P2X7 Receptor Regulates Collagen Expression in Human Intestinal Fibroblasts: Relevance in Intestinal Fibrosis.

Intestinal fibrosis is a common complication that affects more than 50% of Crohn´s Disease (CD) patients. There is no pharmacological treatment against this complication, with surgery being the only option. Due to the unknown role of P2X7 in intestinal fibrosis, we aim to analyze the relevance of this receptor in CD complications. Surgical resections from CD and non-Inflammatory Bowel Disease (IBD) patients were obtained. Intestinal fibrosis was induced with two different murine models: heterotopic transplant model and chronic-DSS colitis in wild-type and P2X7-/- mice. Human small intestine fibroblasts (HSIFs) were transfected with an siRNA against P2X7 and treated with TGF-ß. A gene and protein expression of P2X7 receptor was significantly increased in CD compared to non-IBD patients. The lack of P2X7 in mice provoked an enhanced collagen deposition and increased expression of several profibrotic markers in both murine models of intestinal fibrosis. Furthermore, P2X7-/- mice exhibited a higher expression of proinflammatory cytokines and a lower expression of M2 macrophage markers. Moreover, the transient silencing of the P2X7 receptor in HSIFs significantly induced the expression of Col1a1 and potentiated the expression of Col4 and Col5a1 after TGF-ß treatment. P2X7 regulates collagen expression in human intestinal fibroblasts, while the lack of this receptor aggravates intestinal fibrosis.

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.

pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis.

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD.

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.

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-cell proliferation. We also studied the capacity of EVMSC-T-HIFC to dampen the inflammatory response of activated endothelium and modulate fibrosis. Finally, we tested the therapeutic capacity of EVMSC-T-HIFC in an acute colitis model. EVMSC-T-HIFc induced the repolarization of monocytes from Mφ1 to an Mφ2-like phenotype, which was accompanied by reduced inflammatory cytokine release. EVMSC-T-HIFc-treated Mφ1 had similar effects of immunosuppression on activated peripheral blood mononuclear cells (PBMC) as Mφ2, and reduced the adhesion of PBMCs to activated endothelium. EVMSC-T-HIFc also prevented myofibroblast differentiation of TGF-ß-treated fibroblasts. Finally, administration of EVMSC-T-HIFc promoted healing in a TNBS-induced mouse colitis model in terms of preserving colon length and intestinal mucosa architecture and altering the ratio of Mφ1/ Mφ2 infiltration. In conclusion, EVMSC-T-HIFC have effective anti-inflammatory properties, making them potential therapeutic agents in cell free-based therapies for the treatment of Crohn's disease and likely other immune-mediated inflammatory diseases.

Hypoxia Reduces the Transcription of Fibrotic Markers in the Intestinal Mucosa.

INTRODUCTION: Intestinal fibrosis, characterized by excessive deposition of extracellular matrix proteins, is a common and severe clinical complication of inflammatory bowel disease (IBD). However, the mechanisms underlying fibrosis remain elusive, and currently, there are limited effective pharmacologic treatments that target the development of fibrosis. Hypoxia is one of the key microenvironmental factors influencing intestinal inflammation and has been linked to fibrosis. OBJECTIVE: In the present study, we sought to elucidate the impact of hypoxia on fibrotic gene expression in the intestinal mucosa. METHODS: Human volunteers, IBD patients, and dextran sulphate sodium-treated mice were exposed to hypoxia, and colonic biopsies were collected. The human intestinal epithelial cell line Caco-2, human THP-1 macrophages, and primary human gut fibroblasts were subjected to hypoxia, and changes in fibrotic gene expression were assessed. RESULTS: Human volunteers subjected to hypoxia presented reduced transcriptional levels of fibrotic and epithelial-mesenchymal transition markers in the intestinal mucosa. IBD patients showed a trend towards a decrease in tissue inhibitor of metalloproteinase 1 protein expression. In mice, hypoxic conditions reduced the colonic expression of several collagens and matrix metalloproteinases. Hypoxic Caco-2 cells, THP-1 cells, and primary gut fibroblasts showed a significant downregulation in the expression of fibrotic and tissue remodelling factors. CONCLUSIONS: Stabilization of hypoxia-inducible factors might represent a novel therapeutic approach for the treatment of IBD-associated fibrosis.

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 untargeted metabolomics from serum, faecal, urine and biopsy samples.

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 treatment.

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.

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

The pathogenesis of Crohn's disease-associated fibrostenosis and fistulas imply the epithelial-to-mesenchymal transition (EMT) process. As succinate and its receptor (SUCNR1) are involved in intestinal inflammation and fibrosis, we investigated their relevance in EMT and Crohn's disease (CD) fistulas. Succinate levels and SUCNR1-expression were analyzed in intestinal resections from non-Inflammatory Bowel Disease (non-IBD) subjects and CD patients with stenosing-B2 or penetrating-B3 complications and in a murine heterotopic-transplant model of intestinal fibrosis. EMT, as increased expression of Snail1, Snail2 and vimentin and reduction in E-cadherin, was analyzed in tissues and succinate-treated HT29 cells. The role played by SUCNR1 was studied by silencing its gene. Succinate levels and SUCNR1 expression are increased in B3-CD patients and correlate with EMT markers. SUCNR1 is detected in transitional cells lining the fistula tract and in surrounding mesenchymal cells. Grafts from wild type (WT) mice present increased succinate levels, SUCNR1 up-regulation and EMT activation, effects not observed in SUCNR1-/- tissues. SUCNR1 activation induces the expression of Wnt ligands, activates WNT signaling and induces a WNT-mediated EMT in HT29 cells. In conclusion, succinate and its receptor are up-regulated around CD-fistulas and activate Wnt signaling and EMT in intestinal epithelial cells. These results point to SUCNR1 as a novel pharmacological target for fistula prevention.

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.

Activation of pH-Sensing Receptor OGR1 (GPR68) Induces ER Stress Via the IRE1α/JNK Pathway in an Intestinal Epithelial Cell Model.

Proton-sensing ovarian cancer G-protein coupled receptor (OGR1) plays an important role in pH homeostasis. Acidosis occurs at sites of intestinal inflammation and can induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), an evolutionary mechanism that enables cells to cope with stressful conditions. ER stress activates autophagy, and both play important roles in gut homeostasis and contribute to the pathogenesis of inflammatory bowel disease (IBD). Using a human intestinal epithelial cell model, we investigated whether our previously observed protective effects of OGR1 deficiency in experimental colitis are associated with a differential regulation of ER stress, the UPR and autophagy. Caco-2 cells stably overexpressing OGR1 were subjected to an acidic pH shift. pH-dependent OGR1-mediated signalling led to a significant upregulation in the ER stress markers, binding immunoglobulin protein (BiP) and phospho-inositol required 1α (IRE1α), which was reversed by a novel OGR1 inhibitor and a c-Jun N-terminal kinase (JNK) inhibitor. Proton-activated OGR1-mediated signalling failed to induce apoptosis, but triggered accumulation of total microtubule-associated protein 1 A/1B-light chain 3, suggesting blockage of late stage autophagy. Our results show novel functions for OGR1 in the regulation of ER stress through the IRE1α-JNK signalling pathway, as well as blockage of autophagosomal degradation. OGR1 inhibition might represent a novel therapeutic approach in IBD.

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.

Macrophages as an Emerging Source of Wnt Ligands: Relevance in Mucosal Integrity.

The Wnt signaling pathway is a conserved pathway involved in important cellular processes such as the control of embryonic development, cellular polarity, cellular migration, and cell proliferation. In addition to playing a central role during embryogenesis, this pathway is also an essential part of adult homeostasis. Indeed, it controls the proliferation of epithelial cells in different organs such as intestine, lung, and kidney, and guarantees the maintenance of the mucosa in physiological conditions. The origin of this molecular pathway is the binding between Wnt ligands (belonging to a family of 19 different homologous secreted glycoproteins) and their specific membrane receptors, from the Frizzled receptor family. This specific interaction triggers the activation of the signaling cascade, which in turn activates or suppresses the expression of different genes in order to change the behavior of the cell. On the other hand, alterations of this pathway have been described in pathological conditions such as inflammation, fibrosis, and cancer. In recent years, macrophages-among other cell types-have emerged as a potential source of Wnt ligands. Due to their high plasticity, macrophages, which are central to the innate immune response, are capable of adopting different phenotypes depending on their microenvironment. In the past, two different phenotypes were described: a proinflammatory phenotype-M1 macrophages-and an anti-inflammatory phenotype-M2 macrophages-and a selective expression of Wnt ligands has been associated with said phenotypes. However, nowadays it is assumed that macrophages in vivo move through a continual spectrum of functional phenotypes. In both physiological and pathological (inflammation, fibrosis and cancer) conditions, the accumulation and polarization of macrophages conditions the future of the tissue, facilitating various scenarios, such as resolution of inflammation, activation of fibrosis, and cancer development due to the modulation of the Wnt signaling pathway, in autocrine and paracrine manner. In this work, we provide an overview of studies that have explored the role of macrophages and how they act as a source of Wnt ligands and as mediators of mucosal integrity.

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.

Iron Prevents Hypoxia-Associated Inflammation Through the Regulation of Nuclear Factor-κB in the Intestinal Epithelium.

BACKGROUND & AIMS: Hypoxia-associated pathways influence the development of inflammatory bowel disease. Adaptive responses to hypoxia are mediated through hypoxia-inducible factors, which are regulated by iron-dependent hydroxylases. Signals reflecting oxygen tension and iron levels in enterocytes regulate iron metabolism. Conversely, iron availability modulates responses to hypoxia. In the present study we sought to elucidate how iron influences the responses to hypoxia in the intestinal epithelium. METHODS: Human subjects were exposed to hypoxia, and colonic biopsy specimens and serum samples were collected. HT-29, Caco-2, and T84 cells were subjected to normoxia or hypoxia in the presence of iron or the iron chelator deferoxamine. Changes in inflammatory gene expression and signaling were assessed by quantitative polymerase chain reaction and Western blot. Chromatin immunoprecipitation was performed using antibodies against nuclear factor (NF)-κB and primers for the promoter of tumor necrosis factor (TNF) and interleukin (IL)1ß. RESULTS: Human subjects presented reduced levels of ferritin in the intestinal epithelium after hypoxia. Hypoxia reduced iron deprivation-associated TNF and IL1ß expression in HT-29 cells through the induction of autophagy. Contrarily, hypoxia triggered TNF and IL1ß expression, and NF-κB activation in Caco-2 and T84 cells. Iron blocked autophagy in Caco-2 cells, while reducing hypoxia-associated TNF and IL1ß expression through the inhibition of NF-κB binding to the promoter of TNF and IL1ß. CONCLUSIONS: Hypoxia promotes iron mobilization from the intestinal epithelium. Hypoxia-associated autophagy reduces inflammatory processes in HT-29 cells. In Caco-2 cells, iron uptake is essential to counteract hypoxia-induced inflammation. Iron mobilization into enterocytes may be a vital protective mechanism in the hypoxic inflamed mucosa.

The impact of the rs8005161 polymorphism on G protein-coupled receptor GPR65 (TDAG8) pH-associated activation in intestinal inflammation.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases (IBD) is associated with a decrease in local pH. The gene encoding G-protein-coupled receptor 65 (GPR65) has recently been reported to be a genetic risk factor for IBD. In response to extracellular acidification, proton activation of GPR65 stimulates cAMP and Rho signalling pathways. We aimed to analyse the clinical and functional relevance of the GPR65 associated single nucleotide polymorphism (SNP) rs8005161. METHODS: 1138 individuals from a mixed cohort of IBD patients and healthy volunteers were genotyped for SNPs associated with GPR65 (rs8005161, rs3742704) and galactosylceramidase (rs1805078) by Taqman SNP assays. 2300 patients from the Swiss IBD Cohort Study (SIBDC) were genotyped for rs8005161 by mass spectrometry based SNP genotyping. IBD patients from the SIBDC carrying rs8005161 TT, CT, CC and non-IBD controls (CC) were recruited for functional studies. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift, cAMP accumulation and RhoA activation were measured. RESULTS: In our mixed cohort, but not in SIBDC patients, the minor variant rs8005161 was significantly associated with UC. In SIBDC patients, we observed a consistent trend in increased disease severity in patients carrying the rs8005161-TT and rs8005161-CT alleles. No significant differences were observed in the pH associated activation of cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon an acidic extracellular pH shift. However, we observed significantly impaired RhoA activation after an extracellular acidic pH shift in IBD patients, irrespective of the rs8005161 allele. CONCLUSIONS: The T allele of rs8005161 might confer a more severe disease course in IBD patients. Human monocytes from IBD patients showed impaired pH associated RhoA activation upon an acidic pH shift.

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.

Lack of the pH-sensing Receptor TDAG8 [GPR65] in Macrophages Plays a Detrimental Role in Murine Models of Inflammatory Bowel Disease.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. METHODS: The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. RESULTS: mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naïve T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. CONCLUSIONS: TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.