The gut commensal Blautia maintains colonic mucus function under low-fiber consumption through secretion of short-chain fatty acids.

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.

Intestinal α-Defensins Play a Minor Role in Modulating the Small Intestinal Microbiota Composition as Compared to Diet.

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.

Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection.

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.

Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress.

Colonic goblet cells are specialized epithelial cells that secrete mucus to physically separate the host and its microbiota, thus preventing bacterial invasion and inflammation. How goblet cells control the amount of mucus they secrete is unclear. We found that constitutive activation of autophagy in mice via Beclin 1 enables the production of a thicker and less penetrable mucus layer by reducing endoplasmic reticulum (ER) stress. Accordingly, genetically inhibiting Beclin 1-induced autophagy impairs mucus secretion, while pharmacologically alleviating ER stress results in excessive mucus production. This ER-stress-mediated regulation of mucus secretion is microbiota dependent and requires the Crohn's-disease-risk gene Nod2. Overproduction of mucus alters the gut microbiome, specifically expanding mucus-utilizing bacteria, such as Akkermansia muciniphila, and protects against chemical and microbial-driven intestinal inflammation. Thus, ER stress is a cell-intrinsic switch that limits mucus secretion, whereas autophagy maintains intestinal homeostasis by relieving ER stress.

Obesity-associated microbiota contributes to mucus layer defects in genetically obese mice.

The intestinal mucus layer is a physical barrier separating the tremendous number of gut bacteria from the host epithelium. Defects in the mucus layer have been linked to metabolic diseases, but previous studies predominantly investigated mucus function during high-caloric/low-fiber dietary interventions, thus making it difficult to separate effects mediated directly through diet quality from potential obesity-dependent effects. As such, we decided to examine mucus function in mouse models with metabolic disease to distinguish these factors. Here we show that, in contrast to their lean littermates, genetically obese (ob/ob) mice have a defective inner colonic mucus layer that is characterized by increased penetrability and a reduced mucus growth rate. Exploiting the coprophagic behavior of mice, we next co-housed ob/ob and lean mice to investigate if the gut microbiota contributed to these phenotypes. Co-housing rescued the defect of the mucus growth rate, whereas mucus penetrability displayed an intermediate phenotype in both mouse groups. Of note, non-obese diabetic mice with high blood glucose levels displayed a healthy colonic mucus barrier, indicating that the mucus defect is obesity- rather than glucose-mediated. Thus, our data suggest that the gut microbiota community of obesity-prone mice may regulate obesity-associated defects in the colonic mucosal barrier, even in the presence of dietary fiber.

Does an Apple a Day Also Keep the Microbes Away? The Interplay Between Diet, Microbiota, and Host Defense Peptides at the Intestinal Mucosal Barrier.

A crucial mechanism of intestinal defense includes the production and secretion of host defense peptides (HDPs). HDPs control pathogens and commensals at the intestinal interface by direct killing, by sequestering vital ions, or by causing bacterial cells to aggregate in the mucus layer. Accordingly, the combined activity of various HDPs neutralizes gut bacteria before reaching the mucosa and thus helps to maintain the homeostatic balance between the host and its microbes at the mucosal barrier. Defects in the mucosal barrier have been associated with various diseases that are on the rise in the Western world. These include metabolic diseases, such as obesity and type 2 diabetes, and inflammatory intestinal disorders, including ulcerative colitis and Crohn's disease, the two major entities of inflammatory bowel disease. While the etiology of these diseases is multifactorial, highly processed Western-style diet (WSD) that is rich in carbohydrates and fat and low in dietary fiber content, is considered to be a contributing lifestyle factor. As such, WSD does not only profoundly affect the resident microbes in the intestine, but can also directly alter HDP function, thereby potentially contributing to intestinal mucosal barrier dysfunction. In this review we aim to decipher the complex interaction between diet, microbiota, and HDPs. We discuss how HDP expression can be modulated by specific microbes and their metabolites as well as by dietary factors, including fibers, lipids, polyphenols and vitamins. We identify several dietary compounds that lead to reduced HDP function, but also factors that stimulate HDP production in the intestine. Furthermore, we argue that the effect of HDPs against commensal bacteria has been understudied when compared to pathogens, and that local environmental conditions also need to be considered. In addition, we discuss the known molecular mechanisms behind HDP modulation. We believe that a better understanding of the diet-microbiota-HDP interdependence will provide insights into factors underlying modern diseases and will help to identify potential dietary interventions or probiotic supplementation that can promote HDP-mediated intestinal barrier function in the Western gut.

Endogenous FGF21-signaling controls paradoxical obesity resistance of UCP1-deficient mice.

Uncoupling protein 1 (UCP1) executes thermogenesis in brown adipose tissue, which is a major focus of human obesity research. Although the UCP1-knockout (UCP1 KO) mouse represents the most frequently applied animal model to judge the anti-obesity effects of UCP1, the assessment is confounded by unknown anti-obesity factors causing paradoxical obesity resistance below thermoneutral temperatures. Here we identify the enigmatic factor as endogenous FGF21, which is primarily mediating obesity resistance. The generation of UCP1/FGF21 double-knockout mice (dKO) fully reverses obesity resistance. Within mild differences in energy metabolism, urine metabolomics uncover increased secretion of acyl-carnitines in UCP1 KOs, suggesting metabolic reprogramming. Strikingly, transcriptomics of metabolically important organs reveal enhanced lipid and oxidative metabolism in specifically white adipose tissue that is fully reversed in dKO mice. Collectively, this study characterizes the effects of endogenous FGF21 that acts as master regulator to protect from diet-induced obesity in the absence of UCP1.

Microbiota and mucosal defense in IBD: an update.

Introduction: Inflammatory bowel diseases (IBD) are on the rise worldwide. This review covers the current concepts of the etiology of Crohn´s disease and ulcerative colitis by focusing on an unbalanced interaction between the intestinal microbiota and the mucosal barrier. Understanding these issues is of paramount importance for the development of targeted therapies aiming at the disease cause.Area covered: Gut microbiota alterations and a dysfunctional intestinal mucosa are associated with IBD. Here we focus on specific defense structures of the mucosal barrier, namely antimicrobial peptides and the mucus layer, which keep the gut microbiota at a distance under healthy conditions and are defective in IBD.Expert commentary: The microbiology of both forms of IBD is different but characterized by a reduced bacterial diversity and richness. Abundance of certain bacterial species is altered, and the compositional changes are related to disease activity. In IBD the mucus layer above the epithelium is contaminated by bacteria and the immune reaction is dominated by the antibacterial response. Human genetics suggest that many of the basic deficiencies in the mucosal response, due to Paneth cell, defensin and mucus defects, are primary. Nutrition may also be important but so far there is no therapy targeting the mucosal barrier.

The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function.

The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled Nlrp6-/- , detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in Il18-/- mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus Adlercrutzia consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.

Proteolytic Degradation of reduced Human Beta Defensin 1 generates a Novel Antibiotic Octapeptide.

Microbial resistance against clinical used antibiotics is on the rise. Accordingly, there is a high demand for new innovative antimicrobial strategies. The host-defense peptide human beta-defensin 1 (hBD-1) is produced continuously by epithelial cells and exhibits compelling antimicrobial activity after reduction of its disulphide bridges. Here we report that proteolysis of reduced hBD-1 by gastrointestinal proteases as well as human duodenal secretions produces an eight-amino acid carboxy-terminal fragment. The generated octapeptide retains antibiotic activity, yet with distinct characteristics differing from the full-length peptide. We modified the octapeptide by stabilizing its termini and by using non-natural D-amino acids. The native and modified peptide variants showed antibiotic activity against pathogenic as well as antibiotic-resistant microorganisms, including E. coli, P. aeruginosa and C. albicans. Moreover, in an in vitro C. albicans infection model the tested peptides demonstrated effective amelioration of C. albicans infection without showing cytotoxity on human cells. In summary, protease degradation of hBD-1 provides a yet unknown mechanism to broaden antimicrobial host defense, which could be used to develop defensin-derived therapeutic applications.

Fight them or feed them: how the intestinal mucus layer manages the gut microbiota.

The intestinal tract is inhabited by a tremendous number of microorganisms, termed the gut microbiota. These microorganisms live in a mutualistic relationship with their host and assist in the degradation of complex carbohydrates. Although the gut microbiota is generally considered beneficial, the vast number of microbial cells also form a permanent threat to the host. Thus, the intestinal epithelium is covered with a dense layer of mucus to prevent translocation of the gut microbiota into underlying tissues. Intestinal mucus is an organized glycoprotein network with a host-specific glycan structure. While the mucus layer has long been considered a passive, host-designed barrier, recent studies showed that maturation and function of the mucus layer are strongly influenced by the gut microbiota. In return, the glycan repertoire of mucins can select for distinct mucosa-associated bacteria that are able to bind or degrade specific mucin glycans as a nutrient source. Because the intestinal mucus layer is at the crucial interface between host and microbes, its breakdown leads to gut bacterial encroachment that can eventually cause inflammation and infection. Accordingly, a dysfunctional mucus layer has been observed in colitis in mice and humans. Moreover, the increased consumption of a low-fiber Western-style diet in our modern society has recently been demonstrated to cause bacteria-mediated defects of the intestinal mucus layer. Here, I will review current knowledge on the interaction between gut bacteria and the intestinal mucus layer in health and disease. Understanding the molecular details of this host-microbe interaction may contribute to the development of novel treatment options for diseases involving a dysfunctional mucus layer, such as ulcerative colitis.

Dietary destabilisation of the balance between the microbiota and the colonic mucus barrier.

It has long been acknowledged that dietary fibres are important to maintain a healthy gut. Over the past decade, several studies have shown that loss of complex polysaccharides from the Western diet has resulted in alterations to our colonic microbiota. The concurrent increase in the incidence of inflammatory bowel disease in the Western world has driven us to explore the potential mechanistic link between diet, the microbiota and the host defence systems that normally prevent inflammation. Using mice fed a low fibre Western-style diet and robust live tissue analytical methods we have now provided evidence that this diet impairs the colonic inner mucus layer that normally separates bacteria from host cells. Western societies urgently need to develop their understanding of the molecular mechanisms of the diet-microbiota-mucus axis and its implications for inflammatory diseases.

Bifidobacteria or Fiber Protects against Diet-Induced Microbiota-Mediated Colonic Mucus Deterioration.

Diet strongly affects gut microbiota composition, and gut bacteria can influence the colonic mucus layer, a physical barrier that separates trillions of gut bacteria from the host. However, the interplay between a Western style diet (WSD), gut microbiota composition, and the intestinal mucus layer is less clear. Here we show that mice fed a WSD have an altered colonic microbiota composition that causes increased penetrability and a reduced growth rate of the inner mucus layer. Both barrier defects can be prevented by transplanting microbiota from chow-fed mice. In addition, we found that administration of Bifidobacterium longum was sufficient to restore mucus growth, whereas administration of the fiber inulin prevented increased mucus penetrability in WSD-fed mice. We hypothesize that the presence of distinct bacteria is crucial for proper mucus function. If confirmed in humans, these findings may help to better understand diseases with an affected mucus layer, such as ulcerative colitis.

Gram-positive bacteria are held at a distance in the colon mucus by the lectin-like protein ZG16.

The distal colon functions as a bioreactor and harbors an enormous amount of bacteria in a mutualistic relationship with the host. The microbiota have to be kept at a safe distance to prevent inflammation, something that is achieved by a dense inner mucus layer that lines the epithelial cells. The large polymeric nets made up by the heavily O-glycosylated MUC2 mucin forms this physical barrier. Proteomic analyses of mucus have identified the lectin-like protein ZG16 (zymogen granulae protein 16) as an abundant mucus component. To elucidate the function of ZG16, we generated recombinant ZG16 and studied Zg16-/- mice. ZG16 bound to and aggregated Gram-positive bacteria via binding to the bacterial cell wall peptidoglycan. Zg16-/- mice have a distal colon mucus layer with normal thickness, but with bacteria closer to the epithelium. Using distal colon explants mounted in a horizontal perfusion chamber we demonstrated that treatment of bacteria with recombinant ZG16 hindered bacterial penetration into the mucus. The inner colon mucus of Zg16-/- animals had a higher load of Gram-positive bacteria and showed bacteria with higher motility in the mucus close to the host epithelium compared with cohoused littermate Zg16+/+ The more penetrable Zg16-/- mucus allowed Gram-positive bacteria to translocate to systemic tissues. Viable bacteria were found in spleen and were associated with increased abdominal fat pad mass in Zg16-/- animals. The function of ZG16 reveals a mechanism for keeping bacteria further away from the host colon epithelium.

Signals from the gut microbiota to distant organs in physiology and disease.

The ecosystem of the human gut consists of trillions of bacteria forming a bioreactor that is fueled by dietary macronutrients to produce bioactive compounds. These microbiota-derived metabolites signal to distant organs in the body, which enables the gut bacteria to connect to the immune and hormone system, to the brain (the gut-brain axis) and to host metabolism, as well as other functions of the host. This microbe-host communication is essential to maintain vital functions of the healthy host. Recently, however, the gut microbiota has been associated with a number of diseases, ranging from obesity and inflammatory diseases to behavioral and physiological abnormalities associated with neurodevelopmental disorders. In this Review, we will discuss microbiota-host cross-talk and intestinal microbiome signaling to extraintestinal organs. We will review mechanisms of how this communication might contribute to host physiology and discuss how misconfigured signaling might contribute to different diseases.

Disulphide-reduced psoriasin is a human apoptosis-inducing broad-spectrum fungicide.

The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.

Gastric antimicrobial peptides fail to eradicate Helicobacter pylori infection due to selective induction and resistance.

BACKGROUND: Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection. MATERIALS AND METHODS: We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy. RESULTS: H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples. CONCLUSION: These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.

Antimicrobial activity of high-mobility-group box 2: a new function to a well-known protein.

The human intestinal tract is highly colonized by a vast number of microorganisms. Despite this permanent challenge, infections remain rare, due to a very effective barrier defense system. Essential effectors of this system are antimicrobial peptides and proteins (AMPs), which are secreted by intestinal epithelial and lymphoid cells, balance the gut microbial community, and prevent the translocation of microorganisms. Several antimicrobial proteins have already been identified in the gut. Nonetheless, we hypothesized that additional AMPs are yet to be discovered in this setting. Using biological screening based on antimicrobial function, here we identified competent antibacterial activity of high-mobility-group box 2 (HMGB2) against Escherichia coli. By recombinant expression, we confirmed this biologically new antimicrobial activity against different commensal and pathogenic bacteria. In addition, we demonstrated that the two DNA-binding domains (HMG boxes A and B) are crucial for the antibiotic function. We detected HMGB2 in several gastrointestinal tissues by mRNA analysis and immunohistochemical staining. In addition to the nuclei, we also observed HMGB2 in the cytoplasm of intestinal epithelial cells. Furthermore, HMGB2 was detectable in vitro in the supernatants of two different cell types, supporting an extracellular function. HMGB2 expression was not changed in inflammatory bowel disease but was detected in certain stool samples of patients, whereas it was absent from control individuals. Taken together, we characterized HMGB2 as an antimicrobial protein in intestinal tissue, complementing the diverse repertoire of gut mucosal defense molecules.