Milk fat globule-epidermal growth factor 8 (MFGE8) prevents intestinal fibrosis.

OBJECTIVE: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues. DESIGN: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models. RESULTS: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvß5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo. CONCLUSION: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures.

The Postbiotic Butyrate Mitigates Gut Mucosal Disruption Caused by Acute Ethanol Exposure.

We aimed to test how the postbiotic butyrate impacts select gut bacteria, small intestinal epithelial integrity, and microvascular endothelial activation during acute ethanol exposure in mice and primary human intestinal microvascular endothelial cells (HIMECs). Supplementation during an acute ethanol challenge with or without tributyrin, a butyrate prodrug, was delivered to C57BL/6 mice. A separate group of mice received 3 days of clindamycin prior to the acute ethanol challenge. Upon euthanasia, blood endotoxin, cecal bacteria, jejunal barrier integrity, and small intestinal lamina propria dendritic cells were assessed. HIMECs were tested for activation following exposure to ethanol ± lipopolysaccharide (LPS) and sodium butyrate. Tributyrin supplementation protected a butyrate-generating microbe during ethanol and antibiotic exposure. Tributyrin rescued ethanol-induced disruption in jejunal epithelial barrier, elevated plasma endotoxin, and increased mucosal vascular addressin cell-adhesion molecule-1 (MAdCAM-1) expression in intestinal microvascular endothelium. These protective effects of tributyrin coincided with a tolerogenic dendritic response in the intestinal lamina propria. Lastly, sodium butyrate pre- and co-treatment attenuated the direct effects of ethanol and LPS on MAdCAM-1 induction in the HIMECs from a patient with ulcerative colitis. Tributyrin supplementation protects small intestinal epithelial and microvascular barrier integrity and modulates microvascular endothelial activation and dendritic tolerizing function during a state of gut dysbiosis and acute ethanol challenge.

Genomes of Bacteroides ovatus, B. cellulosilyticus, B. uniformis, Phocaeicola vulgatus, and P. dorei isolated from gut cavernous fistulous tract micropathologies in Crohn's disease.

Surgically removed bowels from Crohn's disease patients exhibit a novel form of micropathologies known as cavernous fistulous tract microlesions (CavFT), resembling fissures. We announce the genomes/plasmids and antimicrobial resistance genes of six CavFT bacterial isolates representing the Bacteroidota genera Bacteroides and Phocaeicola. Plasmids were identified in Bacteroides cellulosilyticus and Phocaeicola vulgatus.

Clonal Parabacteroides from Gut Microfistulous Tracts as Transmissible Cytotoxic Succinate-Commensal Model of Crohn's Disease Complications.

Crohn's disease (CD) has been traditionally viewed as a chronic inflammatory disease that cause gut wall thickening and complications, including fistulas, by mechanisms not understood. By focusing on Parabacteroides distasonis (presumed modern succinate-producing commensal probiotic), recovered from intestinal microfistulous tracts (cavernous fistulous micropathologies CavFT proposed as intermediate between 'mucosal fissures' and 'fistulas') in two patients that required surgery to remove CD-damaged ilea, we demonstrate that such isolates exert pathogenic/pathobiont roles in mouse models of CD. Our isolates are clonally-related; potentially emerging as transmissible in the community and mice; proinflammatory and adapted to the ileum of germ-free mice prone to CD-like ileitis (SAMP1/YitFc) but not healthy mice (C57BL/6J), and cytotoxic/ATP-depleting to HoxB8-immortalized bone marrow derived myeloid cells from SAMP1/YitFc mice when concurrently exposed to succinate and extracts from CavFT-derived E. coli , but not to cells from healthy mice. With unique genomic features supporting recent genetic exchange with Bacteroides fragilis -BGF539, evidence of international presence in primarily human metagenome databases, these CavFT Pdis isolates could represent to a new opportunistic Parabacteroides species, or subspecies (' cavitamuralis' ) adapted to microfistulous niches in CD.

Fibrosis in IBD: from pathogenesis to therapeutic targets.

BACKGROUND: Intestinal fibrosis resulting in stricture formation and obstruction in Crohn's disease (CD) and increased wall stiffness leading to symptoms in ulcerative colitis (UC) is among the largest unmet needs in inflammatory bowel disease (IBD). Fibrosis is caused by a multifactorial and complex process involving immune and non-immune cells, their soluble mediators and exposure to luminal contents, such as microbiota and environmental factors. To date, no antifibrotic therapy is available. Some progress has been made in creating consensus definitions and measurements to quantify stricture morphology for clinical practice and trials, but approaches to determine the degree of fibrosis within a stricture are still lacking. OBJECTIVE: We herein describe the current state of stricture pathogenesis, measuring tools and clinical trial endpoints development. DESIGN: Data presented and discussed in this review derive from the past and recent literature and the authors' own research and experience. RESULTS AND CONCLUSIONS: Significant progress has been made in better understanding the pathogenesis of fibrosis, but additional studies and preclinical developments are needed to define specific therapeutic targets.

Single-cell isolation from full-thickness human intestinal tissue resections for single-cell RNA sequencing.

Single-cell isolation techniques allow the investigation of physical and functional relationships between individual cells within a complex cell population. Here, we present a protocol for single-cell isolation from full-thickness intestinal tissue resections. We describe steps for pre-processing specimens, isolation of lamina propria and muscular layers, and red blood cell lysis. We then detail fixation of isolated cells and assessment of cell quality. The resulting cell suspension can be subjected to RNA sequencing on the 10× Chromium platform. For complete details on the use and execution of this protocol, please refer to Mukherjee et al.1.

Omics and Multi-Omics in IBD: No Integration, No Breakthroughs.

The recent advent of sophisticated technologies like sequencing and mass spectroscopy platforms combined with artificial intelligence-powered analytic tools has initiated a new era of "big data" research in various complex diseases of still-undetermined cause and mechanisms. The investigation of these diseases was, until recently, limited to traditional in vitro and in vivo biological experimentation, but a clear switch to in silico methodologies is now under way. This review tries to provide a comprehensive assessment of state-of-the-art knowledge on omes, omics and multi-omics in inflammatory bowel disease (IBD). The notion and importance of omes, omics and multi-omics in both health and complex diseases like IBD is introduced, followed by a discussion of the various omics believed to be relevant to IBD pathogenesis, and how multi-omics "big data" can generate new insights translatable into useful clinical tools in IBD such as biomarker identification, prediction of remission and relapse, response to therapy, and precision medicine. The pitfalls and limitations of current IBD multi-omics studies are critically analyzed, revealing that, regardless of the types of omes being analyzed, the majority of current reports are still based on simple associations of descriptive retrospective data from cross-sectional patient cohorts rather than more powerful longitudinally collected prospective datasets. Given this limitation, some suggestions are provided on how IBD multi-omics data may be optimized for greater clinical and therapeutic benefit. The review concludes by forecasting the upcoming incorporation of multi-omics analyses in the routine management of IBD.

Stricturing Crohn's Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions.

BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. METHODS: We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models. RESULTS: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis. CONCLUSIONS: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.

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.

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions.

Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

Human intestinal myofibroblasts deposited collagen VI enhances adhesiveness for T cells - A novel mechanism for maintenance of intestinal inflammation.

OBJECTIVE: Inflammatory bowel diseases (IBD) cause chronic intestinal damage and extracellular matrix (ECM) remodeling. The ECM may play an active role in inflammation by modulating immune cell functions, including cell adhesion, but this hypothesis has not been tested in IBD. DESIGN: Primary human intestinal myofibroblast (HIMF)-derived ECM from IBD and controls, 3D decellularized colon or ECM molecule-coated scaffolds were tested for their adhesiveness for T cells. Matrisome was analysed via proteomics. Functional integrin blockade was used to investigate the underlying mechanism. Analysis of the pediatric Crohn's disease (CD) RISK inception cohort was used to explore an altered ECM gene expression as a potential predictor for a future complicated disease course. RESULTS: HIMF-derived ECM and 3D decellularized colonic ECM from IBD bound more T cells compared to control. Control HIMFs exposed to the pro-inflammatory cytokines Iinterleukin-1ß (IL-1ß) and tumor necrosis factor (TNF) increased, and to transforming growth factor-ß1 (TGF-ß1) decreased ECM adhesiveness to T cells. Matrisome analysis of the HIMF-derived ECM revealed collagen VI as a major culprit for differences in T cell adhesion. Collagen VI knockdown in HIMF reduced adhesion T cell as did the blockage of integrin αvß1. Elevated gene expression of collagen VI in biopsies of pediatric CD patients was linked to risk for future stricturing disease. CONCLUSION: HIMF-derived ECM in IBD binds a remarkably enhanced number of T cells, which is dependent on Collagen VI and integrin αvß1. Collagen VI expression is a risk factor for a future complicated CD course. Blocking immune cells retention may represent a novel approach to treatment in IBD.

Geosocial Features and Loss of Biodiversity Underlie Variable Rates of Inflammatory Bowel Disease in a Large Developing Country: A Population-Based Study.

BACKGROUND: The epidemiology of inflammatory bowel disease (IBD) in developing countries may uncover etiopathogenic factors. We investigated IBD prevalence in Brazil by investigating its geographic, spatial, and temporal distribution, and attempted to identify factors associated with its recent increase. METHODS: A drug prescription database was queried longitudinally to identify patients and verify population distribution and density, race, urbanicity, sanitation, and Human Development Index. Prevalence was calculated using the number of IBD patients and the population estimated during the same decade. Data were matched to indices using linear regression analyses. RESULTS: We identified 162 894 IBD patients, 59% with ulcerative colitis (UC) and 41% with Crohn's disease (CD). The overall prevalence of IBD was 80 per 100 000, with 46 per 100 000 for UC and 36 per 100 000 for CD. Estimated rates adjusted to total population showed that IBD more than triplicated from 2008 to 2017. The distribution of IBD demonstrated a South-to-North gradient that generally followed population apportionment. However, marked regional differences and disease clusters were identified that did not fit with conventionally accepted IBD epidemiological associations, revealing that the rise of IBD was variable. In some areas, loss of biodiversity was associated with high IBD prevalence. CONCLUSIONS: When distribution is considered in the context of IBD prevalence, marked regional differences become evident. Despite a background of Westernization, hotspots of IBD are recognized that are not explained by population density, urbanicity, sanitation, or other indices but apparently are explained by biodiversity loss. Thus, the rise of IBD in developing countries is not uniform, but rather is one that varies depending on yet unexplored factors like geoecological conditions.

The analysis of a large population of inflammatory bowel disease (IBD) patients in a developing country reveals that the rising prevalence of IBD is not uniform and is linked to factors not traditionally associated with IBD, such as geosocial features and loss of biodiversity.

Activated intestinal muscle cells promote preadipocyte migration: a novel mechanism for creeping fat formation in Crohn's disease.

OBJECTIVE: Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn's disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. DESIGN: Tissues from normal, UC, non-strictured and strictured Crohn's disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals. RESULTS: Crohn's disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-ß1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5ß1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix. CONCLUSION: Crohn's disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

Review article: the sphingosine 1 phosphate/sphingosine 1 phosphate receptor axis - a unique therapeutic target in inflammatory bowel disease.

BACKGROUND: Ozanimod, a high selective sphingosine 1 phosphate (S1P) receptor (S1PR) 1/5 modulator was approved by the Food and Drug Administration for the treatment of adult patients with moderately to severely active ulcerative colitis. Additional S1PR modulators are being tested in clinical development programmes for both ulcerative colitis and Crohn's disease. AIM: To provide an overview of advances in understanding S1PRs biology and summarise preclinical and clinical investigations of S1P receptor modulators in chronic inflammatory disease with special emphasis on inflammatory bowel diseases (IBD). METHODS: We performed a narrative review using PubMed and ClinicalTrials.gov. RESULTS: Through S1PRs, S1P regulates multiple cellular processes, including proliferation, migration, survival, and vascular barrier integrity. The S1PRs function of regulating lymphocyte trafficking is well known, but new functions of S1PRs expand our knowledge of S1PRs biology. Several S1PR modulators are in clinical development for both ulcerative colitis and Crohn's disease and have shown promise in phase II and III studies with ozanimod now being approved for ulcerative colitis. CONCLUSIONS: S1P receptor modulators constitute a novel, promising, safe, and convenient strategy for the treatment of IBD.

Breaking the therapeutic ceiling in drug development in ulcerative colitis.

Increased knowledge of the intricate pathogenesis of ulcerative colitis has triggered an advance in drug development during the past two decades, resulting in the advent of several biological agents and small-molecule therapies. Although the increase in therapeutic options is positive, remission rates of patients with ulcerative colitis given new therapeutic agents in induction trials remain at a modest 20-30%, seemingly facing a so-called therapeutic ceiling. This therapeutic ceiling requires a critical appraisal and highlights the need for alternative strategies for drug development. In this Review, we objectively itemise the boundaries of therapeutic efficacy in ulcerative colitis, provide possible explanations for the shortcomings of current strategies, and propose solutions to achieve better therapeutic outcomes in ulcerative colitis.

Novel mechanisms and clinical trial endpoints in intestinal fibrosis.

The incidence of inflammatory bowel diseases (IBD) worldwide has resulted in a global public health challenge. Intestinal fibrosis leading to stricture formation and bowel obstruction is a frequent complication in Crohn's disease (CD), and the lack of anti-fibrotic therapies makes elucidation of fibrosis mechanisms a priority. Progress has shown that mesenchymal cells, cytokines, microbial products, and mesenteric adipocytes are jointly implicated in the pathogenesis of intestinal fibrosis. This recent information puts prevention or reversal of intestinal strictures within reach through innovative therapies validated by reliable clinical trial endpoints. Here, we review the role of immune and non-immune components of the pathogenesis of intestinal fibrosis, including new cell clusters, cytokine networks, host-microbiome interactions, creeping fat, and their translation for endpoint development in anti-fibrotic clinical trials.

Results of the Seventh Scientific Workshop of ECCO: Precision Medicine in IBD-What, Why, and How.

Many diseases that affect modern humans fall in the category of complex diseases, thus called because they result from a combination of multiple aetiological and pathogenic factors. Regardless of the organ or system affected, complex diseases present major challenges in diagnosis, classification, and management. Current forms of therapy are usually applied in an indiscriminate fashion based on clinical information, but even the most advanced drugs only benefit a limited number of patients and to a variable and unpredictable degree. This 'one measure does not fit all' situation has spurred the notion that therapy for complex disease should be tailored to individual patients or groups of patients, giving rise to the notion of 'precision medicine' [PM]. Inflammatory bowel disease [IBD] is a prototypical complex disease where the need for PM has become increasingly clear. This prompted the European Crohn's and Colitis Organisation to focus the Seventh Scientific Workshop on this emerging theme. The articles in this special issue of the Journal address the various complementary aspects of PM in IBD, including what PM is; why it is needed and how it can be used; how PM can contribute to prediction and prevention of IBD; how IBD PM can aid in prognosis and improve response to therapy; and the challenges and future directions of PM in IBD. This first article of this series is structured on three simple concepts [what, why, and how] and addresses the definition of PM, discusses the rationale for the need of PM in IBD, and outlines the methodology required to implement PM in IBD in a correct and clinically meaningful way.

IL-36 in chronic inflammation and fibrosis - bridging the gap?

IL-36 is a member of the IL-1 superfamily and consists of three agonists and one receptor antagonist (IL-36Ra). The three endogenous agonists, IL-36α, -ß, and -γ, act primarily as proinflammatory cytokines, and their signaling through the IL-36 receptor (IL-36R) promotes immune cell infiltration and secretion of inflammatory and chemotactic molecules. However, IL-36 signaling also fosters secretion of profibrotic soluble mediators, suggesting a role in fibrotic disorders. IL-36 isoforms and IL-36 have been implicated in inflammatory diseases including psoriasis, arthritis, inflammatory bowel diseases, and allergic rhinitis. Moreover, IL-36 has been connected to fibrotic disorders affecting the kidney, lung, and intestines. This review summarizes the expression, cellular source, and function of IL-36 in inflammation and fibrosis in various organs, and proposes that IL-36 modulation may prove valuable in preventing or treating inflammatory and fibrotic diseases and may reveal a mechanistic link between inflammation and fibrosis.