IL-27 promotes T cell-dependent colitis through multiple mechanisms

Abstract

Interleukin-27 (IL-27) is a cytokine known to have both proinflammatory and immunoregulatory functions. The latter appear to dominate in vivo, where IL-27 suppresses TH17 responses and promotes the differentiation of Tr1 cells expressing interferon-γ and IL-10 and lacking forkhead box P3 (Foxp3). Accordingly, IL-27 receptor α (Il27ra)-deficient mice suffer from exacerbated immune pathology when infected with various parasites or challenged with autoantigens. Because the role of IL-27 in human and experimental mouse colitis is controversial, we studied the consequences of Il27ra deletion in the mouse T cell transfer model of colitis and unexpectedly discovered a proinflammatory role of IL-27. Absence of Il27ra on transferred T cells resulted in diminished weight loss and reduced colonic inflammation. A greater fraction of transferred T cells assumed a Foxp3(+) phenotype in the absence of Il27ra, suggesting that IL-27 functions to restrain regulatory T cell (T(reg)) development. Indeed, IL-27 suppressed Foxp3 induction in vitro and in an ovalbumin-dependent tolerization model in vivo. Furthermore, effector cell proliferation and IFN-γ production were reduced in the absence of Il27ra. Collectively, we describe a proinflammatory role of IL-27 in T cell-dependent intestinal inflammation and provide a rationale for targeting this cytokine in pathological situations that result from a breakdown in peripheral immune tolerance.

Figure 1.

Decreased severity of CD45Rb^hi colitis in the absence of T cell–derived IL-27R. (A) Relative weight loss after transfer of CD4⁺CD45Rb^hi or unsorted CD4⁺ cells from WT or Il27ra^−/− (KO) mice into CB17-SCID recipients. (B) Weight loss relative to initial weight at 11 wk after transfer of CD4⁺CD45Rb^hi or unsorted CD4⁺ cells from WT or KO mice. (C) Colon length measurements at 11 wk after transfer. (D) Histological scoring of colitis severity (E) Representative hematoxylin and eosin staining of colons from mice transferred with WT CD45Rb^hi, Il27ra^−/− CD45Rb^hi, or Il27ra^−/− unsorted CD4⁺ cells. Data are from two experiments representing four individual experiments. Bars, 100 µm. *, P < 0.05.

Figure 2.

Il27ra^−/− CD45Rb^hi cells preferentially assume a Foxp3⁺ phenotype. (A) Time course of the percentage of Foxp3⁺ cells relative to CD4⁺ cells in peripheral blood of mice transferred with CD45Rb^hi cells from WT or Il27ra^−/− donor mice. (B) Representative Foxp3 staining of splenocytes gated on CD4⁺ cells at 12 wk after transfer of CD45Rb^hi cells. (C and D) Frequencies of Foxp3⁺ CD4⁺ T cells (C) and total CD4⁺ cells (D) obtained from the spleen, mLN, and lamina propria at 12 wk after transfer. (E) Absolute number of CD4⁺ T cells in spleen, mLN, and lamina propria at 12 wk after transfer. Data are representative of three individual experiments. *, P < 0.05; ***, P < 0.001.

Figure 3.

IL-27 inhibits the induction of OVA-specific T regulatory cells in vitro. (A) DO11.10⁺Rag2^−/− CD4⁺ T cells were isolated from WT or Il27ra^−/− (KO) mice and confirmed to be Foxp3⁻CD25⁻. (B and C) DO11.10⁺CD4⁺ cells from WT or Il27ra^−/− mice were incubated with CD11c⁺MHCII⁺ DCs isolated from the colonic lamina propria in the presence of OVA_323-339 and TGF-β, with or without IL-27, and stained for Foxp3 and CD25 (B) and CD69 (C). Ctrl, Control; β+27, TGF-β + IL-27; β, TGF-β; Act., Activated. Data are representative of two individual experiments.

Figure 4.

Il27ra^−/− DO11.10⁺ T cells convert more readily to T regulatory cells in vivo. (A) Representative Foxp3 staining of WT or Il27ra^−/− (KO) DO11.10⁺ cells after transfer to balb/c mice and oral administration of 1.5% OVA in water for 5 d. (B and C) Quantitative analysis of percentage (B) and absolute numbers (C) of Foxp3⁺ cells in the mLNs and spleens of mice receiving WT or Il27ra^−/− DO11.10⁺ T cells and fed 1.5% OVA or control water. (D-E) Intracellular cytokine staining analysis of IL-2 (D) and IFN-γ (E) in the mLNs from OVA-fed or control mice. (F) Absolute numbers of Foxp3⁺ DO11.10⁺ cells in the mLN of control or OVA-treated mice that had received 3 × 10⁵ CD45Rb^hi cells 4 wk earlier. Data are from a single experiment representing two individual experiments. *, P < 0.05; ***, P < 0.001.

Figure 5.

Reduced TH1 and enhanced TH17 polarization after transfer of Il27ra^−/− CD45Rb^hi cells. (A) Representative IFN-γ and IL-22 intracellular cytokine staining of lamina propria isolates from CB17-SCID mice injected with WT or Il27ra^−/− (KO) CD45Rb^hi CD4⁺ T cells stimulated with PMA, ionomycin, and brefeldin A. Samples are gated on CD4⁺ cells. (B–E) Quantitative analysis of intracellular IFN-γ (B), IL-17A (C), IL-22 (D), and IL-13 (E) staining in splenocytes, mLNs, and colonic lamina propria lymphocytes from CB17-SCID mice transferred with WT or Il27ra^−/− CD45Rb^hi cells. Data are representative of three individual experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Neutrophil content in the lamina propria of WT and Il27ra^−/− CD45Rb^hi recipient mice. (A) Representative flow cytometry of colonic lamina propria isolates 12 wk after transfer of CD45Rb^hi cells. Lamina propria leukocytes were surface stained with anti-Gr1 and anti-CD11b. Quantification of percentage (B) and absolute number (C) of Gr1^hiCD11b⁺ cells. Data are from one single experiment.