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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.

Specialized defense mechanisms at mucosal barriers along the gastrointestinal tract constantly protect us against the trillions of microorganisms living inside the human body. These mechanisms include a mucus layer as a physical barrier that prevents bacteria from reaching the epithelium and the production of antimicrobial peptides (AMPs) as a chemical barrier that helps neutralize or lyse these bacteria. On the other hand, many intestinal bacteria benefit human health by providing colonization resistance against pathogenic bacteria, helping produce vitamins, aiding in the digestion of complex carbohydrates, and producing anti-inflammatory short-chain fatty acids. Therefore, the intestinal mucosal barrier has the challenging task of maintaining a homeostatic interaction between the host and the intestinal microbiota. 

Alterations in the integrity of the mucus barrier and the expression of AMPs have been associated with inflammatory bowel disease and obesity. This thesis investigates how the intake of a high-fat and low-fiber Western-style diet (WSD) as an exogenous factor can affect the protective function of the mucus barrier and intestinal AMPs in mice with or without modulation of the microbiota.

In paper 1 “Intestinal α-Defensins Play a Minor Role in Modulating the Small Intestinal Microbiota Composition as Compared to Diet” we fed wild-type and Mmp7^-/- mice, which lack active a-defensins, the major family of AMPs in the small intestine, a control or a WSD.  We found that diet had a stronger impact on modulating small intestinal microbiota composition, while defensins only modulated the abundance of specific bacteria. In addition, defensins protected against metabolic dysfunction induced by the intake of a WSD.

In paper 2 “Investigating the link between antimicrobial defense, gut microbiota and metabolic dysfunction at the small intestinal mucosal barrier” we investigated the effect of obesogenic diets (Western diet or a high fat diet), obesity itself and other variables, including microbiota composition and sex, on small intestinal AMP expression. We observed that prolonged intake of a WSD had a stronger impact on AMP expression than genetic obesity, and determined that experimental set-up defined by mouse vendor and diet type, may have a larger influence than the specific dietary disturbances.

In paper 3 “Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection” we determined that the mucus layer of the jejunum formed aggregates and became more penetrable to bacteria-sized beads following the intake of a WSD. Both Muc2^-/- and WSD-fed mice had an altered microbiota composition and increased susceptibility to enteric infection with Citrobacter rodentium in the jejunum, highlighting the role of the mucus layer as a microbiota- supporting niche that mediates colonization resistance against infection.

In summary, our work investigates the mechanisms by which a WSD changes the small intestinal microbiota composition at different intestinal sites while simultaneously disrupting mucus and AMP function. Our findings can aid the development of potential therapeutic avenues for addressing obesity and inflammatory bowel diseases through targeted modulation of mucus function, AMP expression or microbial composition.

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.

The intestinal microbiota is at the interface between the host and its environment and thus under constant exposure to host-derived and external modulators. While diet is considered to be an important external factor modulating microbiota composition, intestinal defensins, one of the major classes of antimicrobial peptides, have been described as key host effectors that shape the gut microbial community. However, since dietary compounds can affect defensin expression, thereby indirectly modulating the intestinal microbiota, their individual contribution to shaping gut microbiota composition remains to be defined. To disentangle the complex interaction among diet, defensins, and small-intestinal microbiota, we fed wild-type (WT) mice and mice lacking functionally active α-defensins (Mmp7-/- mice) either a control diet or a Western-style diet (WSD) that is rich in saturated fat and simple carbohydrates but low in dietary fiber. 16S rDNA sequencing and robust statistical analyses identified that bacterial composition was strongly affected by diet while defensins had only a minor impact. These findings were independent of sample location, with consistent results between the lumen and mucosa of the jejunum and ileum, in both mouse genotypes. However, distinct microbial taxa were also modulated by α-defensins, which was supported by differential antimicrobial activity of ileal protein extracts. As the combination of WSD and defensin deficiency exacerbated glucose metabolism, we conclude that defensins only have a fine-tuning role in shaping the small-intestinal bacterial composition and might instead be important in protecting the host against the development of diet-induced metabolic dysfunction.

IMPORTANCE: Alterations in the gut microbial community composition are associated with many diseases, and therefore identifying factors that shape the microbial community under homeostatic and diseased conditions may contribute to the development of strategies to correct a dysbiotic microbiota. Here, we demonstrate that a Western-style diet, as an extrinsic parameter, had a stronger impact on shaping the small intestinal bacterial composition than intestinal defensins, as an intrinsic parameter. While defensins have been previously shown to modulate bacterial composition in young mice, our study supplements these findings by showing that defensins may be less important in adult mice that harbor a mature microbial community. Nevertheless, we observed that defensins did affect the abundance of distinct bacterial taxa in adult mice and protected the host from aggravated diet-induced glucose impairments. Consequently, our study uncovers a new angle on the role of intestinal defensins in the development of metabolic diseases in adult mice.

Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospecific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the regiospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colonization, but is highly sensitive to disruption by exposure to a WSD.