Improved mouse models of the small intestine microbiota using region-specific sampling from humans.

Our understanding of region-specific microbial function within the gut is limited due to reliance on stool. Using a recently developed capsule device, we exploit regional sampling from the human intestines to develop models for interrogating small intestine (SI) microbiota composition and function. In vitro culturing of human intestinal contents produced stable, representative communities that robustly colonize the SI of germ-free mice. During mouse colonization, the combination of SI and stool microbes altered gut microbiota composition, functional capacity, and response to diet, resulting in increased diversity and reproducibility of SI colonization relative to stool microbes alone. Using a diverse strain library representative of the human SI microbiota, we constructed defined communities with taxa that largely exhibited the expected regional preferences. Response to a fiber-deficient diet was region-specific and reflected strain-specific fiber-processing and host mucus-degrading capabilities, suggesting that dietary fiber is critical for maintaining SI microbiota homeostasis. These tools should advance mechanistic modeling of the human SI microbiota and its role in disease and dietary responses.

Gut microbial carbohydrate metabolism contributes to insulin resistance.

Insulin resistance is the primary pathophysiology underlying metabolic syndrome and type 2 diabetes1,2. Previous metagenomic studies have described the characteristics of gut microbiota and their roles in metabolizing major nutrients in insulin resistance3-9. In particular, carbohydrate metabolism of commensals has been proposed to contribute up to 10% of the host's overall energy extraction10, thereby playing a role in the pathogenesis of obesity and prediabetes3,4,6. Nevertheless, the underlying mechanism remains unclear. Here we investigate this relationship using a comprehensive multi-omics strategy in humans. We combine unbiased faecal metabolomics with metagenomics, host metabolomics and transcriptomics data to profile the involvement of the microbiome in insulin resistance. These data reveal that faecal carbohydrates, particularly host-accessible monosaccharides, are increased in individuals with insulin resistance and are associated with microbial carbohydrate metabolisms and host inflammatory cytokines. We identify gut bacteria associated with insulin resistance and insulin sensitivity that show a distinct pattern of carbohydrate metabolism, and demonstrate that insulin-sensitivity-associated bacteria ameliorate host phenotypes of insulin resistance in a mouse model. Our study, which provides a comprehensive view of the host-microorganism relationships in insulin resistance, reveals the impact of carbohydrate metabolism by microbiota, suggesting a potential therapeutic target for ameliorating insulin resistance.

Fatty acid overproduction by gut commensal microbiota exacerbates obesity.

Although recent studies have highlighted the impact of gut microbes on the progression of obesity and its comorbidities, it is not fully understood how these microbes promote these disorders, especially in terms of the role of microbial metabolites. Here, we report that Fusimonas intestini, a commensal species of the family Lachnospiraceae, is highly colonized in both humans and mice with obesity and hyperglycemia, produces long-chain fatty acids such as elaidate, and consequently facilitates diet-induced obesity. High fat intake altered the expression of microbial genes involved in lipid production, such as the fatty acid metabolism regulator fadR. Monocolonization with a FadR-overexpressing Escherichia coli exacerbated the metabolic phenotypes, suggesting that the change in bacterial lipid metabolism is causally involved in disease progression. Mechanistically, the microbe-derived fatty acids impaired intestinal epithelial integrity to promote metabolic endotoxemia. Our study thus provides a mechanistic linkage between gut commensals and obesity through the overproduction of microbe-derived lipids.

Human Gut Microbiota and Its Metabolites Impact Immune Responses in COVID-19 and Its Complications.

BACKGROUND & AIMS: We investigate interrelationships between gut microbes, metabolites, and cytokines that characterize COVID-19 and its complications, and we validate the results with follow-up, the Japanese 4D (Disease, Drug, Diet, Daily Life) microbiome cohort, and non-Japanese data sets. METHODS: We performed shotgun metagenomic sequencing and metabolomics on stools and cytokine measurements on plasma from 112 hospitalized patients with SARS-CoV-2 infection and 112 non-COVID-19 control individuals matched by important confounders. RESULTS: Multiple correlations were found between COVID-19-related microbes (eg, oral microbes and short-chain fatty acid producers) and gut metabolites (eg, branched-chain and aromatic amino acids, short-chain fatty acids, carbohydrates, neurotransmitters, and vitamin B6). Both were also linked to inflammatory cytokine dynamics (eg, interferon γ, interferon λ3, interleukin 6, CXCL-9, and CXCL-10). Such interrelationships were detected highly in severe disease and pneumonia; moderately in the high D-dimer level, kidney dysfunction, and liver dysfunction groups; but rarely in the diarrhea group. We confirmed concordances of altered metabolites (eg, branched-chain amino acids, spermidine, putrescine, and vitamin B6) in COVID-19 with their corresponding microbial functional genes. Results in microbial and metabolomic alterations with severe disease from the cross-sectional data set were partly concordant with those from the follow-up data set. Microbial signatures for COVID-19 were distinct from diabetes, inflammatory bowel disease, and proton-pump inhibitors but overlapping for rheumatoid arthritis. Random forest classifier models using microbiomes can highly predict COVID-19 and severe disease. The microbial signatures for COVID-19 showed moderate concordance between Hong Kong and Japan. CONCLUSIONS: Multiomics analysis revealed multiple gut microbe-metabolite-cytokine interrelationships in COVID-19 and COVID-19related complications but few in gastrointestinal complications, suggesting microbiota-mediated immune responses distinct between the organ sites. Our results underscore the existence of a gut-lung axis in COVID-19.

Analysis of Peripherally Derived Treg in the Intestine.

Elucidation of the symbiotic relationship between the host and its gut microbiota is critically important for understanding host pathophysiology. Peripherally derived regulatory T cells (pTregs) are recognized as central to immune homeostasis in the intestine. Moreover, the gut microbiota nourishes the intestinal and systemic immune systems, including pTreg, via their metabolites and other components. Therefore, methods to detect pTreg as well as to analyze the interactions between the gut microbiota and pTreg are important for better understanding of the symbiotic relationship with these microorganisms. Here, we describe a protocol to isolate colonic lamina propria cells and analyze pTregs in mice.

IgA in human health and diseases: Potential regulator of commensal microbiota.

Gut microbiota has extensive and tremendous impacts on human physiology and pathology. The regulation of microbiota is therefore a cardinal problem for the mutualistic relationship, as both microbial overgrowth and excessive immune reactions toward them could potentially be detrimental to host homeostasis. Growing evidence suggests that IgA, the most dominant secretory immunoglobulin in the intestine, regulates the colonization of commensal microbiota, and consequently, the microbiota-mediated intestinal and extra-intestinal diseases. In this review, we discuss the interactions between IgA and gut microbiota particularly relevant to human pathophysiology. We review current knowledge about how IgA regulates gut microbiota in humans and about the molecular mechanisms behind this interaction. We further discuss the potential role of IgA in regulating human diseases by extrapolating experimental findings, suggesting that IgA can be a future therapeutic strategy that functionally modulates gut microbiota.

A Japanese Herbal Formula, Daikenchuto, Alleviates Experimental Colitis by Reshaping Microbial Profiles and Enhancing Group 3 Innate Lymphoid Cells.

Daikenchuto (DKT) is one of the most widely used Japanese herbal formulae for various gastrointestinal disorders. It consists of Zanthoxylum Fructus (Japanese pepper), Zingiberis Siccatum Rhizoma (processed ginger), Ginseng radix, and maltose powder. However, the use of DKT in clinical settings is still controversial due to the limited molecular evidence and largely unknown therapeutic effects. Here, we investigated the anti-inflammatory actions of DKT in the dextran sodium sulfate (DSS)-induced colitis model in mice. We observed that DKT remarkably attenuated the severity of experimental colitis while maintaining the members of the symbiotic microbiota such as family Lactobacillaceae and increasing levels of propionate, an immunomodulatory microbial metabolite, in the colon. DKT also protected colonic epithelial integrity by upregulating the fucosyltransferase gene Fut2 and the antimicrobial peptide gene Reg3g. More remarkably, DKT restored the reduced colonic group 3 innate lymphoid cells (ILC3s), mainly RORγthigh-ILC3s, in DSS-induced colitis. We further demonstrated that ILC3-deficient mice showed increased mortality during experimental colitis, suggesting that ILC3s play a protective function on colonic inflammation. These findings demonstrate that DKT possesses anti-inflammatory activity, partly via ILC3 function, to maintain the colonic microenvironment. Our study also provides insights into the molecular basis of herbal medicine effects, promotes more profound mechanistic studies towards herbal formulae and contributes to future drug development.

High-throughput identification and quantification of single bacterial cells in the microbiota.

The bacterial microbiota works as a community that consists of many individual organisms, i.e., cells. To fully understand the function of bacterial microbiota, individual cells must be identified; however, it is difficult with current techniques. Here, we develop a method, Barcoding Bacteria for Identification and Quantification (BarBIQ), which classifies single bacterial cells into taxa-named herein cell-based operational taxonomy units (cOTUs)-based on cellularly barcoded 16S rRNA sequences with single-base accuracy, and quantifies the cell number for each cOTU in the microbiota in a high-throughput manner. We apply BarBIQ to murine cecal microbiotas and quantify in total 3.4 × 105 bacterial cells containing 810 cOTUs. Interestingly, we find location-dependent global differences in the cecal microbiota depending on the dietary vitamin A deficiency, and more differentially abundant cOTUs at the proximal location than the distal location. Importantly, these location differences are not clearly shown by conventional 16S rRNA gene-amplicon sequencing methods, which quantify the 16S rRNA genes, not the cells. Thus, BarBIQ enables microbiota characterization with the identification and quantification of individual constituent bacteria, which is a cornerstone for microbiota studies.

Reciprocal regulation of IgA and the gut microbiota: a key mutualism in the intestine.

The mammalian intestine is home to trillions of microbes, and their colonization contributes to host physiology through the production of indispensable metabolites and competition against pathogens. However, it is also important to balance this symbiotic relationship, as overgrowth and translocation of microbes could trigger a fatal infection. IgA is the major immunoglobulin class produced and secreted in the intestine and is considered to play a pivotal role in maintaining homeostasis. In this review, we summarize recent studies exploring the interactions between IgA and the gut microbiota and explain how different types of IgA could coexist to regulate the gut microbiota. In particular, we discuss two important aspects of IgA in controlling the gut microbes: function and specificity. Differences in these two aspects appear attributable to how IgA is induced and are associated with the functions of IgA as well. Together, our review delineates a recent understanding of IgA-microbiome interactions and proposes a future direction to clarify its complexity.

Acetate differentially regulates IgA reactivity to commensal bacteria.

The balance between bacterial colonization and its containment in the intestine is indispensable for the symbiotic relationship between humans and their bacteria. One component to maintain homeostasis at the mucosal surfaces is immunoglobulin A (IgA), the most abundant immunoglobulin in mammals1,2. Several studies have revealed important characteristics of poly-reactive IgA3,4, which is produced naturally without commensal bacteria. Considering the dynamic changes within the gut environment, however, it remains uncertain how the commensal-reactive IgA pool is shaped and how such IgA affects the microbial community. Here we show that acetate-one of the major gut microbial metabolites-not only increases the production of IgA in the colon, but also alters the capacity of the IgA pool to bind to specific microorganisms including Enterobacterales. Induction of commensal-reactive IgA and changes in the IgA repertoire by acetate were observed in mice monocolonized with Escherichia coli, which belongs to Enterobacterales, but not with the major commensal Bacteroides thetaiotaomicron, which suggests that acetate directs selective IgA binding to certain microorganisms. Mechanistically, acetate orchestrated the interactions between epithelial and immune cells, induced microbially stimulated CD4 T cells to support T-cell-dependent IgA production and, as a consequence, altered the localization of these bacteria within the colon. Collectively, we identified a role for gut microbial metabolites in the regulation of differential IgA production to maintain mucosal homeostasis.

CD8+ regulatory T cells are critical in prevention of autoimmune-mediated diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic ß-cells are destroyed. Intestinal helminths can cause asymptomatic chronic and immunosuppressive infections and suppress disease in rodent models of T1D. However, the underlying regulatory mechanisms for this protection are unclear. Here, we report that CD8+ regulatory T (Treg) cells prevent the onset of streptozotocin -induced diabetes by a rodent intestinal nematode. Trehalose derived from nematodes affects the intestinal microbiota and increases the abundance of Ruminococcus spp., resulting in the induction of CD8+ Treg cells. Furthermore, trehalose has therapeutic effects on both streptozotocin-induced diabetes and in the NOD mouse model of T1D. In addition, compared with healthy volunteers, patients with T1D have fewer CD8+ Treg cells, and the abundance of intestinal Ruminococcus positively correlates with the number of CD8+ Treg cells in humans.

Recurrent cardiovascular events in patients with newly diagnosed acute coronary syndrome: Influence of diabetes and its management with medication.

AIMS: The effects of type 2 diabetes mellitus (T2DM) medications on secondary prevention after acute coronary syndrome (ACS) remain unclear. We evaluated recurrent cardiovascular disease (CVD) after primary diagnosis of ACS in T2DM patients. METHODS: This retrospective cohort study included 569 patients with newly diagnosed ACS from 2007 to 2012. The endpoint was recurrent CVD up to a five-year maximum follow-up until 2016. Kaplan-Meier analysis and Cox proportional hazard regressions were performed to examine the association between T2DM diagnosis, different antidiabetic drugs, and recurrent CVD. RESULTS: Among 569 patients, 198 had T2DM. The mean follow-up was 1540 (interquartile range, 864-2157) days. Patients with diabetes showed higher risk of recurrent cardiovascular event compared with those without (P = 0.004). Patients with diabetes treated with metformin (65 patients) showed longer event-free survival, compared with those on other antidiabetic medications (P = 0.005). Multivariable analysis confirmed a reduced risk of recurrent CVD associated with metformin (hazard ratio, 0.33; 95% confidence interval, 0.12-0.91), while lower hemoglobin A1c levels on admission were not associated with better CVD outcomes. CONCLUSIONS: T2DM increases risk of recurrent CVD after first ACS episode regardless of glycemic control on admission, while use of metformin may reduce recurrence.

Understanding the immune signature fingerprint of peritoneal dialysis-related peritonitis.

Although acute peritonitis is a common and severe complication associated with peritoneal dialysis, the culture-based test used as the diagnostic criterion for this disease is often too slow to allow appropriate point-of-care diagnosis of specific bacterial infection. To address this problem, Zhang et al. report the efficacy of a novel set of immune biomarkers derived from a machine-learning algorithm applied to patient data. This fingerprint could predict major pathogenic causes of peritonitis.

Comparison of cardiovascular disease risk associated with 3 lipid measures in Japanese adults.

BACKGROUND: To assess dyslipidemia, measurement of low-density lipoprotein cholesterol via either Friedewald equation (LDL-F) or direct assay (LDL-D), and non-high-density lipoprotein cholesterol (non-HDL-C) are recommended with some guidelines showing preference to direct over calculated measurements. However, direct comparisons of their respective associations with cardiovascular disease (CVD) risk are currently unavailable. OBJECTIVE: In this study, we evaluated the clinical effectiveness of LDL-F and non-HDL-C vs LDL-D and their associations with CVD. METHODS: This retrospective cohort study comprised apparently healthy Japanese individuals who underwent an annual health check-up between 2005 and 2007 and completed a 5-year follow-up visit. The incidence of CVD, including coronary and cerebrovascular diseases, during a 5-year follow-up period was evaluated using multivariate logistic regression. RESULTS: At baseline, 26,739 participants (mean age, 47 years; 49.0% men) were enrolled, and 292 (1.09%) incidents of CVD were identified at follow-up. Baseline LDL-F, LDL-D, and non-HDL-C were all significantly associated with CVD, although the effect appeared higher for LDL-F, particularly for coronary heart disease. Increased risks of CVD were observed for high LDL-F (≥130 mg/dL), despite being categorized into the lower LDL category based on LDL-D (odds ratio [OR], 1.85; 95% confidence interval [CI], 1.19-2.87) and non-HDL-C (OR, 1.75; 95% CI, 1.22-2.52). Without high LDL-F, no CVD associations were found for high LDL-D (P = .62) or non-HDL-C (P = .93). CONCLUSION: Despite growing availability of direct assays and increasing evidence of non-HDL-C utility, the Friedewald equation may offer better clinical utility for CVD prevention, especially in the screening of apparently healthy individuals.

Friction-mediated dynamic disordering of phospholipid membrane by mechanical motions of photoresponsive molecular glue: activation of ion permeation.

A water-soluble photoresponsive molecular glue, Azo-(18)Glue, consisting of a photochromic azobenzene core and two adhesive dendritic wedges with a total of 18 peripheral guanidinium ion (Gu(+)) pendants tightly adheres to the surface of a phospholipid membrane, even in buffer, via a multivalent salt-bridge formation with phosphate anions. A photomechanical motion of adhering Azo-(18)Glue possibly gives rise to dynamic structural disordering of the phospholipid membrane and activates transmembrane ion permeation. In sharp contrast, no activation of ion permeation results when poorly adhesive Azo-(6)Glue carrying only six Gu(+) pendants is used in place of Azo-(18)Glue.

Treatment of autoimmune pancreatitis with the anecdotes of the first report.

The first case that led researchers to put forward a new concept of autoimmune pancreatitis (AIP) was treated with steroids by gastroenterologists in Tokyo Women's Medical University. It is important to differentiate AIP from pancreatic cancer before treatment with steroids is started. Today, steroids are standard therapy for AIP worldwide. In the Japanese consensus guidelines, steroid therapy is indicated for symptomatic AIP. After management of glucose levels and obstructive jaundice, oral prednisolone is initiated at 0.6 mg/kg/day for 2-4 weeks and is gradually tapered to a maintenance dose of 2.5-5 mg/day over 2-3 months. To prevent relapse, maintenance therapy with low-dose prednisolone is used. For relapsed AIP, readministration or increased doses of steroids are effective. The presence of proximal bile duct stenosis and elevated serum IgG4 levels may be predictive of relapse of AIP. It is necessary to verify the validity of the Japanese regimen of steroid therapy for AIP. The necessity, drugs, and duration of maintenance therapy for AIP need to be clarified by prospective studies.

Cancer metastasis is accelerated through immunosuppression during Snail-induced EMT of cancer cells.

Epithelial-mesenchymal transition (EMT) is a key step toward cancer metastasis, and Snail is a major transcription factor governing EMT. Here, we demonstrate that Snail-induced EMT accelerates cancer metastasis through not only enhanced invasion but also induction of immunosuppression. Murine and human melanoma cells with typical EMT features after snail transduction induced regulatory T cells and impaired dendritic cells in vitro and in vivo partly through TSP1 production. Although Snail(+) melanoma did not respond to immunotherapy, intratumoral injection with snail-specific siRNA or anti-TSP1 monoclonal antibody significantly inhibited tumor growth and metastasis following increase of tumor-specific tumor-infiltrating lymphocytes and systemic immune responses. These results suggest that inhibition of Snail-induced EMT could simultaneously suppress both tumor metastasis and immunosuppression in cancer patients.

'In a certain sense Dr. William Osler has been my best mentor'. An interview with Prof. Tadashi Takeuchi by Martin E. Fernandez-Zapico.

Tadashi Takeuchi is a Professor Emeritus of the Tokyo Women's Hospital University, one of the founders of the Japan Pancreas Society and first Editor-in-Chief of its official journal. His research contributed enormously to the understanding of the role of gastrointestinal hormones in pancreatic physiology as well as disease. In this interview, Professor Takeuchi discusses the importance of mentorship during career development.