Proximal colon-derived O-glycosylated mucus encapsulates and modulates the microbiota

Abstract

Colon mucus segregates the intestinal microbiota from host tissues, but how it organizes to function throughout the colon is unclear. In mice, we found that colon mucus consists of two distinct O-glycosylated entities of Muc2: a major form produced by the proximal colon, which encapsulates the fecal material including the microbiota, and a minor form derived from the distal colon, which adheres to the major form. The microbiota directs its own encapsulation by inducing Muc2 production from proximal colon goblet cells. In turn, O-glycans on proximal colon-derived Muc2 modulate the structure and function of the microbiota as well as transcription in the colon mucosa. Our work shows how proximal colon control of mucin production is an important element in the regulation of host-microbiota symbiosis.

Fig. 1.. The proximal colon-derived mucus encapsulates microbiota-containing fecal pellets.

(A) Tiled immunofluorescent image of a WT whole colon coil section (left) with goblet cells highlighted (blue boxed images, right). Prox, Mid, and Dis represents proximal, middle, and distal colon, respectively. (B) Schematic showing different colon regions (left); Muc2⁺MALII^- goblet cell numbers (GCs, middle); and schematic GC Muc2 and MALII expression profile (right). (C) The number of GCs per crypt (left), and Muc2 expression in colon epithelium (right, qPCR normalized to villin). (D) Magnified images of red boxed areas in (A). (E) ddPCR plot of bacterial 16S rRNA gene of samples from WT distal colon with (P) or without pellets (inter-pellet, IP). H₂O, negative control. +, positive control. (F) Methods of analyzing mucus on fecal pellets in tissue or excreted fecal pellet wrapped in peritoneum and their Alcian blue-stained images. (G) Quantification of mucus thickness stained by Alcian blue (median, red; quartiles, black). (H) Z-stacked confocal images of colon cross-sections with pellets or excreted pellets wrapped with peritoneum and corresponding y/z plane (right to each image). Bottom, single-colored images of white-boxed regions. (I) Thickness of the mucus layers of (H). (J) New model of the mucus layers. The niche and the barrier layer are associated with the fecal pellets. The barrier layer consists of the b1 and b2 layers. The proximal colon produces the niche and the b1 layer. The distal colon forms the minor b2 layer. Scale bars, 4 mm (A); 500 μm (F); 20 μm (A insets, D, F inserts, H). n≥ 10 mice, 3 – 5 months old, both sexes. *P < 0.05; **P < 0.01, vs. b2 layer.

Fig. 2.. Proximal colon-derived mucin-type O-glycans govern the composition and function of the mucus barrier.

(A) Diagrams depicting simplified forms of core 1- or core 3-derived O-glycans or Tn antigen (GalNAc) in the different colon regions of mouse lines used for results below. (B) Representative images of colon sections stained with Alcian blue. Arrows, mucus barrier layer. (C) Proportion of Alcian blue⁺ goblet cells in different colon regions (top), and median mucus thickness in distal colons (bottom). (D) Z-stack confocal images of colon sections (top). b1, b2, and niche identify the different sublayers of the mucus. (E) Mean mucus thickness in distal colons (D). (F) Histogram showing frequency distribution of individual bacterial distances to the bottom of the distal mucosa surface represented in (D). (G) Confocal tiling images of cross-sections of fecal pellets stained with the lectin UEA1. Arrows mark the mucus layer. (H) Violin plot of mucus thickness of entire mucus layer surrounding the fecal pellet represented in (G). (I) Blended Z-stack confocal images of proximal colon cross sections. (J) Representative images of H&E-stained colon cross sections. (K) Graph depicts histologic colitis scores. n≥ 5 mice, 3 – 5 months old, both sexes. Scale bars, 20 μm (B, D, and I); 50 μm (J); 500 μm (G). *P < 0.05, as indicated, and vs. b2 layer in (D).

Fig. 3.. The microbiota directs its own encapsulation by inducing Muc2 production in the proximal colon.

(A) Gross picture of colon (top) and Alcian blue-stained mucus and goblet cells in colon sections (bottom) of mice with and without a complex microbiota. Arrows mark mucus barrier layer. (B) Median mucus barrier layer thickness in distal colon sections represented in (A). (C) The formation of the fecal mucus barrier over time in ExGF mice. Images show Alcian blue-stained excreted fecal sections. Inset, magnified image of mucus layer (arrows). (D) Violin plot of mucus thickness over time in (C). (E) Median mucus barrier layer thickness of excreted feces from multiple ExGF mice. (F) Confocal Z-stack images of distal colon (top) and excreted fecal pellet sections (bottom). Arrow, b2 layer. Line, spans thickness of the b1 layer. Right, mean thickness of b1 and b2 layers. (G) Western blot of tissue Muc2 separated by composite AgPAGE (top), and densitometry of Muc2 signal (bottom). GAPDH, loading control. (H) Alcian blue-stained WT colon sections. Arrowheads mark mucus secretion. (I) Confocal Z-stack images of WT colon sections. Arrowheads, secreted mucus. (J) Tiled confocal images of excreted fecal pellet sections from mice pre- or post-inoculation with a complex microbiota. Scale bars, 10 μm (C insets); 20 μm (A, F, H, I); 500 μm (C); 1000 μm (J). *P < 0.05, as indicated in (B and E), vs. D0 in (D), and vs. GF b1 layer in (F).

Fig. 4.. Loss of O-glycans in the proximal colon alters microbiota community structure and the host mucosal transcriptome throughout the colon.

(A) Principal component analysis (PCA) of species-level counts. The first three principal components (PC1, PC2, PC3) and percent of variance explained are shown. n = 5 mice/group. (B) Relative abundance for selected taxa showing highest PC3 loadings in (A). y axis, variance-stabilized data computed from species-level counts (Bracken classifier). (C) Partial Least Squares Discriminant Analysis (PLS-DA) of microbial metabolites. (D) Metabolite correlation networks between control (n = 5) and TM-DKO^Prox (n = 5). (E) Venn diagram of pair-wise correlated metabolites. (F) Gene expression signature showing 53 significantly different genes between pooled control and TM-DKO^Prox mice. For clarity, only Group 1 is shown. Green boxes, maximal enrichment for cell-cycle, proliferation genes (hypergeometric P-value<1e-12). Marked boxes, genes governing epithelial function. Genes are sorted (left to right) by significance (LTR P-value, table S4). Grey gradient highlights P-values in decreasing significance. (G) Barplot showing genes uniquely different in TM-DKO^Prox distal colon. Left (black/white heatmap): Gene class assigned to each gene according to its expression profile in indicated single-cell epithelial subtypes (black boxes, see Methods); Middle: Barplots of gene expression fold changes in each group of mice for both proximal and distal colon; Right: selected top-ranked genes by global fold-change grouped by their molecular function. *P < 0.05, as indicated.