Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases
. Here we show that centenarians have a distinct gut microbiome that is ...enriched in microorganisms that are capable of generating unique secondary bile acids, including various isoforms of lithocholic acid (LCA): iso-, 3-oxo-, allo-, 3-oxoallo- and isoallolithocholic acid. Among these bile acids, the biosynthetic pathway for isoalloLCA had not been described previously. By screening 68 bacterial isolates from the faecal microbiota of a centenarian, we identified Odoribacteraceae strains as effective producers of isoalloLCA both in vitro and in vivo. Furthermore, we found that the enzymes 5α-reductase (5AR) and 3β-hydroxysteroid dehydrogenase (3β-HSDH) were responsible for the production of isoalloLCA. IsoalloLCA exerted potent antimicrobial effects against Gram-positive (but not Gram-negative) multidrug-resistant pathogens, including Clostridioides difficile and Enterococcus faecium. These findings suggest that the metabolism of specific bile acids may be involved in reducing the risk of infection with pathobionts, thereby potentially contributing to the maintenance of intestinal homeostasis.
Intestinal colonization by bacteria of oral origin has been correlated with several negative health outcomes, including inflammatory bowel disease. However, a causal role of oral bacteria ectopically ...colonizing the intestine remains unclear. Using gnotobiotic techniques, we show that strains of Klebsiella spp. isolated from the salivary microbiota are strong inducers of T helper 1 (TH1) cells when they colonize in the gut. These Klebsiella strains are resistant to multiple antibiotics, tend to colonize when the intestinal microbiota is dysbiotic, and elicit a severe gut inflammation in the context of a genetically susceptible host. Our findings suggest that the oral cavity may serve as a reservoir for potential intestinal pathobionts that can exacerbate intestinal disease.
Manipulation of the gut microbiota holds great promise for the treatment of inflammatory and allergic diseases. Although numerous probiotic microorganisms have been identified, there remains a ...compelling need to discover organisms that elicit more robust therapeutic responses, are compatible with the host, and can affect a specific arm of the host immune system in a well-controlled, physiological manner. Here we use a rational approach to isolate CD4(+)FOXP3(+) regulatory T (Treg)-cell-inducing bacterial strains from the human indigenous microbiota. Starting with a healthy human faecal sample, a sequence of selection steps was applied to obtain mice colonized with human microbiota enriched in Treg-cell-inducing species. From these mice, we isolated and selected 17 strains of bacteria on the basis of their high potency in enhancing Treg cell abundance and inducing important anti-inflammatory molecules--including interleukin-10 (IL-) and inducible T-cell co-stimulator (ICOS)--in Treg cells upon inoculation into germ-free mice. Genome sequencing revealed that the 17 strains fall within clusters IV, XIVa and XVIII of Clostridia, which lack prominent toxins and virulence factors. The 17 strains act as a community to provide bacterial antigens and a TGF-β-rich environment to help expansion and differentiation of Treg cells. Oral administration of the combination of 17 strains to adult mice attenuated disease in models of colitis and allergic diarrhoea. Use of the isolated strains may allow for tailored therapeutic manipulation of human immune disorders.
Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. ...Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.
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•A strong correlation between epithelial adhesion and Th17 induction by SFB and EHEC•Twenty Th17-inducing strains isolated from human feces show epithelial-adhesive property•Bacterial adhesion elicits a Th17-inducing gene-expression program in epithelium
The adhesion of specific members of the gut microbiome to intestinal epithelial cells is found to be essential for the induction of Th17 cells, thus highlighting location as an additional layer of regulation for the adaptive immune system.
Commensal bacteria influence host physiology, without invading host tissues. We show that proteins from segmented filamentous bacteria (SFB) are transferred into intestinal epithelial cells (IECs) ...through adhesion-directed endocytosis that is distinct from the clathrin-dependent endocytosis of invasive pathogens. This process transfers microbial cell wall-associated proteins, including an antigen that stimulates mucosal T helper 17 (T
17) cell differentiation, into the cytosol of IECs in a cell division control protein 42 homolog (CDC42)-dependent manner. Removal of CDC42 activity in vivo led to disruption of endocytosis induced by SFB and decreased epithelial antigen acquisition, with consequent loss of mucosal T
17 cells. Our findings demonstrate direct communication between a resident gut microbe and the host and show that under physiological conditions, IECs acquire antigens from commensal bacteria for generation of T cell responses to the resident microbiota.
Although most bile acids (BAs) in feces are present in noncovalent forms that can be extracted with ethanol, non-negligible amounts of saponifiable BAs are also present. It is a major concern that ...such saponifiable BAs are routinely omitted from fecal BA measurements. We compared the BA profiles of healthy stools that were obtained with/without alkaline hydrolysis and found that as much as 29.7% (2.1–67.7%) of total BAs were saponifiable. Specifically, alkaline treatment led to significant elevations of isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) concentrations, suggesting that considerable proportions of isoDCA and isoLCA were esterified. Precursor ion scan data from LC/MS suggested the presence of long-chain FA-linked BAs. We chemically synthesized a series of fatty acid 3β-acyl conjugates of isoDCA and isoLCA as analytical standards and analyzed their fecal profiles from newborns to adults (n = 64) by LC/MS. FA-conjugated isobile acids (FA-isoBAs) were constantly present from 2 years of age to adulthood. C16- and C18-chain FA-isoBA esters were predominantly found regardless of age, but small amounts of acetic acid esters were also found. FA-isoBA concentrations were not correlated to fecal FA concentrations. Interestingly, there were some adults who did not have FA-isoBAs. Gut bacteria involved in the production of FA-isoBAs have not been identified yet. The present study provides insight into the establishment of early gut microbiota and the interactive development of esterified BAs.The contribution of FA-isoBAs to gut physiology and their role in pathophysiologic conditions such as inflammatory bowel disease are currently under investigation.
Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. ...Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (T
17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to T
17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the T
17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.
Segmented filamentous bacteria (SFB) are Gram-positive, anaerobic, spore-forming commensals that reside in the gut of many animal species. Described more than forty years ago, SFB have recently ...gained interest due to their unique ability to modulate the host immune system through induction of IgA and Th17 cells. Here, we describe a collection of methods to detect and quantify SFB and SFB adhesion in intestinal mucosa, as well as SFB-specific CD4 T cells in the lamina propria. In addition, we describe methods for purification of SFB from fecal material of SFB-monoassociated gnotobiotic mice. Using these methods we examine the kinetics of SFB colonization and Th17 cell induction. We also show that SFB colonize unevenly the intestinal mucosa and that SFB adherence occurs predominantly in the terminal ileum and correlates with an increased proportion of SFB-specific Th17 cells.
•SFB colonization and Th17 cell induction follow defined kinetics.•SFB are distributed unevenly along the length of the small intestine.•SFB-specific Th17 cells are enriched in the terminal ileum.•Th17 cell induction in the small intestine correlates with SFB adherence.
Dysbiotic microbiota contributes to the pathogenesis of Crohn's disease (CD) by regulating the immune system. Although pro-inflammatory microbes are probably enriched in the small intestinal (SI) ...mucosa, most studies have focused on fecal microbiota. This study aimed to examine jejunal and ileal mucosal specimens from patients with CD via double-balloon enteroscopy. Comparative microbiome analysis revealed that the microbiota composition of CD SI mucosa differs from that of non-CD controls, with an increased population of several families, including Enterobacteriaceae, Ruminococcaceae, and Bacteroidaceae. Upon anaerobic culturing of the CD SI mucosa, 80 bacterial strains were isolated, from which 9 strains representing 9 distinct species (Escherichia coli, Ruminococcus gnavus, Klebsiella pneumoniae, Erysipelatoclostridium ramosum, Bacteroides dorei, B. fragilis, B. uniformis, Parabacteroides distasonis, and Streptococcus pasteurianus) were selected on the basis of their significant association with CD. The colonization of germ-free (GF) mice with the 9 strains enhanced the accumulation of T
H
1 cells and, to a lesser extent, T
H
17 cells in the intestine, among which an E. coli strain displayed high potential to induce T
H
1 cells and intestinal inflammation in a strain-specific manner. The present results indicate that the CD SI mucosa harbors unique pro-inflammatory microbiota, including T
H
1 cell-inducing E. coli, which could be a potential therapeutic target.
Certain proteases derived from house dust mites and plants are considered to trigger initiation of allergic airway inflammation by disrupting tight junctions between epithelial cells. It is known ...that inhalation of proteases such as house dust mite-derived Der p1 and/or papaya-derived papain caused airway eosinophilia in naïve mice and even in Rag-deficient mice that lack acquired immune cells such as T, B and NKT cells. In contrast, little is known regarding the possible involvement of proteases derived from Aspergillus species (fungal-associated proteases; FAP), which are ubiquitous saprophytic fungi in the environment, in the development of allergic airway eosinophilia. Here, we found that inhalation of FAP by naïve mice led to airway eosinophilia that was dependent on protease-activated receptor-2 (PAR2), but not TLR2 and TLR4. Those findings suggest that the protease activity of FAP, but not endotoxins in FAP, are important in the setting. In addition, development of that eosinophilia was mediated by innate immune cells (ILCs) such as innate lymphoid cells, but not by acquired immune cells such as T, B and NKT cells. Whereas IL-33, IL-25 and thymic stromal lymphopoietin (TSLP) are involved in induction of FAP-induced ILC-mediated airway eosinophilia, IL-33-rather than IL-25 and/or TSLP-was critical for the eosinophilia in our model. Our findings improve our understanding of the molecular mechanisms involved in induction of airway inflammation by FAP.
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