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How bacterial strains within a complex human microbiota collectively shape intestinal T cell homeostasis is not well-understood. Methods that quickly identify effector strains or species that drive specific mucosal T cell phenotypes are needed to define general principles for how the microbiota modulates host immunity. We colonize germ-free mice with defined communities of cultured strains and profile antigen-specific responses directed towards individual strains ex vivo. We find that lamina propria T cells are specific to bacterial strains at the species level and can discriminate between strains of the same species. Ex vivo restimulations consistently identify the strains within complex communities that induce Th17 responses in vivo providing the potential to shape baseline immune tone via community composition. Using an adoptive transfer model of colitis, we find that lamina propria T cells respond to different bacterial strains in conditions of inflammation versus homeostasis. Collectively, our approach represents a novel method for efficiently predicting the relative impact of individual bacterial strains within a complex community and for parsing microbiota-dependent phenotypes into component fractions.SIGNIFICANCEDetermining the mechanisms by which the gut microbiome modulates the host immune system has translational potential for treating or preventing immune mediated disease. A key challenge is identifying the immunogenic bacterial strains in the setting of a complex microbiota. We use a combination of anaerobic culturing, in vitro T cell assays, gnotobiotic mouse models, and ex vivo T cell restimulations to explore the influence of species and strain diversity on the specificity of mucosal T cells. Our approach efficiently predicts the relative impact of individual bacterial strains within a complex community and can be used to parse microbiota-dependent phenotypes into component fractions.

Despite the fundamental role of strain variation in gut microbiota function, the number of unique strains of a species that can stably colonize the human gut is still unknown. In this work, we determine the strain richness of common gut species using thousands of sequenced bacterial isolates and metagenomes. We find that strain richness varies across species, is transferable by fecal microbiota transplantation, and is low in the gut compared to other environments. Therapeutic administration of supraphysiologic numbers of strains per species only temporarily increases recipient strain richness, which subsequently converges back to the population average. These results suggest that properties of the gut ecosystem govern the number of strains of each species colonizing the gut and provide a theoretical framework for strain engraftment and replacement in fecal microbiota transplantation and defined live biotherapeutic products.

Immunoglobulin(Ig) A antibodies are the most abundant antibodies supporting mucosal immune homeostasis and host-microbiota interactions. Driven by gut commensal microbes, IgA-secreting plasma cells (PC) differentiate through T cell-dependent (Td) or T cell independent (Ti) mechanisms. While commensal bacteria within the microbiota are known for their ability to promote IgA, the role of non-bacterial commensal microbes on the induction of IgA remains elusive. Here, we demonstrate that permanent colonization with the protozoan commensal Tritrichomonas musculis (T.mu) promotes T-cell dependent, IgA class-switch recombination and intestinal accumulation of IgA-secreting PC. T.mu colonization specifically drives the expansion of T follicular helper cells and a unique ICOS+ non-Tfh cell population, accompanied by an increase in germinal center B cells. Blockade of ICOS:ICOSL co-stimulation or MHCII-expression on B cells are central for the induction of IgA following colonization by T.mu, implicating a previously underappreciated mode of IgA induction following protozoan commensal colonization. Finally, the commensal T.mu further improves the induction of IgA-secreting plasma cells and their peripheral dissemination, even against non-protozoan, orally ingested antigens, identifying T.mu as natural adjuvant for IgA. Collectively, these findings propose a previously unknown, protozoa-driven mode of IgA induction that supports intestinal immune homeostasis even against non-microbial antigens.

Fecal microbiota transplantation (FMT) has been successfully applied to treat recurrent Clostridium difficile infection in humans, but a precise method to measure which bacterial strains stably engraft in recipients and evaluate their association with clinical outcomes is lacking. We assembled a collection of >1,000 different bacterial strains that were cultured from the fecal samples of 22 FMT donors and recipients. Using our strain collection combined with metagenomic sequencing data from the same samples, we developed a statistical approach named Strainer for the detection and tracking of bacterial strains from metagenomic sequencing data. We applied Strainer to evaluate a cohort of 13 FMT longitudinal clinical interventions and detected stable engraftment of 71% of donor microbiota strains in recipients up to 5 years post-FMT. We found that 80% of recipient gut bacterial strains pre-FMT were eliminated by FMT and that post-FMT the strains present persisted up to 5 years later, together with environmentally acquired strains. Quantification of donor bacterial strain engraftment in recipients independently explained (precision 100%, recall 95%) the clinical outcomes (relapse or success) after initial and repeat FMT. We report a compendium of bacterial species and strains that consistently engraft in recipients over time that could be used in defined live biotherapeutic products as an alternative to FMT. Our analytical framework and Strainer can be applied to systematically evaluate either FMT or defined live bacterial therapeutic studies by quantification of strain engraftment in recipients. Quantification of gut bacterial strains after fecal microbiome transplantation using the Strainer algorithm delineates long-term stable engraftment that explains patient outcomes.

Despite being the most abundantly secreted immunoglobulin isotype, the pattern of reactivity of immunoglobulin A (IgA) antibodies toward each individual’s own gut commensal bacteria still remains elusive. By colonizing germ-free mice with defined commensal bacteria, we found that the binding specificity of bulk fecal and serum IgA toward resident gut bacteria resolves well at the species level and has modest strain-level specificity. IgA hybridomas generated from lamina propria B cells of gnotobiotic mice showed that most IgA clones recognized a single bacterial species, whereas a small portion displayed cross-reactivity. Orally administered hybridoma-produced IgAs still retained bacterial antigen binding capability, implying the potential for a new class of therapeutic antibodies. Species-specific IgAs had a range of strain specificities. Given the distinctive bacterial species and strain composition found in each individual’s gut, our findings suggest the IgA antibody repertoire is shaped uniquely to bind “self” gut bacteria.

A growing body of experimental data suggests that microbes in the gut influence behavior and can alter brain physiology and neurochemistry. Although promising, researchers are only starting to understand the potential of the gut microbiota for use in neurological disease. Recent evidence demonstrated that gastrointestinal activities are linked to mood disorders such as anxiety, depression, and most recently, cognitive functions in age-related neurodegenerative disorders. Studies from our group and others are uncovering new evidence suggesting that the gut microbiota plays a crucial role in the metabolism and bioavailability of certain dietary compounds and synthetic drugs. Based on this evidence, this review article will discuss the implications of the gut microbiota in mechanisms of bioavailability and biotransformation with an emphasis on dietary polyphenol compounds. This will be followed by a survey of ongoing innovative research identifying the ability of individual gut bacteria to enhance the bioavailability of gut-derived, brain-penetrating, bioactive polyphenol metabolites that ultimately influence mechanisms associated with the promotion of resilience against psychological and cognitive impairment in response to stress. Lastly, current research initiatives aimed at promoting the generation of brain bioactive polyphenol metabolites by specialized gut microbes will be discussed, specifically the use of gnotobiotic mice to develop bioengineered second generation probiotics. We propose that leveraging the gut microbial ecosystem to generate brain targeted bioactive metabolites from dietary polyphenols can attenuate lifestyle risk factors and promote resilience against age-related cognitive decline.

ImportanceThe effect of rationally defined nonpathogenic, nontoxigenic, commensal strains of Clostridia on prevention of Clostridioides difficile infection (CDI) is unknown.ObjectiveTo determine the efficacy of VE303, a defined bacterial consortium of 8 strains of commensal Clostridia, in adults at high risk for CDI recurrence. The primary objective was to determine the recommended VE303 dosing for a phase 3 trial.Design, Setting, and ParticipantsPhase 2, randomized, double-blind, placebo-controlled, dose-ranging study conducted from February 2019 to September 2021 at 27 sites in the US and Canada. The study included 79 participants aged 18 years or older who were diagnosed with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence (defined as aged ≥75 years or ≥65 years with ≥1 risk factors: creatinine clearance 2, proton pump inhibitor use, remote [>6 months earlier] CDI history).InterventionsParticipants were randomly assigned to high-dose VE303 (8.0 × 109 colony-forming units [CFUs]) (n = 30), low-dose VE303 (1.6 × 109 CFUs) (n = 27), or placebo capsules (n = 22) orally once daily for 14 days.Main Outcomes and MeasuresThe primary efficacy end point was the proportion of participants with CDI recurrence at 8 weeks using a combined clinical and laboratory definition. The primary efficacy end point was analyzed in 3 prespecified analyses, using successively broader definitions for an on-study CDI recurrence: (1) diarrhea consistent with CDI plus a toxin-positive stool sample; (2) diarrhea consistent with CDI plus a toxin-positive, polymerase chain reaction–positive, or toxigenic culture–positive stool sample; and (3) diarrhea consistent with CDI plus laboratory confirmation or (in the absence of a stool sample) treatment with a CDI-targeted antibiotic.ResultsBaseline characteristics were similar across the high-dose VE303 (n = 29; 1 additional participant excluded from efficacy analysis), low-dose VE303 (n = 27), and placebo (n = 22) groups. The participants’ median age was 63.5 years (range, 24-96); 70.5% were female; and 1.3% were Asian, 1.3% Black, 2.6% Hispanic, and 96.2% White. CDI recurrence rates through week 8 (using the efficacy analysis 3 definition) were 13.8% (4/29) for high-dose VE303, 37.0% (10/27) for low-dose VE303, and 45.5% (10/22) for placebo (P = .006, high-dose VE303 vs placebo).Conclusions and RelevanceAmong adults with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence, high-dose VE303 prevented recurrent CDI compared with placebo. A larger, phase 3 study is needed to confirm these findings.Trial RegistrationClinicalTrials.gov Identifier: NCT03788434

Background and aims: Immune dysregulation caused by SARS-CoV-2 infection is thought to play a pathogenic role in COVID-19. SARS-CoV-2 can infect a variety of host cells, including intestinal epithelial cells. We sought to characterize the role of the gastrointestinal immune system in the pathogenesis of the inflammatory response associated with COVID-19. Methods: We measured cytokines, inflammatory markers, viral RNA, microbiome composition and antibody responses in stool and serum samples from a prospectively enrolled cohort of 44 hospitalized COVID-19 patients. Results: SARS-CoV-2 RNA was detected in stool of 41% of patients and was found more frequently in patients with diarrhea than those without (16[44%] vs 5[19%], p=0.06). Patients who survived had lower median viral genome copies than those who did not (p=0.021). Compared to uninfected controls, COVID-19 patients had higher median fecal levels of IL-8 (166.5 vs 286.5 pg/mg; p=0.05) and lower levels of fecal IL-10 (678 vs 194 pg/mg; p

Medullary thymic epithelial cells (mTECs) induce T cell tolerance in the thymus through the elimination of self-reactive thymocytes. Commensal bacteria are also critical for shaping T cell responses in the gut and distal organs. We previously showed that mice depleted of mTECs (Traf6ΔTEC) generated autoreactive T cells and developed autoimmune hepatitis (AIH). In this report, we found that Toll-like receptor (TLR)-mediated microbial sensing on liver hematopoietic cells and the gut microbiota contributed to AIH development in Traf6ΔTEC mice. While adoptive transfer of thymic Traf6ΔTEC T cells in immune-deficient mice was sufficient for AIH development, colonization of germ-free mice with Traf6ΔTEC microbiota failed to induce AIH, suggesting that the gut microbiota contributes to but is not sufficient for AIH development. Microbiota-mediated exacerbation of AIH associated with increased numbers of hepatic Foxp3(+) T cells and their increase was proportional to the degree of inflammation. The contribution of the gut microbiota to AIH development associated with an altered microbial signature whose composition was influenced by the qualitative nature of the thymic T cell compartment. These results suggest that aberrant selection of T cells in the thymus can induce changes in the gut microbiota that lead to exacerbation of organ-specific autoimmunity and AIH. Our results add to our understanding of the mechanisms of AIH development and create a platform towards developing novel therapeutic approaches for treating this disease.

Significance Composition of gut microbiota is altered in many human diseases, including inflammatory bowel disease. Some hope that restoring microbiota to a healthy state could help treat such diseases. We have used mice colonized with microbiotas from humans with inflammatory bowel disease to study what happens when these mice receive a microbiota transplant from a set of healthy humans. We find that the mouse gut immune system is changed by microbiota transplants, becoming broadly less inflammatory and protecting mice from colitis. By culturing bacteria from these microbiotas, we identify one strain that induces inflammatory responses in mice and show that it is modified by microbiota transplant. We also show that increases in the density of microbiota following transplant may be antiinflammatory., The building evidence for the contribution of microbiota to human disease has spurred an effort to develop therapies that target the gut microbiota. This is particularly evident in inflammatory bowel diseases (IBDs), where clinical trials of fecal microbiota transplantation have shown some efficacy. To aid the development of novel microbiota-targeted therapies and to better understand the biology underpinning such treatments, we have used gnotobiotic mice to model microbiota manipulations in the context of microbiotas from humans with inflammatory bowel disease. Mice colonized with IBD donor-derived microbiotas exhibit a stereotypical set of phenotypes, characterized by abundant mucosal Th17 cells, a deficit in the tolerogenic RORγt+ regulatory T (Treg) cell subset, and susceptibility to disease in colitis models. Transplanting healthy donor-derived microbiotas into mice colonized with human IBD microbiotas led to induction of RORγt+ Treg cells, which was associated with an increase in the density of the microbiotas following transplant. Microbiota transplant reduced gut Th17 cells in mice colonized with a microbiota from a donor with Crohn’s disease. By culturing strains from this microbiota and screening them in vivo, we identified a specific strain that potently induces Th17 cells. Microbiota transplants reduced the relative abundance of this strain in the gut microbiota, which was correlated with a reduction in Th17 cells and protection from colitis.

Despite identification of numerous associations between microbiomes and diseases, the complexity of the human microbiome has hindered identification of individual species and strains that are causative in host phenotype or disease. Uncovering causative microbes is vital to fully understand disease processes and to harness the potential therapeutic benefits of microbiota manipulation. Developments in sequencing technology, animal models, and bacterial culturing have facilitated the discovery of specific microbes that impact the host and are beginning to advance the characterization of host-microbiome interaction mechanisms. We summarize the historical and contemporary experimental approaches taken to uncover microbes from the microbiota that affect host biology and describe examples of commensals that have specific effects on the immune system, inflammation, and metabolism. There is still much to learn, and we lay out challenges faced by the field and suggest potential remedies for common pitfalls encountered in the hunt for causative commensal microbes.

SUMMARYThe gut microbiota enhances systemic immunoglobulin G (IgG) responses to vaccines. However, it is unknown whether this effect involves IgA, a mucosal antibody that coats intestinal microbes. Here we found that gut IgA increased peripheral IgG responses to pneumococcal vaccines, as these responses were profoundly impaired in mice with global or mucosa-restricted IgA deficiency. The positive effect of IgA on vaccine-induced IgG production implicated gut bacteria. Indeed, IgG responses to pneumococcal vaccines were also defective in ex-germ free mice recolonized with gut microbes from mouse or human IgA-deficient donors. IgA exerted this IgG-enhancing effect by constraining the systemic translocation of intestinal commensal antigens, which caused chronic immune activation, including T cell overexpression of programmed death-1. This immune inhibitory receptor hindered vaccine-specific IgG production by eliciting functional B cell unresponsiveness, which was reverted by anti-programmed death-1 treatment. Thus, gut IgA is functionally interconnected with systemic IgG via intestinal microbes.

The intestinal microbiota actively converts dietary flavanols into phenolic acids, some of which are bioavailable in vivo and may promote resilience to select neurological disorders by interfering with key pathologic mechanisms. Since every person harbors a unique set of gut bacteria, we investigated the influence of the gut microbiota's interpersonal heterogeneity on the production and bioavailability of flavonoid metabolites that may interfere with the misfolding of alpha (α)-synuclein, a process that plays a central role in Parkinson's disease and other α-synucleinopathies. We generated two experimental groups of humanized gnotobiotic mice with compositionally diverse gut bacteria and orally treated the mice with a flavanol-rich preparation (FRP). The two gnotobiotic mouse groups exhibited distinct differences in the generation and bioavailability of FRP-derived microbial phenolic acid metabolites that have bioactivity towards interfering with α-synuclein misfolding or inflammation. We also demonstrated that these bioactive phenolic acids are effective in modulating the development and progression of motor dysfunction in a Drosophila model of α-synucleinopathy. Lastly, through in vitro bacterial fermentation studies, we identified select bacteria that are capable of supporting the generation of these bioavailable and bioactive phenolic acids. Outcomes from our studies provide a better understanding of how interpersonal heterogeneity in the gut microbiota differentially modulates the efficacy of dietary flavanols to protect against select pathologic mechanisms. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies and other neurological disorders involving protein misfolding and/or inflammation.

Accumulating evidence indicates that the health impact of dietary phenolic compounds, including the principal grape-derived polyphenols, ( + )-catechin and ( − )-epicatechin, is exerted by not only the parent compounds but also their phenolic metabolites generated by the gut microbiota. In this work, a new high-throughput, sensitive and reproducible analytical method was developed employing ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) for the simultaneous analysis of 16 microbial-generated phenolic acid metabolites (PAMs) along with their precursors, catechin and epicatechin. Following optimizing the solvent system, LC conditions and MS parameters, method validation was carried out to evaluate the sensitivity, selectivity, accuracy and precision of the proposed method, and to ensure promising recovery of all analytes extracted from the matrix prior to bioanalysis. Results showed that the optimized analytical method allowed successful confirmation and quantitation of all analytes under dynamic multiple reaction monitoring mode using trans-cinnamic acid-d(7) as an internal standard (I.S.). Excellent sensitivity and linearity were obtained for all analytes, with lower limits of detection (LLODs) and lower limits of quantification (LLOQs) in the ranges of 0.225–2.053 ng/mL and 0.698–8.116 ng/mL, respectively. By examining blank matrix spiked with standard mixture at different concentration levels, promising recoveries at two spiking levels (low level, 91.2–115%; high level 90.2–121%), and excellent precision (RSD < 10%) were obtained. This method was then successfully applied to an in vitro study where catechin/epicatechin-enriched broth samples were anaerobically fermented with gut microbes procured from healthy human donors. All sources of bacteria employed showed remarkable activity in metabolizing grape polyphenols and distinct variations in the production of PAMs. The successful application of this method in the in vitro fermentation assays demonstrates its suitability for high-throughput analysis of polyphenol metabolites, particularly catechin/epicatechin-derived PAMs, in biological studies.

Despite being the most abundantly secreted immunoglobulin isotype, the reactivity of IgA antibodies towards each individual’s own gut commensal bacteria still remains elusive. By colonizing germ-free mice with defined commensal bacteria, we found the binding specificity of bulk fecal and serum IgA towards resident gut bacteria resolves well at the species level and has modest strain level specificity. IgA hybridomas generated from lamina propria B cells of gnotobiotic mice showed that most IgA clones recognized a single bacterial species, while a small portion displayed polyreactivity. Species-specific IgAs had a range of strain specificities. Given the unique bacterial species and strain composition in each individual’s gut, our findings suggest the IgA repertoire is uniquely shaped to bind our self gut bacteria.

SummaryThe population structure of strains within a bacterial species is poorly defined, despite the functional importance of strain variation in the human gut microbiota on health. Here we analyzed >1000 sequenced bacterial strains from the fecal microbiota of 47 individuals from two countries and combined them with >150,000 bacterial genomes from NCBI to quantify the strain population size of different bacterial species, as well as the frequency of finding the same strain colonized in unrelated individuals who had no opportunities for direct microbial strain transmission. Strain population sizes ranged from tens to over one-hundred thousand per species. Prevalent strains in common gut microbiota species with small population sizes were the most likely to be harbored in two or more unrelated individuals. The finite strain population size of certain species creates the opportunity to comprehensively sequence the entirety of these species’ prevalent strains and associate their presence in different individuals with health outcomes.

Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of coronavirus disease 2019 (COVID-19), we investigated the impact of GI infection on disease pathogenesis in three large cohorts of patients in the United States and Europe. Unexpectedly, we observed that GI involvement was associated with a significant reduction in disease severity and mortality, with an accompanying reduction in key inflammatory proteins including IL-6, CXCL8, IL-17A and CCL28 in circulation. In a fourth cohort of COVID-19 patients in which GI biopsies were obtained, we identified severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) within small intestinal enterocytes for the first timein vivobut failed to obtain culturable virus. High dimensional analyses of GI tissues confirmed low levels of cellular inflammation in the GI lamina propria and an active downregulation of key inflammatory genes includingIFNG, CXCL8, CXCL2andIL1Bamong others. These data draw attention to organ-level heterogeneity in disease pathogenesis and highlight the role of the GI tract in attenuating SARS-CoV-2-associated inflammation with related mortality benefit.

Fecal Microbiota Transplantation (FMT), while successful for the treatment of recurrent Clostridioides difficile (rCDI) infection, lacks a quantitative identification of the discrete bacterial strains that transmit and stably engraft in recipients, and their association with clinical outcomes. Using >1,000 unique bacterial strains isolated and sequenced from a combination of 22 FMT donors and recipients, we develop a statistical approach Strainer to detect and track sequenced bacterial strains from low depth metagenomic sequencing data. On application to 14 FMT interventions, we detect stable and high engraftment of ∼71% of gut microbiota strains in recipients at even 5-years post-transplant, a remarkably durable therapeutic from a single administration. We found differential transmission and engraftment efficacy across bacterial taxonomic groups over short and long-time scales. Although ∼80% of the original pre-FMT recipient strains were eliminated by the FMT, those strains that remain persist even 5 years later, along with newer strains acquired from the environment. The precise quantification of donor bacterial strains in recipients independently explained the clinical outcomes of early and late relapse. Our framework identifies the consistently engrafting discrete bacterial strains for use in Live Biotherapeutic Products (LBP) as a safer, scalable alternative to FMT and enables systematic evaluation of different FMT and LBP study designs.

Recent studies demonstrate that gut microbiota regulate tumor response to immune checkpoint blockade. Still, the mechanisms by which microbiota control tumor response to immunotherapy remain unclear. We colonized germ-free mice with cultured human-derived microbiota prior to tumor inoculation. While no human donor microbiota altered tumor growth, two distinct gut microbiota inhibited tumor response to anti-PD-L1. Colonization with non-responder microbiota led to reduced tumor immune cell infiltration and modified antigen presenting cell phenotype. RNA sequencing of tumor-infiltrating CD8+ T cells revealed enrichment for stem cell-like genes in non-responders and reduced effector-like expression conferring cytotoxic potential. Antibiotic depletion and microbiota transplant restored anti-PD-L1 response in non-responders, with expansion of effector cells and cytotoxicity. Concomitant blockade of TNFα similarly improved response to anti-PD-L1 and increased cytotoxicity. These results demonstrate inhibitory roles for the microbiota in checkpoint blockade and reveal the potential for microbiota transplant and TNF blockade to overcome microbiota-mediated resistance.

The coronavirus disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide, igniting an unprecedented effort from the scientific community to understand the biological underpinning of COVID19 pathophysiology. In this review, we summarize the current state of knowledge of innate and adaptive immune responses elicited by SARS-CoV-2 infection and the immunological pathways that likely contribute to disease severity and death. We also discuss the rationale and clinical outcome of current therapeutic strategies as well as prospective clinical trials to prevent or treat SARS-CoV-2 infection.

The gut mounts secretory immunoglobulin A (SIgA) responses to commensal bacteria through nonredundant T cell-dependent (TD) and T cell-independent (TI) pathways that promote the establishment of mutualistic host-microbiota interactions. SIgAs from the TD pathway target penetrant bacteria, and their induction requires engagement of CD40 on B cells by CD40 ligand on T follicular helper cells. In contrast, SIgAs from the TI pathway bind a larger spectrum of bacteria, but the mechanism underpinning their production remains elusive. Here, we show that the intestinal TI pathway required CD40-independent B cell-activating signals from TACI, a receptor for the innate CD40 ligand-like factors BAFF and APRIL. TACI-induced SIgA responses targeted a fraction of the gut microbiota without shaping its overall composition. Of note, TACI was dispensable for TD induction of IgA in gut-associated lymphoid organs. Thus, BAFF/APRIL signals acting on TACI orchestrate commensal bacteria-specific SIgA responses through an intestinal TI program.

The building evidence for the contribution of microbiota to human disease has spurred an effort to develop therapies that target the gut microbiota. This is particularly evident in inflammatory bowel diseases, where clinical trials of fecal microbiota transplant have shown some efficacy. To aid the development of novel microbiota-targeted therapies and to better understand the biology underpinning such treatments, we have used gnotobiotic mice to model microbiota manipulations in the context of microbiotas from humans with inflammatory bowel disease. Mice colonized with IBD donor-derived microbiotas exhibit a stereotypical set of phenotypes, characterized by abundant mucosal Th17 cells and a deficit in the tolerogenic RORγt+ Treg cell subset. Transplanting healthy donor-derived microbiota into mice colonized with human IBD microbiotas lead to induction of RORγt+ Treg cells, which was associated with an increase in the density of the microbiotas following transplant. Microbiota transplant reduced gut Th17 cells in mice colonized with a microbiota from a donor with Crohn’s disease. By culturing strains from this microbiota and screening them in vivo, we identified a specific strain that potently induces Th17 cells. Microbiota transplants reduced the relative abundance of this strain in the gut microbiota, correlated with a reduction in Th17 cells.

Neonatal inflammatory diseases are associated with severe morbidity, but the inflammatory factors underlying them and their potential effector mechanisms are poorly defined. Here we show that necrotizing enterocolitis in neonate mice is accompanied by elevation of IL-23 and IL-22 and decreased production of pancreatic enzymes. These phenotypes are mirrored in neonate mice overexpressing IL-23 in CX3CR1+ myeloid cells or in keratinocytes. The mice fail to grow and die prematurely, displaying systemic inflammation, nutrient malabsorption and decreased expression of intestinal and pancreatic genes mediating digestion and absorption of carbohydrates, proteins, and lipids. Germ-free environment improves, and genetic ablation of IL-22 restores normal growth in mice overexpressing IL-23. Mechanistically, IL-22 acts directly at the level of pancreatic acinar cells to decrease expression of the pancreas associated transcription factor 1a (PTF1a). These results show that augmented production of IL-23 and IL-22 in early life has a negative impact on pancreatic enzyme secretion and food absorption., Necrotizing enterocolitis (NEC) is associated with severe neonatal morbidity. Here the authors show, mirroring the NEC phenotype, that IL-23 overexpression in neonates causes malabsorption and decreased expression of intestinal and pancreatic genes mediating food digestion and uptake through IL-22, which directly suppresses pancreatic cell differentiation.

Background & Aims Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance. Methods Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions. Results COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms. Conclusions These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2–associated inflammation needs to be further examined.

A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.

The development of a given botanical preparation for eventual clinical application requires extensive, detailed characterizations of the chemical composition, as well as the biological availability, biological activity, and safety profiles of the botanical. These issues are typically addressed using diverse experimental protocols and model systems. Based on this consideration, in this study we established a comprehensive database and analysis framework for the collection, collation, and integrative analysis of diverse, multiscale data sets. Using this framework, we conducted an integrative analysis of heterogeneous data from in vivo and in vitro investigation of a complex bioactive dietary polyphenol-rich preparation (BDPP) and built an integrated network linking data sets generated from this multitude of diverse experimental paradigms. We established a comprehensive database and analysis framework as well as a systematic and logical means to catalogue and collate the diverse array of information gathered, which is securely stored and added to in a standardized manner to enable fast query. We demonstrated the utility of the database in (1) a statistical ranking scheme to prioritize response to treatments and (2) in depth reconstruction of functionality studies. By examination of these data sets, the system allows analytical querying of heterogeneous data and the access of information related to interactions, mechanism of actions, functions, etc., which ultimately provide a global overview of complex biological responses. Collectively, we present an integrative analysis framework that leads to novel insights on the biological activities of a complex botanical such as BDPP that is based on data-driven characterizations of interactions between BDPP-derived phenolic metabolites and their mechanisms of action, as well as synergism and/or potential cancellation of biological functions. Out integrative analytical approach provides novel means for a systematic integrative analysis of heterogeneous data types in the development of complex botanicals such as polyphenols for eventual clinical and translational applications.

Small intestinal bacterial overgrowth (SIBO) has been implicated in symptoms associated with functional gastrointestinal disorders (FGIDs), though mechanisms remain poorly defined and treatment involves non-specific antibiotics. Here we show that SIBO based on duodenal aspirate culture reflects an overgrowth of anaerobes, does not correspond with patient symptoms, and may be a result of dietary preferences. Small intestinal microbial composition, on the other hand, is significantly altered in symptomatic patients and does not correspond with aspirate culture results. In a pilot interventional study we found that switching from a high fiber diet to a low fiber, high simple sugar diet triggered FGID-related symptoms and decreased small intestinal microbial diversity while increasing small intestinal permeability. Our findings demonstrate that characterizing small intestinal microbiomes in patients with gastrointestinal symptoms may allow a more targeted antibacterial or a diet-based approach to treatment.

Preclinical human IBD mechanisms is part of five focus areas of the Challenges in IBD research document, which also include environmental triggers, novel technologies, precision medicine and pragmatic clinical research. The Challenges in IBD research document provides a comprehensive overview of current gaps in inflammatory bowel diseases (IBD) research and delivers actionable approaches to address them. It is the result of a multidisciplinary input from scientists, clinicians, patients, and funders, and represents a valuable resource for patient centric research prioritization. In particular, the preclinical human IBD mechanisms manuscript is focused on highlighting the main research gaps in the pathophysiological understanding of human IBD. These research gap areas include: 1) triggers of immune responses; 2) intestinal epithelial homeostasis and wound repair; 3) age-specific pathophysiology; 4) disease complications; 5) heterogeneous response to treatments; and 6) determination of disease location. As an approach to address these research gaps, the prioritization of reverse translation studies is proposed in which clinical observations are the foundation for experimental IBD research in the lab, and for the identification of new therapeutic targets and biomarkers. The use of human samples in validating basic research findings and development of precision medicine solutions is also proposed. This prioritization aims to put emphasis on relevant biochemical pathways and humanized in vitro and in vivo models that extrapolate meaningfully to human IBD, to eventually yield first-in-class and effective therapies.

Background Recurrent and severe Clostridium difficile infections (CDI) are treated with fecal microbiota transplant (FMT). Uncertainty exists regarding FMT effectiveness for CDI with underlying inflammatory bowel disease (IBD) and regarding its effects on disease activity and effectiveness in transferring the donor microbiota to patients with and without IBD. Methods Subjects with and without IBD who underwent FMT for recurrent or severe CDI between 2013 and 2016 at The Mount Sinai Hospital were followed for up to 6 months. The primary outcome was CDI recurrence 6 months after FMT. Secondary outcomes were (1) CDI recurrence 2 months after FMT; (2) frequency of IBD flare after FMT; (3) microbiota engraftment after FMT; (and 4) predictors of CDI recurrence. Results One hundred thirty-four patients, 46 with IBD, were treated with FMT. Follow-up was available in 83 and 118 patients at 6 and 2 months, respectively. There was no difference in recurrence in patients with and without IBD at 6 months (38.7% vs 36.5%; P > 0.99) and 2 months (22.5% vs 17.9%; P = 0.63). Proton pump inhibitor use, severe CDI, and comorbid conditions were predictors of recurrence. Pre-FMT microbiota was not predictive of CDI recurrence. Subjects with active disease requiring medication escalation had reduced engraftment, with no difference in engraftment based on CDI recurrence or IBD endoscopic severity at FMT. Conclusions Inflammatory bowel disease did not affect CDI recurrence rates 6 months after FMT. Pre-FMT microbiota was not predictive of recurrence, and microbial engraftment was impacted in those requiring IBD treatment escalation, though not by CDI recurrence or IBD disease severity.

Dietary polyphenols promote memory in models of sleep deprivation (SD), stress, and neurodegeneration. The biological properties of dietary polyphenols greatly depend upon the bioavailability of their phenolic metabolites derivatives, which are modulated by gut microbiota. We recently demonstrated that supplementation with grape-derived bioactive dietary polyphenol preparation (BDPP) improves SD-induced cognitive impairment. This study examined the role of the gut microbiota in the ability of BDPP to prevent memory impairment in response to SD. C57BL6/J mice, treated with antibiotics mix (ABX) or BDPP or both, were sleep-deprived at the end of a fear conditioning training session and fear memory was assessed the next day. Gut microbiota composition was analyzed in fecal samples and BDPP-driven phenolic acid metabolites extraction was measured in plasma. We report that the beneficial effect of BDPP on memory in SD is attenuated by ABX-induced dysbiosis. We identified specific communities of fecal microbiota that are associated with the bioavailability of BDPP-derived phenolic acids, which in turn, are associated with memory promotion. These results suggest the gut microbiota composition significantly affects the bioavailability of phenolic acids that drive the dietary polyphenols’ cognitive resilience property. Our findings provide a preclinical model with which to test the causal association of gut microbiota-polyphenols, with the ultimate goal of potential developing dietary polyphenols for the prevention/treatment of cognitive impairment.

Fecal IgA production depends on colonization by a gut microbiota. However, the bacterial strains that drive gut IgA production remain largely unknown. By accessing the IgA-inducing capacity of a diverse set of human gut microbial strains, we identified Bacteroides ovatus as the species that best induced gut IgA production. However, this induction varied biomodally across different B. ovatus strains. The high IgA-inducing B. ovatus strains preferentially elicited more IgA production in the large intestine through both T-cell-dependent and T-cell-independent B cell-activation pathways. Remarkably, a low-IgA phenotype in mice could be robustly and consistently converted into a high-IgA phenotype by transplanting a multiplex cocktail of high IgA-inducing B. ovatus strains but not individual ones. Thus, microbial strain specificity is essential for the optimal induction of high-IgA responses in the gut. Our results highlight the critical importance of microbial strains in driving phenotype variation in the mucosal immune system and provide a strategy to robustly modify a gut immune phenotype, including IgA production.

Background and aimsPrenatal and early life bacterial colonisation is thought to play a major role in shaping the immune system. Furthermore, accumulating evidence links early life exposures to the risk of developing IBD later in life. We aimed to assess the effect of maternal IBD on the composition of the microbiome during pregnancy and on the offspring’s microbiome.MethodsWe prospectively examined the diversity and taxonomy of the microbiome of pregnant women with and without IBD and their babies at multiple time points. We evaluated the role of maternal IBD diagnosis, the mode of delivery, antibiotic use and feeding behaviour on the microbiome composition during early life. To assess the effects of IBD-associated maternal and infant microbiota on the enteric immune system, we inoculated germ-free mice (GFM) with the respective stool and profiled adaptive and innate immune cell populations in the murine intestines.ResultsPregnant women with IBD and their offspring presented with lower bacterial diversity and altered bacterial composition compared with control women and their babies. Maternal IBD was the main predictor of the microbiota diversity in the infant gut at 7, 14, 30, 60 and 90 days of life. Babies born to mothers with IBD demonstrated enrichment in Gammaproteobacteria and depletion in Bifidobacteria. Finally, GFM inoculated with third trimester IBD mother and 90-day infant stools showed significantly reduced microbial diversity and fewer class-switched memory B cells and regulatory T cells in the colon.ConclusionAberrant gut microbiota composition persists during pregnancy with IBD and alters the bacterial diversity and abundance in the infant stool. The dysbiotic microbiota triggered abnormal imprinting of the intestinal immune system in GFM.