Creeping Fat-Derived Free Fatty Acids Induce Hyperplasia of Intestinal Muscularis Propria Muscle Cells: A Novel Link Between Fat and Intestinal Stricture Formation in Crohn's Disease.

Background & Aims: In Crohn's disease, wrapping of mesenteric fat around the bowel wall, so-called "creeping fat," is highly associated with strictures. The strongest contributor to luminal narrowing in strictures is a thickening of the human intestinal muscularis propria (MP). We investigated creeping fat-derived factors and their effect on mechanisms of human intestinal MP smooth muscle cell (HIMC) hyperplasia.

Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism.

Background: Metabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function.

Genomes of , , , , and 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 genera and . Plasmids were identified in and .

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.

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.

Clonal 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 (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 , but not to cells from healthy mice.

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.

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.

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.

35kDa hyaluronan ameliorates ethanol driven loss of anti-microbial defense and intestinal barrier integrity in a TLR4-dependent manner.

Acute and chronic alcohol exposure compromise intestinal epithelial integrity, due to reduced expression of anti-microbial peptides (AMP) and loss of tight junction integrity. Ameliorating gut damage is beneficial in preventing associated distant organ pathologies. Orally administered purified hyaluronan (HA) polymers with an average size of 35 kDa have multiple protective effects in the gut and are well-tolerated in humans.

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.

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.

Selective deletion of MyD88 signaling in α-SMA positive cells ameliorates experimental intestinal fibrosis via post-transcriptional regulation.

Intestinal fibrosis leading to strictures remains a significant clinical problem in inflammatory bowel diseases (IBD). The role of bacterial components in activating intestinal mesenchymal cells and driving fibrogenesis is largely unexplored. Tamoxifen-inducible α-SMA promoter Cre mice crossed with floxed MyD88 mice were subjected to chronic dextran sodium sulfate colitis.

Complete Genome Sequence of a Parabacteroides distasonis Strain (CavFT hAR46) Isolated from a Gut Wall-Cavitating Microlesion in a Patient with Severe Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) of the digestive tract in humans. There is evidence that could contribute to IBD. Here, we present the complete genome sequence of a strain designated CavFT-hAR46, which was isolated from a gut intramural cavernous fistulous tract (CavFT) microlesion in a CD patient.

Mutual Regulation of TLR/NLR and CEACAM1 in the Intestinal Microvasculature: Implications for IBD Pathogenesis and Therapy.

Background: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) displays multiple activities, among which pathogen binding and angiogenesis are particularly prominent. These same functions are also exerted by Toll- and NOD-like receptors (TLRs and NLRs), which are critical mediators of innate immune responses. We investigated whether a functional inter-relationship exists between CEACAM1 and TLRs and NLRs and its potential impact on induction of intestinal angiogenesis.

Layilin is critical for mediating hyaluronan 35kDa-induced intestinal epithelial tight junction protein ZO-1 in vitro and in vivo.

Tight junction proteins are critical in maintaining homeostatic intestinal permeability. Multiple intestinal inflammatory diseases are correlated with reduced expression of tight junction proteins. We have recently reported that oral treatment of mice with Hyaluronan 35kDa (HA35) increases colonic expression of tight junction protein zonula occludens-1 (ZO-1).

Crohn's Disease Fibroblasts Overproduce the Novel Protein KIAA1199 to Create Proinflammatory Hyaluronan Fragments.

Background & Aims: Crohn's Disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Fibrosis, a serious complication of CD, occurs when activated intestinal fibroblasts deposit excessive amounts of extracellular matrix (ECM) in affected areas. A major component of the ECM is high-molecular-weight hyaluronan (HA) that, when depolymerized to low-molecular-weight fragments, becomes proinflammatory and profibrotic.

Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis.

The endothelial-to-mesenchymal transition (EndoMT) is a crucial cellular process during heart development necessary to the formation of cardiac valves. This embryonic process reappears in several pathological situations, such as vascular injury or organ fibrosis of various etiologies, as a mediator of extracellular matrix-producing cells. Because radiation induces both vascular damage and fibrosis, we investigated whether radiation exposure induces EndoMT in primary human intestinal microvascular endothelial cells (HIMECs) and whether EndoMT contributes to radiation-induced rectal damage in humans and in a preclinical model of radiation proctitis in mice.

Chromosome-associated protein D3 promotes bacterial clearance in human intestinal epithelial cells by repressing expression of amino acid transporters.

Background & Aims: Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The Drosophila chromosome-associated protein D3 (dCAP-D3) regulates responses to bacterial infection.

The neurotensin-HIF-1α-VEGFα axis orchestrates hypoxia, colonic inflammation, and intestinal angiogenesis.

The expression of neurotensin (NT) and its receptor (NTR1) is up-regulated in experimental colitis and inflammatory bowel disease; NT/NTR1 interactions regulate gut inflammation. During active inflammation, metabolic shifts toward hypoxia lead to the activation of hypoxia-inducible factor (HIF)-1, which enhances vascular endothelial growth factor (VEGF) expression, promoting angiogenesis. We hypothesized that NT/NTR1 signaling regulates intestinal manifestations of hypoxia and angiogenesis by promoting HIF-1 transcriptional activity and VEGFα expression in experimental colitis.

IL-13Rα2-bearing, type II NKT cells reactive to sulfatide self-antigen populate the mucosa of ulcerative colitis.

Objective: Previous studies have shown that ulcerative colitis (UC) is associated with the presence of lamina propria non-invariant (Type II) NKT cells producing IL-13 and mediating epithelial cell cytotoxicity. Here we sought to define the antigen(s) stimulating the NKT cells and to quantitate these cells in the UC lamina propria.

Design: Detection of Type II NKT cells in UC lamina propria mononuclear cells (LPMC) with lyso-sulfatide loaded tetramer and quantum dot-based flow cytometry and staining.

T-helper 2 cytokines, transforming growth factor β1, and eosinophil products induce fibrogenesis and alter muscle motility in patients with eosinophilic esophagitis.

Background & Aims: Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEFs), human esophageal muscle cells (HEMCs), and esophageal muscle strips to eosinophil-derived products.

Methods: Biopsy specimens were collected via endoscopy from the upper, middle, and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls).

Cytokine-induced chromatin modifications of the type I collagen alpha 2 gene during intestinal endothelial-to-mesenchymal transition.

Background: Fibrosis of the intestine is currently an irreversible complication of inflammatory bowel disease; yet, little is understood of the underlying pathogenesis and antifibrotic strategies remain elusive. To develop effective therapies, knowledge of the mechanism of transcription and excessive deposition of type I collagen, a hallmark of fibrosis, is needed. We have shown previously that endothelial-to-mesenchymal transition (EndoMT) contributes to the pool of intestinal fibrotic cells and that a cytokine cocktail (interleukin 1-β, tumor necrosis factor α, and transforming growth factor β) induces collagen I alpha 2 (COL1A2) mRNA and protein.

Pro-angiogenic activity of TLRs and NLRs: a novel link between gut microbiota and intestinal angiogenesis.

Background & Aims: In intestinal inflammation the gut microbiota induces an innate immune response by activating epithelial and immune cells that initiate or maintain inflammation. We investigated whether the microbiota also can activate local microvascular cells and induce angiogenesis.

Methods: Human intestinal microvascular endothelial cells (HIMEC) and human intestinal fibroblasts (HIF) were exposed to bacterial ligands specific for Toll-like receptor (TLR)2/6 and 4, and NOD1 and NOD2, and cell proliferation, migration, transmigration, tube formation, and production of pro-angiogenic factors were measured.