Microbiota therapeutics for inflammatory bowel disease: the way forward.

Microbiota therapeutics that transplant faecal material from healthy donors to people with mild-to-moderate ulcerative colitis have shown the potential to induce remission in about 30% of participants in small, phase 2 clinical trials. Despite this substantial achievement, the field needs to leverage the insights gained from these trials and progress towards phase 3 clinical trials and drug approval, while identifying the distinct clinical niche for this new therapeutic modality within inflammatory bowel disease (IBD) therapeutics. We describe the lessons that can be learned from past studies of microbiota therapeutics, from full spectrum donor stool to defined products manufactured in vitro. We explore the actionable insights these lessons provide on the design of near-term studies and future trajectories for the integration of microbiota therapeutics in the treatment of IBD. If successful, microbiota therapeutics will provide a powerful orthogonal approach (complementing or in combination with existing immunomodulatory drugs) to raise the therapeutic ceiling for the many non-responders and partial responders within the IBD patient population.

Gut-associated lymphoid tissue attrition associates with response to anti-α4ß7 therapy in ulcerative colitis.

Vedolizumab (VDZ) is a first-line treatment in ulcerative colitis (UC) that targets the α4ß7- mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) axis. To determine the mechanisms of action of VDZ, we examined five distinct cohorts of patients with UC. A decrease in naïve B and T cells in the intestines and gut-homing (ß7+) plasmablasts in circulation of VDZ-treated patients suggested that VDZ targets gut-associated lymphoid tissue (GALT). Anti-α4ß7 blockade in wild-type and photoconvertible (KikGR) mice confirmed a loss of GALT size and cellularity because of impaired cellular entry. In VDZ-treated patients with UC, treatment responders demonstrated reduced intestinal lymphoid aggregate size and follicle organization and a reduction of ß7+IgG+ plasmablasts in circulation, as well as IgG+ plasma cells and FcγR-dependent signaling in the intestine. GALT targeting represents a previously unappreciated mechanism of action of α4ß7-targeted therapies, with major implications for this therapeutic paradigm in UC.

Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes.

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.

VE303, a Defined Bacterial Consortium, for Prevention of Recurrent Clostridioides difficile Infection: A Randomized Clinical Trial.

Importance: The effect of rationally defined nonpathogenic, nontoxigenic, commensal strains of Clostridia on prevention of Clostridioides difficile infection (CDI) is unknown. Objective: To 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 Participants: Phase 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 <60 mL/min/1.73 m2, proton pump inhibitor use, remote [>6 months earlier] CDI history). Interventions: Participants 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 Measures: The 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. Results: Baseline 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 Relevance: Among 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 Registration: ClinicalTrials.gov Identifier: NCT03788434.

Seminars in immunology special issue: Nutrition, microbiota and immunity The unexplored microbes in health and disease.

Functional characterization of the microbiome's influence on host physiology has been dominated by a few characteristic example strains that have been studied in detail. However, the extensive development of methods for high-throughput bacterial isolation and culture over the past decade is enabling functional characterization of the broader microbiota that may impact human health. Characterizing the understudied majority of human microbes and expanding our functional understanding of the diversity of the gut microbiota could enable new insights into diseases with unknown etiology, provide disease-predictive microbiome signatures, and advance microbial therapeutics. We summarize high-throughput culture-dependent platforms for characterizing bacterial strain function and host-interactions. We elaborate on the importance of these technologies in facilitating mechanistic studies of previously unexplored microbes, highlight new opportunities for large-scale in vitro screens of host-relevant microbial functions, and discuss the potential translational applications for microbiome science.

To Fiber or Not to Fiber: The Swinging Pendulum of Fiber Supplementation in Patients with Inflammatory Bowel Disease.

Evidence-based dietary guidance around dietary fiber in inflammatory bowel disease (IBD) has been limited owing to insufficient reproducibility in intervention trials. However, the pendulum has swung because of our increased understanding of the importance of fibers in maintaining a health-associated microbiome. Preliminary evidence suggests that dietary fiber can alter the gut microbiome, improve IBD symptoms, balance inflammation, and enhance health-related quality of life. Therefore, it is now more vital than ever to examine how fiber could be used as a therapeutic strategy to manage and prevent disease relapse. At present, there is limited knowledge about which fibers are optimal and in what form and quantity they should be consumed to benefit patients with IBD. Additionally, individual microbiomes play a strong role in determining the outcomes and necessitate a more personalized nutritional approach to implementing dietary changes, as dietary fiber may not be as benign as once thought in a dysbiotic microbiome. This review describes dietary fibers and their mechanism of action within the microbiome, details novel fiber sources, including resistant starches and polyphenols, and concludes with potential future directions in fiber research, including the move toward precision nutrition.

Species-specific CD4+ T cells enable prediction of mucosal immune phenotypes from microbiota composition.

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 toward 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 unique method for efficiently predicting the relative impact of individual bacterial strains within a complex community and for parsing microbiota-dependent phenotypes into component fractions.

Mining the microbiota to identify gut commensals modulating neuroinflammation in a mouse model of multiple sclerosis.

BACKGROUND: The gut microbiome plays an important role in autoimmunity including multiple sclerosis and its mouse model called experimental autoimmune encephalomyelitis (EAE). Prior studies have demonstrated that the multiple sclerosis gut microbiota can contribute to disease, hence making it a potential therapeutic target. In addition, antibiotic treatment has been shown to ameliorate disease in the EAE mouse model of multiple sclerosis. Yet, to this date, the mechanisms mediating these antibiotic effects are not understood. Furthermore, there is no consensus on the gut-derived bacterial strains that drive neuroinflammation in multiple sclerosis. RESULTS: Here, we characterized the gut microbiome of untreated and vancomycin-treated EAE mice over time to identify bacteria with neuroimmunomodulatory potential. We observed alterations in the gut microbiota composition following EAE induction. We found that vancomycin treatment ameliorates EAE, and that this protective effect is mediated via the microbiota. Notably, we observed increased abundance of bacteria known to be strong inducers of regulatory T cells, including members of Clostridium clusters XIVa and XVIII in vancomycin-treated mice during the presymptomatic phase of EAE, as well as at disease peak. We identified 50 bacterial taxa that correlate with EAE severity. Interestingly, several of these taxa exist in the human gut, and some of them have been implicated in multiple sclerosis including Anaerotruncus colihominis, a butyrate producer, which had a positive correlation with disease severity. We found that Anaerotruncus colihominis ameliorates EAE, and this is associated with induction of RORγt+ regulatory T cells in the mesenteric lymph nodes. CONCLUSIONS: We identified vancomycin as a potent modulator of the gut-brain axis by promoting the proliferation of bacterial species that induce regulatory T cells. In addition, our findings reveal 50 gut commensals as regulator of the gut-brain axis that can be used to further characterize pathogenic and beneficial host-microbiota interactions in multiple sclerosis patients. Our findings suggest that elevated Anaerotruncus colihominis in multiple sclerosis patients may represent a protective mechanism associated with recovery from the disease. Video Abstract.

Human gut microbiota stimulate defined innate immune responses that vary from phylum to strain.

The potential of commensal bacteria to modulate host immunity remains largely uncharacterized, largely due to the vast number of strains that comprise the human gut microbiota. We have developed a screening platform to measure the innate immune responses of myeloid cells to 277 bacterial strains isolated from the gut microbiota of healthy individuals and those with inflammatory bowel diseases. The innate immune responses to gut-derived bacteria are as strong as those toward pathogenic bacteria, and they vary from phylum to strain. Myeloid cells differentially rely upon innate receptors TLR2 or TLR4 to sense taxa, with differential sensing of Bacteroidetes and Proteobacteria that predict in vivo functions. These innate immune responses can be modeled using combinations of up to 8 Toll-like receptor (TLR) agonists. Furthermore, the immunogenicity of strains is stable over time and following fecal microbiota transplantation into new human recipients. Collectively, this high-throughput approach provides an insight into how commensal microorganisms shape innate immune phenotypes.

Immunoglobulin A antibody composition is sculpted to bind the self gut microbiome.

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.

Impaired central tolerance induces changes in the gut microbiota that exacerbate autoimmune hepatitis.

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.

Causative Microbes in Host-Microbiome Interactions.

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.

Enterococcus faecalis Glucosamine Metabolism Exacerbates Experimental Colitis.

BACKGROUND & AIMS: The inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, are caused in part by aberrant immune responses to resident intestinal bacteria. Certain dietary components, including carbohydrates, are associated with IBDs and alter intestinal bacterial composition. However, the effects of luminal carbohydrates on the composition and colitogenic potential of intestinal bacteria are incompletely understood. We hypothesize that carbohydrate metabolism by resident proinflammatory intestinal bacteria enhances their growth and worsens intestinal inflammation. METHODS: We colonized germ-free, wild-type, and colitis-susceptible interleukin-10 knockout mice (Il10-/-) with a consortium of resident intestinal bacterial strains and quantified colon inflammation using blinded histologic scoring and spontaneous secretion of IL12/23p40 by colon explants. We measured luminal bacterial composition using real-time 16S polymerase chain reaction, bacterial gene expression using RNA sequencing and real-time polymerase chain reaction, and luminal glucosamine levels using gas chromatography-mass spectrometry. RESULTS: We show that a consortium of 8 bacterial strains induces severe colitis in Il10-/- mice and up-regulates genes associated with carbohydrate metabolism during colitis. Specifically, Enterococcus faecalis strain OG1RF is proinflammatory and strongly up-regulates OG1RF_11616-11610, an operon that encodes genes of a previously undescribed phosphotransferase system that we show imports glucosamine. Experimental colitis is associated with increased levels of luminal glucosamine and OG1RF_11616 causes worse colitis, not by increasing E faecalis numbers, but rather by mechanisms that require the presence of complex microbiota. CONCLUSIONS: Further studies of luminal carbohydrate levels and bacterial carbohydrate metabolism during intestinal inflammation will improve our understanding of the pathogenesis of IBDs and may lead to the development of novel therapies for these diseases.

Limited intestinal inflammation despite diarrhea, fecal viral RNA and SARS-CoV-2-specific IgA in patients with acute COVID-19.

Gastrointestinal symptoms are common in COVID-19 patients but the nature of the gut immune response to SARS-CoV-2 remains poorly characterized, partly due to the difficulty of obtaining biopsy specimens from infected individuals. In lieu of tissue samples, we measured cytokines, inflammatory markers, viral RNA, microbiome composition, and antibody responses in stool samples from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.

Food colorants metabolized by commensal bacteria promote colitis in mice with dysregulated expression of interleukin-23.

Both genetic predisposition and environmental factors appear to play a role in inflammatory bowel disease (IBD) development. Genetic studies in humans have linked the interleukin (IL)-23 signaling pathway with IBD, but the environmental factors contributing to disease have remained elusive. Here, we show that the azo dyes Red 40 and Yellow 6, the most abundant food colorants in the world, can trigger an IBD-like colitis in mice conditionally expressing IL-23, or in two additional animal models in which IL-23 expression was augmented. Increased IL-23 expression led to generation of activated CD4+ T cells that expressed interferon-γ and transferred disease to mice exposed to Red 40. Colitis induction was dependent on the commensal microbiota promoting the azo reduction of Red 40 and generation of a metabolite, 1-amino-2-naphthol-6-sulfonate sodium salt. Together these findings suggest that specific food colorants represent novel risk factors for development of colitis in mice with increased IL-23 signaling.

Intestinal Host Response to SARS-CoV-2 Infection and COVID-19 Outcomes in Patients With Gastrointestinal Symptoms.

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.

Modeling dysbiosis of human NASH in mice: Loss of gut microbiome diversity and overgrowth of Erysipelotrichales.

BACKGROUND & AIM: Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD) that is responsible for a growing fraction of cirrhosis and liver cancer cases worldwide. Changes in the gut microbiome have been implicated in NASH pathogenesis, but the lack of suitable murine models has been a barrier to progress. We have therefore characterized the microbiome in a well-validated murine NASH model to establish its value in modeling human disease. METHODS: The composition of intestinal microbiota was monitored in mice on a 12- or 24-week NASH protocol consisting of high fat, high sugar Western Diet (WD) plus once weekly i.p injection of low-dose CCl4. Additional mice were subjected to WD-only or CCl4-only conditions to assess the independent effect of these variables on the microbiome. RESULTS: There was substantial remodeling of the intestinal microbiome in NASH mice, characterized by declines in both species diversity and bacterial abundance. Based on changes to beta diversity, microbiota from NASH mice clustered separately from controls in principal coordinate analyses. A comparison between WD-only and CCl4-only controls with the NASH model identified WD as the primary driver of early changes to the microbiome, resulting in loss of diversity within the 1st week. A NASH signature emerged progressively at weeks 6 and 12, including, most notably, a reproducible bloom of the Firmicute order Erysipelotrichales. CONCLUSIONS: We have established a valuable model to study the role of gut microbes in NASH, enabling us to identify a new NASH gut microbiome signature.

Gastrointestinal involvement attenuates COVID-19 severity and mortality.

Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of coronavirus disease 2019 (COVID-19), we investigated intestinal infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its effect on disease pathogenesis. SARS-CoV-2 was detected in small intestinal enterocytes by immunofluorescence staining or electron microscopy, in 13 of 15 patients studied. High dimensional analyses of GI tissues revealed low levels of inflammation in general, including active downregulation of key inflammatory genes such as IFNG, CXCL8, CXCL2 and IL1B and reduced frequencies of proinflammatory dendritic cell subsets. To evaluate the clinical significance of these findings, examination of two large, independent cohorts of hospitalized patients in the United States and Europe revealed a significant reduction in disease severity and mortality that was independent of gender, age, and examined co-morbid illnesses. The observed mortality reduction in COVID-19 patients with GI symptoms was associated with reduced levels of key inflammatory proteins including IL-6, CXCL8, IL-17A and CCL28 in circulation but was not associated with significant differences in nasopharyngeal viral loads. 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. ONE SENTENCE SUMMARY: Intestinal infection with SARS-CoV-2 is associated with a mild inflammatory response and improved clinical outcomes.