- Email: [email protected]
Sa1430 Immunochip Analysis Identifies Multiple Susceptibility Loci for Collagenous Colitis
AB-mediated rise in liPA is detrimental to the large intestinal barrier and may therefore be of pathophysiological relevance in the development of IBD in susceptible individuals. Methods and Results: Oral application of a mixture of vancomycin and metronidazole (V/M) resulted in a rapid and long lasting major increase (~20x) of the murine liPA whereas ampicillin treatment did not. Analogous to GF mice, cecal supernatants of V/M treated mice (V/M-cs) contain significantly higher levels of host proteases, mostly trypsin (>20x), than control (ctr) mice, as revealed by LC-MS/MS analysis. In contrast to cecal supernatants from ctr mice (ctr-cs), apical administration of V/M-cs or GF-cs significantly reduced transepithelial electrical resistance (TEER) and increased permeability towards fluorescein in transwell experiments using a large intestinal epithelial cell line (PTK6) and in Ussing chamber experiments using cecal tissue of ctr mice. Preincubation of V/M-cs or GF-cs with PMSF, a serine protease inhibitor, abrogated these detrimental effects. Short term V/M treatment (2 d) of wildtype (wt) or IL10-/- mice resulted in high liPA that was associated with increased penetration of orally administered FITC dextran (4 kDa) to the systemic circulation as well as with significantly reduced TEER and increased permeability of large intestinal tissue sections compared to the respective untreated control mice. In spite of the acute detrimental impact of V/M on the large intestinal barrier, repeated short term V/M treatments (for 7 d, at 4 and 8 weeks) did not change susceptibility of wt mice towards DSS-induced colitis (at 12 weeks). However, preliminary data from analogously V/M treated IL10-/- mice, showing significantly reduced weight gain, increased spleen to weight ratio and increased MLN weight, indicate that the repeated short term V/M treatments promote colitis development in this spontaneous IBD model. Conclusion and Outlook: V/M treatment mediates a rapid and major increase in liPA and is detrimental to the large intestinal barrier. In vitro and ex vivo data indicate that the high serine protease activity is causal for the reduced large intestinal barrier function, which needs to be confirmed in vivo by using targeted serine protease inhibition. Our results suggest that AB-mediated transient leakage of the large intestinal barrier might promote the development of IBD in susceptible individuals.
Gene Expression and miRNA Profiling in Ileal Mucosa of SERT Knock-Out Mouse Using Microarray Approaches Christopher R. Manzella, Megha Singhal, Anoop Kumar, Hayley Coffing, Alexander Ticho, Shubha Priyamvada, Arivarasu Natarajan Anbazhagan, Seema Saksena, Waddah A. Alrefai, Pradeep K. Dudeja, Ravinder K. Gill The serotonin transporter (SERT, SLC6A4), belonging to Na+ and Cl- dependent transporters, functions to regulate the availability of serotonin (5-HT) in various tissues including brain, lungs, platelets and intestine. Alterations in serotonin levels and/or SERT expression have been implicated in pathophysiological conditions including anxiety like disorders, obesity, pulmonary hypertension, cardiovascular disorders and GI disorders such as IBD, IBS and diarrhea. Accordingly, SERT-deficiency in mice is pleiotropic with many different phenotypic traits. Transgenic mice with global deletion of SERT exhibit increased anxiety, abnormal GI motility, obesity associated with hyperglycemia. Additionally, these mice are more susceptible to intestinal inflammation. How SERT deficiency underlies GI related disturbances and exacerbates the severity of gut inflammation is not known. We hypothesized that SERT deficiency causes aberrant miRNA expression and alters expression of genes implicated in regulatory networks. Thus, we evaluated global mRNA and miRNA expression profile to identify potential intestinal targets that contribute to disease pathogenesis, when SERT gene is non-functional. For these studies, RNA was extracted from ileum mucosa from age matched (9-10 wk) control and SERT KO mice by Trizol and was subjected to Phalanx Mouse miRNA and mRNA OneArray Profiling. Results demonstrated that 29 miRNAs were significantly upregulated in the SERT knockout mice ileum. Notable, upregulated miRNAs were mmulet-7c-5p, mmu-miR-16-5p, mmu-miR-31-5p, mmu-miR-143-5p, mmu-miR-6918-5p many of which have been implicated in Crohn's disease patients and in obesity. Our microarray data revealed 492 significantly upregulated and 293 downregulated genes in SERT KO mouse ileum. Pathway analysis revealed significant enrichment in cell cycle, signaling, chemotaxis, and epithelial transporters. Genes of interest were validated by RT-PCR in the ileum, which included CYP1A1 (~20 fold decrease, p < 0.05), CLDN8 (~30% decrease, p < 0.05), and HMGCS2 (~7 fold increase, p < 0.05). Similar alterations in these genes were observed in the colon of SERT KO mice by RT-PCR. A remarkable decrease in expression of Cyp1a1, encoding for a cytochrome P450 enzyme, provides novel role of serotonergic pathways in drug and xenobiotic detoxification. CLDN8 encodes for claudin 8, found to be downregulated in patients with Crohn's disease and HMGCS2, the mitochondrial 3hydroxy-3-methylglutaryl CoA synthase, deficiency of which is linked to hypoglycemia. Our data provides novel clues for differential expression of genes affected by 5-HT and/or SERT deficiency in the intestine. These studies also form the future platform to identify potential miRNA-mRNA regulatory networks in the development of 5-HT related disorders.
Sa1431 Intestinal Hypoxia and Reperfusion: Role for Eph-ephrin System Valentina Vivo, Simona Bertoni, Irene Zini, Lisa Flammini, Massimiliano Tognolini, Vigilio Ballabeni, Elisabetta Barocelli Background and aim: Mesenteric ischemia-reperfusion (I/R) is a life-threatening clinical problem consisting in the temporary deprivation of blood flow to the gut. The ensuing local organ damage is followed by the development of a systemic inflammatory response, eventually leading to multiple organ failure. Several neuronal, paracrine and plasmatic messengers are involved in mesenteric I/R injury. However, up to now, no studies have been performed on Eph receptors, the largest family of tyrosine kinase receptors, and their cell-bound ephrin ligands, whose role in cell adhesion-based processes during inflammation and immunity is growingly emerging. Eph-ephrin interaction generates a bidirectional signaling affecting both the receptor bearing (forward signaling) and the ligand bearing cells (reverse signaling). Our aim was therefore to investigate the effects produced by unidirectional activation of forward signaling (administration of chimeric protein ephrinA1-Fc; 50, 100, 200microg/kg), of reverse signaling (EphA2-Fc; 180microg/kg), or inhibition of both signals (monomeric EphA2 at equimolar doses of ephrinA1-Fc) on the inflammatory responses caused by mesenteric I/R in mice. Methods: Eph-ephrin proteins were intravenously administered to Swiss mice 5 min prior to 45 min occlusion of the superior mesenteric artery (SMA) followed by 5h reperfusion: intestinal and lung myeloperoxidase (MPO) activity, index of neutrophil recruitment, gut malondialdehyde (MDA) levels, marker of lipoperoxidation, and edema, index of increased vascular permeability, were compared to those of control I/R mice, receiving only vehicle, and of sham operated (SO) mice, subjected to the same manipulation without SMA occlusion and receiving vehicle. All experiments were performed according to the guidelines for the Care and Use of Animals (DL26/2014). Results: I/R mice displayed increased gut (45.3±4.7 vs 0.8±0.5 U/g, P<0.001) and lung (289.9±21.0 vs 144.6±45.1 U/g, P<0.05) MPO activity and higher MDA levels (595.0±87.3 vs 203.4±62.6 nmol/g dry tissue, P<0.05) and edema (4.4±0.1 vs 3.4±0.3 wet to dry weight ratio, P<0.01) compared to SO. EphrinA1Fc markedly reduced leukocyte infiltration in the gut (24.9±2.9 U/g) at 100microg/kg and in the lung (128.0±19.1 U/g) at 200microg/kg, while monomeric EphA2 significantly lowered intestinal edema formation (3.9±0.1g/g, P<0.05 vs I/R) at the highest dose. Neither EphA2Fc nor equimolar doses of IgG-Fc alone were effective in modifying I/R-induced inflammatory responses. Conclusions: These preliminary findings indicate that pharmacological modulation of Eph-ephrin system may be advantageous in limiting the inflammatory responses elicited by mesenteric I/R: forward signaling activation attenuates local and systemic leukocytes enrolment and reverse signaling silencing contrasts local changes in vascular permeability.
Sa1430 Immunochip Analysis Identifies Multiple Susceptibility Loci for Collagenous Colitis Giulia Roda, Jianzhong Hu, Zhongyang Zhang, Joana Torres, Anli Chen, Robert E. Petras, Darrell Pardi, Alina Iuga, Gabriel Levi, Wenqing Cao, Florian Rieder, Ilyssa Gordon, Ke Hao, Noam Harpaz, Judy H. Cho, Inga Peter, Jean-Frederic Colombel Background: Collagenous colitis is an increasingly recognized cause for chronic, non-bloody diarrhea. The etiology of CC remains unknown. A role for dysregulated immune responses and genetic factors has been proposed. Recently, Westerlind et al. genotyped around 300 CC cases showing the important role of HLA related immune mechanisms in CC pathogenesis [Westerlind et al. Gut 2015]. We have carried out the largest genome-wide association study of CC with the aim of identifying genetic loci that are closely associated with CC. Methods: DNA extracted from 1056 formalin-fixed, paraffin-embedded (FFPE) tissue of patients with histologically confirmed CC was genotyped using the Illumina Immunochip. Existing Immunochip genotyping data on IBD-free individuals (N=1,300) provided by the NIDDK IBD Genetics Consortium were obtained as controls. After applying quality controls filters, a total of 618 cases and 1199 controls with genotype data on 64535 single nucleotide polymorphisms (SNPs) were included in analysis. HLA alleles were imputed by SNP2HLA for samples passing QC. In addition, we performed a meta-analysis combining our association results with those recently published by Westerlind et al. for overlapping typed SNP markers and imputed HLA alleles using METAL. Results: The meta-analysis on SNPs revealed five markers, which passed the genome-wide significance level for association with CC (p-value<5e-8) and showed consistent direction of effect between the two studies. They all hits the HLA region, suggesting a role in antigen presentation and T-cell responses. In particular, based on the meta-analysis for imputed HLA alleles, the most significant association (p-value < 1e-5) with consistent increased risk was present at HLA-B*08:01, HLA-DQA1*05:01, HLADRB1*03:01 and HLA-DQB1*02:01. Moreover, a significant association (p-value < 1e-5) with consistent effect was also identified for PTPN2 on chromosome 18. This gene has a pivotal role in T-cell responses and has also been implicated in the pathogenesis of Crohn's disease. Conclusion: These findings further support and confirm the hypothesis that CC has a genetic component and HLA-related immune mechanisms are implicated on its pathogenesis. Moreover, some pathways involved in T-cell responses overlap with those of IBD, supporting the important role of T cells in CC pathogenesis.
Sa1432 CFTR Knockout Induces Oxidative Stress, Inflammation and Mitochondrial Dysfunctions in Caco-2/15 Intestinal Epithelial Cells Marie-Laure Kleme Amani, Alain Théophile Sané, Gilles Gouspillou, Jean-Philippe LeducGaudet, Carole Garofalo, David Saint-Pierre, Yves Berthiaume, Ernest G. Seidman, Emile Levy Cystic fibrosis (CF) is the most lethal of genetic diseases in Caucasians. It is due to mutations of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. This defect leads to a multisystemic disease in which oxidative stress (OxS) and inflammation play a central role. If OxS is generally referred to intestinal antioxidant malabsorption, sustained inflammation is considered a response to colonisation of airways by bacterial pathogens. However, recent studies underlined the presence of OxS and inflammation markers in airways without any infection. HYPOTHESIS: We put forward that CFTR defects directly induce OxS and inflammation in intestinal epithelial cells as severe gastrointestinal disorders occur in CF. OBJECTIVE: The aim of this study is to determine whether total CFTR ablation in Caco2/15 cells leads to oxidative and inflammatory processes via intracellular mechanisms implicating mitochondria dysfunctions. METHODS: A total deletion of the CFTR gene was performed in Caco-2/15 cells with Zing-Finger Nuclease constructs to mimic homozygous CF status. Then, markers of OxS and inflammation were assessed under basal conditions
Sa1430a Antibiotic Mediated Rise in Large Intestinal Proteolytic Activity Is Paralleled by Reduced Large Intestinal Barrier Function and May Be a Risk Factor for IBD Development in Susceptible Organisms Hongsup Yoon, Monika Schaubeck, Dirk Haller, Gabriele Hörmannsperger Background: Antibiotic (AB) therapy, especially metronidazole and fluorochinolone treatment, has recently been associated with increased risk for the development of Crohns Disease. In experimental and human studies, specific ABs have been found to abrogate the physiological inactivation of pancreatic proteases by the large intestinal microbiota, resulting in a major increase of the large intestinal protease activity (liPA). We hypothesize that the
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Gene Expression and miRNA Profiling in Ileal Mucosa of SERT Knock-Out Mouse Using Microarray Approaches Christopher R. Manzella, Megha Singhal, Anoop Kumar, Hayley Coffing, Alexander Ticho, Shubha Priyamvada, Arivarasu Natarajan Anbazhagan, Seema Saksena, Waddah A. Alrefai, Pradeep K. Dudeja, Ravinder K. Gill The serotonin transporter (SERT, SLC6A4), belonging to Na+ and Cl- dependent transporters, functions to regulate the availability of serotonin (5-HT) in various tissues including brain, lungs, platelets and intestine. Alterations in serotonin levels and/or SERT expression have been implicated in pathophysiological conditions including anxiety like disorders, obesity, pulmonary hypertension, cardiovascular disorders and GI disorders such as IBD, IBS and diarrhea. Accordingly, SERT-deficiency in mice is pleiotropic with many different phenotypic traits. Transgenic mice with global deletion of SERT exhibit increased anxiety, abnormal GI motility, obesity associated with hyperglycemia. Additionally, these mice are more susceptible to intestinal inflammation. How SERT deficiency underlies GI related disturbances and exacerbates the severity of gut inflammation is not known. We hypothesized that SERT deficiency causes aberrant miRNA expression and alters expression of genes implicated in regulatory networks. Thus, we evaluated global mRNA and miRNA expression profile to identify potential intestinal targets that contribute to disease pathogenesis, when SERT gene is non-functional. For these studies, RNA was extracted from ileum mucosa from age matched (9-10 wk) control and SERT KO mice by Trizol and was subjected to Phalanx Mouse miRNA and mRNA OneArray Profiling. Results demonstrated that 29 miRNAs were significantly upregulated in the SERT knockout mice ileum. Notable, upregulated miRNAs were mmulet-7c-5p, mmu-miR-16-5p, mmu-miR-31-5p, mmu-miR-143-5p, mmu-miR-6918-5p many of which have been implicated in Crohn's disease patients and in obesity. Our microarray data revealed 492 significantly upregulated and 293 downregulated genes in SERT KO mouse ileum. Pathway analysis revealed significant enrichment in cell cycle, signaling, chemotaxis, and epithelial transporters. Genes of interest were validated by RT-PCR in the ileum, which included CYP1A1 (~20 fold decrease, p < 0.05), CLDN8 (~30% decrease, p < 0.05), and HMGCS2 (~7 fold increase, p < 0.05). Similar alterations in these genes were observed in the colon of SERT KO mice by RT-PCR. A remarkable decrease in expression of Cyp1a1, encoding for a cytochrome P450 enzyme, provides novel role of serotonergic pathways in drug and xenobiotic detoxification. CLDN8 encodes for claudin 8, found to be downregulated in patients with Crohn's disease and HMGCS2, the mitochondrial 3hydroxy-3-methylglutaryl CoA synthase, deficiency of which is linked to hypoglycemia. Our data provides novel clues for differential expression of genes affected by 5-HT and/or SERT deficiency in the intestine. These studies also form the future platform to identify potential miRNA-mRNA regulatory networks in the development of 5-HT related disorders.
Sa1431 Intestinal Hypoxia and Reperfusion: Role for Eph-ephrin System Valentina Vivo, Simona Bertoni, Irene Zini, Lisa Flammini, Massimiliano Tognolini, Vigilio Ballabeni, Elisabetta Barocelli Background and aim: Mesenteric ischemia-reperfusion (I/R) is a life-threatening clinical problem consisting in the temporary deprivation of blood flow to the gut. The ensuing local organ damage is followed by the development of a systemic inflammatory response, eventually leading to multiple organ failure. Several neuronal, paracrine and plasmatic messengers are involved in mesenteric I/R injury. However, up to now, no studies have been performed on Eph receptors, the largest family of tyrosine kinase receptors, and their cell-bound ephrin ligands, whose role in cell adhesion-based processes during inflammation and immunity is growingly emerging. Eph-ephrin interaction generates a bidirectional signaling affecting both the receptor bearing (forward signaling) and the ligand bearing cells (reverse signaling). Our aim was therefore to investigate the effects produced by unidirectional activation of forward signaling (administration of chimeric protein ephrinA1-Fc; 50, 100, 200microg/kg), of reverse signaling (EphA2-Fc; 180microg/kg), or inhibition of both signals (monomeric EphA2 at equimolar doses of ephrinA1-Fc) on the inflammatory responses caused by mesenteric I/R in mice. Methods: Eph-ephrin proteins were intravenously administered to Swiss mice 5 min prior to 45 min occlusion of the superior mesenteric artery (SMA) followed by 5h reperfusion: intestinal and lung myeloperoxidase (MPO) activity, index of neutrophil recruitment, gut malondialdehyde (MDA) levels, marker of lipoperoxidation, and edema, index of increased vascular permeability, were compared to those of control I/R mice, receiving only vehicle, and of sham operated (SO) mice, subjected to the same manipulation without SMA occlusion and receiving vehicle. All experiments were performed according to the guidelines for the Care and Use of Animals (DL26/2014). Results: I/R mice displayed increased gut (45.3±4.7 vs 0.8±0.5 U/g, P<0.001) and lung (289.9±21.0 vs 144.6±45.1 U/g, P<0.05) MPO activity and higher MDA levels (595.0±87.3 vs 203.4±62.6 nmol/g dry tissue, P<0.05) and edema (4.4±0.1 vs 3.4±0.3 wet to dry weight ratio, P<0.01) compared to SO. EphrinA1Fc markedly reduced leukocyte infiltration in the gut (24.9±2.9 U/g) at 100microg/kg and in the lung (128.0±19.1 U/g) at 200microg/kg, while monomeric EphA2 significantly lowered intestinal edema formation (3.9±0.1g/g, P<0.05 vs I/R) at the highest dose. Neither EphA2Fc nor equimolar doses of IgG-Fc alone were effective in modifying I/R-induced inflammatory responses. Conclusions: These preliminary findings indicate that pharmacological modulation of Eph-ephrin system may be advantageous in limiting the inflammatory responses elicited by mesenteric I/R: forward signaling activation attenuates local and systemic leukocytes enrolment and reverse signaling silencing contrasts local changes in vascular permeability.
Sa1430 Immunochip Analysis Identifies Multiple Susceptibility Loci for Collagenous Colitis Giulia Roda, Jianzhong Hu, Zhongyang Zhang, Joana Torres, Anli Chen, Robert E. Petras, Darrell Pardi, Alina Iuga, Gabriel Levi, Wenqing Cao, Florian Rieder, Ilyssa Gordon, Ke Hao, Noam Harpaz, Judy H. Cho, Inga Peter, Jean-Frederic Colombel Background: Collagenous colitis is an increasingly recognized cause for chronic, non-bloody diarrhea. The etiology of CC remains unknown. A role for dysregulated immune responses and genetic factors has been proposed. Recently, Westerlind et al. genotyped around 300 CC cases showing the important role of HLA related immune mechanisms in CC pathogenesis [Westerlind et al. Gut 2015]. We have carried out the largest genome-wide association study of CC with the aim of identifying genetic loci that are closely associated with CC. Methods: DNA extracted from 1056 formalin-fixed, paraffin-embedded (FFPE) tissue of patients with histologically confirmed CC was genotyped using the Illumina Immunochip. Existing Immunochip genotyping data on IBD-free individuals (N=1,300) provided by the NIDDK IBD Genetics Consortium were obtained as controls. After applying quality controls filters, a total of 618 cases and 1199 controls with genotype data on 64535 single nucleotide polymorphisms (SNPs) were included in analysis. HLA alleles were imputed by SNP2HLA for samples passing QC. In addition, we performed a meta-analysis combining our association results with those recently published by Westerlind et al. for overlapping typed SNP markers and imputed HLA alleles using METAL. Results: The meta-analysis on SNPs revealed five markers, which passed the genome-wide significance level for association with CC (p-value<5e-8) and showed consistent direction of effect between the two studies. They all hits the HLA region, suggesting a role in antigen presentation and T-cell responses. In particular, based on the meta-analysis for imputed HLA alleles, the most significant association (p-value < 1e-5) with consistent increased risk was present at HLA-B*08:01, HLA-DQA1*05:01, HLADRB1*03:01 and HLA-DQB1*02:01. Moreover, a significant association (p-value < 1e-5) with consistent effect was also identified for PTPN2 on chromosome 18. This gene has a pivotal role in T-cell responses and has also been implicated in the pathogenesis of Crohn's disease. Conclusion: These findings further support and confirm the hypothesis that CC has a genetic component and HLA-related immune mechanisms are implicated on its pathogenesis. Moreover, some pathways involved in T-cell responses overlap with those of IBD, supporting the important role of T cells in CC pathogenesis.
Sa1432 CFTR Knockout Induces Oxidative Stress, Inflammation and Mitochondrial Dysfunctions in Caco-2/15 Intestinal Epithelial Cells Marie-Laure Kleme Amani, Alain Théophile Sané, Gilles Gouspillou, Jean-Philippe LeducGaudet, Carole Garofalo, David Saint-Pierre, Yves Berthiaume, Ernest G. Seidman, Emile Levy Cystic fibrosis (CF) is the most lethal of genetic diseases in Caucasians. It is due to mutations of Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. This defect leads to a multisystemic disease in which oxidative stress (OxS) and inflammation play a central role. If OxS is generally referred to intestinal antioxidant malabsorption, sustained inflammation is considered a response to colonisation of airways by bacterial pathogens. However, recent studies underlined the presence of OxS and inflammation markers in airways without any infection. HYPOTHESIS: We put forward that CFTR defects directly induce OxS and inflammation in intestinal epithelial cells as severe gastrointestinal disorders occur in CF. OBJECTIVE: The aim of this study is to determine whether total CFTR ablation in Caco2/15 cells leads to oxidative and inflammatory processes via intracellular mechanisms implicating mitochondria dysfunctions. METHODS: A total deletion of the CFTR gene was performed in Caco-2/15 cells with Zing-Finger Nuclease constructs to mimic homozygous CF status. Then, markers of OxS and inflammation were assessed under basal conditions
Sa1430a Antibiotic Mediated Rise in Large Intestinal Proteolytic Activity Is Paralleled by Reduced Large Intestinal Barrier Function and May Be a Risk Factor for IBD Development in Susceptible Organisms Hongsup Yoon, Monika Schaubeck, Dirk Haller, Gabriele Hörmannsperger Background: Antibiotic (AB) therapy, especially metronidazole and fluorochinolone treatment, has recently been associated with increased risk for the development of Crohns Disease. In experimental and human studies, specific ABs have been found to abrogate the physiological inactivation of pancreatic proteases by the large intestinal microbiota, resulting in a major increase of the large intestinal protease activity (liPA). We hypothesize that the
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Sa1429