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.

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.

Precise quantification of bacterial strains after fecal microbiota transplantation delineates long-term engraftment and explains outcomes.

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.

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.

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.

A LAT-Based Signaling Complex in the Immunological Synapse as Determined with Live Cell Imaging Is Less Stable in T Cells with Regulatory Capability.

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIß inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.

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.

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.

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

We sought to characterize the role of the gastrointestinal immune system in the pathogenesis of the inflammatory response associated with COVID-19. We measured cytokines, inflammatory markers, viral RNA, microbiome composition and antibody responses in stool 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.

Defined microbiota transplant restores Th17/RORγt+ regulatory T cell balance in mice colonized with inflammatory bowel disease microbiotas.

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.

Immunology of COVID-19: Current State of the Science.

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.

Chromatin Priming Renders T Cell Tolerance-Associated Genes Sensitive to Activation below the Signaling Threshold for Immune Response Genes.

Immunological homeostasis in T cells is maintained by a tightly regulated signaling and transcriptional network. Full engagement of effector T cells occurs only when signaling exceeds a critical threshold that enables induction of immune response genes carrying an epigenetic memory of prior activation. Here we investigate the underlying mechanisms causing the suppression of normal immune responses when T cells are rendered anergic by tolerance induction. By performing an integrated analysis of signaling, epigenetic modifications, and gene expression, we demonstrate that immunological tolerance is established when both signaling to and chromatin priming of immune response genes are weakened. In parallel, chromatin priming of immune-repressive genes becomes boosted, rendering them sensitive to low levels of signaling below the threshold needed to activate immune response genes. Our study reveals how repeated exposure to antigens causes an altered epigenetic state leading to T cell anergy and tolerance, representing a basis for treating auto-immune diseases.

Infants born to mothers with IBD present with altered gut microbiome that transfers abnormalities of the adaptive immune system to germ-free mice.

BACKGROUND AND AIMS: Prenatal 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. METHODS: We 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. RESULTS: Pregnant 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. CONCLUSION: Aberrant 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.

Gut microbiota density influences host physiology and is shaped by host and microbial factors.

To identify factors that regulate gut microbiota density and the impact of varied microbiota density on health, we assayed this fundamental ecosystem property in fecal samples across mammals, human disease, and therapeutic interventions. Physiologic features of the host (carrying capacity) and the fitness of the gut microbiota shape microbiota density. Therapeutic manipulation of microbiota density in mice altered host metabolic and immune homeostasis. In humans, gut microbiota density was reduced in Crohn's disease, ulcerative colitis, and ileal pouch-anal anastomosis. The gut microbiota in recurrent Clostridium difficile infection had lower density and reduced fitness that were restored by fecal microbiota transplantation. Understanding the interplay between microbiota and disease in terms of microbiota density, host carrying capacity, and microbiota fitness provide new insights into microbiome structure and microbiome targeted therapeutics. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Microbiotas from Humans with Inflammatory Bowel Disease Alter the Balance of Gut Th17 and RORγt+ Regulatory T Cells and Exacerbate Colitis in Mice.

Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of RORγt+ Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T cells into Rag1-/- mice. The proportions of Th17 and RORγt+ Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1-/- colitis model. Thus, an impact on intestinal Th17 and RORγt+ Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis.

Interactions Between Diet and the Intestinal Microbiota Alter Intestinal Permeability and Colitis Severity in Mice.

BACKGROUND & AIMS: It is not clear how the complex interactions between diet and the intestinal microbiota affect development of mucosal inflammation or inflammatory bowel disease. We investigated interactions between dietary ingredients, nutrients, and the microbiota in specific pathogen-free (SPF) and germ-free (GF) mice given more than 40 unique diets; we quantified individual and synergistic effects of dietary macronutrients and the microbiota on intestinal health and development of colitis. METHODS: C56BL/6J SPF and GF mice were placed on custom diets containing different concentrations and sources of protein, fat, digestible carbohydrates, and indigestible carbohydrates (fiber). After 1 week, SPF and GF mice were given dextran sulfate sodium (DSS) to induce colitis. Disease severity was determined based on the percent weight change from baseline, and modeled as a function of the concentration of each macronutrient in the diet. In unchallenged mice, we measured intestinal permeability by feeding mice labeled dextran and measuring levels in blood. Feces were collected and microbiota were analyzed by 16S rDNA sequencing. We collected colons from mice and performed transcriptome analyses. RESULTS: Fecal microbiota varied with diet; the concentration of protein and fiber had the strongest effect on colitis development. Among 9 fiber sources tested, psyllium, pectin, and cellulose fiber reduced the severity of colitis in SPF mice, whereas methylcellulose increased severity. Increasing dietary protein increased the density of the fecal microbiota and the severity of colitis in SPF mice, but not in GF mice or mice given antibiotics. Psyllium fiber reduced the severity of colitis through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary casein protein and psyllium fiber in parallel accounted for most variation in gut microbial density and intestinal permeability in unchallenged mice, as well as the severity of DSS-induced colitis; changes in 1 ingredient could be offset by changes in another. CONCLUSIONS: In an analysis of the effects of different dietary components and the gut microbiota on mice with and without DSS-induced colitis, we found complex mixtures of nutrients affect intestinal permeability, gut microbial density, and development of intestinal inflammation.

Protein kinase C theta is required for efficient induction of IL-10-secreting T cells.

Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance.