- Email: [email protected]
Tu1839 miR-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis
intestinal macrophages. Changes in OGR1 expression in MM6 cells under hypoxia were determined on activation by tumor necrosis factor (TNF), with or without nuclear factor (NF)-kB inhibitors. Results: OGR1 expression was significantly higher in tumor tissue compared to normal murine colon tissue. Hypoxia positively regulated the expression of OGR1 in MM6 cells, primary human intestinal macrophages and in colonic tissue from inflammatory bowel disease (IBD) patients compared to healthy subjects. In MM6 cells, hypoxia enhanced TNF induced OGR1 expression was reversed by NF- kB inhibition. In addition to the effect of TNF and hypoxia, OGR1 mRNA expression was elevated at low pH, suggesting positive feed-forward regulation of OGR1 activity in acidic conditions. Conclusion: OGR1 expression is induced in cells of human myleoid lineage not only by TNF, and by hypoxia, but also by extracellular acidic pH. Hypoxia, known to cross-talk with the NF-kB pathway, enhances the TNF- mediated induction of OGR1 expression. The induction of OGR1 expression by TNF and hypoxia, and subsequent pH-sensing activity, may play a role in IBD pathogenesis.
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Background & aims: MicroRNAs (miRs) are small RNAs that negatively modulate the expression of various genes. We have reported that miR-31-3p is induced by SP stimulation in NCM460 colonocytes overexpressing the SP, neurokinin-1 receptor (NCM460-NK-1R) (Fang K et al, CMGH In press). We also reported during DDW 2015 that mucosal expression of miR-31-3p is increased during experimental colitis and ulcerative colitis colon tissue and modulates RhoA expression in NCM460-NK-1R cells in response to SP. However, the role of miR-31-3p during colitis has never been investigated. Our aim was to determine the importance of miR-31-3p in inflammatory signaling and experimental colitis. Methods: NCM460-NK-1R cells were transfected with anti-miR-31-3p or anti-miR-control using siPORT NeoFX (Ambion). Constitutively active RhoA was expressed in vitro using an adenoviral system (Cell Biolabs). Acute mouse colitis was induced by intracolonic TNBS administration or addition of DSS in the drinking water, respectively. The role of miR-31-3p was examined by administrating antisense (as)-miR-31-3p or as-miR-control intracolonicaly into DSS-exposed mice, and the in vivo function of miR-31-3p during the healing phase of colitis by administrating (as)-miR-31-3p or as-miR-control intracolonicaly during the 10 days of healing following 5 days of DSS treatment. Results: Bioinformatics analysis indicated that RhoA was a possible downstream target of miR-31-3p. Indeed, transfection with a miR-313p mimic decreased the protein level and activity of RhoA in NCM460-NK-1R cells following SP exposure. Expression of constitutively active RhoA also increased SP-induced CCL2, IL6, TNFa, and CXCL10 expression in NCM460-NK-1R cells (n=3, p<0.05), while miR-31-3p silencing increased CCL2 (by 54%) and IL6 (by 2.1 fold) mRNA (n=3, p<0.05). Overexpression of miR-31-3p inhibited TNF a, CCL2, IL6 and IL1 b mRNA expression induced by a cytokine-cocktail (10µg/ml of TNFa, IFN-g, IL6 and IL8) in NCM460 cells (n=3, p<0.05). NK-1R deficient TNBS-exposed mice had reduced miR-31-3p levels, compared to wild-type colitic mice, suggesting a SP-dependent response. Intracolonic as-miR-31-3p administration increased colitis histopathology score as well as colonic expression of CCL2, TNF aand Cxcl10 mRNA (n=8, p<0.05) 5 days after DSS administration, but had no effect on the healing phase of DSS-induced colitis. Conclusions: This is the first demonstration that the SP-induced miR-31-3p regulates inflammatory gene expression and colitis, representing a novel anti-inflammatory regulator for colitis and IBD. Supported by NIDDK RO-1 DK 47343 (CP), a CURE: DDRC P30 DK 41301 Pilot and Feasibility study (KF), T32 DK07180-40 (DP), and by a Research Fellowship from the Crohn's and Colitis Foundation (IKML).
Tu1837 The Role of ROS/NF-kB and Notch Signaling Pathways in the Effect of DAPT on Inflammation in BAR-T Cells Cheng Feng, Jun Zhang, Dong Liu, Yumei Luo, Jing Wu, Yuanyuan Nian, Rong Zhang Background: Chronic inflammation plays an important role in the carcinogenesis and development from Barrett's esophagus(BE) to esophageal adenocarcinoma(EAC). Notch signaling is a well-conserved signaling pathway involved in cell fate decisions, proliferation and apoptosis. DAPT is the g-secretase inhibitor which can inhibit Notch signaling. The present study aimed to investigate the effect of DAPT on the inflammation factors expression, the generation of reactive oxygen(ROS), apoptosis and NF- kB activation in DCA-treated BART cells in vitro. Methods: 1.BAR-T cells were treated with deoxycholic acid (DCA) to build inflammation model. 2.MTT and Annexin V-FITC/PI assays were performed to investigate the proliferation and apoptosis rate of BAR-T cells. The intra-cellular ROS levels were detected using fluorescence microscopy and flow cytometry. The TNF-a, IL-8, IL-6,IL-1b,Bcl-2 mRNA and protein levels were detected by real-time PCR and ELISA respectively. The Notch1, Notch2, Notch3 and Notch4 mRNA levels were also detected by real-time PCR. Western blot assays were used to demonstrate the NF- kB activation and underlying signaling pathway in DAPT-treated BAR-T cells. Results: 1.Treating with 250mM, 30min DCA could up-regulate the TNF-a, IL-8, IL-6,IL-1b and Bcl-2 expression, intracellular ROS and NF- kB activation in BAR-T cells. DCA could decrease Notch1,Notch2, Notch3 and Notch4 mRNA expressions in BAR-T cells. 2.DAPT(in a time-dose dependent manner) could inhibit Notch signaling pathway, expression of inflammatory factors, intracellular ROS and NF- kB activation in DCAtreated BAR-T cells. Pretreatment with NAC or PDTC dramatically attenuated expression of inflammatory factors , intracellular ROS and NF- kB activation induced by DCA in BAR-T cells. 3.DAPT could induce BAR-T cells apoptosis in a time-dose dependent manner, and down-regulate Bcl-2 expression in an ROS/NF-kB-dependent manner in BAR-T cells. Pretreatment with NAC(a ROS scavenger) or PDTC(a NF- kB inhibitor) could induce apoptosis and down-regulate Bcl-2 expression in DCA-treated BAR-T cells. Conclusion: DAPT could inhibit expression of inflammatory factors induced by DCA in an ROS/NF- kB signaling pathway dependent manner in BAR-T cells. DAPT also could inhibit proliferation and induce BART cells apoptosis through ROS/NF-kB/Bcl-2 signaling pathway, which indicated that DAPT could be a potential therapeutic target for BE.
Tu1840 Epithelial Cell Junction Regulation by the Stress-Associated miR-150, miR335, and miR-142-3p in the Absence or Presence of Chronic Dexamethasone Sarah K. Abey, Jeffrey Robinson, Nicolaas H. Fourie, Dan Wang, Natnael Kenea, Tatyana Vishnyakova, Wendy A. Henderson Disruption of epithelial integrity through cell junction remodeling is a potential gut-stress response. We identified miRNAs potentially involved in regulation of epithelial integrity, miR-150, miR-335, and miR-142-3p, based on Zona Occludens-1 (ZO1) and Occludin (OCLN) target-site predictions. Each miRNA targets cytoskeleton and we have previously reported their alterations in patients with chronic stress-induced gastrointestinal dysfunction. Using a miR-mimic transfection strategy, we transiently overexpressed miR-150, miR-335, and miR-142-3p, alone and in combination in colon epithelial cells. Confocal microscopy and immunofluorescence staining found that miR-142-3p overexpression had the effect of removing Occludin, but not ZO-1, from its location at the cell junction. miR-150 and miR-335 also disrupted the overall appearance of cell junctions. Trans-epithelial electrical resistance (TEER) measurements in polarized colon epithelial cells confirmed that miR-1423p overexpression resulted in significant perturbations across the epithelial barrier. qPCR for endogenous OCLN transcript levels showed a slight increase following miR-142-3p expression. Interestingly, combined overexpression of miR-150 with miR-142-3p rescued miR-142-3p-induced epithelial barrier effects. Based on these observations, we hypothesized that miR-150, miR-335, and miR-142-3p were mutually involved in a regulatory network. qPCR was used to test for altered expression of each endogenous miRNA following miR mimic overexpression. The endogenous miR-150 levels were found to be significantly induced by miR-335, and to a lesser extent by miR-142-3p. Finally, to link this network and its potential functional implications to gut-stress response, we tested the effects of individual and combination miRNA overexpression under chronic exposure to the glucocorticoid dexamethasone, known to induce alterations in cultured epithelial cell junctions. Chronic dexamethasone exposure resulted in attenuation of TEER following individual and combination miRNA overexpression. Together, our data showed that miR-150, miR-335, and miR142-3p exerted individual and combined effects on the epithelial integrity, partly through modulation of endogenous ZO-1 and OCLN expressions and localization within cell junctions. miRNA effects were modulated by chronic exposure of colon epithelial cells to dexamethasone, suggesting a potential integrated network for gut-stress response pathway.
Tu1838 Pharmacological Inhibition of Phosphatase and Tensin Homologue (Pten) Elicits Colitis in IL-10-KO Mice Sang H. Rhee, Charalabos Pothoulakis BACKGROUND & AIMS: Phosphatase and tensin homologue (Pten) hydrolyzes the 3phosphate from phosphatidylinositol 3,4,5-trisphosphate (PIP3) to generate PIP2. Although Pten is one of the well-known tumor suppressor genes associated with various tumor development, emerging evidence indicates that Pten is able to regulate immune responses initiated by microbes in the intestine. Indeed, we previously demonstrated that Pten gene deletion in the intestinal epithelium resulted in altered gut microbiome, leading to enhanced susceptibility to spontaneous colitis development in IL-10-KO mice. In line with such findings, here, we further dissect the involvement of Pten in the patients with ulcerative colitis (UC), and demonstrate that the pharmacological inhibition of Pten not only changes gut microbiome, but also induces pan-colitis in IL-10-KO mice. METHODS: Human colonic mucosa tissues were collected from the patients with UC and normal subjects. Gene expression was measured by quantitative real-time PCR (qPCR). We used the vanadium complex VO-OHpic that is a potent, allosteric inhibitor of Pten. Age-matched (5 weeks old) IL-10KO, IL-10 Receptor B (IL-10RB)-KO, and C57BL/6 mice were treated with oral gavage of VO-OHpic (100 µM/mouse) or DMSO (5%)-containing water (once per every day) for 1 month. Pten enzymatic activity was measured using the mouse intestinal tissues. Inflammation- or immune responses-related genes were evaluated by gene array and qPCR. Fecal microbiome was analyzed by sequencing 16S rRNA gene. RESULTS: Pten mRNA expression level was reduced (55% reduction in fold, P=0.0032) in the colonic mucosal tissues from UC patients (n=9) compared to normal colon tissues (n=19). VO-OHpic treatment inhibited Toll-like receptor (TLR) 5-induced signaling pathways in the human colonic epithelial NCM460 cells. Oral administration of VO-OHpic resulted in diminished PIP2 level in the mouse intestine. The abundance of Bacteroides in the feces was increased in VO-OHpictreated mice (~50%) compared to vehicle-treated mice (~20%) (P=0.01, n=8/group). VOOHpic administration in IL-10-KO mice elicited pan-colitis characterized with massive neutrophil infiltration and necrosis throughout the colonic mucosa, while vehicle-treated IL-10-KO mice exhibited normal physiology in the colon. The Pten inhibitor treatment did not cause any abnormality in IL-10RB-KO or C57BL/6 mice. CONCLUSIONS: Oral administration of the Pten inhibitor in mice suppressed Pten enzymatic activity in the intestine, leading to changed fecal microbiome and enhanced susceptibility to colitis development in an IL-10 deficient environment. Thus, our study suggests an immune modulatory effect of Pten in the intestine, associated with colitis development in a genetically susceptible condition to colitis.
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miR-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis Kai Fang, Ivy Ka Man Law, Aristea Sideri, Vanessa Huang, David M. Padua, Dimitrios Iliopoulos, Charalabos Pothoulakis
Tu1839
Background & aims: MicroRNAs (miRs) are small RNAs that negatively modulate the expression of various genes. We have reported that miR-31-3p is induced by SP stimulation in NCM460 colonocytes overexpressing the SP, neurokinin-1 receptor (NCM460-NK-1R) (Fang K et al, CMGH In press). We also reported during DDW 2015 that mucosal expression of miR-31-3p is increased during experimental colitis and ulcerative colitis colon tissue and modulates RhoA expression in NCM460-NK-1R cells in response to SP. However, the role of miR-31-3p during colitis has never been investigated. Our aim was to determine the importance of miR-31-3p in inflammatory signaling and experimental colitis. Methods: NCM460-NK-1R cells were transfected with anti-miR-31-3p or anti-miR-control using siPORT NeoFX (Ambion). Constitutively active RhoA was expressed in vitro using an adenoviral system (Cell Biolabs). Acute mouse colitis was induced by intracolonic TNBS administration or addition of DSS in the drinking water, respectively. The role of miR-31-3p was examined by administrating antisense (as)-miR-31-3p or as-miR-control intracolonicaly into DSS-exposed mice, and the in vivo function of miR-31-3p during the healing phase of colitis by administrating (as)-miR-31-3p or as-miR-control intracolonicaly during the 10 days of healing following 5 days of DSS treatment. Results: Bioinformatics analysis indicated that RhoA was a possible downstream target of miR-31-3p. Indeed, transfection with a miR-313p mimic decreased the protein level and activity of RhoA in NCM460-NK-1R cells following SP exposure. Expression of constitutively active RhoA also increased SP-induced CCL2, IL6, TNFa, and CXCL10 expression in NCM460-NK-1R cells (n=3, p<0.05), while miR-31-3p silencing increased CCL2 (by 54%) and IL6 (by 2.1 fold) mRNA (n=3, p<0.05). Overexpression of miR-31-3p inhibited TNF a, CCL2, IL6 and IL1 b mRNA expression induced by a cytokine-cocktail (10µg/ml of TNFa, IFN-g, IL6 and IL8) in NCM460 cells (n=3, p<0.05). NK-1R deficient TNBS-exposed mice had reduced miR-31-3p levels, compared to wild-type colitic mice, suggesting a SP-dependent response. Intracolonic as-miR-31-3p administration increased colitis histopathology score as well as colonic expression of CCL2, TNF aand Cxcl10 mRNA (n=8, p<0.05) 5 days after DSS administration, but had no effect on the healing phase of DSS-induced colitis. Conclusions: This is the first demonstration that the SP-induced miR-31-3p regulates inflammatory gene expression and colitis, representing a novel anti-inflammatory regulator for colitis and IBD. Supported by NIDDK RO-1 DK 47343 (CP), a CURE: DDRC P30 DK 41301 Pilot and Feasibility study (KF), T32 DK07180-40 (DP), and by a Research Fellowship from the Crohn's and Colitis Foundation (IKML).
Tu1837 The Role of ROS/NF-kB and Notch Signaling Pathways in the Effect of DAPT on Inflammation in BAR-T Cells Cheng Feng, Jun Zhang, Dong Liu, Yumei Luo, Jing Wu, Yuanyuan Nian, Rong Zhang Background: Chronic inflammation plays an important role in the carcinogenesis and development from Barrett's esophagus(BE) to esophageal adenocarcinoma(EAC). Notch signaling is a well-conserved signaling pathway involved in cell fate decisions, proliferation and apoptosis. DAPT is the g-secretase inhibitor which can inhibit Notch signaling. The present study aimed to investigate the effect of DAPT on the inflammation factors expression, the generation of reactive oxygen(ROS), apoptosis and NF- kB activation in DCA-treated BART cells in vitro. Methods: 1.BAR-T cells were treated with deoxycholic acid (DCA) to build inflammation model. 2.MTT and Annexin V-FITC/PI assays were performed to investigate the proliferation and apoptosis rate of BAR-T cells. The intra-cellular ROS levels were detected using fluorescence microscopy and flow cytometry. The TNF-a, IL-8, IL-6,IL-1b,Bcl-2 mRNA and protein levels were detected by real-time PCR and ELISA respectively. The Notch1, Notch2, Notch3 and Notch4 mRNA levels were also detected by real-time PCR. Western blot assays were used to demonstrate the NF- kB activation and underlying signaling pathway in DAPT-treated BAR-T cells. Results: 1.Treating with 250mM, 30min DCA could up-regulate the TNF-a, IL-8, IL-6,IL-1b and Bcl-2 expression, intracellular ROS and NF- kB activation in BAR-T cells. DCA could decrease Notch1,Notch2, Notch3 and Notch4 mRNA expressions in BAR-T cells. 2.DAPT(in a time-dose dependent manner) could inhibit Notch signaling pathway, expression of inflammatory factors, intracellular ROS and NF- kB activation in DCAtreated BAR-T cells. Pretreatment with NAC or PDTC dramatically attenuated expression of inflammatory factors , intracellular ROS and NF- kB activation induced by DCA in BAR-T cells. 3.DAPT could induce BAR-T cells apoptosis in a time-dose dependent manner, and down-regulate Bcl-2 expression in an ROS/NF-kB-dependent manner in BAR-T cells. Pretreatment with NAC(a ROS scavenger) or PDTC(a NF- kB inhibitor) could induce apoptosis and down-regulate Bcl-2 expression in DCA-treated BAR-T cells. Conclusion: DAPT could inhibit expression of inflammatory factors induced by DCA in an ROS/NF- kB signaling pathway dependent manner in BAR-T cells. DAPT also could inhibit proliferation and induce BART cells apoptosis through ROS/NF-kB/Bcl-2 signaling pathway, which indicated that DAPT could be a potential therapeutic target for BE.
Tu1840 Epithelial Cell Junction Regulation by the Stress-Associated miR-150, miR335, and miR-142-3p in the Absence or Presence of Chronic Dexamethasone Sarah K. Abey, Jeffrey Robinson, Nicolaas H. Fourie, Dan Wang, Natnael Kenea, Tatyana Vishnyakova, Wendy A. Henderson Disruption of epithelial integrity through cell junction remodeling is a potential gut-stress response. We identified miRNAs potentially involved in regulation of epithelial integrity, miR-150, miR-335, and miR-142-3p, based on Zona Occludens-1 (ZO1) and Occludin (OCLN) target-site predictions. Each miRNA targets cytoskeleton and we have previously reported their alterations in patients with chronic stress-induced gastrointestinal dysfunction. Using a miR-mimic transfection strategy, we transiently overexpressed miR-150, miR-335, and miR-142-3p, alone and in combination in colon epithelial cells. Confocal microscopy and immunofluorescence staining found that miR-142-3p overexpression had the effect of removing Occludin, but not ZO-1, from its location at the cell junction. miR-150 and miR-335 also disrupted the overall appearance of cell junctions. Trans-epithelial electrical resistance (TEER) measurements in polarized colon epithelial cells confirmed that miR-1423p overexpression resulted in significant perturbations across the epithelial barrier. qPCR for endogenous OCLN transcript levels showed a slight increase following miR-142-3p expression. Interestingly, combined overexpression of miR-150 with miR-142-3p rescued miR-142-3p-induced epithelial barrier effects. Based on these observations, we hypothesized that miR-150, miR-335, and miR-142-3p were mutually involved in a regulatory network. qPCR was used to test for altered expression of each endogenous miRNA following miR mimic overexpression. The endogenous miR-150 levels were found to be significantly induced by miR-335, and to a lesser extent by miR-142-3p. Finally, to link this network and its potential functional implications to gut-stress response, we tested the effects of individual and combination miRNA overexpression under chronic exposure to the glucocorticoid dexamethasone, known to induce alterations in cultured epithelial cell junctions. Chronic dexamethasone exposure resulted in attenuation of TEER following individual and combination miRNA overexpression. Together, our data showed that miR-150, miR-335, and miR142-3p exerted individual and combined effects on the epithelial integrity, partly through modulation of endogenous ZO-1 and OCLN expressions and localization within cell junctions. miRNA effects were modulated by chronic exposure of colon epithelial cells to dexamethasone, suggesting a potential integrated network for gut-stress response pathway.
Tu1838 Pharmacological Inhibition of Phosphatase and Tensin Homologue (Pten) Elicits Colitis in IL-10-KO Mice Sang H. Rhee, Charalabos Pothoulakis BACKGROUND & AIMS: Phosphatase and tensin homologue (Pten) hydrolyzes the 3phosphate from phosphatidylinositol 3,4,5-trisphosphate (PIP3) to generate PIP2. Although Pten is one of the well-known tumor suppressor genes associated with various tumor development, emerging evidence indicates that Pten is able to regulate immune responses initiated by microbes in the intestine. Indeed, we previously demonstrated that Pten gene deletion in the intestinal epithelium resulted in altered gut microbiome, leading to enhanced susceptibility to spontaneous colitis development in IL-10-KO mice. In line with such findings, here, we further dissect the involvement of Pten in the patients with ulcerative colitis (UC), and demonstrate that the pharmacological inhibition of Pten not only changes gut microbiome, but also induces pan-colitis in IL-10-KO mice. METHODS: Human colonic mucosa tissues were collected from the patients with UC and normal subjects. Gene expression was measured by quantitative real-time PCR (qPCR). We used the vanadium complex VO-OHpic that is a potent, allosteric inhibitor of Pten. Age-matched (5 weeks old) IL-10KO, IL-10 Receptor B (IL-10RB)-KO, and C57BL/6 mice were treated with oral gavage of VO-OHpic (100 µM/mouse) or DMSO (5%)-containing water (once per every day) for 1 month. Pten enzymatic activity was measured using the mouse intestinal tissues. Inflammation- or immune responses-related genes were evaluated by gene array and qPCR. Fecal microbiome was analyzed by sequencing 16S rRNA gene. RESULTS: Pten mRNA expression level was reduced (55% reduction in fold, P=0.0032) in the colonic mucosal tissues from UC patients (n=9) compared to normal colon tissues (n=19). VO-OHpic treatment inhibited Toll-like receptor (TLR) 5-induced signaling pathways in the human colonic epithelial NCM460 cells. Oral administration of VO-OHpic resulted in diminished PIP2 level in the mouse intestine. The abundance of Bacteroides in the feces was increased in VO-OHpictreated mice (~50%) compared to vehicle-treated mice (~20%) (P=0.01, n=8/group). VOOHpic administration in IL-10-KO mice elicited pan-colitis characterized with massive neutrophil infiltration and necrosis throughout the colonic mucosa, while vehicle-treated IL-10-KO mice exhibited normal physiology in the colon. The Pten inhibitor treatment did not cause any abnormality in IL-10RB-KO or C57BL/6 mice. CONCLUSIONS: Oral administration of the Pten inhibitor in mice suppressed Pten enzymatic activity in the intestine, leading to changed fecal microbiome and enhanced susceptibility to colitis development in an IL-10 deficient environment. Thus, our study suggests an immune modulatory effect of Pten in the intestine, associated with colitis development in a genetically susceptible condition to colitis.
S-957
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Page 957
AGA Abstracts
AGA Abstracts
miR-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis Kai Fang, Ivy Ka Man Law, Aristea Sideri, Vanessa Huang, David M. Padua, Dimitrios Iliopoulos, Charalabos Pothoulakis