Mucosal Single-Cell Profiling of Crohn's-Like Disease of the Pouch Reveals Unique Pathogenesis and Therapeutic Targets.

BACKGROUND & AIMS: The pathophysiology of Crohn's-like disease of the ileal pouch-anal anastomosis (CDP) in patients with a history of ulcerative colitis (UC) is unknown. We examined mucosal cells from patients with and without CDP using single cell analyses. METHODS: Endoscopic samples were collected from pouch body and pre-pouch ileum (pouch/ileum) of 50 patients with an IPAA. Single-cell RNA sequencing (scRNA-seq) was performed on pouch/ileal tissues of patients with normal pouch/ileum and CDP. Mass cytometry was performed on mucosal immune cells from patients with UC with normal pouch/ileum, CDP, pouchitis, as well as those with familial adenomatous polyposis (FAP) after pouch formation. Findings were independently validated using immunohistochemistry. RESULTS: The cell populations/states in pouch body differed from those in pre-pouch ileum, likely secondary to increased microbial burden. Compared to FAP pouch, UC pouch was enriched in colitogenic immune cells even without inflammation. CDP was characterized by increases in Th17 cells, inflammatory fibroblasts, inflammatory monocytes, TREM1+ monocytes, clonal expansion of effector T cells, and overexpression of Th17-inducing cytokine genes such as IL23, IL1B and IL6 by mononuclear phagocytes (MNPs). Ligand-receptor analysis further revealed a stromal-MNP-lymphocyte circuit in CDP. Integrated analysis showed that upregulated immune mediators in CDP were similar to those in CD and pouchitis, but not UC. Additionally, CDP pouch/ileum exhibited heightened endoplasmic reticulum (ER) stress across all major cell compartments. CONCLUSIONS: CDP likely represents a distinct entity of inflammatory bowel disease with heightened ER stress in both immune and non-immune cells, which may become a novel diagnostic biomarker and therapeutic target for CDP.

First-in-Human Assessment of Gut Permeability in Crohn's Disease Patients Using Fluorophore Technology.

Background and Aims: The dual sugar absorption test as a classic measure of human intestinal permeability has limited clinical utility due to lengthy and cumbersome urine collection, assay variability, and long turnaround. We aimed to determine if the orally administered fluorophore MB-102 (relmapirazin) (molecular weight [MW] = 372) compares to lactulose (L) (MW = 342) and rhamnose (R) (MW = 164)-based dual sugar absorption test as a measure of gut permeability in people with a spectrum of permeability including those with Crohn's disease (CD). Methods: We performed a single-center, randomized, open-label, crossover study comparing orally administered MB-102 (1.5 or 3.0 mg/kg) to L (1000 mg) and R (200 mg). Adults with active small bowel CD on magnetic resonance enterography (cases) and healthy adults (controls) were randomized to receive either MB-102 or L and R on study day 1, and the other tracer 3 to 7 days later. Urine was collected at baseline and 1, 2, 4, 6, 8, 10, and 12 hours after tracer ingestion to calculate the cumulative urinary percent excretion of MB-102 and L and R. Results: Nine cases and 10 controls completed the study without serious adverse events. Urinary recovery of administered MB-102 correlated with recovery of lactulose (r-squared = 0.83) for all participants. MB-102 urine recovery was also tracked with the L:R ratio urine recovery (r-squared = 0.57). In controls, the percentages of L and MB-102 recovered were similar within a narrow range, unlike in CD patients. Conclusion: This first-in-human study of an orally administered fluorophore to quantify gastrointestinal permeability in adults with CD demonstrates that MB-102 is well tolerated, and its recovery in urine mirrors that of percent L and the L:R ratio.

Comprehensive Association Analyses of Extraintestinal Manifestations in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) frequently develop extraintestinal manifestations (EIMs) that contribute substantially to morbidity. We assembled the largest multicohort data set to date to investigate the clinical, serologic, and genetic factors associated with EIM complications in IBD. METHODS: Data were available in 12,083 unrelated European ancestry IBD cases with presence or absence of EIMs (eg, ankylosing spondylitis [ankylosing spondylitis and sacroiliitis], primary sclerosing cholangitis [PSC], peripheral arthritis, and skin and ocular manifestations) across 4 cohorts (Cedars-Sinai Medical Center, National Institute for Diabetes and Digestive and Kidney Diseases IBD Genetics Consortium, Sinai Helmsley Alliance for Research Excellence Consortium, and Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort). Clinical and serologic parameters were analyzed by means of univariable and multivariable regression analyses using a mixed-effects model. Within-case logistic regression was performed to assess genetic associations. RESULTS: Most EIMs occurred more commonly in female subjects (overall EIM: P = 9.0E-05, odds ratio [OR], 1.2; 95% CI, 1.1-1.4), with CD (especially colonic disease location; P = 9.8E-09, OR, 1.7; 95% CI, 1.4-2.0), and in subjects who required surgery (both CD and UC; P = 3.6E-19, OR, 1.7; 95% CI, 1.5-1.9). Smoking increased risk of EIMs except for PSC, where there was a "protective" effect. Multiple serologic associations were observed, including with PSC (anti-nuclear cytoplasmic antibody; IgG and IgA, anti-Saccharomyces cerevisiae antibodies; and anti-flagellin) and any EIM (anti-nuclear cytoplasmic antibody; IgG and IgA, anti-Saccharomyces cerevisiae antibodies; and anti-Pseudomonas fluorescens-associated sequence). We identified genome-wide significant associations within major histocompatibility complex (ankylosing spondylitis and sacroiliitis, P = 1.4E-15; OR, 2.5; 95% CI, 2.0-3.1; PSC, P = 2.7E-10; OR, 2.8; 95% CI, 2.0-3.8; ocular, P = 2E-08, OR, 3.6; 95% CI, 2.3-5.6; and overall EIM, P = 8.4E-09; OR, 2.2; 95% CI, 1.7-2.9) and CPEB4 (skin, P = 2.7E-08; OR, 1.5; 95% CI, 1.3-1.8). Genetic associations implicated tumor necrosis factor, JAK-STAT, and IL6 as potential targets for EIMs. Contrary to previous reports, only 2% of our subjects had multiple EIMs and most co-occurrences were negatively correlated. CONCLUSIONS: We have identified demographic, clinical, and genetic associations with EIMs that revealed underlying mechanisms and implicated novel and existing drug targets-important steps toward a more personalized approach to IBD management.

Intergenerational protective anti-gut commensal immunoglobulin G originates in early life.

Maternal immunoglobulins of the class G (IgGs) protect offspring from enteric infection, but when, where, and how these antibodies are physiologically generated and confer protection remains enigmatic. We found that circulating IgGs in adult mice preferentially bind early-life gut commensal bacteria over their own adult gut commensal bacteria. IgG-secreting plasma cells specific for early-life gut bacteria appear in the intestine soon after weaning, where they remain into adulthood. Manipulating exposure to gut bacteria or plasma cell development before, but not after, weaning reduced IgG-secreting plasma cells targeting early-life gut bacteria throughout life. Further, the development of this anti-gut commensal IgG response coincides with the early-life interval in which goblet cell-associated antigen passages (GAPs) are present in the colon. Offspring of dams "perturbed" by B cell ablation or reduced bacterial exposure in early life were more susceptible to enteric pathogen challenge. In contrast to current concepts, protective maternal IgGs targeted translocating gut commensals in the offspring, not the enteric pathogen. These early-life events affecting anti-commensal IgG production have intergenerational effects for protection of the offspring.

Small Intestinal Goblet Cells Control Humoral Immune Responses and Mobilization During Enteric Infection.

Humoral immune responses within the gut play diverse roles including pathogen clearance during enteric infections, maintaining tolerance, and facilitating the assemblage and stability of the gut microbiota. How these humoral immune responses are initiated and contribute to these processes are well studied. However, the signals promoting the expansion of these responses and their rapid mobilization to the gut mucosa are less well understood. Intestinal goblet cells form goblet cell-associated antigen passages (GAPs) to deliver luminal antigens to the underlying immune system and facilitate tolerance. GAPs are rapidly inhibited during enteric infection to prevent inflammatory responses to innocuous luminal antigens. Here we interrogate GAP inhibition as a key physiological response required for effective humoral immunity. Independent of infection, GAP inhibition resulted in enrichment of transcripts representing B cell recruitment, expansion, and differentiation into plasma cells in the small intestine (SI), which were confirmed by flow cytometry and ELISpot assays. Further we observed an expansion of isolated lymphoid follicles within the SI, as well as expansion of plasma cells in the bone marrow upon GAP inhibition. S1PR1-induced blockade of leukocyte trafficking during GAP inhibition resulted in a blunting of SI plasma cell expansion, suggesting that mobilization of plasma cells from the bone marrow contributes to their expansion in the gut. However, luminal IgA secretion was only observed in the presence of S. typhimurium infection, suggesting that although GAP inhibition mobilizes a mucosal humoral immune response, a second signal is required for full effector function. Overriding GAP inhibition during enteric infection abrogated the expansion of laminar propria IgA+ plasma cells. We conclude that GAP inhibition is a required physiological response for efficiently mobilizing mucosal humoral immunity in response to enteric infection.

Gut microbiota induces weight gain and inflammation in the gut and adipose tissue independent of manipulations in diet, genetics, and immune development.

Obesity and the metabolic syndrome are complex disorders resulting from multiple factors including genetics, diet, activity, inflammation, and gut microbes. Animal studies have identified roles for each of these, however the contribution(s) specifically attributed to the gut microbiota remain unclear, as studies have used combinations of genetically altered mice, high fat diet, and/or colonization of germ-free mice, which have an underdeveloped immune system. We investigated the role(s) of the gut microbiota driving obesity and inflammation independent of manipulations in diet and genetics in mice with fully developed immune systems. We demonstrate that the human obese gut microbiota alone was sufficient to drive weight gain, systemic, adipose tissue, and intestinal inflammation, but did not promote intestinal barrier leak. The obese microbiota induced gene expression promoting caloric uptake/harvest but was less effective at inducing genes associated with mucosal immune responses. Thus, the obese gut microbiota is sufficient to induce weight gain and inflammation.

Vancomycin-induced gut microbial dysbiosis alters enteric neuron-macrophage interactions during a critical period of postnatal development.

Vancomycin is a broad-spectrum antibiotic widely used in cases of suspected sepsis in premature neonates. While appropriate and potentially lifesaving in this setting, early-life antibiotic exposure alters the developing microbiome and is associated with an increased risk of deadly complications, including late-onset sepsis (LOS) and necrotizing enterocolitis (NEC). Recent studies show that neonatal vancomycin treatment disrupts postnatal enteric nervous system (ENS) development in mouse pups, which is in part dependent upon neuroimmune interactions. This suggests that early-life antibiotic exposure could disrupt these interactions in the neonatal gut. Notably, a subset of tissue-resident intestinal macrophages, muscularis macrophages, has been identified as important contributors to the development of postnatal ENS. We hypothesized that vancomycin-induced neonatal dysbiosis impacts postnatal ENS development through its effects on macrophages. Using a mouse model, we found that exposure to vancomycin in the first 10 days of life, but not in adult mice, resulted in an expansion of pro-inflammatory colonic macrophages by increasing the recruitment of bone-marrow-derived macrophages. Single-cell RNA sequencing of neonatal colonic macrophages revealed that early-life vancomycin exposure was associated with an increase in immature and inflammatory macrophages, consistent with an influx of circulating monocytes differentiating into macrophages. Lineage tracing confirmed that vancomycin significantly increased the non-yolk-sac-derived macrophage population. Consistent with these results, early-life vancomycin exposure did not expand the colonic macrophage population nor decrease enteric neuron density in CCR2-deficient mice. Collectively, these findings demonstrate that early-life vancomycin exposure alters macrophage number and phenotypes in distinct ways compared with vancomycin exposure in adult mice and results in altered ENS development.

Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans: report of an expert meeting.

The present report summarizes the United States Department of Veterans Affairs (VA) field-based meeting titled "Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans." Our Veteran patient population experiences a high incidence of service-related chronic physical and mental health problems, such as infection, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), various forms of hematological and non-hematological malignancies, neurologic conditions, end-stage organ failure, requiring transplantation, and posttraumatic stress disorder (PTSD). We report the views of a group of scientists who focus on the current state of scientific knowledge elucidating the mechanisms underlying the aforementioned disorders, novel therapeutic targets, and development of new approaches for clinical intervention. In conclusion, we dovetailed on four research areas of interest: 1) microbiome interaction with immune cells after hematopoietic cell and/or solid organ transplantation, graft-versus-host disease (GVHD) and graft rejection, 2) intestinal inflammation and its modification in IBD and cancer, 3) microbiome-neuron-immunity interplay in mental and physical health, and 4) microbiome-micronutrient-immune interactions during homeostasis and infectious diseases. At this VA field-based meeting, we proposed to explore a multi-disciplinary, multi-institutional, collaborative strategy to initiate a roadmap, specifically focusing on host microbiome-immune interactions among those with service-related chronic diseases to potentially identify novel and translatable therapeutic targets.

Polygenic risk score predicts risk of primary sclerosing cholangitis in inflammatory bowel disease.

BACKGROUND: Forty distinct primary sclerosing cholangitis (PSC) genomic loci have been identified through multiancestry meta-analyses. The polygenic risk score (PRS) could serve as a promising tool to discover unique disease behaviour, like PSC, underlying inflammatory bowel disease (IBD). AIM: To test whether PRS indicates PSC risk in patients with IBD. MATERIALS AND METHODS: Mayo Clinic and Washington University at St Louis IBD cohorts were used to test our hypothesis. PRS was modelled through the published PSC loci and weighted with their corresponding effect size. Logistic regression was applied to predict the PSC risk. RESULTS: In total, 63 (5.6%) among 1130 patients with IBD of European ancestry had PSC. Among 381 ulcerative colitis (UC), 12% had PSC; in contrast to 1.4% in 761 Crohn disease (CD). Compared with IBD alone, IBD-PSC had significantly higher PRS (PSC risk: 3.0% at the lowest PRS quartile vs 7.2% at the highest PRS quartile, Ptrend =.03). In IBD subphenotypes subgroup analysis, multivariate analysis shows that UC-PSC is associated with more extensive UC disease (OR, 5.60; p=0.002) and younger age at diagnosis (p=0.02). In CD, multivariate analysis suggests that CD-PSC is associated with colorectal cancer (OR, 50; p=0.005). CONCLUSIONS: We found evidence that patients with IBD with PSC presented with a clinical course difference from that of patients with IBD alone. PRS can influence PSC risk in patients with IBD. Once validated in an independent cohort, this may help identify patients with the highest likelihood of developing PSC.

Novel Genetic Risk Variants and Clinical Predictors Associated With Primary Sclerosing Cholangitis in Patients With Ulcerative Colitis.

INTRODUCTION: Patients with ulcerative colitis (UC) who are likely to have primary sclerosing cholangitis (PSC) should be identified because PSC can influence UC clinical behavior and outcomes.The aim of this study was to establish a model incorporating clinical and genetic risk predictors that identifies patients with UC at risk of developing PSC. METHODS: We conducted a retrospective case-control study. Inflammatory bowel disease cohorts from multiple institutions were used as discovery and replicate datasets. Quality control criteria, including minor allele frequency, call rates, Hardy-Weinberg equilibrium, cryptic relatedness, and population stratification (through principal components), were used. Discriminative accuracy was evaluated with area under the receiver operating characteristic curve. RESULTS: Fifty-seven of 581 patients (9.8%) with UC had PSC. Multivariate analysis showed that patients with UC-PSC had more extensive disease (odds ratio [OR], 5.42; P = 1.57E-04), younger diagnosis age (younger than 20 years; OR, 2.22; P = 0.02), and less smoking (OR, 0.42; P = 0.02) than those with UC. After linkage disequilibrium pruning and multivariate analyses, 3 SNPs (rs3131621 at 6p21.33; rs9275596 and rs11244 at 6p21.32) at the HLA region were found associated with a 2- to 3-fold increased risk of PSC. Our model demonstrated good discriminatory power (area under the receiver operating characteristic curve, 88%). DISCUSSION: Three variants in HLA (6p21.3) region significantly distinguished patients with UC-PSC from patients with UC alone. Once further validated in an independent large cohort, our model could be used to identify patients with UC at risk of PSC, and it could also help guide disease management.

Antigen-driven colonic inflammation is associated with development of dysplasia in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an immune-mediated disease of the bile ducts that co-occurs with inflammatory bowel disease (IBD) in almost 90% of cases. Colorectal cancer is a major complication of patients with PSC and IBD, and these patients are at a much greater risk compared to patients with IBD without concomitant PSC. Combining flow cytometry, bulk and single-cell transcriptomics, and T and B cell receptor repertoire analysis of right colon tissue from 65 patients with PSC, 108 patients with IBD and 48 healthy individuals we identified a unique adaptive inflammatory transcriptional signature associated with greater risk and shorter time to dysplasia in patients with PSC. This inflammatory signature is characterized by antigen-driven interleukin-17A (IL-17A)+ forkhead box P3 (FOXP3)+ CD4 T cells that express a pathogenic IL-17 signature, as well as an expansion of IgG-secreting plasma cells. These results suggest that the mechanisms that drive the emergence of dysplasia in PSC and IBD are distinct and provide molecular insights that could guide prevention of colorectal cancer in individuals with PSC.

Genetic coding variant in complement factor B (CFB) is associated with increased risk for perianal Crohn's disease and leads to impaired CFB cleavage and phagocytosis.

OBJECTIVE: Perianal Crohn's disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B (CFB). DESIGN: Immunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry. RESULTS: Perianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB, in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum. CONCLUSION: pCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Neonatal development of intestinal neuroimmune interactions.

Interactions between the enteric nervous system (ENS), immune system, and gut microbiota regulate intestinal homeostasis in adults, but their development and role(s) in early life are relatively underexplored. In early life, these interactions are dynamic, because the mucosal immune system, microbiota, and the ENS are developing and influencing each other. Moreover, disrupting gut microbiota and gut immune system development, and potentially ENS development, by early-life antibiotic exposure increases the risk of diseases affecting the gut. Here, we review the development of the ENS and immune/epithelial cells, and identify potential critical periods for their interactions and development. We also highlight knowledge gaps that, when addressed, may help promote intestinal homeostasis, including in the settings of early-life antibiotic exposure.

Identification of Gut Bacteria such as Lactobacillus johnsonii that Disseminate to Systemic Tissues of Wild Type and MyD88-/- Mice.

In healthy hosts the gut microbiota is restricted to gut tissues by several barriers some of which require MyD88-dependent innate immune sensor pathways. Nevertheless, some gut taxa have been reported to disseminate to systemic tissues. However, the extent to which this normally occurs during homeostasis in healthy organisms is still unknown. In this study, we recovered viable gut bacteria from systemic tissues of healthy wild type (WT) and MyD88-/- mice. Shotgun metagenomic-sequencing revealed a marked increase in the relative abundance of L. johnsonii in intestinal tissues of MyD88-/- mice compared to WT mice. Lactobacillus johnsonii was detected most frequently from multiple systemic tissues and at higher levels in MyD88-/- mice compared to WT mice. Viable L. johnsonii strains were recovered from different cell types sorted from intestinal and systemic tissues of WT and MyD88-/- mice. L. johnsonii could persist in dendritic cells and may represent murine immunomodulatory endosymbionts.

Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis.

Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis - in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances.

Cells in the gut need to be protected against the many harmful microbes which inhabit this environment. Yet the immune system also needs to 'keep an eye' on intestinal contents to maintain tolerance to innocuous substances, such as those from the diet. The 'goblet cells' that are part of the gut lining do both: they create a mucus barrier that stops germs from invading the body, but they also can pass on molecules from the intestine to immune cells deep in the tissue to promote tolerance. This is achieved through a 'GAP' mechanism. A chemical messenger called acetylcholine can trigger both mucus release and the GAP process in goblet cells. Gustafsson et al. investigated how the cells could take on these two seemingly opposing roles in response to the same signal. A fluorescent molecule was introduced into the intestines of mice, and monitored as it pass through the goblet cells. This revealed how the GAP process took place: the cells were able to capture molecules from the intestines, wrap them in internal sack-like vesicles and then transport them across the entire cell. To explore the role of acetylcholine, Gustafsson et al. blocked the receptors that detect the messenger at the surface of goblet cells. Different receptors and therefore different cascades of molecular events were found to control mucus secretion and GAP formation; this explains how the two processes can be performed in parallel and independently from each other. Understanding how cells relay molecules to the immune system is relevant to other tissues in contact with the environment, such as the eyes, the airways, or the inside of the genital and urinary tracts. Understanding, and then ultimately harnessing this mechanism could help design of new ways to deliver drugs to the immune system and alter immune outcomes.

The microbial one-hit wonder.

Intestinal inflammation, in the absence of infection, occurs from contributions by genetics and environment. Chen et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210324) challenge this concept by demonstrating that a dominant transmissible dysbiotic microbial community predisposes to intestinal inflammation in absence of genetic alterations.

PIR-B Regulates CD4+ IL17a+ T-Cell Survival and Restricts T-Cell-Dependent Intestinal Inflammatory Responses.

BACKGROUND & AIMS: CD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype. METHODS: We used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non-inflammatory bowel disease patients and sorted human memory CD4+ T cells. RESULTS: We identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B- Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1-dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population. CONCLUSIONS: Our findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.

RNF20 and RNF40 regulate vitamin D receptor-dependent signaling in inflammatory bowel disease.

Despite the identification of several genetic factors linked to increased susceptibility to inflammatory bowel disease (IBD), underlying molecular mechanisms remain to be elucidated in detail. The ubiquitin ligases RNF20 and RNF40 mediate the monoubiquitination of histone H2B at lysine 120 (H2Bub1) and were shown to play context-dependent roles in the development of inflammation. Here, we aimed to examine the function of the RNF20/RNF40/H2Bub1 axis in intestinal inflammation in IBD patients and mouse models. For this purpose, intestinal sections from IBD patients were immunohistochemically stained for H2Bub1. Rnf20 or Rnf40 were conditionally deleted in the mouse intestine and mice were monitored for inflammation-associated symptoms. Using mRNA-seq and chromatin immunoprecipitation (ChIP)-seq, we analyzed underlying molecular pathways in primary intestinal epithelial cells (IECs) isolated from these animals and confirmed these findings in IBD resection specimens using ChIP-seq.The majority (80%) of IBD patients displayed a loss of H2Bub1 levels in inflamed areas and the intestine-specific deletion of Rnf20 or Rnf40 resulted in spontaneous colorectal inflammation in mice. Consistently, deletion of Rnf20 or Rnf40 promoted IBD-associated gene expression programs, including deregulation of various IBD risk genes in these animals. Further analysis of murine IECs revealed that H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes is RNF20/40-dependent. Finally, these effects were confirmed in a subgroup of Crohn's disease patients which displayed epigenetic and expression changes in RNF20/40-dependent gene signatures. Our findings reveal that loss of H2B monoubiquitination promotes intestinal inflammation via decreased VDR activity thereby identifying RNF20 and RNF40 as critical regulators of IBD.