Commensal-dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine

Abstract

Alterations in the composition of intestinal commensal bacteria are associated with enhanced susceptibility to multiple inflammatory diseases, including those conditions associated with interleukin (IL)-17-producing CD4(+) T helper (Th17) cells. However, the relationship between commensal bacteria and the expression of proinflammatory cytokines remains unclear. Using germ-free mice, we show that the frequency of Th17 cells in the large intestine is significantly elevated in the absence of commensal bacteria. Commensal-dependent expression of the IL-17 family member IL-25 (IL-17E) by intestinal epithelial cells limits the expansion of Th17 cells in the intestine by inhibiting expression of macrophage-derived IL-23. We propose that acquisition of, or alterations in, commensal bacteria influences intestinal immune homeostasis via direct regulation of the IL-25-IL-23-IL-17 axis.

Figure 1.

Enteric commensal bacteria are required to limit the frequency of Th17 cells in the intestine. (A) Expression of IL-17 by CD4⁺ T cells in the Peyer's patches, cecal patch, and LP of the small or large intestine of CNV and GF mice was analyzed by flow cytometry. Flow cytometry plots depict log10 fluorescence. (B–D) Expression of mRNA for Il17 and Rorc (B), Il6 and Tgfb (C), or Il23a and Il12b (D) in the large intestine of CNV and GF mice was analyzed by quantitative real-time PCR. (E and F) Expression of mRNA for Il23a (E) and Il17 (F) in the large intestine of CNV and antibiotic-treated (Tx) CNV mice was analyzed by quantitative real-time PCR. Results are from 3 experiments (n = 6–9). Rel. units, relative units. *, P < 0.05. Error bars indicate the SEM.

Figure 2.

Equivalent regulatory T cell and Th1 cell responses in CNV and GF mice. (A) Cells isolated from the spleen, mLN, cecal patch, or large intestinal LP were stained for expression of CD4 and Foxp3 and analyzed by flow cytometry. (B–E) Splenocytes from CNV or GF mice were stimulated with anti-CD3 and -CD28 in the absence (B and C) or presence (D and E) of 1 ng/ml IL-12 for 72 h, followed by incubation with PMA, ionomycin, and brefeldin A for the final 5 h. Harvested cells were stained for expression of CD4 and IFN-γ by flow cytometry (B and D), and cell-free supernatants were analyzed by ELISA (C and E). Flow cytometry plots depict log10 fluorescence. Data are representative of 2 experiments (n = 8). Error bars indicate the SEM.

Figure 3.

Commensal-dependent expression of IL-25 inhibits IL-23 and the frequency of Th17 cells in the large intestine. (A) Expression of mRNA for Il27p28 and Ebi3 in the large intestine of CNV and GF mice was analyzed by quantitative real-time PCR. (B and C) Expression of mRNA for Il25 in the large intestine (B) and purified IECs (C) of CNV and GF mice was analyzed by quantitative real-time PCR. Data are from 3 experiments (n = 12). (D–F) Expression of mRNA for Il17 (D), the frequency of IL-17⁺ CD4⁺ cells (E), and expression of mRNA for Il23a (F) in the large intestine of CNV mice, GF mice, or GF mice treated with IL-25 (0.5 μg daily for 7 d) was analyzed by quantitative real-time PCR. Data are from 2 experiments (n = 6). (G–I) Expression of mRNA for Il17 (G) and Rorc (H) and the frequency of IL-17⁺ CD4⁺ cells (I) in the large intestine of CNV mice, GF mice, or GF mice treated with a neutralizing monoclonal antibody against IL-23p19 (αp19; 1 mg daily for 7 d) was analyzed by quantitative real-time PCR. Data are from 2 experiments (n = 4–6). Rel. units, relative units. *, P < 0.05. Error bars indicate the SEM.

Figure 4.

Endogenous IL-25 is required to limit IL-23 and -17 expression in the large intestine. (A–C) Expression of mRNA for Il17 (A), Il23a (B), and Il12b (C) in the large intestine of CNV and GF mice was analyzed by quantitative real-time PCR. (D) Expression of IL-17 by CD4⁺ T cells in the large intestinal LP was analyzed by flow cytometry. Flow cytometry plots depict log10 fluorescence. Data are from 3 experiments (n = 6–9). Rel. units, relative units. *, P < 0.05. Error bars indicate the SEM.

Figure 5.

IL-25 limits accessory cell-derived IL-23 independently of STAT6. (A) Increased expression of Il23a and Il12b by CD11b⁺ cells isolated from the large intestinal LP of GF mice. (B–F) Expression of mRNA for Il23a (B), Il12a (C), Tgfb (D), Il6 (E), and Il10 (F) in macrophages 4 h after stimulation with LPS in the absence or presence of IL-25 was analyzed by quantitative real-time PCR. Results are from four experiments. (G) mRNA expression of IL-23p19 (Il23a) in WT or Stat6^−/− macrophages was analyzed by real-time PCR after stimulation with media alone, LPS, or LPS and IL-25. Results are representative of two experiments. *, P < 0.05. Error bars indicate the SEM.