Role of the epithelial barrier in intestinal fibrosis associated with inflammatory bowel disease: relevance of the epithelial-to mesenchymal transition.

In inflammatory bowel disease (IBD), chronic inflammation in the gastrointestinal tract can lead to tissue damage and remodelling, which can ultimately result in fibrosis. Prolonged injury and inflammation can trigger the activation of fibroblasts and extracellular matrix (ECM) components. As fibrosis progresses, the tissue becomes increasingly stiff and less functional, which can lead to complications such as intestinal strictures, obstructive symptoms, and eventually, organ dysfunction. Epithelial cells play a key role in fibrosis, as they secrete cytokines and growth factors that promote fibroblast activation and ECM deposition. Additionally, epithelial cells can undergo a process called epithelial-mesenchymal transition, in which they acquire a more mesenchymal-like phenotype and contribute directly to fibroblast activation and ECM deposition. Overall, the interactions between epithelial cells, immune cells, and fibroblasts play a critical role in the development and progression of fibrosis in IBD. Understanding these complex interactions may provide new targets for therapeutic interventions to prevent or treat fibrosis in IBD. In this review, we have collected and discussed the recent literature highlighting the contribution of epithelial cells to the pathogenesis of the fibrotic complications of IBD, including evidence of EMT, the epigenetic control of the EMT, the potential influence of the intestinal microbiome in EMT, and the possible therapeutic strategies to target EMT. Finally we discuss the pro-fibrotic interactions epithelial-immune cells and epithelial-fibroblasts cells.

Autophagy in intestinal fibrosis: relevance in inflammatory bowel disease.

Chronic inflammation is often associated with fibrotic disorders in which an excessive deposition of extracellular matrix is a hallmark. Long-term fibrosis starts with tissue hypofunction and finally ends in organ failure. Intestinal fibrosis is not an exception, and it is a frequent complication of inflammatory bowel disease (IBD). Several studies have confirmed the link between deregulated autophagy and fibrosis and the presence of common prognostic markers; indeed, both up- and downregulation of autophagy are presumed to be implicated in the progression of fibrosis. A better knowledge of the role of autophagy in fibrosis may lead to it becoming a potential target of antifibrotic therapy. In this review we explore novel advances in the field that highlight the relevance of autophagy in fibrosis, and give special focus to fibrosis in IBD patients.

IFNγ-Treated Macrophages Induce EMT through the WNT Pathway: Relevance in Crohn's Disease.

BACKGROUND: Fibrosis is a common complication of Crohn's disease (CD) in which macrophages play a central role. Epithelial-mesenchymal transition (EMT) and the WNT pathway have been associated with fibrosis. We aim to analyse the relevance of the tissue microenvironment in macrophage phenotype and the EMT process. METHODS: Intestinal surgical resections are obtained from control and CD patients with stenotic or penetrating behaviour. Cytokine's expression, macrophage phenotype, EMT markers and WNT signalling pathway are determined by WB, RT-PCR, ELISA or Cytometry. U937 cells are treated with IFNγ, TNFα, IL1ß, IL4 or IL10 and co-cultured with HT29 cells and, in some cases, are treated with XAV939 or miFZD4. The expression of macrophage, EMT and WNT pathway markers in U937 or HT29 cells is analysed by WB or RT-PCR. RESULTS: IFNγ, WNT6, CD16 and CD86 are increased in the intestinal tissue of CD patients. IFNγ-treated U937 activated the EMT process and WNT pathway in HT29 cells, and the EMT process is mediated by FZD4. CONCLUSIONS: An IFNγ-rich microenvironment polarises macrophages, which induces EMT through the WNT pathway.

SUCNR1 Mediates the Priming Step of the Inflammasome in Intestinal Epithelial Cells: Relevance in Ulcerative Colitis.

Intestinal epithelial cells (IECs) constitute a defensive physical barrier in mucosal tissues and their disruption is involved in the etiopathogenesis of several inflammatory pathologies, such as Ulcerative Colitis (UC). Recently, the succinate receptor SUCNR1 was associated with the activation of inflammatory pathways in several cell types, but little is known about its role in IECs. We aimed to analyze the role of SUCNR1 in the inflammasome priming and its relevance in UC. Inflammatory and inflammasome markers and SUCNR1 were analyzed in HT29 cells treated with succinate and/or an inflammatory cocktail and transfected with SUCNR1 siRNA in a murine DSS model, and in intestinal resections from 15 UC and non-IBD patients. Results showed that this receptor mediated the inflammasome, priming both in vitro in HT29 cells and in vivo in a murine chronic DSS-colitis model. Moreover, SUNCR1 was also found to be involved in the activation of the inflammatory pathways NFкB and ERK pathways, even in basal conditions, since the transient knock-down of this receptor significantly reduced the constitutive levels of pERK-1/2 and pNFкB and impaired LPS-induced inflammation. Finally, UC patients showed a significant increase in the expression of SUCNR1 and several inflammasome components which correlated positively and significantly. Therefore, our results demonstrated a role for SUCNR1 in basal and stimulated inflammatory pathways in intestinal epithelial cells and suggested a pivotal role for this receptor in inflammasome activation in UC.

The vitamin D receptor Taq I polymorphism is associated with reduced VDR and increased PDIA3 protein levels in human intestinal fibroblasts.

The synonymous single nucleotide polymorphism (SNP) rs731236, located in the vitamin D receptor (VDR) gene (Taq I) has been associated with both decreased levels of the protein in peripheral blood mononuclear cells and a fibrosis-related complication in Crohn´s disease (CD). Interactions between VDR and a protein-disulfide isomerase-associated 3 (PDIA3) in the regulation of extracellular matrix have been reported and we aim to analyze the relevance of the VDR genotypes and the effects of Vitamin D (VD) in the expression of VDR, PDIA3 and proliferation of intestinal fibroblasts. Human intestinal fibroblasts were isolated from the non-affected surgical resections of colorectal patients and classified according to the VDR genotype. In some cases, cells were transfected with specific PDIA3 siRNA. Basal and VD-stimulated expression of VDR, PDIA3 and Collagen 1A1 (COL1A1) as well as fibroblast migration/proliferation were analyzed. Our data show that intestinal fibroblasts homozygous for the C allele in the VDR gene exhibited lower VDR protein levels and higher proliferation than cells homozygous for the T allele. VD increased VDR and attenuated the accelerated proliferation of CC fibroblasts. The diminished VDR level detected in CC cells was associated with increased levels of both PDIA3 and COL1A1 expression and the transient silencing of PDIA3 significantly reduced COL1A1 expression. We conclude that intestinal fibroblasts homozygous for the C allele in the VDR gene exhibited: reduced VDR protein levels, increased proliferation and increased PDIA3/COL1A1 expression. Treatment with VD increased VDR and attenuated proliferation of these cells.

Diminished Vitamin D Receptor Protein Levels in Crohn's Disease Fibroblasts: Effects of Vitamin D.

Vitamin D (VD) deficiency has been associated to Crohn's disease (CD) pathogenesis, and the exogenous administration of VD improves the course of the disease, but the mechanistic basis of these observations remains unknown. Vitamin D receptor (VDR) mediates most of the biological functions of this hormone, and we aim to analyze here the expression of VDR in intestinal tissue, epithelial cells, and fibroblasts from CD patients. The effects of VD on a fibroblast wound healing assay and murine intestinal fibrosis are also analyzed. Our data show diminished VDR protein levels in surgical resections and epithelial cells from CD patients. In intestinal fibroblasts isolated from damaged tissue of CD patients, we detected enhanced migration and decreased VDR expression compared with both fibroblasts from non-damaged tissue of the same CD patient or control fibroblasts. Treatment with VD increased VDR protein levels, avoided the accelerated migration in CD fibroblasts, and prevented murine intestinal fibrosis induced by the heterotopic transplant model. In conclusion, our study demonstrates diminished VDR protein levels associated with enhanced migration in intestinal fibroblasts from damaged tissue of CD patients. In these cells, VD accumulates VDR and normalizes migration, which supports that CD patients would benefit from the VD anti-fibrotic therapeutic value that we demonstrate in a murine experimental model.

WNT2b Activates Epithelial-mesenchymal Transition Through FZD4: Relevance in Penetrating Crohn´s Disease.

BACKGROUND AND AIMS: Epithelial-mesenchymal transition [EMT] has been related to fibrosis and fistula formation, common complications associated with Crohn´s disease [CD]. The WNT signalling pathway mediates EMT, and specific WNT/FZD interactions have been related to the activation of this process in several diseases. We aim to analyse the relevance of EMT and WNT ligands and receptors in the penetrating behaviour of CD. METHODS: Intestinal surgical resections were obtained from control and CD patients with a stenotic or penetrating behaviour. Fibrosis was determined by the histological analysis of collagen deposition and EMT by confocal microscopy. The expression of WNT ligands, inhibitors, and FZD receptors was analysed by RT-PCR, WB, IH, and IF studies. The effects of WNT2b and the role of FZD4 in EMT were analysed in HT29 epithelial cells. RESULTS: Fibrosis and expression of EMT markers were detected in samples from CD patients irrespective of the clinical behaviour. However, an increased colocalisation of E-CADHERIN and VIMENTIN, an increased number of cells expressing WNT2b, and a higher expression of FZD4 and WNT2b/FZD4 interaction, were detected in intestinal tissue from the penetrating compared with the stenotic CD behaviour. WNT2b induced EMT in HT29 cells through FZD4 activation. CONCLUSIONS: An increased EMT, associated with increased WNT2b/FZD4 interaction, was detected in intestinal tissue from CD patients with a penetrating behaviour. WNT2b, through FZD4 activation, induces EMT in vitro which points to a novel pharmacological target to prevent intestinal penetrating complications of CD.

Autophagy Stimulation as a Potential Strategy Against Intestinal Fibrosis.

We recently observed reduced autophagy in Crohn's disease patients and an anti-inflammatory effect of autophagy stimulation in murine colitis, but both anti- and pro-fibrotic effects are associated with autophagy stimulation in different tissues, and fibrosis is a frequent complication of Crohn's disease. Thus, we analyzed the effects of pharmacological modulation of autophagy in a murine model of intestinal fibrosis and detected that autophagy inhibition aggravates, while autophagy stimulation prevents, fibrosis. These effects are associated with changes in inflammation and in collagen degradation in primary fibroblasts. Thus, pharmacological stimulation of autophagy may be useful against intestinal fibrosis.

Succinate receptor mediates intestinal inflammation and fibrosis.

Succinate, an intermediate of the tricarboxylic acid cycle, is accumulated in inflamed areas and its signaling through succinate receptor (SUCNR1) regulates immune function. We analyze SUCNR1 expression in the intestine of Crohn's disease patients and its role in murine intestinal inflammation and fibrosis. We show that both serum and intestinal succinate levels and SUCNR1 expression in intestinal surgical resections were higher in CD patients than in controls. SUCNR1 co-localized with CD86, CD206, and α-SMA+ cells in human intestine and we found a positive and significant correlation between SUCNR1 and α-SMA expression. In human isolated fibroblasts from CD patients SUCNR1 expression was higher than in those from controls and treatment with succinate increased SUCNR1 expression, fibrotic markers and inflammatory cytokines through SUCNR1. This receptor modulated the expression of pro-inflammatory cytokines in resting murine macrophages, macrophage polarization and fibroblast activation and Sucnr1-/- mice were protected against both acute TNBS-colitis and intestinal fibrosis induced by the heterotopic transplant of colonic tissue. We demonstrate increased succinate levels in serum and SUCNR1 expression in intestinal tissue of CD patients and show a role for SUCNR1 in murine intestinal inflammation and fibrosis.

Stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis.

BACKGROUND AND PURPOSE: Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis. EXPERIMENTAL APPROACH: Mice were treated with intrarectal administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (3.5 mg·20 g-1 ) and body weight was measured daily. Histological damage was scored 2 or 4 days after treatment. Some mice received trehalose (3% in drinking water 3 weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg·kg-1 , i.p.), betanin (1 g·kg-1 , i.p.) or betanin + 3-methyladenine (3MA) (10 mg·kg-1 , i.p.). Protein levels of p-mTOR, p62, LC3, BCL10, NFκB, IκBα and p-IκBα in mucosa were determined by Western blots and mRNA expression of TNFα, IL1ß, IL6, IL10, COX2, CCR7, CD11c, inducible NOS and CD86 by qRT-PCR. KEY RESULTS: Impaired autophagy associated with body weight loss and intestinal damage was detected in the mucosa of TNBS-treated mice. Administration of trehalose, rapamycin or betanin prevented the impaired autophagic flux induced by TNBS and decreased mucosal protein levels of BCL10, p-IκBα and NFκB-p65 and the expression of pro-inflammatory cytokines and M1 macrophage markers. Blockade of autophagosome formation by treatment with 3MA, prevented the reduction in protein levels of p62, BCL10, p-IκBα and NFκB-p65 induced by betanin in TNBS-treated mice and weakened the protective effects of betanin on murine colitis. CONCLUSIONS AND IMPLICATIONS: Pharmacological stimulation of mucosal autophagy reduced intestinal inflammation and improved murine colitis.

The flesh ethanolic extract of Hylocereus polyrhizus exerts anti-inflammatory effects and prevents murine colitis.

BACKGROUND & AIMS: IBD is a chronic disorder of the gastrointestinal tract characterized by mucosal inflammation and epithelial damage. Biologic therapy has significantly improved the course of the disease but there are still a high percentage of patients that do not respond to current therapies. We aim to determine the effects of the flesh ethanolic extract of Hylocereus polyrhizus (EH) in a mice model of colitis induced by TNBS. METHODS: Balb/c mice received TNBS (175 mg/kg, 100 µl, i.r.) and six and thirty hours later were administered with EH (1 g/kg, i.p.). Mice were weighted daily and after sacrificing (2 and 4 days after TNBS) we analyzed mucosal histology, myeloperoxidase activity (MPO), the expression of pro-inflammatory molecules (qPCR) and NF-κB and Iκß-α protein levels. The chemical characterization of the EH was determined by LC-MS/MS. RESULTS: The administration of EH to TNBS-treated mice prevented (P < 0.05) the loss of body weight and significantly reduced in the colon: a) histological damage score, b) MPO enzymatic activity c) the expression of pro-inflammatory molecules and d) Iκß-α degradation and nuclear NF-κß protein levels. The LC-MS analysis detected metabolites such as polyphenols and fatty acids. CONCLUSION: Systemic administration of the ethanolic extract of H. polyrhizus exerts an anti-inflammatory effect and prevents murine colitis induced by TNBS.