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.

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.

A defined commensal consortium elicits CD8 T cells and anti-cancer immunity.

There is a growing appreciation for the importance of the gut microbiota as a therapeutic target in various diseases. However, there are only a handful of known commensal strains that can potentially be used to manipulate host physiological functions. Here we isolate a consortium of 11 bacterial strains from healthy human donor faeces that is capable of robustly inducing interferon-γ-producing CD8 T cells in the intestine. These 11 strains act together to mediate the induction without causing inflammation in a manner that is dependent on CD103+ dendritic cells and major histocompatibility (MHC) class Ia molecules. Colonization of mice with the 11-strain mixture enhances both host resistance against Listeria monocytogenes infection and the therapeutic efficacy of immune checkpoint inhibitors in syngeneic tumour models. The 11 strains primarily represent rare, low-abundance components of the human microbiome, and thus have great potential as broadly effective biotherapeutics.

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.

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.

Clinical impact of pre-transplant gut microbial diversity on outcomes of allogeneic hematopoietic stem cell transplantation.

Post-transplant microbial diversity in the gastrointestinal tract is closely associated with clinical outcomes following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, little is known about the impact of the fecal microbiota before allo-HSCT. We analyzed fecal samples approximately 2 weeks before conditioning among 107 allo-HSCT recipients between 2013 and 2015. Microbial analysis was performed using 16S rRNA gene sequencing. Operational taxonomic unit-based microbial diversity was estimated by calculating the Shannon index. Patients were classified into three groups based on the diversity index: low (<2), intermediate (2, 3), and high (>3) diversity (18 (16.8%), 48 (44.9%), and 41 (38.3%) patients, respectively). There were no significant differences in the 20-month overall survival, cumulative incidence of relapse, and non-relapse mortality among three groups. The cumulative incidence of grade II to IV acute graft-versus-host disease (aGVHD) was similar among the three groups (low 55.6%; intermediate 35.4%; high 48.8%, p = 0.339, at day 100). Furthermore, we found no differences in the cumulative incidence of grade II to IV acute gastrointestinal GVHD among the three groups (low 38.9%; intermediate 21.3%; high 24.4%, p = 0.778, at day 100). Regarding the composition of microbiota before allo-HSCT, aGVHD patients showed a significantly higher abundance of phylum Firmicutes (p < 0.01) and a lower tendency for Bacteroidetes (p = 0.106) than non-aGVHD patients. Maintenance of Bacteroidetes throughout allo-HSCT may be a strategy to prevent aGVHD.

Bifidobacterium-Rich Fecal Donor May Be a Positive Predictor for Successful Fecal Microbiota Transplantation in Patients with Irritable Bowel Syndrome.

BACKGROUND/AIMS: Dysbiosis is associated with various systemic disorders including irritable bowel syndrome (IBS). Fecal microbiota transplantation (FMT) might restore intestinal microbial balance. The study aimed to determine the safety and efficacy of FMT in IBS patients, as well as also positive predictors for FMT. METHODS: This was a single-arm, open-label study. Eligible patients were diagnosed based on Rome III Diagnostic Criteria. Fecal materials were administered to the patient via colonoscopy. The primary end point was a change in the Bristol stool form scale at 4 weeks after FMT. Recovery to types 3-4 was considered a clinical response. The secondary end point was a change in intestinal microbiota and psychological status using the Hamilton Rating Scale. RESULTS: Ten patients were enrolled. Six patients achieved a clinical response. The diversity of patients 4 weeks after FMT increased significantly compared with patients before FMT, and that of responding patients was significantly higher than non-responder patients. The abundance of Bifidobacterium in effective donors was significantly higher than in ineffective donors and patients. Psychological status of all patients was significantly improved after FMT. CONCLUSIONS: FMT for patients with IBS is safe, and relatively effective. Bifidobacterium-rich fecal donor may be a positive predictor for successful FMT. Key Summary: (1) Dysbiosis is associated with various gastrointestinal disorders including IBS. (2) FMT has potential to restore intestinal microbial balance. (3) We showed that FMT improved stool form and psychological status of IBS patients. (4) Bifidobacterium-rich donor efficiently induced symbiosis in IBS patients.

Clinical Efficacy and Safety of Oral Qing-Dai in Patients with Ulcerative Colitis: A Single-Center Open-Label Prospective Study.

BACKGROUND/AIMS: Chinese herbal medicine Qing-Dai (also known as indigo naturalis) has been used to treat various inflammatory conditions. However, not much has been studied about the use of oral Qing-Dai in the treatment for ulcerative colitis (UC) patients. Studies exploring alternative treatments for UC are of considerable interest. In this study, we aimed at prospectively evaluating the safety and efficacy of Qing-Dai for UC patients. METHODS: The open-label, prospective pilot study was conducted at Keio University Hospital. A total of 20 patients with moderate UC activity were enrolled. Oral Qing-Dai in capsule form was taken twice a day (daily dose, 2 g) for 8 weeks. RESULTS: At week 8, the rates of clinical response, clinical remission, and mucosal healing were 72, 33, and 61%, respectively. The clinical and endoscopic scores, CRP levels, and fecal occult blood results were also significantly improved. We observed 2 patients with mild liver dysfunction; 1 patient discontinued due to infectious colitis and 1 patient discontinued due to mild nausea. CONCLUSION: This is the first prospective study indicating that oral Qing-Dai is effective for inducing remission in patients with moderate UC activity and can be tolerated. Thus, Qing-Dai may be considered an alternative treatment for patients, although further investigation is warranted.

Ulcerative colitis endoscopic index of severity is associated with long-term prognosis in ulcerative colitis patients treated with infliximab.

BACKGROUND AND AIM: Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is characterized by periods of remission and episodes of relapse. Mucosal healing is an emerging therapeutic target in UC and various scoring systems have been used. The UC endoscopic index of severity (UCEIS) is the only validated endoscopic index at present, with minimum interobserver variation. Correlation of UCEIS scores after treatment and clinical outcomes of UC has not been examined. In the present study, we aimed to evaluate the usefulness of UCEIS after treatment with infliximab. METHODS: The medical records of 82 UC patients, treated with infliximab at Keio University Hospital between October 2010 and July 2013, were reviewed retrospectively. Endoscopic findings were evaluated based on the UCEIS. RESULTS: Mean pre-therapeutic UCEIS score was 5.1. Pre-therapeutic UCEIS scores were not associated with short-term outcomes. Forty-five patients underwent colonoscopy at 3-12 months after starting treatment; mean post-therapeutic UCEIS score was 2.4, with a score of 0-1 in 16 (35.6%) patients, 2-4 in 19 (42.2%) patients, and 5-8 in 10 (22.2%) patients. Importantly, a post-therapeutic UCEIS score of 0 or 1 after treatment was associated with a favorable long-term outcome. CONCLUSION: UCEIS score is a useful instrument for evaluating endoscopic improvement in UC patients treated with infliximab, and mucosal healing may be defined with a UCEIS score of 0 or 1.

Dysregulation of the Intestinal Microbiome in Patients With Haploinsufficiency of A20.

INTRODUCTION: Haploinsufficiency of A20 (HA20) is a form of inborn errors of immunity (IEI). IEIs are genetically occurring diseases, some of which cause intestinal dysbiosis. Due to the dysregulation of regulatory T cells (Tregs) observed in patients with HA20, gut dysbiosis was associated with Tregs in intestinal lamina propria. METHODS: Stool samples were obtained from 16 patients with HA20 and 15 of their family members. Infant samples and/or samples with recent antibiotics use were excluded; hence, 26 samples from 13 patients and 13 family members were analyzed. The 16S sequencing process was conducted to assess the microbial composition of samples. Combined with clinical information, the relationship between the microbiome and the disease activity was statistically analyzed. RESULTS: The composition of gut microbiota in patients with HA20 was disturbed compared with that in healthy family members. Age, disease severity, and use of immunosuppressants corresponded to dysbiosis. However, other explanatory factors, such as abdominal symptoms and probiotic treatment, were not associated. The overall composition at the phylum level was stable, but some genera were significantly increased or decreased. Furthermore, among the seven operational taxonomic units (OTUs) that increased, two OTUs, Streptococcus mutans and Lactobacillus salivarius, considerably increased in patients with autoantibodies than those without autoantibodies. DISCUSSION: Detailed interaction on intestinal epithelium remains unknown; the relationship between the disease and stool composition change helps us understand the mechanism of an immunological reaction to microorganisms.

Hematopoietic Cell Transplantation Rescues Inflammatory Bowel Disease and Dysbiosis of Gut Microbiota in XIAP Deficiency.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) deficiency is an infrequent inborn error of immunity that is often associated with refractory inflammatory bowel disease (IBD). The natural course of XIAP deficiency is typically associated with poor prognosis, and hematopoietic cell transplantation (HCT) is the only curative treatment. OBJECTIVE: To study (1) the effect of HCT on patients with XIAP deficiency undergoing HCT, (2) the status of XIAP deficiency-associated IBD after HCT, and (3) the gut microbiota of XIAP deficiency-associated IBD before and after HCT. METHODS: A nationwide survey of patients with XIAP deficiency was conducted. A spreadsheet questionnaire was collected from the physicians. Feces samples collected from the patients before and after HCT and their healthy family members were analyzed. RESULTS: Twenty-six patients with XIAP deficiency underwent HCT by the end of March 2020, and 22 patients (84.6%) survived. All the survivors underwent a fludarabine-based reduced-intensity condition regimen. Acute graft-versus-host disease was observed in 17 patients (65.4%). Nineteen patients experienced refractory IBD before undergoing HCT. IBD improved remarkably after HCT. After HCT, the colonoscopic and pathological symptoms were restored to normal, and the pediatric ulcerative colitis activity index improved significantly. Gut microbiota indicated dysbiosis before HCT; however, it was improved to resemble that of the healthy family members after HCT. CONCLUSIONS: This study revealed that HCT has a favorable outcome for XIAP deficiency. HCT rescues gut inflammation and dysbiosis in patients with XIAP deficiency.

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.

Fecal microbiota transplantation for recurrent Clostridium difficile infection in a patient with ulcerative colitis.

Fecal microbiota transplantation (FMT) has been reported as a safe and effective therapy in patients with refractory and recurrent Clostridium difficile infection (CDI). FMT has also been reported as a promising therapy in patients with ulcerative colitis (UC). Both, CDI and UC, are believed to be caused by dysbiosis, such as altered compositions or decreased diversity of the intestinal microbiota. This report describes a patient with UC in remission with a second recurrent episode of CDI, who was treated with FMT. A single FMT performed via colonoscopy completely resolved the patient's diarrhea and eradicated C. difficile bacteriologically without any severe complications. Molecular biological analysis of the patient's fecal microbiota showed that FMT could dramatically change the altered composition of intestinal microbiota and restore its diversity. Despite the restoration of the intestinal microbiota, FMT could not prevent a relapse of UC in this patient. However, it improved the intestinal symptoms of CDI and could prevent further recurrences of CDI.

Single fecal microbiota transplantation failed to change intestinal microbiota and had limited effectiveness against ulcerative colitis in Japanese patients.

BACKGROUND/AIMS: Recent developments in analytical techniques including next-generation sequencing have clarified the correlation between intestinal microbiota and inflammatory bowel disease. Fecal microbiota transplantation (FMT) for patients with ulcerative colitis (UC) is proposed as a potential approach to resolving their dysbiosis; however, its safety and efficacy have not been confirmed. This single-arm, open-label, non-randomized study aimed to evaluate the safety and efficacy of FMT for Japanese patients with UC as the first registered clinical trial in Japan. METHODS: We enrolled 10 patients with active UC despite medical therapy. The donors were the patients' relatives and were carefully screened for infectious diseases. Fecal material was administered via colonoscopy, and the primary endpoint was the presence or absence of serious adverse events related to FMT. The secondary endpoint was a change in partial Mayo score at 12 weeks post-FMT. Scores ≤2 were considered a clinical response. Fecal samples were collected to follow changes in gut microbiota, while extracted complementary DNA were analyzed by a next-generation sequencer. We obtained written informed consent from all patients and donors. This study was approved by our Institutional Review Board and is registered in the University hospital Medical Information Network (UMIN) Clinical Trials Registry (UMIN 000012814). RESULTS: Five patients with moderate disease and five with severe disease were enrolled. No severe adverse effects were observed. One patient achieved clinical response; however, none of the patients' microbiota diversity recovered to the donor levels. CONCLUSIONS: The use of single FMT for UC was safe; however, we failed to show its clinical efficacy and potential to change the intestinal microbiota.

A Single Species of Clostridium Subcluster XIVa Decreased in Ulcerative Colitis Patients.

BACKGROUND: Imbalance of the intestinal microbiota is associated with gastrointestinal disease and autoimmune disease and metabolic syndrome. Analysis of the intestinal microbiota has recently progressed, and the association with inflammatory bowel disease has been reported at the species level. Such findings suggest that the recovery of homeostasis in the intestinal microbiota could cure inflammatory bowel disease. We aimed to search new probiotic candidates for inflammatory bowel disease through translational research by analysis of ulcerative colitis (UC) patients' intestinal microbiota and clarify the effects of them on inflammation. Here, we focused on Fusicatenibacter saccharivorans, which belongs to Clostridium subcluster XIVa and was successfully isolated and cultured in 2013. We analyzed the association of F. saccharivorans to UC patients' activity and inflammation for the first time. METHODS: Feces from UC patients and healthy controls were analyzed by 16S ribosomal RNA gene sequences. F. saccharivorans was administered to murine colitis model. Colitic lamina propria mononuclear cells from UC patients and mice were stimulated with F. saccharivorans. RESULTS: The whole fecal bacteria in active UC patients were less than that in quiescent UC patients. Furthermore, F. saccharivorans was decreased in active UC patients and increased in quiescent. The administration of F. saccharivorans improved murine colitis. F. saccharivorans induced interleukin 10 production by lamina propria mononuclear cells from not only colitis model mice but also UC patients. CONCLUSIONS: F. saccharivorans decreased in correlation to UC activity and suppresses intestinal inflammation. These results suggest that F. saccharivorans could lead to a novel UC treatment.

Inhibition of Dectin-1 Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine.

Dectin-1, the receptor for ß-glucans, protects the host against fungal infection; however, its role in intestinal immunity is incompletely understood. We found that Dectin-1-deficient (Clec7a(-/-)) mice were refractory to both dextran sodium sulfate (DSS)- and CD45RB(high)CD4(+) T cell-induced colitis, and that this resistance was associated with an increase in regulatory T (Treg) cells. The proportion of lactobacilli, especially Lactobacillus murinus, in the commensal microflora was increased in Clec7a(-/-) mouse colons, and accompanied by a decrease in antimicrobial peptides induced by Dectin-1 signaling. L. murinus colonization increased Treg cells in the colon. Oral administration of laminarin, a Dectin-1 antagonist, suppressed the development of DSS-colitis, associated with an increase of L. murinus and Treg cells. Human patients with inflammatory bowel disease were found to have a decreased proportion of closely related Lactobacillus species. These observations suggest that Dectin-1 regulates the homeostasis of intestinal immunity by controlling Treg cell differentiation through modification of microbiota.

Classical Th1 cells obtain colitogenicity by co-existence of RORγt-expressing T cells in experimental colitis.

BACKGROUND: Both Th1 and Th17 cell types are involved in the pathogenesis of chronic intestinal inflammation. We recently demonstrated that retinoid-related orphan receptor gamma t (RORγt)-expressing Th17 cells are progenitor cells for alternative Th1 cells, which have the potential to induce colitis. However, the involvement of classical Th1 (cTh1) cells generated directly from naive T cells without RORγt expression in the pathogenesis of colitis remains poorly understood. METHODS: We performed a series of in vivo experiments using a murine chronic colitis model induced by adoptive transfer of splenic CD4CD45RB(high) T cells obtained from wild-type, RORγt(gfp/gfp), or RORγt(gfp/gfp) mice into RAG-2(-/-) mice. RESULTS: RAG-2(-/-) mice receiving transfer of in vitro-manipulated RORγt(gfp/gfp) Th1 cells developed colitis. RAG-2(-/-) mice co-transferred with splenic CD4CD45RB(high) T cells obtained from wild-type mice and RORγt(gfp/gfp) mice developed colitis with a significant increase in RORγt cTh1 cell numbers when compared with noncolitic mice transferred with splenic CD4CD45RB(high) T cells obtained from RORγt(gfp/gfp) mice. Furthermore, RAG-2(-/-) mice transferred with in vivo-manipulated RORγt(gfp/gfp) cTh1 cells developed colitis with a significant increase in RORγt(gfp/gfp) cTh1 cell numbers. CONCLUSIONS: These findings indicate that both alternative Th1 cells and cTh1 cells have the potential to be colitogenic in an adaptive transfer model. The development of cTh1 cells was dependent on the co-existence of RORγt-expressing T cells, suggesting a critical role for the interactions of these cell types in the development of chronic intestinal inflammation.