individual mouse. Below: Representative microphotographs of colonic sections from Il23a^−/−Rag1^−/− mice transferred with WT (score 2) or DN CD4⁺CD45RB^hi T cells (score 5). The scale bars represent 200 μm. (B) IL-17 and IFN-γ production in Il23a^−/−Rag1^−/− mice transferred with wild-type (WT) or dnTGFβRII (DN) naive T cells. The figure shows representative FACS plots of IL-17 and IFN-γ production by MLN lymphocytes gated on CD4⁺ cells and the concentrations of IL-17 and IFN-γ (mean + SEM) in the colon of transferred Il23a^−/−Rag1^−/− mice. (C) Foxp3 expression in CD4⁺ cells after transfer. Left: Representative FACS plots showing Foxp3 frequency in MLN from transferred Il23a^−/−Rag1^−/− mice. Plots are gated on CD4⁺ TCRβ⁺ cells. Right: Percentage of Foxp3⁺ among MLN CD4⁺ T cells of transferred Il23a^−/−Rag1^−/−. Each point represents an individual mouse. Data are pooled from three independent experiments. ^∗, p < 0.05; ^∗∗, p < 0.01; ^∗∗∗, p < 0.001.

Figure 4

CD45RB^hi-Derived Foxp3⁺ Cells Are Increased in the Colon after Naive T Cell Transfer into Il23a^−/−Rag1^−/− Mice (A) Frequency of Foxp3⁺ cells among CD4⁺ T cells from spleen, MLN, and colonic LPL from IL-23-deficient or -sufficient Rag1^−/− recipients transferred with CD4⁺ CD25⁻ CD45RB^hi naive T cells. Each point represents an individual mouse; data are pooled from two independent experiments. (B) Design of the congenic-transfer experiment. A mixture of 99% sorted CD45.1⁺ CD4⁺ CD25⁻ CD45RB^hi naive T cells and 1% CD45.2⁺ CD4⁺ CD25⁺ CD45RB^low regulatory T cells was injected into Rag1^−/− recipients. FACS plots show representative Foxp3 staining of sorted naive (left) and regulatory (right) populations, gated on CD4⁺ cells. (C) Approximately 2 months after transfer, cells from spleen, MLN, and colonic LP were stained for Foxp3 and the congenic marker CD45.2. Left: Representative FACS plot showing Foxp3 versus CD45.2 expression in the spleen of transferred Il23a^−/−Rag1^−/−. The plot is gated on CD3⁺ CD4⁺ cells. Right: Percentage of CD45.2⁺ Foxp3⁺ cells in the CD3⁺ CD4⁺ population in the spleen, MLN, and LP of transferred IL-23-sufficient or -deficient Rag1^−/− recipients. Each point represents an individual mouse. (D) Percentage of CD45.2⁻ Foxp3⁺ cells in the CD3⁺ CD4⁺ population of the mice analyzed in (C). ^∗, p < 0.05; ^∗∗, p < 0.01; ^∗∗∗, p < 0.001; ns, not significant.

Figure 5

Foxp3-Deficient Naive T Cells Induce Colitis in Il23a^−/−Rag1^−/− Mice (A) Colitis in mice transferred with Foxp3-deficient naive T cells. Left: Colitis score of Rag1^−/− (circles) or Il23a^−/−Rag1^−/− (triangles) transferred with wild-type or Foxp3-deficient naive T cells. Each point represents an individual mouse. Right: Representative microphotographs of colonic sections from Rag1^−/− mice transferred with wild-type (score 8) or Foxp3^−/− naive T cells (score 10) or from Il23a^−/−Rag1^−/− mice transferred with wild-type (score 4) or Foxp3^−/− naive T cells (score 9). The scale bars represent 200 μm. ^∗∗∗, p < 0.001. (B) Left: Percentage of IL-17-secreting cells in the CD4⁺ population from spleen, MLN, and colon of Il23a^−/−Rag1^−/− mice transferred with wild-type or Foxp3^−/− naive T cells. Each point represents an individual mouse. Right: Amounts of IL-17 in colon homogenate from Il23a^−/−Rag1^−/− mice transferred with wild-type or Foxp3^−/− naive T cells. Data show the mean + SEM of nine or ten mice. Differences were not statistically significant. Data are pooled from two independent experiments.

Figure 6

Cytokine Expression in Inflamed Colons from Il23a^−/−Rag1^−/− Mice (A) Foxp3 expression after culture of CD4⁺CD45RB^hi T cells with TGF-β, IL-23, and/or IL-21. Left: Representative FACS plots. Cells are gated on forward and side scatter to exclude dead cells. Right: Percentage of Foxp3⁺ cells in the CD4⁺ population. Data show mean + SEM of three replicates and are representative of three independent experiments. ns, not significant. (B) Relative mRNA expression of IL-6, IL-21, IL-27p28, and EBI3 in colon homogenates after naive T cell transfer with wild-type (empty columns) or dnTGFβRII or Foxp3^−/− CD4⁺ T cells (filled columns). Data from mice transferred with dnTGFβRII or Foxp3^−/− T cells were pooled because they yielded similar values. Data were normalized to HPRT for each sample. The average value for Il23a^−/−Rag1^−/− mice transferred with wild-type T cells (noninflamed) was set as one. Data show mean + SEM of between seven and 11 mice per group. ^∗, p < 0.05; ^∗∗, p < 0.01; ^∗∗∗, p < 0.001; ns, not significant. (C) Amounts of proinflammatory cytokines in colon homogenates of Rag1^−/− or Il23a^−/−Rag1^−/− transferred with Foxp3^−/− naive T cells. Data show mean + SEM of between five and ten mice from two independent experiments. None of the differences were significant.