Mucin O-glycans facilitate symbiosynthesis to maintain gut immune homeostasis

Abstract

Background: The dysbiosis of gut microbiota has been implicated in the pathogenesis of inflammatory bowel diseases; however, the underlying mechanisms have not yet been elucidated. Heavily glycosylated mucin establishes a first-line barrier against pathogens and serves as a niche for microbial growth.

Methods: To elucidate relationships among dysbiosis, abnormal mucin utilisation, and microbial metabolic dysfunction, we analysed short-chain fatty acids (SCFAs) and mucin components in stool samples of 40 healthy subjects, 49 ulcerative colitis (UC) patients, and 44 Crohn's disease (CD) patients from Japan.

Findings: Levels of n-butyrate were significantly lower in stools of both CD and UC patients than in stools of healthy subjects. Correlation analysis identified seven bacterial species positively correlated with n-butyrate levels; the major n-butyrate producer, Faecalibacterium prausnitzii, was particularly underrepresented in CD patients, but not in UC patients. In UC patients, there were inverse correlations between mucin O-glycan levels and the production of SCFAs, such as n-butyrate, suggesting that mucin O-glycans serve as an endogenous fermentation substrate for n-butyrate production. Indeed, mucin-fed rodents exhibited enhanced n-butyrate production, leading to the expansion of RORgt⁺Treg cells and IgA-producing cells in colonic lamina propria. Microbial utilisation of mucin-associated O-glycans was significantly reduced in n-butyrate-deficient UC patients.

Interpretation: Mucin O-glycans facilitate symbiosynthesis of n-butyrate by gut microbiota. Abnormal mucin utilisation may lead to reduced n-butyrate production in UC patients. FUND: Japan Society for the Promotion of Science, Health Labour Sciences Research Grant, AMED-Crest, AMED, Yakult Foundation, Keio Gijuku Academic Development Funds, The Aashi Grass Foundation, and The Canon Foundation.

Keywords: Butyrate; Inflammatory bowel disease; Microbiota; Mucin.

Fig. 1

Faecal organic acid concentrations in inflammatory bowel disease patients and healthy subjects. Faecal organic acid concentrations were analysed in healthy subjects, patients with active (UC-A) or remissive (UC-R) ulcerative colitis (UC), and patients with active (CD-A) or remissive (CD-R) Crohn's disease (CD). Data represent the mean ± standard deviation (n = 12–43/group). ^⁎p < 0·05, ^⁎⁎p < 0·01, and ^⁎⁎⁎p < 0·001 (analysis of variance followed by Tukey's multiple comparison test or the Kruskal–Wallis test followed by Dunn's multiple comparison test).

Fig. 2

Microbial signatures of inflammatory bowel disease patients and healthy subjects. (A) Principal coordinate analysis of unweighted UniFrac distances among the microbiota of inflammatory bowel disease (IBD) patients and healthy subjects. (B) Species richness (Chao 1) of the microbiota of IBD patients and healthy subjects. (C, D) Discriminating taxa between the microbiota of (C) Crohn's disease (CD) or (D) ulcerative colitis (UC) patients and healthy subjects, as determined by LEfSe [38] analysis. Discriminating taxa for the indicated groups are annotated on phylogenetic trees. (E) Venn diagram showing criteria for butyrate-associated bacteria. (F, G) Relative abundances of butyrate-associated bacteria at the genus (F) and species (G) levels. The left panels represent the total abundances of butyrate-associated bacteria, and the right panels indicate the abundances of individual bacterial genera or species. Each boxplot represents the median, interquartile range (IQR), and the lowest and highest values within 1·5 IQRs of the first and third quartiles (n = 7–23/group). Outliers are not shown. Different letters over boxplots indicate significant differences (p < 0·05; Kruskal–Wallis test followed by Dunn's multiple test).

Fig. 3

Stool mucin components of inflammatory bowel disease patients and healthy subjects. (A, B) Levels of (A) mucin and (B) mucin-associated protein and O-glycan were analysed in the stools of inflammatory bowel disease (IBD) patients and healthy subjects. Data represent the mean ± standard deviation (n = 5–38/group). *p < 0·05 and **p < 0·01 (analysis of variance followed by Tukey's multiple comparison test or the Kruskal–Wallis test followed by Dunn's multiple comparison test). (C) Correlation network of the levels of short-chain fatty acids (SCFAs), mucin components, and bacteria in stool samples. Nodes represent SCFAs, mucin components, or bacterial genera with average levels higher than 1·0%. Node colours reflect average levels in ulcerative colitis (UC) patients relative to those in healthy subjects. Red and blue lines indicate positive (Spearman's correlation coefficient > 0·3) and negative (correlation coefficient < −0·3) correlations, respectively. Edge thickness represents the strength of the correlation. Line colour intensity reflects the extent of the correlation. (D) Scatter density plots of faecal n-butyrate levels versus mucin O-glycan levels for individual disease groups. Increasing intensity of each colour (white to black, blue, or red) reflects scatter plot density. Spearman coefficients (rho) and FDRs are shown. (E) UC and Crohn's disease (CD) patients were classified tinto two groups each, based on faecal n-butyrate detection, to compare faecal levels of mucin O-glycans. Patients with n-butyrate concentrations below 0·1 μmol/g were classified as ‘-’, while the remaining patients were classified as ‘+’. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Stool mucinase activity in ulcerative colitis patients and healthy subjects. (A) Stool samples of ulcerative colitis (UC) patients and healthy subjects were analysed for mucinase activity. Boxplots represent the median, interquartile range (IQR), and the lowest and highest values within 1·5 IQRs of the first and third quartiles (n = 10–11/group). (B, C) Scatter plots of mucinase activity and (B) total short-chain fatty acids (SCFAs) and (C) n-butyrate concentrations. Spearman's correlation coefficient (rho) and false discovery rate (FDR) are shown. Box plots represent stool concentrations of total SCFAs (B) and butyrate (C) in the two groups (n = 6–10/group). ^⁎p < 0·05 and ^⁎⁎p < 0·01 (Wilcoxon rank-sum test).

Fig. 5

Effects of mucin supplementation on short-chain fatty acid production by gut microbiota. (A) Rat caecal contents were cultured in an in vitro fermentation system in the presence or absence of porcine gastric mucin and short-chain fatty acid (SCFA) concentrations were measured at the indicated time points. Data are representative of two independent experiments with similar results. (B–D) Caecal SCFA levels (B), O-glycan levels (C), and mucinase activity (D) in the stools of rats fed a control diet or diets containing 0·3% or 0·9% porcine stomach mucin. Data represent the mean ± standard deviation (n = 5–6/group). ^⁎p < 0·05 and ^⁎⁎p < 0·01 (analysis of variance followed by Dunnett's multiple comparison test or the Kruskal–Wallis test followed by Dunn's multiple comparison test, compared with the control group).

Fig. 6

Immune modulation by a mucin-containing diet. (A–E) Caecal SCFA levels (A), frequencies of Foxp3⁺RORγt⁺ cells and Foxp3⁺RORγt⁻ cells within CD4⁺ T cells (B and C), and frequencies of IgA⁺B220⁻ cells within CD45⁺ cells (D and E) in the colonic mucosa of mice fed a control diet or a diet containing 1·5% porcine stomach mucin for 3 weeks. Representative flow cytometry plots are shown in B and D. Data represent the mean ± standard deviation (n = 5/group). ^⁎p < 0·05, ^⁎⁎p < 0·01, and ^⁎⁎⁎p < 0·001 (two-tailed Student's t-test).