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Dietary lipids disturb colonic microbiota–mucus interactions and microbial transformation of host-derived bile acids. This altered bile acid pool triggers increased lipid absorption in the small intestine, revealing an unexpected link between the colonic mucus niche and small intestinal lipid metabolism.

A mucus hydrogel covers the intestinal epithelium, protecting the host from passing food and resident gut microbiota. The mucus consists of a highly organized glycoprotein network, mainly produced by goblet cells. In the distal colon, where microbial abundance is highest, goblet cells continuously secrete mucus, which “grows” by expansion, forming a gradient from an inner nearly sterile layer to an outer loose layer where bacteria reside. This pushes microbes away from the host epithelium, thereby reducing the risk of infection and inflammation.

The gut microbiota is predominantly composed of bacteria and is strongly influenced by diet. Individuals in industrialized societies exhibit reduced microbial diversity compared to those in non-industrialized societies – a difference partly attributed to the Western-style diet (WSD), which is low in dietary fiber and high in simple sugars and saturated fat. In mice fed a WSD, microbiota diversity is reduced, and the mucus barrier is weakened, as seen by a slower mucus growth rate and increased bacterial penetration, which raises the risk for harmful microbial interactions. Similar microbial and mucus alterations are observed in patients with inflammatory bowel disease (IBD). While mucus properties are microbiota-dependent, the underlying regulatory mechanisms are still largely elusive. This thesis aims to clarify these mechanisms by studying the colonic mucus barrier in a WSD environment, with both human and mouse microbiota. Moreover, the effects of structures previously untested for their mucus-influencing properties during WSD feeding are evaluated in mice.

In study 1, the impact of diet-induced changes in the human microbiota on the colonic mucus barrier was studied by transplanting fecal samples into microbiota-depleted mice. Healthy participants increased their fiber intake over three months, and their microbiota was collected before and after the intervention. When transplanted into mice, only the high-fiber-derived microbiota maintained mucus growth under WSD feeding and reduced the pathogen load during intestinal infection. The bacterial taxon Blautia was enriched in the high-fiber group, and Blautia coccoides emerged as a key regulator of mucus integrity through the production of the short-chain fatty acids (SCFAs) acetate and propionate. These metabolites were shown to stimulate mucus growth via the free-fatty acid receptor 2 (Ffar2), revealing a previously unrecognized mechanism by which microbial metabolites directly impact mucus integrity.

In studies 2 and 3, the mucus-influencing effects of bovine milk-derived casein glycomacropeptide (CGMP) and human milk oligosaccharide (HMO) structures were examined in WSD-fed mice. CGMP is a glycosylated protein found in cheese whey, while HMOs are breast milk components with over 200 known structures. Both GCMP and HMOs share structural similarities with mucin glycans and may act as decoy substrates for bacterial degradation under low-fiber conditions. In study 2, CGMP structures improved mucus growth rate, where specifically, a highly sialylated CGMP (HSA) increased propionate levels and the relative abundance of Bifidobacteria, a bacterium previously linked to mucus maintenance. In study 3, specific HMO structures enhanced mucus integrity and improved mucus penetrability in a mouse model for IBD. HMO-dependent mucus modulation could be linked to changes in bacterial composition, increased SCFA levels and glycan-targeting enzyme activities. These findings emphasize the structure-specific effects of CGMPs and HMOs and their distinct modulation of microbiota–mucus interactions.

In summary, this thesis reveals new insights into how microbial metabolites regulate the mucus barrier in the Western gut and highlights novel strategies to be exploited for treating mucus-associated disorders such as IBD.

The small intestinal mucosa has the delicate task of allowing absorption of nutrients and limiting microbial colonization at the mucosal surface through production of antimicrobial peptides and proteins (AMPs). However, while environmental factors, including different diets, have been shown to alter AMP expression, the results from the literature are conflicting on their specific impact. Moreover, the interdependence between diet, AMPs, and metabolic health is largely unexplored. The aim of this study was thus to investigate the effect of obesogenic diets, obesity itself, and other variables, including mouse vendor, microbiota composition, and sex, on intestinal AMP expression. By using different dietary interventions in mice, we here show that prolonged intake of an obesogenic Western-style diet had a stronger impact on AMP expression than diet-independent obesity. Additionally, when comparing AMP expression under different diets in mice of both sexes from different vendors, the combined contribution of these factors had the strongest impact on absolute AMP transcript numbers, but also on the variability in small intestinal microbiota composition at the mucosa and content. Finally, we identified a novel host–microbe interaction, in which the gut commensal Faecalibaculum bloomed upon WSD-feeding and specifically induced the expression of the AMP Reg3g. Our findings thus reveal that the experimental setup, defined by mouse vendor, sex, and diet type, has a major influence on small-intestinal AMP expression. These findings could partly explain the discrepancy in the literature regarding the effect of diets on AMP expression, preventing any accurate generalization about the impact of diet on the antimicrobial response.

The colonic mucus layer is the primary interface between the host and the gut microbiota. It serves both as an ecological niche for bacteria and a barrier protecting the host from microbial exposure. Disruption of the mucus layer, particularly under Western-style diet (WSD) feeding, increases the risk of infection and inflammation. Here, we identify casein glycomacropeptide (CGMP), a milk-derived glycopeptide, as a novel dietary supplement capable of preserving mucus function under WSD consumption. Notably, we demonstrate that the sialylation level of CGMP is a key determinant of its protective effects. Supplementation of highly sialylated CGMP not only prevented WSD-induced mucus defects but also altered the gut microbiota composition, enhancing beneficial bacterial genera, particularly Bifidobacterium. Mechanistically, bacterial shifts were associated with increased production of the short-chain fatty acid propionate, which can induce mucus growth. Our findings thus reveal sialylated CGMP as a promising prebiotic supplement to counteract diet-induced mucus dysfunction, highlighting the importance of protein-bound glycan structures in modulating host-microbiota interaction.

Recent evidence indicates that repeated antibiotic usage lowers microbial diversity and ultimately changes the gut microbiota community. However, the physiological effects of repeated–but not recent–antibiotic usage on microbiota-mediated mucosal barrier function are largely unknown. By selecting human individuals from the deeply phenotyped Estonian Microbiome Cohort (EstMB), we here utilized human-to-mouse fecal microbiota transplantation to explore long-term impacts of repeated antibiotic use on intestinal mucus function. While a healthy mucus layer protects the intestinal epithelium against infection and inflammation, using ex vivo mucus function analyses of viable colonic tissue explants, we show that microbiota from humans with a history of repeated antibiotic use causes reduced mucus growth rate and increased mucus penetrability compared to healthy controls in the transplanted mice. Moreover, shotgun metagenomic sequencing identified a significantly altered microbiota composition in the antibiotic-shaped microbial community, with known mucus-utilizing bacteria, including Akkermansia muciniphila and Bacteroides fragilis, dominating in the gut. The altered microbiota composition was further characterized by a distinct metabolite profile, which may be caused by differential mucus degradation capacity. Consequently, our proof-of-concept study suggests that long-term antibiotic use in humans can result in an altered microbial community that has reduced capacity to maintain proper mucus function in the gut.

Beneficial gut bacteria are indispensable for developing colonic mucus and fully establishing its protective function against intestinal microorganisms. Low-fiber diet consumption alters the gut bacterial configuration and disturbs this microbe-mucus interaction, but the specific bacteria and microbial metabolites responsible for maintaining mucus function remain poorly understood. By using human-to-mouse microbiota transplantation and ex vivo analysis of colonic mucus function, we here show as a proof-of-concept that individuals who increase their daily dietary fiber intake can improve the capacity of their gut microbiota to prevent diet-mediated mucus defects. Mucus growth, a critical feature of intact colonic mucus, correlated with the abundance of the gut commensal Blautia, and supplementation of Blautia coccoides to mice confirmed its mucus-stimulating capacity. Mechanistically, B. coccoides stimulated mucus growth through the production of the short-chain fatty acids propionate and acetate via activation of the short-chain fatty acid receptor Ffar2, which could serve as a new target to restore mucus growth during mucus-associated lifestyle diseases.

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.