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…

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…

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 …

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

CD16+ Macrophages Mediate Fibrosis in Inflammatory Bowel Disease.

Background and Aims Fibrosis is a common complication of Crohn's disease [CD], and is related to dysregulated tissular repair following inflammation, in which macrophages play a central role. We have previously observed that STAT6-/- mice present delayed mucosal recovery after 2,4,6-trinitrobenzenesulfonic acid [TNBS]-induced colitis due to a deficiency in reparatory interleukin-4 [IL4]/STAT6-dependent M2 macrophages, which can be reverted by the exogenous transfer of this cell type. In the present study, we analyse the role of STAT6-dependent macrophages in intestinal fibrosis. Methods Colitis was induced by weekly intra-rectal administration of TNBS [6 weeks] to STAT6-/- mice and wild-typ…

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

iNOS-derived nitric oxide mediates the increase in TFF2 expression associated with gastric damage: role of HIF-1.

Trefoil (TFF) peptides are involved in gastrointestinal mucosal restitution. An hypoxia inducible factor 1 (HIF-1)-dependent induction of TFF genes has been reported in gastric epithelial cells. Nitric oxide (NO) is associated with mucosal damage and modulates HIF-1 activity. The aim of the present study was to analyze the role of iNOS-derived NO in HIF-1alpha stabilization and TFF gene expression in damaged gastric mucosa. Aspirin caused gastric injury that peaked 6 h after dosing and returned to normality at 24 h. iNOS mRNA expression occurs in the corpus in parallel with damage. Blockade of iNOS activity did not modify gastric lesions induced by aspirin but delayed mucosal healing. Aspir…

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…

Induction of trefoil factor (TFF)1, TFF2 and TFF3 by hypoxia is mediated by hypoxia inducible factor-1: implications for gastric mucosal healing

Background and purpose:  Mucosal microcirculation is compromised during gastric damage induced by non-steroidal anti-inflammatory drugs, such as aspirin. Consequently, oxygen supply to epithelial cells is decreased. The trefoil factor (TFF) peptides are involved in mechanisms of defence and repair in the gastrointestinal tract but their regulation at sites of gastric injury is unknown. Experimental approach:  Hypoxia and expression of TFF genes and peptides were measured in the damaged stomach of aspirin-treated rats. In a human gastric cell line (AGS cells), the effects of hypoxia and of hypoxia inducible factor (HIF)-1 (through transient transfection of HIF-1α siRNA or over-expression of …

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…

P092 Exogenous administration of IL-4-treated macrophages prevents intestinal fibrosis in Stat6 knockout mice

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 …

Diverse stress signals activate the C1 subgroup MAP kinases ofArabidopsis

AbstractMitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in plants. In Arabidopsis, 20 MAPKs have been identified and classified into four major groups (A–D). Little is known about the role of group C MAPKs. We have studied the activation of Arabidopsis subgroup C1 MAPKs (AtMPK1/AtMPK2) in response to mechanical injury. An increase in their kinase activity was detected in response to wounding that was blocked by cycloheximide. Jasmonic acid (JA) activated AtMPK1/AtMPK2 in the absence of wounding. Wound and JA-induction of AtMPK1/2 kinase activity was not prevented in the JA-insensitive coi1 mutant. Other stress signals, such as abscisic a…

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…

Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A.

Plant cells possess much of the molecular machinery necessary for receptor-mediated endocytosis (RME), but this process still awaits detailed characterization. In order to identify a reliable and well-characterized marker to investigate RME in plant cells, we have expressed the human transferrin receptor (hTfR) in Arabidopsis protoplasts. We have found that hTfR is mainly found in endosomal (Ara7- and FM4-64-positive) compartments, but also at the plasma membrane, where it mediates binding and internalization of its natural ligand transferrin (Tfn). Cell surface expression of hTfR increases upon treatment with tyrphostin A23, which inhibits the interaction between the YTRF endocytosis signa…

A Single Nucleotide Polymorphism in the Vitamin D Receptor Gene Is Associated With Decreased Levels of the Protein and a Penetrating Pattern in Crohn’s Disease

Background: Vitamin D signaling modulates inflammation through the vitamin D receptor (VDR). The synonymous single nucleotide polymorphism (SNP) rs731236, located in the VDR gene, has been associated with a higher risk of Crohn's disease (CD). We analyzed differences in VDR expression levels among CD patients who were homozygous for allelic variants in this SNP and their relevance for disease course. Methods: DNA was extracted from blood samples of CD patients, and SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. Fresh blood from patients was used to isolate peripheral blood mononuclear cells (PBMCs) or to determine the expression of adhesi…

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…

P087 Increased wnt ligands expression in M2c macrophages is associated with fibrosis in Stat6 knockout mice

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…

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

La utilización de la metodología ApS refuerza la adquisición de competencias a largo plazo

[EN] Service-learning (SL) is a methodology that contributes to the formation of values and the citizenship of university students, who can acquire a relevant role in the construction of a more dignified, inclusive, cohesive and equitable citizenship. The objective of the work is to quantify how the SL methodology implemented in a subject improves the competences of the students in the same course of implementation and after one year of implementation. Four experimental groups were formed: a) group LS-course 2017-2018; b) group NO-LS-course 2017-2018; c) group LS-course 2018-2019; d) group NO-LScourse 2018-2019. Each one of the groups was evaluated during the academic year 2018-2019 in the …

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…

Indomethacin Disrupts Autophagic Flux by Inducing Lysosomal Dysfunction in Gastric Cancer Cells and Increases Their Sensitivity to Cytotoxic Drugs

AbstractNSAIDs inhibit tumorigenesis in gastrointestinal tissues and have been proposed as coadjuvant agents to chemotherapy. The ability of cancer epithelial cells to adapt to the tumour environment and to resist cytotoxic agents seems to depend on rescue mechanisms such as autophagy. In the present study we aimed to determine whether an NSAID with sensitizing properties such as indomethacin modulates autophagy in gastric cancer epithelial cells. We observed that indomethacin causes lysosomal dysfunction in AGS cells and promotes the accumulation of autophagy substrates without altering mTOR activity. Indomethacin enhanced the inhibitory effects of the lysosomotropic agent chloroquine on l…

Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana.

†These authors contributed equally to the work. Accession numbers Sequence data from this article can be found in Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: At1g10210 (MPK1), At1g59580 (MPK2), At2g43790 (MPK6), At2g18170 (MPK7), At1g18150 (MPK8), At4g36450 (MPK14), At5g40440 (MKK3), At2g32510 (MAP3K17) and At1g05100 (MAP3K18). SUMMARY Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in AB…

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