Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Phage display of environmental protein toxins and virulence factors reveals the prevalence, persistence, and genetics of antibody responses.

Microbial exposures are crucial environmental factors that impact healthspan by sculpting the immune system and microbiota. Antibody profiling via Phage ImmunoPrecipitation Sequencing (PhIP-Seq) provides a high-throughput, cost-effective approach for detecting exposure and response to microbial protein products. We designed and constructed a library of 95,601 56-amino acid peptide tiles spanning 14,430 proteins with "toxin" or "virulence factor" keyword annotations. We used PhIP-Seq to profile the antibodies of ∼1,000 individuals against this "ToxScan" library. In addition to enumerating immunodominant antibody epitopes, we studied the age-dependent stability of the ToxScan profile and used a genome-wide association study to find that the MHC-II locus modulates bacterial epitope selection. We detected previously described anti-flagellin antibody responses in a Crohn's disease cohort and identified an association between anti-flagellin antibodies and juvenile dermatomyositis. PhIP-Seq with the ToxScan library is thus an effective tool for studying the environmental determinants of health and disease at cohort scale.

Regulation of inflammation by microbiota interactions with the host.

The study of the intestinal microbiota has begun to shift from cataloging individual members of the commensal community to understanding their contributions to the physiology of the host organism in health and disease. Here, we review the effects of the microbiome on innate and adaptive immunological players from epithelial cells and antigen-presenting cells to innate lymphoid cells and regulatory T cells. We discuss recent studies that have identified diverse microbiota-derived bioactive molecules and their effects on inflammation within the intestine and distally at sites as anatomically remote as the brain. Finally, we highlight new insights into how the microbiome influences the host response to infection, vaccination and cancer, as well as susceptibility to autoimmune and neurodegenerative disorders.

Human gut bacteria produce ΤΗ17-modulating bile acid metabolites.

The microbiota modulates gut immune homeostasis. Bacteria influence the development and function of host immune cells, including T helper cells expressing interleukin-17A (TH17 cells). We previously reported that the bile acid metabolite 3-oxolithocholic acid (3-oxoLCA) inhibits TH17 cell differentiation1. Although it was suggested that gut-residing bacteria produce 3-oxoLCA, the identity of such bacteria was unknown, and it was unclear whether 3-oxoLCA and other immunomodulatory bile acids are associated with inflammatory pathologies in humans. Here we identify human gut bacteria and corresponding enzymes that convert the secondary bile acid lithocholic acid into 3-oxoLCA as well as the abundant gut metabolite isolithocholic acid (isoLCA). Similar to 3-oxoLCA, isoLCA suppressed TH17 cell differentiation by inhibiting retinoic acid receptor-related orphan nuclear receptor-γt, a key TH17-cell-promoting transcription factor. The levels of both 3-oxoLCA and isoLCA and the 3α-hydroxysteroid dehydrogenase genes that are required for their biosynthesis were significantly reduced in patients with inflammatory bowel disease. Moreover, the levels of these bile acids were inversely correlated with the expression of TH17-cell-associated genes. Overall, our data suggest that bacterially produced bile acids inhibit TH17 cell function, an activity that may be relevant to the pathophysiology of inflammatory disorders such as inflammatory bowel disease.

Microbiota-Induced TNF-like Ligand 1A Drives Group 3 Innate Lymphoid Cell-Mediated Barrier Protection and Intestinal T Cell Activation during Colitis.

Inflammatory bowel disease (IBD) results from a dysregulated interaction between the microbiota and a genetically susceptible host. Genetic studies have linked TNFSF15 polymorphisms and its protein TNF-like ligand 1A (TL1A) with IBD, but the functional role of TL1A is not known. Here, we found that adherent IBD-associated microbiota induced TL1A release from CX3CR1+ mononuclear phagocytes (MNPs). Using cell-specific genetic deletion models, we identified an essential role for CX3CR1+MNP-derived TL1A in driving group 3 innate lymphoid cell (ILC3) production of interleukin-22 and mucosal healing during acute colitis. In contrast to this protective role in acute colitis, TL1A-dependent expression of co-stimulatory molecule OX40L in MHCII+ ILC3s during colitis led to co-stimulation of antigen-specific T cells that was required for chronic T cell colitis. These results identify a role for ILC3s in activating intestinal T cells and reveal a central role for TL1A in promoting ILC3 barrier immunity during colitis.

Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis.

Thymic development of gut-microbiota-specific T cells.

Humans and their microbiota have coevolved a mutually beneficial relationship in which the human host provides a hospitable environment for the microorganisms and the microbiota provides many advantages for the host, including nutritional benefits and protection from pathogen infection1. Maintaining this relationship requires a careful immune balance to contain commensal microorganisms within the lumen while limiting inflammatory anti-commensal responses1,2. Antigen-specific recognition of intestinal microorganisms by T cells has previously been described3,4. Although the local environment shapes the differentiation of effector cells3-5 it is unclear how microbiota-specific T cells are educated in the thymus. Here we show that intestinal colonization in early life leads to the trafficking of microbial antigens from the intestine to the thymus by intestinal dendritic cells, which then induce the expansion of microbiota-specific T cells. Once in the periphery, microbiota-specific T cells have pathogenic potential or can protect against related pathogens. In this way, the developing microbiota shapes and expands the thymic and peripheral T cell repertoire, allowing for enhanced recognition of intestinal microorganisms and pathogens.

Author Correction: Bile acid metabolites control TH17 and Treg cell differentiation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bile acid metabolites control TH17 and Treg cell differentiation.

Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.

Transferable Immunoglobulin A-Coated Odoribacter splanchnicus in Responders to Fecal Microbiota Transplantation for Ulcerative Colitis Limits Colonic Inflammation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC. METHODS: Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A-coated microbiota (called IgA-seq) was used to define immune-reactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity. RESULTS: Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3+/RORγt+ regulatory T cells, induction of interleukin (IL) 10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models. CONCLUSIONS: This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the efficacy of microbial therapy for UC. Clinicaltrials.gov ID NCT02516384.

The Relationship Between the Endoscopic Healing Index, Fecal Calprotectin, and Magnetic Resonance Enterography in Crohn's Disease.

INTRODUCTION: The serum-based endoscopic healing index (EHI) test identifies endoscopic Crohn's disease (CD) activity. Data are lacking on the relationship between EHI with other endpoints. We assessed the relationship between EHI and the simplified Magnetic Resonance Index of Activity. MATERIALS AND METHODS: Data were prospectively collected on patients with CD with either an EHI or fecal calprotectin (FCAL) within 90 days of magnetic resonance enterography (MRE). Diagnostic accuracy was assessed using area under the receiver operator characteristics. Proportions with any, severe, and terminal ileum MR inflammation were compared above/below identified thresholds for both EHI and FCAL. RESULTS: A total of 241 MREs paired to either EHI or FCAL from 155 patients were included. Both EHI and FCAL had similar accuracy to diagnose inflammation (area under the receiver operator characteristics: EHI: 0.635 to 0.651, FCAL: 0.680 to 0.708). Optimal EHI values were 42 and 26 for inflammation on MRE and endoscopy, respectively. Patients with EHI ≥42 (100% vs. 63%, P=0.002), FCAL >50 µg/g (87% vs. 64%, P<0.001) and FCAL >250 µg/g (90% vs. 75%, P=0.02) had higher rates of simplified Magnetic Resonance Index of Activity ≥1 compared with lower values. EHI differentiated ileitis numerically more than FCAL (delta: 24% to 25% vs. 11% to 21%). Patients with FCAL ≥50 µg/g had higher rates of severe inflammation compared with FCAL <50 µg/g (75% vs. 47%, P<0.001), whereas smaller differentiation existed for EHI threshold of 42 (63% vs. 49%, P=0.35). CONCLUSION: Both EHI and FCAL were specific in their confirmation of inflammation and disease activity on MRE in patients with CD. However, MRE-detected inflammation was frequently present in the presence of low EHI and FCAL in similar proportions.

Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring.

Maternal immune activation (MIA) contributes to behavioural abnormalities associated with neurodevelopmental disorders in both primate and rodent offspring. In humans, epidemiological studies suggest that exposure of fetuses to maternal inflammation increases the likelihood of developing autism spectrum disorder. In pregnant mice, interleukin-17a (IL-17a) produced by T helper 17 (TH17) cells (CD4+ T helper effector cells involved in multiple inflammatory conditions) induces behavioural and cortical abnormalities in the offspring exposed to MIA. However, it is unclear whether other maternal factors are required to promote MIA-associated phenotypes. Moreover, the underlying mechanisms by which MIA leads to T cell activation with increased IL-17a in the maternal circulation are not well understood. Here we show that MIA phenotypes in offspring require maternal intestinal bacteria that promote TH17 cell differentiation. Pregnant mice that had been colonized with mouse commensal segmented filamentous bacteria or human commensal bacteria that induce intestinal TH17 cells were more likely to produce offspring with MIA-associated abnormalities. We also show that small intestine dendritic cells from pregnant, but not from non-pregnant, females secrete IL-1ß, IL-23 and IL-6 and stimulate T cells to produce IL-17a upon exposure to MIA. Overall, our data suggest that defined gut commensal bacteria with a propensity to induce TH17 cells may increase the risk of neurodevelopmental disorders in the offspring of pregnant mothers undergoing immune system activation owing to infections or autoinflammatory syndromes.

Impact of Inflammatory Bowel Disease Therapies on Durability of Humoral Response to SARS-CoV-2 Vaccination.

Immunization against the spike protein of SARS-CoV-2 reduces transmission1,2 and severe outcomes. However, little is known regarding the impact of immune-mediated diseases and immunosuppressive medications on the efficacy of vaccination. Vaccination immunity is transient, with breakthrough cases increasing at longer time intervals since the last dose.3,4 Although there are data on SARS-CoV-2 vaccine on early seroconversion in patients with inflammatory bowel disease (IBD),5 no data in the same cohort exist describing the durability of these antibodies over time. We sought to investigate the impact of IBD and its therapies on postvaccination antibody response and kinetics of immunogenicity decline, because these findings may better inform clinical guidelines and recommendations on precautions and booster vaccination.

Dual Biologic or Small Molecule Therapy for Treatment of Inflammatory Bowel Disease: A Systematic Review and Meta-analysis.

BACKGROUND AND AIMS: We conducted a systematic review and meta-analysis to summarize emerging data on the safety and effectiveness of dual biologic therapy in combination or with tofacitinib in patients with refractory inflammatory bowel disease (IBD). METHODS: Through a systematic search of multiple electronic databases through November 9, 2020, we identified cohort studies or case series (>10 patients) reporting the safety and effectiveness of simultaneous use of biologic agents in combination or with tofacitinib in patients with IBD. Rates of adverse events, clinical remission, and endoscopic remission were synthesized using pooled data, and we identified factors associated with successful dual therapy. RESULTS: We identified 30 studies reporting 288 trials of dual biologic or small molecule therapy in 279 patients (76% Crohn's disease; median duration of treatment 24 weeks (IQR25-IQR75 1332)). The main indications for dual therapy included medically refractory IBD (81%) and concurrent extra-intestinal manifestations or rheumatologic disease (12%). The most common combinations of dual therapy included tumor necrosis factor-α antagonists & anti-integrins (48%), ustekinumab & anti-integrins (19%); 61% of patients had previously failed at least one of the two therapies used in combination. Over a median follow-up of 32 weeks (IQR25-IQR75 24-52), pooled rates of adverse and serious adverse events were 31% (95% CI, 13%-54%) and 6.5% (95% CI, 2.1%-13.1%); pooled rates of clinical and endoscopic remission were 59% (95% CI, 42%-74%), and 34% (95% CI, 23%-46%), respectively. 12% (95% CI, 4%-24%) of patients required surgery. Rates of success were higher in patients on dual therapy due to EIM. Heterogeneity was not significant for endoscopic response (P = .88, I2 = 0%), endoscopic remission (P = .44, I2 = 0%), and malignancy (P = .87, I2 = 0%). However, significant heterogeneity existed for other outcomes. CONCLUSIONS: Dual biologic or small molecule therapy may be a possible option in highly selected, refractory IBD patients at specialized centers. Higher quality combination of therapies with a significant improvement in the quality of data is required prior to more widespread use.

A Quality Improvement Initiative Is Associated With Reduced Time to Administer Biologics and Small Molecules and Emergency Room Visits in Inflammatory Bowel Disease.

BACKGROUND: Delays in biologic or small molecule medication administration are associated with increased adverse events, hospitalization, and surgery in inflammatory bowel disease (IBD). We evaluated the impact of a quality improvement (QI) intervention on the time to administration of biologics or small molecules (TABS) in IBD. METHODS: Data were retrospectively extracted for IBD patients prescribed biologics or small molecules from a convenience sample of providers participating in an accredited QI educational intervention (baseline cohort). Subsequent to the intervention, data were prospectively collected from patients prescribed these medications (postintervention cohort). Dates related to steps between a treatment decision to medication administration were collected. The primary outcome compared TABS in baseline and postintervention cohorts. RESULTS: Eighteen physicians provided survey and patient data for 200 patients in each cohort (n=400). The median time to medication administration (TABS) decreased from baseline to postintervention cohorts (30 vs. 26 d, P=0.04). Emergency room visits before medication administration also decreased (25.5% vs. 12.5%, P=0.001). Similar numerical TABS reductions were observed in subgroups limited to physicians providing patients to both cohorts and for individual medications prescribed. Primary contributors to delays included filling prescriptions subsequent to insurance approval and dispensation subsequent to this. CONCLUSIONS: A QI intervention successfully reduced medication administration times (TABS) by accelerating provider-dependent steps. This intervention was associated with reduced emergency room visits. We propose TABS as a quality metric to assess the effective delivery of therapies in IBD. Further evaluation of QI interventions, patient education on prescription drug insurance, and quality metrics are warranted.

Microbiota restricts trafficking of bacteria to mesenteric lymph nodes by CX(3)CR1(hi) cells.

The intestinal microbiota has a critical role in immune system and metabolic homeostasis, but it must be tolerated by the host to avoid inflammatory responses that can damage the epithelial barrier separating the host from the luminal contents. Breakdown of this regulation and the resulting inappropriate immune response to commensals are thought to lead to the development of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. We proposed that the intestinal immune system is instructed by the microbiota to limit responses to luminal antigens. Here we demonstrate in mice that, at steady state, the microbiota inhibits the transport of both commensal and pathogenic bacteria from the lumen to a key immune inductive site, the mesenteric lymph nodes (MLNs). However, in the absence of Myd88 or under conditions of antibiotic-induced dysbiosis, non-invasive bacteria were trafficked to the MLNs in a CCR7-dependent manner, and induced both T-cell responses and IgA production. Trafficking was carried out by CX(3)CR1(hi) mononuclear phagocytes, an intestinal-cell population previously reported to be non-migratory. These findings define a central role for commensals in regulating the migration to the MLNs of CX(3)CR1(hi) mononuclear phagocytes endowed with the ability to capture luminal bacteria, thereby compartmentalizing the intestinal immune response to avoid inflammation.

Crohn's Disease: Evolution, Epigenetics, and the Emerging Role of Microbiome-Targeted Therapies.

Crohn's disease (CD) is a chronic, systemic, immune-mediated inflammation of the gastrointestinal tract. Originally described in 1932 as non-caseating granulomatous inflammation limited to the terminal ileum, it is now recognized as an expanding group of heterogeneous diseases defined by intestinal location, extent, behavior, and systemic extraintestinal manifestations. Joint diseases, including inflammatory spondyloarthritis and ankylosing spondylitis, are the most common extraintestinal manifestations of CD and share more genetic susceptibility loci than any other inflammatory bowel disease (IBD) trait. The high frequency and overlap with genes associated with infectious diseases, specifically Mendelian susceptibility to mycobacterial diseases (MSMD), suggest that CD may represent an evolutionary adaptation to environmental microbes. Elucidating the diversity of the enteric microbiota and the protean mucosal immune responses in individuals may personalize microbiome-targeted therapies and molecular classifications of CD. This review will focus on CD's natural history and therapies in the context of epigenetics, immunogenetics, and the microbiome.

The functional impact of the intestinal microbiome on mucosal immunity and systemic autoimmunity.

PURPOSE OF REVIEW: This review will highlight recent advances functionally linking the gut microbiome with mucosal and systemic immune cell activation underlying autoimmunity. RECENT FINDINGS: Dynamic interactions between the gut microbiome and environmental cues (including diet and medicines) shape the effector potential of the microbial organ. Key bacteria and viruses have emerged that, in defined microenvironments, play a critical role in regulating effector lymphocyte functions. The coordinated interactions between these different microbial kingdoms - including bacteria, helminths, and viruses (termed transkingdom interactions) - play a key role in shaping immunity. Emerging strategies to identify immunologically relevant microbes with the potential to regulate immune cell functions both at mucosal sites and systemically will likely define diagnostic and therapeutic targets. SUMMARY: The microbiome constitutes a critical microbial organ with coordinated interactions that shape host immunity.