IL-22 promotes mucin-type O-glycosylation and MATH1+ cell-mediated amelioration of intestinal inflammation

Abstract

The interleukin (IL)-22 cytokine can be protective or inflammatory in the intestine. It is unclear if IL-22 receptor (IL-22Ra1)-mediated protection involves a specific type of intestinal epithelial cell (IEC). By using a range of IEC type-specific Il22Ra1 conditional knockout mice and a dextran sulfate sodium (DSS) colitis model, we demonstrate that IL-22Ra1 signaling in MATH1⁺ cells (goblet and progenitor cells) is essential for maintaining the mucosal barrier and intestinal tissue regeneration. The IL-22Ra1 signaling in IECs promotes mucin core-2 O-glycan extension and induces beta-1,3-galactosyltransferase 5 (B3GALT5) expression in the colon. Adenovirus-mediated expression of B3galt5 is sufficient to rescue Il22Ra1^IEC mice from DSS colitis. Additionally, we observe a reduction in the expression of B3GALT5 and the Tn antigen, which indicates defective mucin O-glycan, in the colon tissue of patients with ulcerative colitis. Lastly, IL-22Ra1 signaling in MATH1⁺ progenitor cells promotes organoid regeneration after DSS injury. Our findings suggest that IL-22-dependent protective responses involve O-glycan modification, proliferation, and differentiation in MATH1⁺ progenitor cells.

Keywords: B3GALT5; CP: Immunology; IL-22; IL-22Ra1; MATH1; O-glycan; STAT3; colitis; epithelium; mucin; regeneration.

Figure 1.. Enhanced susceptibility to DSS-induced colitis of intestinal epithelial cell-specific Il22Ra1 knockout mice is independent of LGR5⁺ ISCs

(A) RT-PCR shows Il22Ra1 expression in the distal colon tissue of dextran sulfate sodium (DSS)-treated Il22Ra1^fl/fl and Il22Ra1^IEC mice. (B–D) Analysis of (B) weight loss, (C) colon length, and (D) H&E staining (50 μm, left) and histological score (right) in the distal colon tissue of Il22Ra1^fl/fl and Il22Ra1^IEC mice after DSS treatment. (E and F) Analysis of (E) weight loss with the start of DSS and (F) colon length in Il22^−/− and control (C57BL/6J) mice. (G) RT-PCR analysis of Lgr5 expression in the distal colon tissue of naive and DSS-treated Il22Ra1^fl/fl and Il22Ra1^IEC mice. (H) Schematic representation of tamoxifen and DSS treatment of Il22Ra1^ISC and littermate control mice. (I) RT-PCR analysis for expression of Il22Ra1 and Reg3g genes in the distal colon tissue of naive Il22Ra1^ISC mice with/without 5 days of tamoxifen injection. (J–N) Analysis of (J) weight loss, (K) colon length, (L) H&E staining (50 μm), (M) H&E scoring, and (N) RT-PCR of inflammatory cytokine genes (Il17a, Tnfa, and Il6) and mKi67 expression in distal colon tissue of DSS-treated Il22Ra1^fl/fl and Il22Ra1^ISC mice. (O) RT-PCR analysis of mKi67 expression in the distal colon tissue of naive and DSS-treated Il22Ra1^fl/fl and Il22Ra1^IEC mice. (A)–(C), (G), (J), (K), (N), and (O) are generated from at least 2 independent experiments. (D) and (L) are representative of at least 3 mice per group. Data are presented as mean ± SEM in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-way ANOVA or Mann-Whitney test, two-tailed).

Figure 2.. Dispensable role of Paneth cell-specific (Il22Ra1^Paneth) IL-22Ra1 signaling in DSS-induced colitis

(A and B) Representative image showing immunofluorescence LYZ1-stained cells (left, 50 μm) and their numbers (right) (A) and RT-PCR data for Lyz1 expression (B) in the terminal ileum of DSS-treated Il22Ra1^fl/fl and Il22Ra1^IEC mice. (C and D) Representative image of immunofluorescence LYZ1-stained cells (left, 50 μm) and their numbers (right) (C) and RT-PCR analysis of Lyz1 expression (D) in terminal ileum of DSS-treated Il22Ra1^fl/fl and Il22Ra1^Paneth mice. (E–H) Analysis of (E) weight loss, (F) colon length, (G) H&E staining (left, 50 μm) and scoring (right), and (H) RT-PCR for expression of inflammatory cytokine genes (Il17a, Tnfa, and Il6) in the distal colon tissue of naive and DSS-treated Il22Ra1^fl/fl and Il22Ra1^Paneth mice. (E) and (F) are generated from 3 independent experiments with at least 5 mice per group. (A), (C), and (G) are representative of at least 3 mice per group. Data are presented as mean ± SEM in the graphs. *p < 0.05 (two-way ANOVA or Mann-Whitney test, two-tailed).

Figure 3.. Defect in mucin-type O-glycan and glycosyltransferase expression in Il22Ra1^IEC mice

(A) RT-PCR analysis of expression of various glycosyltransferase enzymes in the distal colon tissue of DSS-treated Il22Ra1^fl/fl and Il22Ra1^IEC mice. (B) Heatmap analysis showing the intensity of various O-glycan core structures (based on m/z ratio) in colon mucin by MALDI-TOF of naive Il22Ra1^fl/fl and Il22Ra1^IEC mice. (C) Heatmap analysis of RNA sequencing data of glycosyltransferase expression in small intestinal organoids of C57BL/6J mice treated with or without rIL-22 (100 ng/mL). (D) RT-PCR analysis of glycosyltransferase expression in colon organoids of Il22Ra1^fl/fl and Il22Ra1^IEC mice with/without IL-22.Fc (100 ng/mL). (E) RT-PCR analysis of glycosyltransferase expression in the distal colon tissue of DSS-treated MyD88 ^fl/fl and MyD88^IEC mice. (A), (D), and (E) are generated from 2–3 independent experiments with at least 4 mice per group. Data are presented as mean ± SEM in the graphs. *p < 0.05, **p < 0.01, and ****p < 0.0001 (Mann-Whitney test, two tailed).

Figure 4.. Administration of Ad-B3galt5 vector protects Il22Ra1^IEC mice from DSS-induced colitis

(A and B) RT-PCR analysis of B3galt5 expression in (A) HEK-293 cell line at different multiplicities of infection and (B) C57BL/6J mice colon organoids transfected with either Ad-B3galt5 or Ad-Y5 (control) vector. (C) Representative image of immunofluorescence adenovirus capsid (green), CLCA1 (red), and DAPI (blue) in colon tissue of Il22Ra1^IEC mice after 5 days of Ad-B3galt5 i.p. injection (20 μm). (D) Representative image of immunofluorescence GFP (green), UEA I (red), and DAPI (blue)-stained colon organoids of C57BL/6J mice transfected with Ad-EGFP or Ad-Y5 vector (50 μm). (E) Schematic representation of Ad-B3galt5/Ad-Y5 and DSS treatment of Il22Ra1^fl/fl and Il22Ra1^IEC mice. (F–H) Analysis of (F) weight loss, (G) colon length, and (H) H&E staining (left, 50 μm) and scoring (right) of distal colon tissue of Il22Ra1^fl/fl and Il22Ra1^IEC mice after adenovirus and DSS treatment. (C) and (D) are representative of 1 independent experiment with at least 2 mice in each group. (F) is representative of 3 independent experiments with at least 5 mice in each group. Data are presented as mean ± SEM in the graphs. *p < 0.05, **p < 0.01, and ****p < 0.0001 (two-way ANOVA or Mann-Whitney test, two-tailed).

Figure 5.. IL-22Ra1 signaling in MATH1⁺ cells plays a significant role in protecting from DSS colitis

(A) Schematic representation of RU-486 and DSS treatment of Il22Ra1^fl/fl and Il22Ra1^Math1-PGR mice. (B–F) Analysis of (B) goblet cell-specific IL-22-inducible gene (Retlnb), (C) weight loss, (D) colon length, (E) H&E staining (left, 50 μm) and scoring (right), and (F) RT-PCR analysis of inflammatory cytokine genes (Il17a, Il6) in the distal colon tissue of DSS-treated Il22Ra1^fl/fl and Il22Ra1^Math1-PGR mice. (G) Representative image of immunohistochemically stained Tn antigen (brown) and Alcian blue (left panel) and their count (right) in the distal colon tissue of DSS-treated Il22Ra1^fl/fl and Il22Ra1^Math1-PGR mice (20 μm). (B)–(G) are representative of 4 independent experiments with at least 4 mice per group. Data are presented as mean ± SEM in the graphs. *p < 0.05 and **p < 0.01 (two-way ANOVA or Mann-Whitney test, two-tailed).

Figure 6.. IL-22 induces B3GALT5 expression in the human colon cell line and patients with UC express immature type O-glycan in colon tissue

(A) Transcriptome analysis (GEO: GSE9452) for expression of glycosyltransferase genes in colon tissue of controls (n = 7), unaffected (non-inflamed) tissue (n = 9), or affected (inflamed) tissue (n = 8) of patients with UC. (B) Transcriptome analysis for expression of glycosyltransferase genes in rectal tissue of controls (n = 20) and patients with UC (n = 206). (C) RT-PCR analysis of B3galt5 and Fut2 genes upon stimulation of HT-29 cell line with human rIL-22 (50 ng/mL). (D) Representative images (3 individual patients) of immunohistochemically stained Tn antigen (brown) and nuclei stained with hematoxylin (left) and their percentage (right) in the colon tissue of diverticulitis (control, unaffected region) and patients with UC (affected region) (50 μm). (C) is representative of 2 independent experiments in triplicate. (D) comprise 5 tissue samples in each group. Data are presented as mean ± SEM in the graphs. *p < 0.05 and **p < 0.01 (Mann-Whitney test, two-tailed, Student’s t test in D).

Figure 7.. IL-22-STAT3 axis induces B3galt5 expression and IL-22Ra1 signaling in MATH1⁺ progenitor cell induces intestinal epithelial regeneration after inflammation

(A) Putative STAT3 binding site analysis by JASPAR in the promoter region of mouse and human B3galt5 gene. (B) Chromatin immunoprecipitation assay in human rIL-22 (50 ng/mL, 24 h)-treated HT-29 cells with anti-pSTAT3 antibody. The pSTAT3-binding DNA was utilized for qPCR to determine the pSTAT3 binding site in the promoter region of the B3GALT5 gene. (C) RT-PCR analysis of B3galt5 gene expression in C57BL/6J mice colon organoids treated with/without mouse IL-22.Fc (50 ng/mL) and/or S3I-201 (STAT3 inhibitor, 10 μM). (D) Schematic representation of colon organoid treatment of Math1-PGR lineage tracer mice. (E) Image of IL-22.Fc- (10 ng/mL, 48 h) and RU-486-treated colon organoids of Math1-PGR lineage tracer mice (150 μm). (F) DSS ± IL-22.Fc (10 ng/mL)-treated colon organoid of Math1-PGR lineage tracer mice on day 9 post-passage with the organoid area on the right (150 μm). Green organoids represent MATH1⁺ progenitor cell-mediated regeneration after DSS treatment. (B) and (C) are representative of 2 independent experiments. Data are presented as mean ± SEM in the graphs. *p < 0.05 (Mann-Whitney test, two-tailed, paired Student’s t test in B). (E) and (F) are representative of 2 independent experiments with 5 mice in each group. Data are presented as mean ± SEM. ****p < 0.0001 (Mann-Whitney test, two-tailed).