P008 Differences in macrophage infiltration and Wnt ligands expression between stricturing and penetrating behaviour in Crohn’s disease

The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD

The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6(-/-) mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also ex…

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 alpha-SMA(+) cells in human intestine and we found a positive and significant correlation between SUCNR1 and alpha-SMA expression. In human isolated fibroblasts from CD patients SUCNR1 expres…

P045 Local inflammation modulates vitamin D receptor protein levels in fibroblasts

HIF-Overexpression and Pro-Inflammatory Priming in Human Mesenchymal Stromal Cells Improves the Healing Properties of Extracellular Vesicles in Experimental Crohn’s Disease

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have therapeutic potential in the treatment of several immune disorders, including ulcerative colitis, owing to their regenerative and immunosuppressive properties. We recently showed that MSCs engineered to overexpress hypoxia-inducible factor 1-alpha and telomerase (MSC-T-HIF) and conditioned with pro-inflammatory stimuli release EVs (EVMSC-T-HIFC) with potent immunomodulatory activity. We tested the efficacy of EVMSC-T-HIFC to repolarize M1 macrophages (Mφ1) to M2-like macrophages (Mφ2-like) by analyzing surface markers and cytokines and performing functional assays in co-culture, including efferocytosis and T-cel…

DOP21 Role of P2X7 in intestinal fibrosis and inflammasome activation: Relevance in Crohn′s Disease

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation. Methods Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtai…

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 cl…

Hypoxia Positively Regulates the Expression of pH-Sensing G-Protein–Coupled Receptor OGR1 (GPR68)

Background & Aims: A novel family of proton-sensing G-proteinâcoupled receptors, including ovarian cancer G-proteinâcoupled receptor 1 (OGR1) (GPR68) has been identified to play a role in pH homeostasis. Hypoxia is known to change tissue pH as a result of anaerobic glucose metabolism through the stabilization of hypoxia-inducible factor-1α. We investigated how hypoxia regulates the expression of OGR1 in the intestinal mucosa and associated cells. Methods: OGR1 expression in murine tumors, human colonic tissue, and myeloid cells was determined by quantitative reverse-transcription polymerase chain reaction. The influence of hypoxia on OGR1 expression was studied in monocytes/macrophages and…

P017 C86/CD16 macrophages accumulate in the mucosa of B3 patients and could mediate EMT in Crohn’s disease

Abstract Background Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their phenotype. Methods The aim of the present study is to analyse the pattern of expression of macrophages, of EMT-related genes and cytokines in surgical resections from Crohn’s disease (CD, n = 43) patients which were categorised according to Montreal classification (B2 or B3); unaffected mucosa of patients with ileocecal cancer was used as control (n = 20). mRNA was isolated from intestinal samples and the expression of macrophage, EMT markers and cytokines were analysed by RT-PCR. PBMCS were isolated from healthy donors and treated during…

P046 Vitamin D decreases PDIA3 and prevents the enhanced migration of fibroblasts from stricturing Crohn’s disease

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD) patients and fibroblasts play an important role in the fibrogenic process. Low vitamin D (VD) levels and a defective VD-signalling pathway have been reported in CD. VD signals through both vitamin D receptor (VDR) and protein disulfide-isomerase A3 (PDIA3) and we have previously demonstrated that VDR protein levels are reduced in fibroblasts isolated from CD patients and that VD increased VDR expression in these cells (A-2080; ECCO 2019). We aim to analyse here the effect of VD on both PDIA3 protein levels and migration in CD fibroblasts. Methods We used intestinal fibroblasts isolated from surgical resections of …

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 …

Decreased Fibrogenesis After Treatment with Pirfenidone in a Newly Developed Mouse Model of Intestinal Fibrosis

BACKGROUND Fibrosis as a common problem in patients with Crohn's disease is a result of an imbalance toward excessive tissue repair. At present, there is no specific treatment option. Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis with both antifibrotic and anti-inflammatory effects. We subsequently investigated the impact of pirfenidone treatment on development of fibrosis in a new mouse model of intestinal fibrosis. METHODS Small bowel resections from donor mice were transplanted subcutaneously into the neck of recipients. Animals received either pirfenidone (100 mg/kg, three times daily, orally) or vehicle. RESULTS After administration of pirfenidone, a signif…

Induction of CD36 and thrombospondin-1 in macrophages by hypoxia-inducible factor 1 and its relevance in the inflammatory process.

Inflammation is part of a complex biological response of vascular tissue to pathogens or damaged cells. First inflammatory cells attempt to remove the injurious stimuli and this is followed by a healing process mediated principally by phagocytosis of senescent cells. Hypoxia and p38-MAPK are associated with inflammation, and hypoxia inducible factor 1 (HIF-1) has been detected in inflamed tissues. We aimed to analyse the role of p38-MAPK and HIF-1 in the transcriptional regulation of CD36, a class B scavenger receptor, and its ligand thrombospondin (TSP-1) in macrophages and to evaluate the involvement of this pathway in phagocytosis of apoptotic neutrophils. We have also assessed HIF-1α, p…

OP03 Inhibition of autophagy exacerbates intestinal fibrosis and EMT

P041 Differences in NOTCH signalling between stricturing and penetrating behaviour in Crohn’s disease

Stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis

Background and pourpose A defective autophagy is involved in the pathogenesis of inflammatory disorders such as IBD. Cross talk interactions between autophagy and inflammation have been reported and we analyse the effects of autophagy stimulators on murine colitis. Experimental approach Mice were treated with intrarectal administration of TNBS (3.5 mg/20 g BW) and body weight was measured every day and histological damage score analysed two or four days after treatment. Some mice received trehalose (3% in drinking water three weeks before TNBS administration) or a daily administration of rapamycin (1.25 mg/kg, i.p.), betanin (1g/kg, i.p.) or betanin + 3MA (10mg/kg, i.p.). Mucosal protein le…

DOP87 SUCNR1 a novel key protagonist in fistula development

Abstract Background Intestinal fistula is a common complication in CD patients whose aetiology is still not well-characterised. It is associated with an exacerbated inflammation and epithelial-to-mesenchymal transition (EMT), a process which allows a switch from epithelial towards a fibrotic behaviour. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis1 but its role in fistula has not yet been analysed. Therefore, we aim to analyse the role of SUCNR1 in EMT and in fistula formation. Methods Intestinal resections were obtained from CD and non-IBD patients. Fistula specimens were identified by the surgeons and collected from B3-CD patients. The expression of S…

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

Vitamin D (VD) deficiency has been associated to Crohn&acute

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

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.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-i…

WNT2b activates epithelial-mesenchymal transition through FZD4: relevance in penetrating Crohns disease.

Abstract 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, inh…

P061 Succinate promotes EMT in intestinal epithelial cells through SUCNR1: Relevance in fistula development

M1 Macrophages Activate Notch Signalling in Epithelial Cells: Relevance in Crohn's Disease

Background: The Notch signalling pathway plays an essential role in mucosal regeneration, which constitutes a key goal of Crohn's disease (CD) treatment. Macrophages coordinate tissue repair and several phenotypes have been reported which differ in the expression of surface proteins, cytokines and hypoxia-inducible factors (HIFs). We analysed the role of HIFs in the expression of Notch ligands in macrophages and the relevance of this pathway in mucosal regeneration. Methods: Human monocytes and U937-derived macrophages were polarized towards the M1 and M2 phenotypes and the expression levels of HIF-1α, HIF-2α, Jagged 1 (Jag1) and delta-like 4 (Dll4) were evaluated. The effects of macrophage…

Hypoxic macrophages impair autophagy in epithelial cells through Wnt1: relevance in IBD.

A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelial autophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1 alpha and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of beta-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial …

Metabolomics as a Promising Resource Identifying Potential Biomarkers for Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a relapsing chronic disorder of the gastrointestinal tract characterized by disruption of epithelial barrier function and excessive immune response to gut microbiota. The lack of biomarkers providing early diagnosis or defining the status of the pathology difficulties an accurate assessment of the disease. Given the different metabolomic profiles observed in IBD patients, metabolomics may reveal prime candidates to be studied, which may help in understanding the pathology and identifying novel therapeutic targets. In this review, we summarize the most current advances describing the promising metabolites such as lipids or amino acids found through untarge…

P051 Macrophages as a source of Notch Ligands in Crohn’s disease: implications in fibrosis

Abstract Background Fibrosis constitute the main complications associated to Crohn’s disease (CD). Notch signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of Notch ligands derived from macrophages in the complications of CD. Methods We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cyt…

P073 An increased autophagy and decreased apoptosis is detected in intestinal fibroblasts from Crohn’s Disease patients

Abstract Background Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) phenotype, with no effective treatment. This process is characterized by a disequilibrium between the production and degradation of the extracellular matrix (ECM), mainly regulated by myofibroblasts. We aim to analyse here, the expression of markers of autophagy, apoptosis and proliferation in intestinal fibroblasts from CD patients. Methods Fibroblasts were isolated from the damaged intestinal mucosa of CD patients with a penetrating and stenotic behaviour. Control cells were obtained from the non-damaged intestine of patients with colorectal cancer. …

P028 The Apa I polymorphism in the Vitamin D receptor gene is associated with a stricturing behaviour in Crohn′s disease patients

Abstract Background Vitamin D receptor (VDR) is a member of the nuclear receptor family of transcription factors that plays an immunomodulatory role in the gastrointestinal tract through binding Vitamin D. Single-nucleotide polymorphisms (SNPs) in the VDR gene have been related to inflammatory bowel disease. Indeed, Crohn′s disease (CD) patients carrying the Taq I polymorphism in VDR gene run a higher risk of developing a penetrating behaviour. We analyse here the association between the VDR SNPs Taq I, Bsm I, Apa I and Fok I and the clinical characteristics of CD. Methods DNA was extracted from blood samples from 80 patients diagnosed with CD from the Hospital of Manises (Valencia). Four p…

Metabolite Sensing GPCRs: Promising Therapeutic Targets for Cancer Treatment?

G-protein-coupled receptors constitute the most diverse and largest receptor family in the human genome, with approximately 800 different members identified. Given the well-known metabolic alterations in cancer development, we will focus specifically in the 19 G-protein-coupled receptors (GPCRs), which can be selectively activated by metabolites. These metabolite sensing GPCRs control crucial processes, such as cell proliferation, differentiation, migration, and survival after their activation. In the present review, we will describe the main functions of these metabolite sensing GPCRs and shed light on the benefits of their potential use as possible pharmacological targets for cancer treat…

Is the Macrophage Phenotype Determinant for Fibrosis Development?

Fibrosis is a pathophysiological process of wound repair that leads to the deposit of connective tissue in the extracellular matrix. This complication is mainly associated with different pathologies affecting several organs such as lung, liver, heart, kidney, and intestine. In this fibrotic process, macrophages play an important role since they can modulate fibrosis due to their high plasticity, being able to adopt different phenotypes depending on the microenvironment in which they are found. In this review, we will try to discuss whether the macrophage phenotype exerts a pivotal role in the fibrosis development in the most important fibrotic scenarios.

P043 Succinate receptor (SUCNR1) mediates leukocyte-endothelial cell interactions induced by TNFa

Abstract Background The Krebs cycle metabolite succinate contributes to inflammatory conditions like arthritis and colitis by activating its receptor SUCNR1. We aimed to analyze whether the succinate-SUCNR1 pathway contributes to the leukocyte-endothelial cell interactions that initiate the inflammatory response. Methods We evaluated leukocyte rolling and adhesion by intravital microscopy in cremaster venules of wild-type (WT) and Sucrn1−/− mice treated, 4h before, with succinate (1 Mm, intraescrotally), combined or not with the common pro-inflammatory cytokine TNFα (500 ng/mice, i.p.), or with their vehicles. We analyzed the activity of the NF-κB signaling pathway in endothelial cells (HUV…

Anti-inflammatory Function of High-Density Lipoproteins via Autophagy of IκB Kinase

Background & Aims: Plasma levels of high-density lipoprotein (HDL) cholesterol are frequently found decreased in patients with inflammatory bowel disease (IBD). Therefore, and because HDL exerts anti-inflammatory activities, we investigated whether HDL and its major protein component apolipoprotein A-I (apoA-I) modulate mucosal inflammatory responses in vitro and in vivo. Methods: The human intestinal epithelial cell line T84 was used as the in vitro model for measuring the effects of HDL on the expression and secretion of tumor necrosis factor (TNF), interleukin-8 (IL-8), and intracellular adhesion molecule (ICAM). Nuclear factor-κB (NF-κB)-responsive promoter activity was studied by …

P003 C86/CD16 macrophages may act as a source of WNT2b in intestinal tissue from B3 Crohn’s disease patients

Mutant HRAS as novel target for MEK and mTOR inhibitors.

HRAS is a frequently mutated oncogene in cancer. However, mutant HRAS as drug target has not been investigated so far. Here, we show that mutant HRAS hyperactivates the RAS and the mTOR pathway in various cancer cell lines including lung, bladder and esophageal cancer. HRAS mutation sensitized toward growth inhibition by the MEK inhibitors AZD6244, MEK162 and PD0325901. Further, we found that MEK inhibitors induce apoptosis in mutant HRAS cell lines but not in cell lines lacking RAS mutations. In addition, knockdown of HRAS by siRNA blocked cell growth in mutant HRAS cell lines. Inhibition of the PI3K pathway alone or in combination with MEK inhibitors did not alter signaling nor had an imp…

P089 IFNγ-macrophages could mediate EMT in Crohn’s disease

Abstract Background Macrophages contribute to fibrosis by releasing different mediators and the pattern of secretion may vary depending on the surrounding environment. We previously described that the mRNA expression of IFNγ was significantly higher in intestinal samples from CD patients. The aim of the present study is to analyze the role of IFNγ-treated macrophages in epithelial mesenchymal transition (EMT). Methods The mRNA and protein expression of IFN in surgical resections from Crohn′s disease. U937 were differentiated to macrophages and then treated with IFNγ (2 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. IFNγ-U937 were coculture with HT29…

P056 Crohn’s Disease associated fibrosis modulates the expression of collagen receptors

Abstract Background Crohn’s Disease (CD) patients often develop stenotic complications as immunomodulatory treatments do not prevent the fibrogenic response in the affected tissues, where a dysregulated activation of stromal cells provokes an excessive deposition of extracellular matrix (ECM). Recent evidences support the notion that local cells can sense the consequent alterations in tissue structure and rigidity through receptors that respond to some ECM components, and this may perpetuate the fibrogenic process even in the absence of inflammation. We aim to analyse the relevance of these signalling pathways in the fibrotic process associated to CD. Methods We obtained fibrotic ileal tiss…

M2 Macrophages Activate WNT Signaling Pathway in Epithelial Cells: Relevance in Ulcerative Colitis

Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analy…

P030 SUCNR1 mediates inflammasome activation: Relevance in Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon1. Inflammasome complex is involved in the intestinal homeostasis regulation, but its role in UC has not been established yet. We have recently reported that SUCNR1 mediates intestinal inflammation and fibrosis2. We aim to analyze the role of SUCNR1 in inflammasome activation and UC. Methods Intestinal resections from UC and non-IBD patients were obtained. HT29 cells were treated with succinate 1mM and an inflammasome activator cocktail (TNF-α 25ng/ml, IFN-γ 20 ng/ml and LPS 1µg/ml) for 24 hours and transfected with SUCNR1 siRNA. Chronic …

Succinate Activates EMT in Intestinal Epithelial Cells through SUCNR1: A Novel Protagonist in Fistula Development

The pathogenesis of Crohn&rsquo