Intestinal epithelial cells express Chi3l1 induced by gut microbiota.

(A) IHC staining to detect Chi3l1 in both ileum and colon from germ-free and wildtype mice. Ctrl (wildtype mice without application of first antibody), WT (wildtype C57B/6J mice). Red arrows indicate Chi3l1-expressing cells. Scale bar, 50μm (Ctrl, Germ-free, WT) and 20μm(zoom). The number of Chi3l1-positive cells in each field of view (FOV) was analyzed. (B) Ileum and colon were collected from wildtype mice and stained with ChgA (green), Chi3l1(red), and nuclear DAPI (blue) in ileum and UEA-1 (green), Chi3l1(red), and nuclear DAPI (blue) in colon. Scale bar, 20μm. Ctrl (without application of first antibody), WT (wildtype C57B/6J mice). (C) Western blot to detect Chi3l1 protein expression in DLD-1 cells after bacteria mix infection for 12 hour. Bacteria mix are total bacteria extracted from feces of wildtype mice. (D) Western blot to detect Chi3l1 protein expression in DLD-1 cells after Staphylococcus Sciuri and E. coli infection for 12 hour. Staphylococcus Sciuri and E. coli are isolated from bacteria mix and verified by 16S rRNA sequencing. Three independent experimental results are showed. (E) Western blot to detect Chi3l1 protein expression in DLD-1 cells after treatment with heat-killed E. coli for 12 hour. Three independent experimental results are showed. (F) Western blot to detect Chi3l1 protein expression in DLD-1 cells after treatment with 100 pg/ml LPS for 12 hour. Three independent experimental results are showed. (G) Immunofluorescence to detect Chi3l1 protein expression in DLD-1 cells after treatment with 100pg/ml LPS for 12 hour. Scale bar, 20μm. The presence of cells in the untreated sample are annotated using white dashed lines based on the overexposure. All data above represent at least three independent experiments. Representative images are shown in A,B, n=3-4 mice/group.

Chi3l1 interact with bacteria via peptidoglycan.

(A) Structural comparison between Chitin and peptidoglycan. Both chitin and peptidoglycan contain N-acetylglucosamine (GlcNAc) and have β-1,4-glycosidic bonds in their structures. However, Chitin is purely a polysaccharide, while peptidoglycan includes a peptide component that forms cross-links between chains. [30] (B) Gram-positive bacteria (E.faecalis, S.saprophyticus) and Gram-negative bacteria (E.coli) were incubated with 1μg of recombinant mouse Chi3l1 protein(rmChi3l1), respectively. Proteins bound to indicated bacteria were precipitated by centrifugation. Western blot was used to detect rmChi3l1 in Pellet, Supernatant (unbound proteins) and Last Wash (last wash unbound proteins). (C) Insoluble Peptidoglycan (PGN) were incubated with either recombinant mouse Chi3l1 protein (rmChi3l1) or Bovine serum albumin (BSA). Proteins bound to PGN were precipitated by centrifugation. Silver staining was used to detect rmChi3l1 in Input, Supernatant (unbound proteins), Pellet and Last Wash (last wash unbound proteins). (D) Insoluble Peptidoglycan (PGN) were incubated with recombinant human Chi3l1 protein (rhChi3l1). Proteins bound to PGN were precipitated by centrifugation. Silver staining was used to detect rhChi3l1 in Input, Supernatant (unbound proteins), Pellet and Last Wash (last wash unbound proteins). All data above represent at least three independent experiments. (E) Insoluble PGN or chitin was incubated with rmChi3l1. Chi3l1 bound to PGN (upper panel) and chitin (lower panel) was precipitated and detected by silver staining. The supernatant represents the last wash, and the pellet contains proteins precipitated by either PGN or chitin. (F) Relative DLD-1 bacterial binding preference after treatment with K12 or GlmM, a PGN synthesis-deficient mutant. Colony-forming units (CFU) were counted, and GlmM CFU were normalized to 1. (G) Relative K12 bacterial adhesion preference after DLD-1 cells were transfected without (Mock), or with scramble shRNA (shCK), or with shChil1. CFU were counted, and the Mock group were normalized to 1.

Intestinal bacteria are disordered in IECΔChil1 mice, especially Gram-positive bacteria.

(A, C, D, G) Female Villin-cre and IECΔChil1 littermates continue to cage together after weaning for 8 weeks. Microbial communities in feces and intestinal lumen were characterized by 16S rRNA sequencing. n=7 or 10/group. (A) Alpha diversity analysis of colon contents between Villin-cre and IECΔChil1 littermates. (B) qPCR analysis of total bacteria in the feces and ileum, colon luminal microbial communities of Villin-cre and IECΔChil1 littermates. Values for each bacterial group are expressed relative to total 16S rRNA levels. Mean ± SEM is displayed. Two-tailed, unpaired student t-test was performed. *P<0.05, ns, not significant. n=5-10/group. (C) Principal Component Analysis of weighed UniFrac distances of 16S community profiles of Villin-cre and IECΔChil1 littermates feces (binary-jaccard). (D) Relative abundance of Gram-positive and Gram-negative bacteria in colon contents of Villin-cre and IECΔChil1 littermates are shown. (E) LTA (green) was detected by immunofluorescence in colon sections of Villin-cre and IECΔChil1 littermates. Nuclei were detected with DAPI. Scale bar, 50μm. The average fluorescence intensity in each field of view (FOV) was analyzed. (F) Fluorescence in situ hybridization (FISH) detection of gram-positive bacteria (red) in the colon of Villin-cre and IECΔChil1 littermates, nuclei were detected with DAPI (blue). Scale bars, 50um. The average fluorescence intensity in each field of view (FOV) was analyzed. (G) Relative abundance of Gram-positive bacteria genera in colon lumen of Villin-cre and IECΔChil1 littermates. (H) Female wildtype and Chil1-/- littermates continue to cage together after weaning for 8 weeks. Microbial communities in feces were characterized by 16S rRNA sequencing. Mean ± SEM is displayed. Two-tailed, unpaired student t-test was performed. ****P<0.0001, ns, not significant. n=4 or 6/group. Representative images are shown in E, F, n= 3/4 mice/group.

Chi3l1 promotes the colonization of Gram-positive bacteria in intestinal mucus layer.

(A) IHC staining to detect Chi3l1 in colon mucus layer from wildtype mice. Ctrl (without application of ant-Chi3l1 antibody), WT (wildtype C57B/6J mice). Black dotted line outlines mucus layer. Scale bar, 50μm (Ctrl, WT). (B) Colons were collected from wildtype mice and stained with UEA-1 (green), Chi3l1(red), and nuclear DAPI (blue). Ctrl (without application of first antibody), WT (wildtype C57B/6J mice). Scale bars, 50um (Ctrl, WT) and 20μm(zoom). (C) Stool, ileum and colon tissues were collected from wildtype mice. Western blot was used to detect Chi3l1 expression in these samples. n=3 mice/sample. (D) Both luminal and mucus-associated proteins of either ileum or colon were extracted. Western blot was used to detect Chi3l1 expression in these samples. lumen (luminal proteins), mucus (mucus-associated proteins). n=3 mice/sample. (E&F) qPCR analysis of specific bacteria in the ileum and colon mucus microbial communities of wildtype and Chil1-/- littermates. (E) qPCR analysis of Gram-positive bacteria is shown. (F) qPCR analysis of Gram-positive bacteria is shown. Values for each bacterial group are expressed relative to total 16S rRNA levels. WT (wildtype C57B/6J mice). Mean ± SEM is displayed. Two-tailed, unpaired student t-test was performed. P value is as indicated in E. *P<0.05, **P<0.01, n=6-9/group. (G) Rectal injection of both wildtype and Chi11-/- mice with FDAA-labeled E. faecalis (a Gram-positive bacteria strain) for 4 hour. Colon sections were collected and colonization of E.faecalis was examined under microscope. Nuclei were stained with DAPI. Representative images are shown in A, B, G, n=3-4 mice/group.

Disordered intestinal bacteria in IECΔChil1 mice contribute to IBD.

(A) Chil1 mRNA relative expression in colon tissues of patients without gut disease (controls, n=35) or with Crohn’s disease (CD, n=40), ulcerative colitis (UC, n=40), (GEO Datasets: SRP303290), Mann-Whitney test was performed. *P < 0.05; ***P < 0.001. (B) Schematic model of the experimental design. Both Villin-cre and IECΔChil1 littermates were fed with 2% DSS in drinking water to induce colitis. (C) Weight change of Villin-cre and IECΔChil1 littermates during DSS feeding. Weight change (%) = Current weight/Initial weight. P values are as indicated. (D) Representative colonic length from Normal and DSS-treated Villin-cre and IECΔChil1 littermates (left) and the statistics of colonic length (right) (**P < 0.01; ***P < 0.001; ****P < 0.0001, ns, not significant). (E) H&E staining of mice colon from Normal and DSS-treated Villin-cre and IECΔChil1 littermates. The inflamed areas are outlined by white dotted line, Scale bars=100um. (F) Schematic of the experimental design. First, antibiotics were used to eliminate gut microbiota for 10 days, and then either fecal microbiota from Villin-cre mice (FMT) or Lactobacillus reuteri were transplanted back to IECΔChil1 mice orally every day for 2 weeks. Finally, colitis mouse model was constructed by 2% DSS feeding in drinking water for another 7 days. (G-I) Villin-cre and IECΔChil1 were only fed with 2% DSS in drinking water for 7days. IECΔChil1+FMT(Villin-cre), and IECΔChil1+Lactobacillus were constructed as described in F. (G) Weight change of Villin-cre, IECΔChil1, IECΔChil1+FMT(Villin-cre), and IECΔChil1+Lactobacillus mice during DSS feeding. (H) Representative colonic length from Villin-cre, IECΔChil1, IECΔChil1+FMT(Villin-cre), and IECΔChil1+Lactobacillus mice (left) and the statistics of colonic length (right). Mean ± SEM is displayed in G, H. one-way ANOVA was performed. P value is as indicated. *P<0.05, ns, not significant, n=3-6/group. (I) H&E staining of mice colon from Villin-cre, IECΔChil1, IECΔChil1+FMT(Villin-cre), and IECΔChil1+Lactobacillus mice after DSS treatment. The inflamed area is outlined by black dotted line, Scale bars=100um. Representative images are shown in C, E, H, I, n=3-6 mice/group.

A schematic working model.

Intestinal epithelial cells are stimulated by the gut microbiota to express Chi3l1. Once expressed, Chi3l1 is secreted into the mucus layer where it interacts with the gut microbiota, specifically through a component of bacterial cell walls called peptidoglycan. This interaction between Chi3l1 and bacteria is beneficial for the colonization of bacteria in the mucus, particularly for gram-positive bacteria like Lactobacillus. Moreover, a deficiency of Chi3l1 leads to an imbalance in the gut microbiota, which exacerbates colitis induced by dextran sodium sulfate (DSS).