Periportal macrophages protect against commensal-driven liver inflammation.

The liver is the main gateway from the gut, and the unidirectional sinusoidal flow from portal to central veins constitutes heterogenous zones, including the periportal vein (PV) and the pericentral vein zones1-5. However, functional differences in the immune system in each zone remain poorly understood. Here intravital imaging revealed that inflammatory responses are suppressed in PV zones. Zone-specific single-cell transcriptomics detected a subset of immunosuppressive macrophages enriched in PV zones that express high levels of interleukin-10 and Marco, a scavenger receptor that sequesters pro-inflammatory pathogen-associated molecular patterns and damage-associated molecular patterns, and consequently suppress immune responses. Induction of Marco+ immunosuppressive macrophages depended on gut microbiota. In particular, a specific bacterial family, Odoribacteraceae, was identified to induce this macrophage subset through its postbiotic isoallolithocholic acid. Intestinal barrier leakage resulted in inflammation in PV zones, which was markedly augmented in Marco-deficient conditions. Chronic liver inflammatory diseases such as primary sclerosing cholangitis (PSC) and non-alcoholic steatohepatitis (NASH) showed decreased numbers of Marco+ macrophages. Functional ablation of Marco+ macrophages led to PSC-like inflammatory phenotypes related to colitis and exacerbated steatosis in NASH in animal experimental models. Collectively, commensal bacteria induce Marco+ immunosuppressive macrophages, which consequently limit excessive inflammation at the gateway of the liver. Failure of this self-limiting system promotes hepatic inflammatory disorders such as PSC and NASH.

Rationally-defined microbial consortia suppress multidrug-resistant proinflammatory Enterobacteriaceae via ecological control.

Persistent colonization and outgrowth of pathogenic organisms in the intestine may occur due to long-term antibiotic usage or inflammatory conditions, which perpetuate dysregulated immunity and tissue damage1,2. Gram-negative Enterobacteriaceae gut pathobionts are particularly recalcitrant to conventional antibiotic treatment3,4, though an emerging body of evidence suggests that manipulation of the commensal microbiota may be a practical alternative therapeutic strategy5-7. In this study, we rationally isolated and down-selected commensal bacterial consortia from healthy human stool samples capable of strongly and specifically suppressing intestinal Enterobacteriaceae. One of the elaborated consortia, consisting of 18 commensal strains, effectively controlled ecological niches by regulating gluconate availability, thereby reestablishing colonization resistance and alleviating antibiotic-resistant Klebsiella-driven intestinal inflammation in mice. Harnessing these microbial activities in the form of live bacterial therapeutics may represent a promising solution to combat the growing threat of proinflammatory, antimicrobial-resistant bacterial infection.

Gut insulin action protects from hepatocarcinogenesis in diabetic mice comorbid with nonalcoholic steatohepatitis.

Diabetes is known to increase the risk of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Here we treat male STAM (STelic Animal Model) mice, which develop diabetes, NASH and HCC associated with dysbiosis upon low-dose streptozotocin and high-fat diet (HFD), with insulin or phlorizin. Although both treatments ameliorate hyperglycemia and NASH, insulin treatment alone lead to suppression of HCC accompanied by improvement of dysbiosis and restoration of antimicrobial peptide production. There are some similarities in changes of microflora from insulin-treated patients comorbid with diabetes and NASH. Insulin treatment, however, fails to suppress HCC in the male STAM mice lacking insulin receptor specifically in intestinal epithelial cells (ieIRKO), which show dysbiosis and impaired gut barrier function. Furthermore, male ieIRKO mice are prone to develop HCC merely on HFD. These data suggest that impaired gut insulin signaling increases the risk of HCC, which can be countered by restoration of insulin action in diabetes.

D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease.

BACKGROUND & AIMS: D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression. METHODS: The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model. RESULTS: The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K. pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E. coli and generation of antibiotic-resistant strains. CONCLUSIONS: D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Diet-mediated constitutive induction of novel IL-4+ ILC2 cells maintains intestinal homeostasis in mice.

Group 2 innate lymphoid cells (ILC2s) expressing IL-5 and IL-13 are localized at various mucosal tissues and play critical roles in the induction of type 2 inflammation, response to helminth infection, and tissue repair. Here, we reveal a unique ILC2 subset in the mouse intestine that constitutively expresses IL-4 together with GATA3, ST2, KLRG1, IL-17RB, and IL-5. In this subset, IL-4 expression is regulated by mechanisms similar to but distinct from those observed in T cells and is partly affected by IL-25 signaling. Although the absence of the microbiota had marginal effects, feeding mice with a vitamin B1-deficient diet compromised the number of intestinal IL-4+ ILC2s. The decrease in the number of IL-4+ ILC2s caused by the vitamin B1 deficiency was accompanied by a reduction in IL-25-producing tuft cells. Our findings reveal that dietary vitamin B1 plays a critical role in maintaining interaction between tuft cells and IL-4+ ILC2s, a previously uncharacterized immune cell population that may contribute to maintaining intestinal homeostasis.

Centenarians have a diverse gut virome with the potential to modulate metabolism and promote healthy lifespan.

Distinct gut microbiome ecology may be implicated in the prevention of aging-related diseases as it influences systemic immune function and resistance to infections. Yet, the viral component of the microbiome throughout different stages in life remains unexplored. Here we present a characterization of the centenarian gut virome using previously published metagenomes from 195 individuals from Japan and Sardinia. Compared with gut viromes of younger adults (>18 yr) and older individuals (>60 yr), centenarians had a more diverse virome including previously undescribed viral genera, such as viruses associated with Clostridia. A population shift towards higher lytic activity was also observed. Finally, we investigated phage-encoded auxiliary functions that influence bacterial physiology, which revealed an enrichment of genes supporting key steps in sulfate metabolic pathways. Phage and bacterial members of the centenarian microbiome displayed an increased potential for converting methionine to homocysteine, sulfate to sulfide and taurine to sulfide. A greater metabolic output of microbial hydrogen sulfide in centenarians may in turn support mucosal integrity and resistance to pathobionts.

An old model with new insights: endogenous retroviruses drive the evolvement toward ASD susceptibility and hijack transcription machinery during development.

The BTBR T+Itpr3tf/J (BTBR/J) strain is one of the most valid models of idiopathic autism, serving as a potent forward genetics tool to dissect the complexity of autism. We found that a sister strain with an intact corpus callosum, BTBR TF/ArtRbrc (BTBR/R), showed more prominent autism core symptoms but moderate ultrasonic communication/normal hippocampus-dependent memory, which may mimic autism in the high functioning spectrum. Intriguingly, disturbed epigenetic silencing mechanism leads to hyperactive endogenous retrovirus (ERV), a mobile genetic element of ancient retroviral infection, which increases de novo copy number variation (CNV) formation in the two BTBR strains. This feature makes the BTBR strain a still evolving multiple-loci model toward higher ASD susceptibility. Furthermore, active ERV, analogous to virus infection, evades the integrated stress response (ISR) of host defense and hijacks the transcriptional machinery during embryonic development in the BTBR strains. These results suggest dual roles of ERV in the pathogenesis of ASD, driving host genome evolution at a long-term scale and managing cellular pathways in response to viral infection, which has immediate effects on embryonic development. The wild-type Draxin expression in BTBR/R also makes this substrain a more precise model to investigate the core etiology of autism without the interference of impaired forebrain bundles as in BTBR/J.

Myeloid-like B cells boost emergency myelopoiesis through IL-10 production during infection.

Emergency myelopoiesis (EM) is a hematopoietic response against systemic infections that quickly supplies innate immune cells. As lymphopoiesis is strongly suppressed during EM, the role of lymphocytes in that process has not received much attention. Here, we found that myeloid-like B cells (M-B cells), which express myeloid markers, emerge in the bone marrow (BM) after the induction of EM. M-B cells were mainly derived from pre-B cells and preferentially expressed IL-10, which directly stimulates hematopoietic progenitors to enhance their survival and myeloid-biased differentiation. Indeed, lacking IL-10 in B cells, blocking IL-10 in the BM with a neutralizing antibody, and deleting the IL-10 receptor in hematopoietic progenitors significantly suppressed EM, which failed to clear microbes in a cecal ligation and puncture model. Thus, a distinct B cell subset generated during infection plays a pivotal role in boosting EM, which suggests the on-demand reinforcement of EM by adaptive immune cells.

Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome.

How intestinal microbes regulate metabolic syndrome is incompletely understood. We show that intestinal microbiota protects against development of obesity, metabolic syndrome, and pre-diabetic phenotypes by inducing commensal-specific Th17 cells. High-fat, high-sugar diet promoted metabolic disease by depleting Th17-inducing microbes, and recovery of commensal Th17 cells restored protection. Microbiota-induced Th17 cells afforded protection by regulating lipid absorption across intestinal epithelium in an IL-17-dependent manner. Diet-induced loss of protective Th17 cells was mediated by the presence of sugar. Eliminating sugar from high-fat diets protected mice from obesity and metabolic syndrome in a manner dependent on commensal-specific Th17 cells. Sugar and ILC3 promoted outgrowth of Faecalibaculum rodentium that displaced Th17-inducing microbiota. These results define dietary and microbiota factors posing risk for metabolic syndrome. They also define a microbiota-dependent mechanism for immuno-pathogenicity of dietary sugar and highlight an elaborate interaction between diet, microbiota, and intestinal immunity in regulation of metabolic disorders.

Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation.

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.

A common epigenetic mechanism across different cellular origins underlies systemic immune dysregulation in an idiopathic autism mouse model.

Immune dysregulation plays a key role in the pathogenesis of autism. Changes occurring at the systemic level, from brain inflammation to disturbed innate/adaptive immune in the periphery, are frequently observed in patients with autism; however, the intrinsic mechanisms behind them remain elusive. We hypothesize a common etiology may lie in progenitors of different types underlying widespread immune dysregulation. By single-cell RNA sequencing (sc-RNA seq), we trace the developmental origins of immune dysregulation in a mouse model of idiopathic autism. It is found that both in aorta-gonad-mesonephros (AGM) and yolk sac (YS) progenitors, the dysregulation of HDAC1-mediated epigenetic machinery alters definitive hematopoiesis during embryogenesis and downregulates the expression of the AP-1 complex for microglia development. Subsequently, these changes result in the dysregulation of the immune system, leading to gut dysbiosis and hyperactive microglia in the brain. We further confirm that dysregulated immune profiles are associated with specific microbiota composition, which may serve as a biomarker to identify autism of immune-dysregulated subtypes. Our findings elucidate a shared mechanism for the origin of immune dysregulation from the brain to the gut in autism and provide new insight to dissecting the heterogeneity of autism, as well as the therapeutic potential of targeting immune-dysregulated autism subtypes.

Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians.

Centenarians have a decreased susceptibility to ageing-associated illnesses, chronic inflammation and infectious diseases1-3. 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 3ß-hydroxysteroid dehydrogenase (3ß-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.

Staphylococcus cohnii is a potentially biotherapeutic skin commensal alleviating skin inflammation.

Host-microbe interactions orchestrate skin homeostasis, the dysregulation of which has been implicated in chronic inflammatory conditions such as atopic dermatitis and psoriasis. Here, we show that Staphylococcus cohnii is a skin commensal capable of beneficially inhibiting skin inflammation. We find that Tmem79-/- mice spontaneously develop interleukin-17 (IL-17)-producing T-cell-driven skin inflammation. Comparative skin microbiome analysis reveals that the disease activity index is negatively associated with S. cohnii. Inoculation with S. cohnii strains isolated from either mouse or human skin microbiota significantly prevents and ameliorates dermatitis in Tmem79-/- mice without affecting pathobiont burden. S. cohnii colonization is accompanied by activation of host glucocorticoid-related pathways and induction of anti-inflammatory genes in the skin and is therefore effective at suppressing inflammation in diverse pathobiont-independent dermatitis models, including chemically induced, type 17, and type 2 immune-driven models. As such, S. cohnii strains have great potential as effective live biotherapeutics for skin inflammation.

Prebiotics protect against acute graft-versus-host disease and preserve the gut microbiota in stem cell transplantation.

Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Therefore, management of aGVHD is important for successful transplantation. Mucosal damage and alteration of the gut microbiota after allo-HSCT are key factors in the development of aGVHD. We conducted a prospective study to evaluate the ability of prebiotics, which can alleviate mucosal damage and manipulate the gut microbiota, to mitigate posttransplantation complications, including aGVHD. Resistant starch (RS) and a commercially available prebiotics mixture, GFO, were administered to allo-HSCT recipients from pretransplantation conditioning to day 28 after allo-HSCT. Prebiotic intake mitigated mucosal injury and reduced the incidence of all aGVHD grades combined and of aGVHD grades 2 to 4. The cumulative incidence of skin aGVHD was markedly decreased by prebiotics intake. Furthermore, the gut microbial diversity was well maintained and butyrate-producing bacterial population were preserved by prebiotics intake. In addition, the posttransplantation fecal butyrate concentration was maintained or increased more frequently in the prebiotics group. These observations indicate that prebiotic intake may be an effective strategy for preventing aGVHD in allo-HSCT, thereby improving treatment outcomes and the clinical utility of stem cell transplantation approaches. This study was registered on the University Hospital Medical Information Network (UMIN) clinical trials registry (https://www.umin.ac.jp/ctr/index.htm) as #UMIN000027563.

TH1 cell-inducing Escherichia coli strain identified from the small intestinal mucosa of patients with Crohn's disease.

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 TH1 cells and, to a lesser extent, TH17 cells in the intestine, among which an E. coli strain displayed high potential to induce TH1 cells and intestinal inflammation in a strain-specific manner. The present results indicate that the CD SI mucosa harbors unique pro-inflammatory microbiota, including TH1 cell-inducing E. coli, which could be a potential therapeutic target.

Endogenous murine microbiota member Faecalibaculum rodentium and its human homologue protect from intestinal tumour growth.

The microbiota has been shown to promote intestinal tumourigenesis, but a possible anti-tumourigenic effect has also been postulated. Here, we demonstrate that changes in the microbiota and mucus composition are concomitant with tumourigenesis. We identified two anti-tumourigenic strains of the microbiota-Faecalibaculum rodentium and its human homologue, Holdemanella biformis-that are strongly under-represented during tumourigenesis. Reconstitution of ApcMin/+ or azoxymethane- and dextran sulfate sodium-treated mice with an isolate of F. rodentium (F. PB1) or its metabolic products reduced tumour growth. Both F. PB1 and H. biformis produced short-chain fatty acids that contributed to control protein acetylation and tumour cell proliferation by inhibiting calcineurin and NFATc3 activation in mouse and human settings. We have thus identified endogenous anti-tumourigenic bacterial strains with strong diagnostic, therapeutic and translational potential.

IL-10 produced by macrophages regulates epithelial integrity in the small intestine.

Macrophages (Mϕs) are known to be major producers of the anti-inflammatory cytokine interleukin-10 (IL-10) in the intestine, thus playing an important role in maintaining gastrointestinal homeostasis. Mϕs that reside in the small intestine (SI) have been previously shown to be regulated by dietary antigens, while colonic Mϕs are regulated by the microbiota. However, the role which resident Mϕs play in SI homeostasis has not yet been fully elucidated. Here, we show that SI Mϕs regulate the integrity of the epithelial barrier via secretion of IL-10. We used an animal model of non-steroidal anti-inflammatory drug (NSAID)-induced SI epithelial injury to show that IL-10 is mainly produced by MHCII+ CD64+ Ly6Clow Mϕs early in injury and that it is involved in the restoration of the epithelial barrier. We found that a lack of IL-10, particularly its secretion by Mϕs, compromised the recovery of SI epithelial barrier. IL-10 production by MHCII+ CD64+ Ly6Clow Mϕs in the SI is not regulated by the gut microbiota, hence depletion of the microbiota did not influence epithelial regeneration in the SI. Collectively, these results highlight the critical role IL-10-producing Mϕs play in recovery from intestinal epithelial injury induced by NSAID.

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

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 (TH17) 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 TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 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.