IL-36R signaling integrates innate and adaptive immune-mediated protection against enteropathogenic bacteria

Abstract

Enteropathogenic bacterial infections are a global health issue associated with high mortality, particularly in developing countries. Efficient host protection against enteropathogenic bacterial infection is characterized by coordinated responses between immune and nonimmune cells. In response to infection in mice, innate immune cells are activated to produce interleukin (IL)-23 and IL-22, which promote antimicrobial peptide (AMP) production and bacterial clearance. IL-36 cytokines are proinflammatory IL-1 superfamily members, yet their role in enteropathogenic bacterial infection remains poorly defined. Using the enteric mouse pathogen, C.rodentium, we demonstrate that signaling via IL-36 receptor (IL-36R) orchestrates a crucial innate-adaptive immune link to control bacterial infection. IL-36R-deficient mice (Il1rl2^-/- ) exhibited significant impairment in expression of IL-22 and AMPs, increased intestinal damage, and failed to contain C. rodentium compared to controls. These defects were associated with failure to induce IL-23 and IL-6, two key IL-22 inducers in the early and late phases of infection, respectively. Treatment of Il1rl2^-/- mice with IL-23 during the early phase of C. rodentium infection rescued IL-22 production from group 3 innate lymphoid cells (ILCs), whereas IL-6 administration during the late phase rescued IL-22-mediated production from CD4⁺ T cell, and both treatments protected Il1rl2^-/- mice from uncontained infection. Furthermore, IL-36R-mediated IL-22 production by CD4⁺ T cells was dependent upon NFκB-p65 and IL-6 expression in dendritic cells (DCs), as well as aryl hydrocarbon receptor (AhR) expression by CD4⁺ T cells. Collectively, these data demonstrate that the IL-36 signaling pathway integrates innate and adaptive immunity leading to host defense against enteropathogenic bacterial infection.

Keywords: adaptive immunity; bacterial infection; innate immunity; interleukin.

Fig. 1.

IL-36 signaling controls C. rodentium infection. Il1rl2^+/+ and Il1rl2^−/− mice infected with bioluminescent C. rodentium (5 to 6 × 10⁹ CFU) by gastric gavage. (A) Serial whole-body imaging at indicated time points. Images are representative of two independent experiments with n = 5 mice per experiment (B–D) (B) Survival rate; (C) average body weight changes; and (D) bacterial shedding in feces of infected Il1rl2^+/+ and Il1rl2^−/− mice at indicated time points. Data are representative of three independent experiments with five mice per group. All data presented as mean ± SEM (multiple t tests, one per row, corrected for multiple comparisons using the Holm–Sidak method; *P < 0.5).

Fig. 2.

IL-36R deficiency results in diminished IL-23, IL-6, and IL-22 expression in the large intestine during C. rodentium infection. (A) Experimental schematic of intestinal bacterial infection by oral infected Il1rl2^+/+ and Il1rl2^−/− mice with 5 to 6 × 10⁹ CFU of C. rodentium. (B and C) PCR array gene expression analyses from large intestine tissues isolated at indicated time points (p.i.) of Il1rl2^+/+ and Il1rl2^−/− mice. (D and E) The top 10 significantly expressed genes with highest up-regulation were obtained from PCR array analysis between C. rodentium-infected Il1rl2^+/+ and Il1rl2^−/− mice at day 4 p.i. (D), and day 8 p.i. (E). (F–H) The time course of (F) IL-22, (G) IL-23, and (H) IL-6 protein expression from colonic tissues isolated from infected mice. Data are representative of two independent experiments with four to five mice per group. All data are presented as mean ± SEM (multiple t tests, one per row, corrected for multiple comparisons using the Holm–Sidak method; *P < 0.5).

Fig. 3.

Early IL-23 administration rescues protective immunity in C. rodentium-infected Il1rl2^−/− mice. (A) Experimental schematic of C. rodentium infection (5 to 6 × 10⁹ CFU) by gastric gavage into Il1rl2^+/+ and Il1rl2^−/− mice, in the presence or absence of IL-23. (B) Serial whole-body imaging of infected mice at indicated time points. Images are representative of two independent experiments with at least five mice/group. (C–E) (C) Survival rate; (D) average body weight change; and (E) bacterial shedding in feces of infected mice at indicated time points. (F and G) (F) The H&E staining and histology scoring of (G) colon sections from infected mice as in A are shown. (H) IL-22 protein expression in the colon at 10 days post C. rodentium infection from Il1rl2^+/+ and Il1rl2^−/− mice. (I) Colonic lamina propria cells of C. rodentium-infected mice at day 4 p.i. were isolated and analyzed by FACS for expression of intracellular IL-22 by Thy1⁺RORγt⁺ gated cells. (J) FACS frequency data of Thy1⁺RORγt⁺IL-22⁺ gated cells of infected mice generated as in I. (K–M) (K) Claudin-2, (L) antimicrobial peptides, and (M) Mucin-2 mRNA expression in colon from infected mice at day 4 p.i. (N) Serial whole-body imaging of C. rodentium-infected Il1rl2^−/− mice in the presence or absence of IL-23 and neutralization antibodies, αCD90 or αCD4 as indicated. Images are representative of two independent experiments. (O–P) (O) Survival rate; (P) average body weight changes; and (Q) bacterial shedding in feces of infected mice as in N at indicated time points. (R) IL-22 protein determined by ELISA of colon of infected mice as in N at indicated time points. Data are representative of three independent experiments with five 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 < 0.0001, ns, not significant).

Fig. 4.

IL-36R regulates CD4⁺ T cell IL-22 production via AhR- and IL-6-mediated signaling. (A) DCs and CD4⁺ T cells from spleen of WT mice were FACS sorted and were cultured either alone or cocultured for 72 h in the presence or absence of IL-36γ. Supernatant were analyzed for IL-22 by ELISA. (B) FACS-sorted naïve CD4⁺ T cells and DCs were cocultured using indicated cells from Il1rl2^+/+ and/or Il1rl2^−/− mice with ± IL-36γ for 72 h. IL-22 protein in supernatant was determined by ELISA. (C) IL-22 protein expression by FACS-sorted coculture DCs and naïve CD4⁺ T cells from ahr^+/+ or ahr^-/− mice in the presence of IL-36γ for 72 h. (D) FACS-sorted naïve CD4⁺ T cells and DCs were cocultured using indicated cells from ahr^+/+ and/or ahr^-/− mice with ± IL-36γ for 72 h. IL-22 protein in supernatant was determined by ELISA. (E) FACS-sorted DCs and CD4⁺ T cells from WT mice were cocultured and stimulated with IL-36γ and αIL-6 antibody for 72 h. IL-22 protein was assessed by ELISA. (F) FACS-sorted naïve CD4⁺ T cells and DCs were cocultured using indicated cells from Il1rl2^+/+ and/or Il1rl2^−/− mice with ± IL-36γ for 72 h. IL-6 protein in supernatant was determined by ELISA. Data are representative of three independent experiments with four to five replicates. (G) BMDCs were generated from WT mice and cultured in the presence or absence of IL-36γ for 24 h, and IL-6 was assessed by ELISA; some cultures were pretreated with NFκB inhibitor, or c-Rel inhibitor, or p50 inhibitor, or p65 inhibitor or with vehicle control for 1 h. (H) ChIP assays for p50, p65, and c-Rel binding to Il6 promoter in BMDCs treated with ± IL-36γ for 8 h. Data are representative of three independent experiments with four to five replicates. All data are presented as mean ± SEM (one-way ANOVA with Tukey’s multiple comparison test. **P < 0.01, ***P < 0.001; ****P < 0.0001; ns, not significant).

Fig. 5.

IL-6 administration accelerates bacterial clearance and restores IL-22 production in C. rodentium-infected Il1rl2^−/− mice. (A) Experimental schematic of C. rodentium infection, in the presence or absence of recombinant IL-6 and/or αCD4. (B) Serial whole-body imaging of infected mice at indicated time points. Images are representative of two independent experiments with at least five mice/group. (C–E) (C) Survival rate; (D) average body weight changes; and (E) bacterial shedding in feces of infected mice at indicated time points. (F and G) (F) H&E staining, and (G) histology scoring of colon sections from infected mice as in A are shown. (H) IL-22 protein expression in colons from infected mice at day 10 p.i. (I) Colonic lamina propria cells of C. rodentium-infected mice as shown in A were isolated on day 10 p.i. and analyzed by FACS for expression of intracellular IL-22 by TCRβ⁺CD4⁺ gated cells. (J) FACS frequency data from TCRβ⁺CD4⁺IL-22⁺ gated cells of C. rodentium-infected mice generated based on I. (K–M) (K) Claudin-2, (L) antimicrobial peptides, and (M) Mucin-2 mRNA expression in colon from infected mice at day 10 p.i. Data are representative of three independent experiments with five 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; ns, not significant).