Malin E. V. Johansson

Regulated host–microbe interactions are a critical aspect of lifelong health. Colonic goblet cells protect from microorganisms via the generation of a mucus barrier structure. Bacteria-sensing sentinel goblet cells provide secondary protection by orchestrating mucus secretion when microbes breach the mucus barrier. Mucus deficiencies in germ-free mice implicate a role for the microbiota in programming barrier generation, but its natural ontogeny remains undefined. We now investigate the mucus barrier and sentinel goblet cell development in relation to postnatal colonization. Combined in vivo and ex vivo analyses demonstrate rapid and sequential microbiota-dependent development of these primary and secondary goblet cell protective functions, with dynamic changes in mucus processing dependent on innate immune signaling via MyD88 and development of functional sentinel goblet cells dependent on the NADPH/dual oxidase family member Duox2. Our findings identify new mechanisms of microbiota–goblet cell regulatory interaction and highlight the critical importance of the pre-weaning period for the normal development of protective systems that are key legislators of host–microbiota interaction.

Mucus in the colon is crucial for intestinal homeostasis by forming a barrier that separates microbes from the epithelium. This is achieved by the structural arrangement of the major mucus proteins, such as MUC2 and FCGBP, both of which are comprised of several von Willebrand D domains (vWD) and assemblies. Numerous disulfide bonds stabilise these domains, and intermolecular bonds generate multimers of MUC2. The oligomeric nature of FCGBP is not known. Human hFCGBP contains 13 vWD domains whereas mouse mFCGBP consists of only 7. We found unpaired cysteines in the vWD1 (human and mouse) and vWD5 (mouse)/vWD11 (human) assemblies which were not involved in disulfide bonds. However, the most C‐terminal vWD domains, vWD7 (mouse)/vWD13 (human), formed disulfide‐linked dimers. The intermolecular bond between C₅₂₈₄ and C₅₄₀₃ of human hFCGBP was observed by using mass spectrometry to generate the dimer. Cryo‐EM structure analysis of recombinant mouse mFCGBP revealed a compact dimer with two symmetric intermolecular disulfide bonds between C₂₄₆₂ and C₂₅₈₁, corresponding to the dimerising cysteines in the human hFCGBP. This compact conformation involves interactions between the vWD assemblies, but although the domains involved at the interface are the same, the nature of the interactions differ. Mouse mFCGBP was also found to exist in a semi‐extended conformation. These different interactions offer insights into the dynamic nature of the FCGBP homodimer.

Objective: To decipher the mechanisms by which the major human milk oligosaccharide (HMO), 2’-fucosyllactose (2’FL), can affect body weight and fat mass gain on high-fat diet (HFD) feeding in mice. We wanted to elucidate whether 2’FL metabolic effects are linked with changes in intestinal mucus production and secretion, mucin glycosylation and degradation, as well as with the modulation of the gut microbiota, faecal proteome and endocannabinoid (eCB) system. Results: 2’FL supplementation reduced HFD-induced obesity and glucose intolerance. These effects were accompanied by several changes in the intestinal mucus layer, including mucus production and composition, and gene expression of secreted and transmembrane mucins, glycosyltransferases and genes involved in mucus secretion. In addition, 2’FL increased bacterial glycosyl hydrolases involved in mucin glycan degradation. These changes were linked to a significant increase and predominance of bacterial genera Akkermansia and Bacteroides , different faecal proteome profile (with an upregulation of proteins involved in carbon, amino acids and fat metabolism and a downregulation of proteins involved in protein digestion and absorption) and, finally, to changes in the eCB system. We also investigated faecal proteomes from lean and obese humans and found similar changes observed comparing lean and obese mice. Conclusion: Our results show that the HMO 2’FL influences host metabolism by modulating the mucus layer, gut microbiota and eCB system and propose the mucus layer as a new potential target for the prevention of obesity and related disorders.

The MUC2 mucin polymer is the main building unit of the intestinal mucus layers separating intestinal microbiota from the host epithelium. The MUC2 mucin is a large glycoprotein with a C-terminal domain similar to the MUC5AC and MUC5B mucins and the von Willebrand factor (VWF). A structural model of the C-terminal part of MUC2, MUC2-C, was generated by combining Cryo-electron microscopy, AlphaFold prediction, information of its glycosylation, and small angle X-ray scattering information. The globular VWD4 assembly in the N-terminal of MUC2-C is followed by 3.5 linear VWC domains that form an extended flexible structure before the C-terminal cystine-knot. All gel-forming mucins and VWF form tail-tail disulfide-bonded dimers in their C-terminal cystine-knot domain, but interestingly the MUC2 mucin has an extra stabilizing disulfide bond on the N-terminal side of the VWD4 domain, likely essential for a stable intestinal mucus barrier.

Goblet cells in the small intestinal crypts contain large numbers of mucin granules that are rapidly discharged to clean bacteria from the crypt. Because acetylcholine released by neuronal and nonneuronal cells controls many aspects of intestinal epithelial function, we used tissue explants and organoids to investigate the response of the small intestinal crypt to cholinergic stimulation. The activation of muscarinic acetylcholine receptors initiated a coordinated and rapid emptying of crypt goblet cells that flushed the crypt contents into the intestinal lumen. Cholinergic stimulation induced an expansion of the granule contents followed by intracellular rupture of the mucin granules. The mucus expanded intracellularly before the rupture of the goblet cell apical membrane and continued to expand after its release into the crypt lumen. The goblet cells recovered from membrane rupture and replenished their stores of mucin granules. Mucus secretion from the goblet cells depended on Ca ²⁺ signaling and the expansion of the mucus in the crypt depended on gap junctions and on ion and water transport by enterocytes adjacent to the goblet cells. This distinctive mode of mucus secretion, which we refer to as “expanding secretion,” efficiently cleans the small intestine crypt through coordinated mucus, ion, and fluid secretion by goblet cells and enterocytes.

The intestinal tract faces numerous challenges that require several layers of defence. The tight epithelium forms a physical barrier that is further protected by a mucus layer, which provides various site-specific protective functions. Mucus is produced by goblet cells, and as a result of single-cell RNA sequencing identifying novel goblet cell subpopulations, our understanding of their various contributions to intestinal homeostasis has improved. Goblet cells not only produce mucus but also are intimately linked to the immune system. Mucus and goblet cell development is tightly regulated during early life and synchronized with microbial colonization. Dysregulation of the developing mucus systems and goblet cells has been associated with infectious and inflammatory conditions and predisposition to chronic disease later in life. Dysfunctional mucus and altered goblet cell profiles are associated with inflammatory conditions in which some mucus system impairments precede inflammation, indicating a role in pathogenesis. In this Review, we present an overview of the current understanding of the role of goblet cells and the mucus layer in maintaining intestinal health during steady-state and how alterations to these systems contribute to inflammatory and infectious disease.

The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.

Dietary fiber is considered a strong intestinal protector, but we do not know whether dietary fiber protects against the long-lasting mucosal damage caused by ionizing radiation. To evaluate whether a fiber-rich diet can ameliorate the long-lasting pathophysiological hallmarks of the irradiated mucosa, C57BL/6J mice on a fiber-rich bioprocessed oat bran diet or a fiber-free diet received 32 Gray in four fractions to the distal colorectum using a linear accelerator and continued on the diets for one, six or 18 weeks. We quantified degenerating crypts, crypt fission, cell proliferation, crypt survival, macrophage density and bacterial infiltration. Crypt loss through crypt degeneration only occurred in the irradiated mice. Initially, it was most frequent in the fiber-deprived group but declined to levels similar to the fiber-consuming group by 18 weeks. The fiber-consuming group had a fast response to irradiation, with crypt fission for growth or healing peaking already at one week post-irradiation, while crypt fission in the fiber-deprived group peaked at six weeks. A fiber-rich diet allowed for a more intense crypt cell proliferation, but the recovery of crypts was eventually lost by 18 weeks. Bacterial infiltration was a late phenomenon, evident in the fiber-deprived animals and intensified manyfold after irradiation. Bacterial infiltration also coincided with a specific pro-inflammatory serum cytokine profile. In contrast, mice on a fiber-rich diet were completely protected from irradiation-induced bacterial infiltration and exhibited a similar serum cytokine profile as sham-irradiated mice on a fiber-rich diet. Our findings provide ample evidence that dietary fiber consumption modifies the onset, timing and intensity of radiation-induced pathophysiological processes in the intestinal mucosa. However, we need more knowledge, not least from clinical studies, before this finding can be introduced to a new and refined clinical practice.

Background and Objectives To test the hypothesis that a predisposition to acquired genetic alterations is associated with ischemic stroke outcome by investigating the association between a polygenic risk score (PRS) for mosaic loss of chromosome Y (mLOY) and outcome in a large international data set. Methods We used data from the genome-wide association study performed within the Genetics of Ischemic Stroke Functional Outcome network, which included 6,165 patients (3,497 men and 2,668 women) with acute ischemic stroke of mainly European ancestry. We assessed a weighted PRS for mLOY and examined possible associations with the modified Rankin Scale (mRS) score 3 months poststroke in logistic regression models. We investigated the whole study sample as well as men and women separately. Results Increasing PRS for mLOY was associated with poor functional outcome (mRS score >2) with an odds ratio (OR) of 1.11 (95% confidence interval [CI] 1.03–1.19) per 1 SD increase in the PRS after adjustment for age, sex, ancestry, stroke severity (NIH Stroke Scale), smoking, and diabetes mellitus. In sex-stratified analyses, we found a statistically significant association in women (adjusted OR 1.20, 95% CI 1.08–1.33). In men, the association was in the same direction (adjusted OR 1.04, 95% CI 0.95–1.14), and we observed no significant genotype-sex interaction. Discussion In this exploratory study, we found associations between genetic variants predisposing to mLOY and stroke outcome. The significant association in women suggests underlying mechanisms related to genomic instability that operate in both sexes. These findings need replication and mechanistic exploration.