A cytokine network involving IL-36γ, IL-23, and IL-22 promotes antimicrobial defense and recovery from intestinal barrier damage

Abstract

The gut epithelium acts to separate host immune cells from unrestricted interactions with the microbiota and other environmental stimuli. In response to epithelial damage or dysfunction, immune cells are activated to produce interleukin (IL)-22, which is involved in repair and protection of barrier surfaces. However, the specific pathways leading to IL-22 and associated antimicrobial peptide (AMP) production in response to intestinal tissue damage remain incompletely understood. Here, we define a critical IL-36/IL-23/IL-22 cytokine network that is instrumental for AMP production and host defense. Using a murine model of intestinal damage and repair, we show that IL-36γ is a potent inducer of IL-23 both in vitro and in vivo. IL-36γ-induced IL-23 required Notch2-dependent (CD11b⁺CD103⁺) dendritic cells (DCs), but not Batf3-dependent (CD11b^-CD103⁺) DCs or CSF1R-dependent macrophages. The intracellular signaling cascade linking IL-36 receptor (IL-36R) to IL-23 production by DCs involved MyD88 and the NF-κB subunits c-Rel and p50. Consistent with in vitro observations, IL-36R- and IL-36γ-deficient mice exhibited dramatically reduced IL-23, IL-22, and AMP levels, and consequently failed to recover from acute intestinal damage. Interestingly, impaired recovery of mice deficient in IL-36R or IL-36γ could be rescued by treatment with exogenous IL-23. This recovery was accompanied by a restoration of IL-22 and AMP expression in the colon. Collectively, these data define a cytokine network involving IL-36γ, IL-23, and IL-22 that is activated in response to intestinal barrier damage and involved in providing critical host defense.

Keywords: inflammatory bowel disease; innate immunity; interleukin; repair.

Fig. 1.

IL-36R deficiency results in impaired IL-23 and IL-22 expression in the colons of DSS-treated mice. (A) PCR array gene expression analyses from colon tissues of Il1rl2^+/+ and Il1rl2^−/− mice treated with DSS for 5 d. The time course of IL-23 mRNA (B) and protein (C) expression in colons from Il1rl2^+/+ and Il1rl2^−/− mice treated with DSS is shown. The time course of IL-22 mRNA (D) and protein (E) expression in colons from Il1rl2^+/+ and Il1rl2^−/− mice treated with DSS is shown. Data are representative of three independent experiments with three to four mice per group. All data are presented as mean ± SEM. *P < 0.5; **P < 0.05; ***P < 0.001.

Fig. 2.

IL-36γ–induced IL-22 production in colonic explants from DSS-treated mice is IL-23–dependent. (A and B) Colonic explants from control (no DSS) or 3-d DSS-treated (3 d DSS) WT mice were cultured for 60 h in the presence or absence of IL-36γ. Supernatants were analyzed for IL-23 (A) and IL-22 (B) by ELISA. (C) Colonic explants from 3-d DSS-treated WT mice were stimulated with IL-36γ and αp19 or αp40 antibodies for 60 h, and IL-22 expression was assessed by ELISA. (D) Colonic explants from 3-d DSS-treated WT (Il12b^+/+) or Il12b^−/− mice stimulated with IL-36γ or IL-23 for 60 h and IL-22 expression were assessed by ELISA. Data are representative of two independent experiments with four to five mice per group. All data are presented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test: **P < 0.01; ***P < 0.001). n.d., not detected; n.s., not significant; stim, stimulation.

Fig. 3.

Notch2-dependent DCs are required for IL-36γ–induced IL-23 and IL-22 expression and recovery from colonic damage. (A) IL-36R (Il1rl2) mRNA expression was analyzed by qPCR in colon tissue isolated from DSS-treated batf3^+/+, batf3^−/−, Notch2^fl/fl, and Notch2^cKO mice directly ex vivo. (B and C) Colonic explants from DSS-treated mice were cultured for 60 h in the presence or absence of IL-36γ or IL-23. Supernatants were analyzed for IL-23 (B) and IL-22 (C) expression by ELISA. (D) DAI of batf3^+/+, batf3^−/−, Notch2^fl/fl, and Notch2^cKO mice treated with DSS for 5 d, followed by normal water. (E) Image and colon length from mice treated as in D, at day 14. (Scale bar, 1 cm.) The expression of IL-23 (F) and IL-22 (G) in colon tissues from DSS mice at day 5 is shown. Data are representative of two independent experiments with three to four mice per group. All data are presented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test: *P < 0.5; **P < 0.05; ***P < 0.001). n.s., not significant.

Fig. 4.

IL-36γ induces IL-23 via signaling through c-Rel and NF-κBp50. (A) BMDCs were generated from c-rel^+/+ and c-rel^−/− mice and cultured in the presence or absence of IL-36γ for 24 h, and IL-23 was assessed by ELISA. (B and C) WT BMDCs were cultured in the presence or absence of IL-36γ for 24 h, and IL-23 was assessed by ELISA. (B) Some cultures were pretreated with the c-Rel inhibitor (IT-603) or with vehicle control (DMSO) for 1 h. (C) Some cultures were pretreated with p50 or p65 inhibitor peptides or control peptides for 1 h. (D) BMDCs were generated from p50^+/+ and p50^−/− mice and cultured in the presence or absence of IL-36γ for 24 h, and IL-23 was assessed by ELISA. (E) ChIP assays for p50 and c-Rel binding to the p19 promoter in BMDCs treated with IL-36γ for 8 h. Data in A–D are representative of at least two independent experiments with n = 5 mice. Data in E are the combined data of two independent experiments with three replicates per experiment. All data are presented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test: **P < 0.01; ***P < 0.001). n.s., not significant.

Fig. 5.

Systemic IL-23 administration induces resolution of DSS-induced colonic damage in Il1rl2^−/− mice. (A) DAI of Il1rl2^+/+ and Il1rl2^−/− mice treated with DSS for 5 d, followed by normal water for 7 d, in the presence or absence of IL-23. (B and C) Image and length of colons from mice treated as in A. (Scale bar, 1 cm.) The H&E staining (D) and histology scoring (E) of colon sections from mice treated as in A are shown. (Scale bar, 100 μm.) Data are representative of three independent experiments with four to five mice per group. All data are presented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test: *P < 0.05; **P < 0.01). n.s., not significant.

Fig. 6.

Systemic IL-23 administration induces IL-22 and AMPs and rescues Il1rl2^−/− mice from DSS-induced colonic damage. (A) IL-22 protein expression in colons from Il1rl2^+/+ and Il1rl2^−/− mice treated with DSS for 5 d in the presence or absence of IL-23. S100A8 (B), S100A9 (C), Reg3α (D), Reg3β (E), and Reg3γ (F) mRNA expression is shown in colons isolated from mice as in A. Data are representative of two independent experiments with five to six mice per group. All data are presented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test: **P < 0.01; ***P < 0.001). n.s., not significant.