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.

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.

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.

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.

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. Therefore, we tested the hypothesis that HA35 ameliorates ethanol-induced gut damage. Specifically, mechanisms that restore epithelial barrier integrity and normalize expression of the Reg3 class of C-type lectin AMPs (i.e. Reg3ß and Reg3γ) were investigated. Chronic ethanol feeding to mice reduced expression of C-type lectin AMPs in the proximal small intestine (jejunum), reduced expression of tight junction proteins and increased bacterial translocation to the mesenteric lymph node. Oral consumption of HA35 during the last 6 days of ethanol exposure ameliorated the effects of chronic ethanol. Similarly, in vitro challenge of isolated intestinal organoids from murine jejunum with ethanol reduced the expression of C-type lectin AMPs and impaired barrier integrity; these ethanol-induced responses were prevented by pre-treatment with HA35. Importantly, HA receptor null jejunum-derived organoids demonstrated that the HA receptor Tlr4, but not Cd44 nor Tlr2, was required for the protective effect of HA35. Consistent with the data from organoids, HA35 did not protect Tlr4-deficient mice from chronic ethanol-induced intestinal injury. Together, these data suggest therapeutic administration of HA35 is beneficial in restoring gut epithelial integrity and defense during the early stages of ethanol-driven intestinal damage.

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.

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.

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. MyD88 was deleted prior to or after induction of colitis. Human intestinal myofibroblasts (HIMF) were exposed to various bacterial components and assessed for fibronectin (FN) and collagen I (Col1) production. RNA sequencing was performed. Post-transcriptional regulation was assessed by polysome profiling assay. Selective deletion of MyD88 in α-SMA-positive cells prior to, but not after induction of, experimental colitis decreased the degree of intestinal fibrosis. HIMF selectively responded to flagellin with enhanced FN or Col1 protein production in a MyD88-dependent manner. RNA sequencing suggested minimal transcriptional changes induced by flagellin in HIMF. Polysome profiling revealed higher proportions of FN and Col1 mRNA in the actively translated fractions of flagellin exposed HIMF, which was mediated by eIF2 alpha and 4EBP1. In conclusion, selectivity of flagellin-induced ECM secretion in HIMF is post-transcriptionally regulated. The results may represent a novel and targetable link between the gut microbiota and intestinal fibrogenesis.

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 Parabacteroides distasonis 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. Methods: This hypothesis was tested using human intestinal microvascular endothelial cells, a unique cell population exposed to microbial products under physiological and pathological conditions. Results: The results show that activation of TLR2/4, TLR4, NOD1, and NOD2 by specific bacterial ligands selectively and differentially upregulates the levels of cellular and soluble CEACAM1 produced by intestinal microvascular endothelial cells. The results also show that CEACAM1 regulates the migration, transmigration, and tube formation of these endothelial cells and mediates vessel sprouting induced by specific TLR and NLR bacterial ligands. Combined, these results demonstrate a close and reciprocal regulatory interaction between CEACAM1 and bacterial products in mediating multiple functions essential to new vessel formation in the gut mucosa. Conclusions: A coordinated and reciprocal interaction of CEACAM1 and microbiota-derived factors is necessary to optimize angiogenesis in the gut mucosa. This suggests that a coordination of endogenous and exogenous innate immune responses is necessary to promote intestinal angiogenesis under physiological and inflammatory conditions such as inflammatory bowel disease.

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). Here, we investigate whether HA35 treatment enhances ZO-1 expression by direct interaction with intestinal epithelium in vitro and have identified the HA receptor responsible for HA35-mediated ZO-1 induction in colonic epithelium in vitro and in vivo. Our results reveal that HA35 treatment increases ZO-1 expression in mouse intestinal epithelial organoids, while large HA 2000kDa is not internalized into the cells. Our immunofluorescence data indicate that layilin, but neither toll-like receptor-4 (TLR-4) nor CD44, mediate the HA35-induced ZO-1 expression in colonic epithelium in vitro and in vivo. Additionally, using layilin null mice we have determined that layilin mediates HA35 induction of ZO-1 in healthy mice and during dextran sulfate sodium (DSS)-induced colitis. Furthermore, we find that while ZO-1 expression levels are reduced, layilin expression levels are equivalent in inflammatory bowel disease (IBD) patients and non-IBD controls. Together, our data suggest that layilin is an important HA receptor, that mediates the effect of oral HA35 treatment on intestinal epithelium. HA35 holds promise as a simple dietary supplement to strengthen gut barrier defense.

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. Mechanisms for HA degradation are incompletely understood, but the novel protein KIAA1199 recently was discovered to degrade HA. We hypothesized that KIAA1199 protein is increased in CD colon fibroblasts and generates HA fragments that foster inflammation and fibrosis. METHODS: Fibroblasts were isolated from explants of surgically resected colon tissue from CD and non-inflammatory bowel disease control (ND) patients. Protein levels and tissue distribution of KIAA1199 were assessed by immunoblot and immunostaining, and functional HA degradation was measured biochemically. RESULTS: Increased levels of KIAA1199 protein were produced and deposited in the ECM by cultured CD fibroblasts compared with controls. Treatment of fibroblasts with the proinflammatory cytokine interleukin (IL) 6 increased deposition of KIAA1199 in the ECM. CD fibroblasts also produce significantly higher levels of IL6 compared with controls, and antibody blockade of IL6 receptors in CD colon fibroblasts decreased the level of KIAA1199 protein in the ECM. Colon fibroblasts degrade HA, however, small interfering RNA silencing of KIAA1199 abrogated that ability. CONCLUSIONS: CD fibroblasts produce increased levels of KIAA1199 primarily through an IL6-driven autocrine mechanism. This leads to excessive degradation of HA and the generation of proinflammatory HA fragments, which contributes to maintenance of gut inflammation and fibrosis.

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. Irradiated HIMECs show phenotypic hallmarks of radiation-induced endothelial cell activation in vitro. Moreover, HIMECs undergo changes in molecular expression pattern compatible with EndoMT, with up-regulation of mesenchymal markers and down-regulation of endothelial markers via transforming growth factor/Smad pathway activation. In vivo, EndoMT readily occurs in the human rectum after radiation therapy for rectal adenocarcinoma. Finally, EndoMT was observed in rectal mucosal and submucosal microvessels in a preclinical model of radiation proctitis in Tie2-green fluorescent protein reporter-expressing mice all along radiation proctitis development, also associated with transforming growth factor/Smad pathway activation. In conclusion, radiation-induced cell activation and tissue inflammation constitute a setting that fosters the phenotypic conversion of endothelial cells into mesenchymal cells. Therefore, EndoMT is identified as a potential participant in radiation-induced gut damage and may represent an interesting therapeutic target in cases of radiation-induced pelvic disease.

The epithelial danger signal IL-1α is a potent activator of fibroblasts and reactivator of intestinal inflammation.

Intestinal epithelial cell (IEC) death is typical of inflammatory bowel disease (IBD). We investigated: i) whether IEC-released necrotic cell products (proinflammatory mediators) amplify mucosal inflammation, ii) the capacity of necrotic cell lysates from HT29 cells or human IECs to induce human intestinal fibroblasts' (HIF) production of IL-6 and IL-8, and iii) whether IL-1α, released by injured colonocytes, exacerbated experimental IBD. Necrotic cell lysates potently induced HIF IL-6 and IL-8 production independent of Toll-like receptors 2 and 4, receptor for advanced glycation end-products, high-mobility group box 1, uric acid, IL-33, or inflammasome activation. IL-1α was the key IEC-derived necrotic cell product involved in HIF cytokine production. IL-1α-positive cells were identified in the epithelium in human IBD and dextran sulfate sodium (DSS)-induced colitis. IL-1α was detected in the stool of colitic mice before IL-1ß. IL-1α enemas reactivated inflammation after DSS colitis recovery, induced IL-1 receptor expression in subepithelial fibroblasts, and activated de novo inflammation even in mice without overt colitis, after the administration of low-dose DSS. IL-1α amplifies gut inflammation by inducing cytokine production by mesenchymal cells. IL-1α-mediated IEC-fibroblast interaction may be involved in amplifying and perpetuating inflammation, even without obvious intestinal damage. IL-1α may be a target for treating early IBD or preventing the reactivation of IBD.

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. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS: Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS: CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, the products of which heterodimerize to form an amino acid transporter in HT-29 cells after bacterial infection; levels of SLC7A5-SLC3A2 were increased in tissues from patients with UC compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5-SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3-deficient cells. CONCLUSIONS: CAP-D3 down-regulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with active UC; strategies to increase its levels might restore mucosal homeostasis to patients with active UC.

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. We studied NTR1 signaling in colitis-associated angiogenesis using 2,4,6-trinitrobenzenesulfonic acid-treated wild-type and NTR1-knockout mice. The effects of NT on HIF-1α and VEGFα were assessed on human colonic epithelial cells overexpressing NTR1 (NCM460-NTR1) and human intestinal microvascular-endothelial cells. NTR1-knockout mice had reduced microvascular density and mucosal integrity score compared with wild-type mice after 2,4,6-trinitrobenzenesulfonic acid treatment. VEGFα mRNA levels were increased in NCM460-NTR1 cells treated with 10(-7) mol/L NT, at 1 and 6 hours post-treatment. NT exposure in NCM460-NTR1 cells caused stabilization, nuclear translocation, and transcriptional activity of HIF-1α in a diacylglycerol kinase-dependent manner. NT did not stimulate tube formation in isolated human intestinal macrovascular endothelial cells but did so in human intestinal macrovascular endothelial cells cocultured with NCM460-NTR1 cells. Our results demonstrate the importance of an NTR1-HIF-1α-VEGFα axis in intestinal angiogenic responses and in the pathophysiology of colitis and inflammatory bowel disease.

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. Culture of UC LPMCs with lyso-sulfatide glycolipid to determine sulfatide induction of epithelial cell cytotoxicity, IL-13 production and IL-13Rα2 expression. Blinded quantum dot-based phenotypic analysis to assess UC LPMC expression of IL-13Rα2, CD161 and IL-13. RESULTS: Approximately 36% of UC LPMC were lyso-sulfatide tetramer positive, whereas few, if any, control LPMCs were positive. When tested, the positive cells were also CD3 and IL-13Rα2 positive. Culture of UC LPMC with lyso-sulfatide glycolipid showed that sulfatide stimulates UC LPMC production of IL-13 and induces UC CD161 LPMC-mediated cytotoxicity of activated epithelial cells; additionally, lyso-sulfatide induces enhanced expression of IL-13Rα2. Finally, blinded phenotypic analysis of UC LP MC using multicolour quantum dot-staining technology showed that approximately 60% of the LPMC bear both IL-13Rα2 and CD161 and most of these cells also produce IL-13. CONCLUSIONS: These studies show that UC lamina propria is replete with Type II NKT cells responsive to lyso-sulfatide glycolipid and bearing IL-13Rα2. Since lyso-sulfatide is a self-antigen, these data suggest that an autoimmune response is involved in UC pathogenesis.