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M1817 Regulation of Intestinal Permeability and Epithelial Cell Tight Junctions by the Ubiquitin-Editing Enzyme TNFAIP3
IEC to control TJ permeability changes induced by TNF. TNFAIP3 acts at a point downstream of MLCK to regulate IEC TJ dynamics. Given that TJ proteins can be controlled by ubiquitination, it is possible that TNFAIP3 acts directly on TJ protein ubiquitination to control intestinal permeability. M1818 Simvastatin Enhances eNOS Expression in Human Intestinal Microvascular Endothelial Cells: Improvement in Crohn's Disease Endothelial Dysfunction Sharon Manley, David G. Binion, Daniel J. Stein, Mary F. Otterson, Parvaneh Rafiee BACKGROUND: Statins, HMGCoA reductase inhibitors, have been shown to possess antiinflammatory effects beyond their lipid-lowering capabilities. The microvasculature undergoes remodeling and adaptation in chronic inflammation, and endothelial dysfunction with enhanced oxyradical mediated activation has been demonstrated in inflammatory bowel disease (IBD; Crohn's disease (CD), ulcerative colitis (UC)). Increased ROCK activity has been implicated in endothelial dysfunction in chronic vascular inflammation. The effect of statin on IBD is unknown. The present study investigated the anti-inflammatory effects of simvastatin on ROCK activation and the role of endothelial nitric oxide synthase (eNOS) in human intestinal microvascular endothelial cells (HIMEC). METHODS: HIMEC monolayers (passage 8-12) from normal and CD tissues were treated with simvastatin (0.1-5 μM). HIMEC expression of cell adhesion molecules (CAM; ICAM-1, VCAM-1, E-selectin), IL-8, IL-6, COX-2 and eNOS were determined using ELISA, Real-time PCR and Western blotting. The reactive oxygen species (ROS) superoxide was detected using hydroethidine (HE) and fluorescence microscopy. HIMEC were activated with TNF-α/LPS. RESULTS: Statin inhibited basal ROS production in CD HIMEC compared to control cells as determined by HE. Statin reduced ROCK activity and upregulated eNOS expression in HIMEC in a dose dependent manner. Statin induction of eNOS was mediated through inhibition of Rho-kinase. Statin blocked TNF-α/LPS induced IL-8, IL-6 and COX-2 mRNA and protein expression in addition to inhibiting activation of STAT-3, MAPKs and NFκB. Similar to the effect of statin, MAPK and NFκB inhibitors significantly decreased TNF-α/LPS induced CAM expression and IL6 protein release in HIMEC. CONCLUSIONS: Statins exert a beneficial effect on intestinal microvascular endothelial cells affected by chronic inflammation in IBD. Future studies evaluating the effect of statins on the intestinal microvasculature and inflammatory responses in patients with CD and UC are warranted.
M1816 Role of TLR2, 4 and 5 Signals in Regulation of B7 Negative Co-Stimulators on Colonic Stromal Cells Iryna V. Pinchuk, Jameel R. Johnson, Ellen Beswick, Jamal I. Saada, Randy C. Mifflin, Victor E. Reyes, Don W. Powell Background: The B7 negative co-stimulators PD-L1 and PD-L2 expressed by antigen presenting cells (APCs) are critical in suppression of activated T cell responses during mucosal homeostasis and suggested to be involved in inflammatory bowel disease (IBD). Human stromal cells, A.k.a. myofibroblasts/fibroblasts (CMFs) are a major PD-L1+ PD-L2+ cell phenotype in normal colonic mucosa and may suppress activated CD4+ T cell proliferation via PD-1 ligands. Upregulation of PD-L1 expression on CMFs during ulcerative colitis (UC) was noted, but mechanisms leading to PD-L1 upregulation are unclear. Aberrant responses to acute bacterial infection may trigger the dysfunctional regulation of immune responses leading to the longstanding shifts in gut flora and development of IBD. Our initial study shown that stimulation of CMFs with Salmonella thyphimurium increase PD-L1 expression linked to TLR4- and TLR5-mediated signaling. We hypothesized that constitutively expressed PD-L1 on CMFs are among key factors in gut mucosal homeostasis and are increased by stimulation of TLRs during IBD. Methods: PD-L1, 2 and TLR 2, 4 and 5 expression on human CMFs from normal mucosa in response to the bacterial pathogen-associated molecular patterns (PAMPs) was analyzedusing real-time RT-PCR and FACS analysis. TLR pathway inhibitors, blocking Abs and siRNA were used to abrogate induction of PD-L1 & PD-L2. Results: CMF stimulation with PAMPs (4-24 h) that signal via the MyD88 adaptor involving pathway TLR 2, 4 and 5, but not TLR6-9, upregulated PD-L1 expression, which was decreased in the presence of appropriate blocking peptide or and antibodies. Blocking of MyD88 expression in CMF by specific siRNA reduced the induction of the PD-L1. Upregulation of PD-L2 was induced by the stimulation of TLR5, but not other TLR requiring MyD88 adaptor and decreased in the presence of the TLR5 peptide antagonist. A positive feedback in the expression of TLR 2, 4 and 5 was observed in response to the stimulation of the CMFs with TLR 2, 4 and 5- specific PAMPs. This suggests that IBD associated shifts in gut flora may trigger strong upregulation of TLRs contributing to the increase in the PD-L1 expression on CMFs during UC progression. Conclusion: Normal colonic mucosa CMFs play an important role in innate immune responses to bacterial PAMPs and may favor suppression of activated CD4+ T cells via PD-L1/2 upregulation. Our data suggest that earlier observed abnormalities in the PD-L1 expression on CMFs in UC colonic mucosa may be due to the stimulation of CMFs through TLR2, 4 and/or TLR5 by PAMPs in subepithelial compartments during IBD-associated inflammation. Supported by AGA, CCFA and NIDDK.
M1819 Actively Inflamed Ulcerative Colitis and Crohn's Colitis are Characterized by Differential Upregulation of the Markers of Erad Activity, Edem1 and Atg5 Rohan Lourie, Thu V. Tran, Michael A. McGuckin, Timothy H. Florin Background: Goblet cell pathology, including accumulation of the MUC2 precursor, occurs in human IBD, and several murine models have linked Muc2 misfolding and endoplasmic reticulum (ER) stress with intestinal inflammation. A number of chaperone molecules detect and direct misfolded proteins through the ER membrane for proteasomal degradation (ERAD). EDEM1 is a short-lived ER chaperone which competes with the endoplasmic reticulum assisted folding system for misfolded substrates. EDEM1 itself is rapidly degraded in a process dependent on the autophagy pathway gene ATG5. Aims: To quantify EDEM1 and ATG5 mRNA in ulcerative colitis and Crohn's colitis by qRT-PCR. Methods: Colonic mucosal biopsies were taken from macroscopically and histologically non-inflamed or inflamed colonic sites of treatment-naïve patients with inflammatory bowel disease (19 Crohn's disease and 25 ulcerative colitis), and 12 age-matched healthy patients undergoing cancer screening. RNA was extracted and qRT-PCR performed with EDEM1, ATG5 and VIL1 specific primers. Analysis: Fold changes for EDEM1 and ATG5 relative to VIL1 (epithelial specific gene encoding villin) were normalized to the mean of the control group and analysis of variance assessed with Kruskal-Wallis testing. Results: EDEM1 was significantly upregulated in inflamed ulcerative colitis, p=0.0002, and in inflamed Crohn's colitis, p=0.002. ATG5 was also significantly upregulated in both inflamed ulcerative colitis, p=0.013 and inflamed Crohn's colitis, p=0.02. The ratio of the fold change of EDEM1 to ATG5 was significantly different between inflamed ulcerative colitis and inflamed Crohn's colitis when assessed by Mann-Whitney testing, with higher levels of EDEM1 to ATG5 mRNA in the ulcerative colitis group. There was no significant difference between either of these groups and the control group. Discussion: EDEM1 mRNA is upregulated in ulcerative colitis, but with an attenuated ATG5 mRNA response compared to Crohn's colitis. Less tightly regulated expression of EDEM1 in goblet cells could lead to diversion of MUC2 to a degradation pathway contributing to the goblet cell pathology and mucus barrier deficiency seen in ulcerative colitis.
M1817 Regulation of Intestinal Permeability and Epithelial Cell Tight Junctions by the Ubiquitin-Editing Enzyme TNFAIP3 James P. Lodolce, Lauren Kolodziej, Jonathan Chang, Jeffrey R. Schneider, Jeannette S. Messer, Jerrold R. Turner, David L. Boone Gut homeostasis depends on the regulation of intestinal epithelial cell (IEC) tight junctions (TJ) necessary for maintaining the semi-permeable barrier of the intestine. Patients with Crohn's disease (CD) have disruption of TJ and impaired barrier function. In addition, CD patients have elevated levels of the pro-inflammatory cytokine TNF and myosin light chain kinase (MLCK) that mediate IEC permeability by regulating TJ. The anti-inflammatory protein TNFAIP3 is required to negatively regulate TNF pro-inflammatory signaling. TNFAIP3 is both a deubiquitinating enzyme and an ubiquitin ligating enzyme. Although TNFAIP3 is expressed in IEC, its role in regulating tight junctions and intestinal permeability remains unknown. METHODS: To determine if TNFAIP3 is required to maintain barrier function In Vivo, we measured FITC-dextran flux in intestinal loops explanted from TNFAIP3 +/+ and -/- mice. To assess TNFAIP3's role in maintaining TJ In Vitro we measured trans-epithelial resistance (TER) and paracellular flux in TNF-treated WT and TNFAIP3 overexpressing IEC monolayers or cells expressing TNFAIP3 shRNA. We investigated MLCK involvement in the TNF-induced decrease in TER by treating cells with PIK, a potent inhibitor of MLCK. To assess MLCK activity, we probed for phosphorylated MLC in TNF-treated cells. RESULTS: TNFAIP3 -/- mice exhibited significantly increased intestinal permeability, indicating TNFAIP3 is required to maintain intestinal barrier integrity In Vivo. In Vitro, IEC overexpressing TNFAIP3 were less susceptible to TNF-induced increases in flux and decreases in TER. TNFAIP3 shRNA expressing cells displayed greater decreases in TER in response to TNF compared to WT (control shRNA) cells. The MLCK inhibitor PIK prevented TER decreases in both control and TNFAIP3 shRNA expressing cells, thus implicating TNFAIP3 in the MLCK pathway of TJ regulation. However, IEC overexpressing TNFAIP3 displayed no difference in the activation of MLCK by TNF. This suggests that TNFAIP3 acts in the MLCK pathway of TJ regulation at a point downstream of MLCK activation. CONCLUSIONS: TNFAIP3 is required for normal intestinal barrier function, and TNFAIP3 can act within
M1820 Activated Toll-Like Receptor 4 (TLR4) Directs Differentiation of Goblet and Paneth Cells Cristhine Pastorini, Rebeca Santaolalla, Masayuki Fukata, John P. Sotolongo, Cecilia Espana, Lory Hayes, Maria T. Abreu Background: Paneth and goblet cells are intestinal secretory cells which play an important role in maintenance of host-microbial homeostasis by regulating microbial density in the small intestine. TLR4 expression in the small intestine increases intestinal proliferation and villus height. We hypothesized that TLR4 signaling could alter differentiation of stem cells into secretory cells. Methods: We developed mice that express constitutively-active TLR4 in intestinal epithelial cells (IECs) under the villin promoter (villin-TLR4 mice). We compared Paneth cell and goblet cell numbers between villin-TLR4 mice and wild-type (WT) littermates using H&E and Alcian blue-Periodic acid-Schiff staining, respectively. Paneth cell maturation was assessed by immunofluorescent detection of lysozyme. Ileal expression of pan-alpha defensin was determined by real time-PCR. Total number of intestinal bacteria was counted by culturing stool pellets from villin-TLR4 and WT mice. Results: The villin-TLR4 mice had a significant reduction in the average number of Paneth cells detected per crypt when compared with WT mice (3.65±0.59 vs 6.92±0.26, p<0.01). Expression of pan-alpha defensin
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AGA Abstracts
AGA Abstracts
UC-CMFs. Myeloid dendritic cells (DC) generated from peripheral blood mononuclear cells by IL-4/GM-CSF treatment were used as control APCs. T cell phenotype was determined by immunostaining followed by FACS analysis. CFSE-proliferation assays, real-time RT-PCR were used to evaluate activation of Treg cells co-cultured with CMFs. Results: Co-culturing of allogeneic TH0 cells with both N- and UC-CMFs leads to the upregulation of FoxP3 mRNA expression. FACS analysis demonstrated that N-CMF induced generation of FoxP3+ T cells from TH0 cells at a rate of 10.2±3.6%, comparable to those co-cultured with DCs. N-CMFs induced FoxP3+ T cells were CD25highCD127-, express IL-10 and TGFβ and possess suppressive activity. Co-culture of the TH0 cells with UC-derived CMFs induced FoxP3 expression in cells bearing CD127+CD25low T effector cell phenotype. Since FoxP3 expression in CD127+CD25low T effectors has been associated with anergy, our data suggest that in contrast to N-CMFs, UC-derived CMFs have a reduced capacity to induce an active Treg and may lead to the induction of anergic FoxP3+ CD4+ T effector cells. Conclusions: These results support the notion that in normal colonic mucosa CMFs have an anti-inflammatory role and contribute to tolerance by supporting the Treg cell function. Our data also suggest that disruption in the capacity of UC-CMFs to induce active Treg may contribute to the progression of UC. Supported by NIDDK, CCFA and AGA.
M1816 Role of TLR2, 4 and 5 Signals in Regulation of B7 Negative Co-Stimulators on Colonic Stromal Cells Iryna V. Pinchuk, Jameel R. Johnson, Ellen Beswick, Jamal I. Saada, Randy C. Mifflin, Victor E. Reyes, Don W. Powell Background: The B7 negative co-stimulators PD-L1 and PD-L2 expressed by antigen presenting cells (APCs) are critical in suppression of activated T cell responses during mucosal homeostasis and suggested to be involved in inflammatory bowel disease (IBD). Human stromal cells, A.k.a. myofibroblasts/fibroblasts (CMFs) are a major PD-L1+ PD-L2+ cell phenotype in normal colonic mucosa and may suppress activated CD4+ T cell proliferation via PD-1 ligands. Upregulation of PD-L1 expression on CMFs during ulcerative colitis (UC) was noted, but mechanisms leading to PD-L1 upregulation are unclear. Aberrant responses to acute bacterial infection may trigger the dysfunctional regulation of immune responses leading to the longstanding shifts in gut flora and development of IBD. Our initial study shown that stimulation of CMFs with Salmonella thyphimurium increase PD-L1 expression linked to TLR4- and TLR5-mediated signaling. We hypothesized that constitutively expressed PD-L1 on CMFs are among key factors in gut mucosal homeostasis and are increased by stimulation of TLRs during IBD. Methods: PD-L1, 2 and TLR 2, 4 and 5 expression on human CMFs from normal mucosa in response to the bacterial pathogen-associated molecular patterns (PAMPs) was analyzedusing real-time RT-PCR and FACS analysis. TLR pathway inhibitors, blocking Abs and siRNA were used to abrogate induction of PD-L1 & PD-L2. Results: CMF stimulation with PAMPs (4-24 h) that signal via the MyD88 adaptor involving pathway TLR 2, 4 and 5, but not TLR6-9, upregulated PD-L1 expression, which was decreased in the presence of appropriate blocking peptide or and antibodies. Blocking of MyD88 expression in CMF by specific siRNA reduced the induction of the PD-L1. Upregulation of PD-L2 was induced by the stimulation of TLR5, but not other TLR requiring MyD88 adaptor and decreased in the presence of the TLR5 peptide antagonist. A positive feedback in the expression of TLR 2, 4 and 5 was observed in response to the stimulation of the CMFs with TLR 2, 4 and 5- specific PAMPs. This suggests that IBD associated shifts in gut flora may trigger strong upregulation of TLRs contributing to the increase in the PD-L1 expression on CMFs during UC progression. Conclusion: Normal colonic mucosa CMFs play an important role in innate immune responses to bacterial PAMPs and may favor suppression of activated CD4+ T cells via PD-L1/2 upregulation. Our data suggest that earlier observed abnormalities in the PD-L1 expression on CMFs in UC colonic mucosa may be due to the stimulation of CMFs through TLR2, 4 and/or TLR5 by PAMPs in subepithelial compartments during IBD-associated inflammation. Supported by AGA, CCFA and NIDDK.
M1819 Actively Inflamed Ulcerative Colitis and Crohn's Colitis are Characterized by Differential Upregulation of the Markers of Erad Activity, Edem1 and Atg5 Rohan Lourie, Thu V. Tran, Michael A. McGuckin, Timothy H. Florin Background: Goblet cell pathology, including accumulation of the MUC2 precursor, occurs in human IBD, and several murine models have linked Muc2 misfolding and endoplasmic reticulum (ER) stress with intestinal inflammation. A number of chaperone molecules detect and direct misfolded proteins through the ER membrane for proteasomal degradation (ERAD). EDEM1 is a short-lived ER chaperone which competes with the endoplasmic reticulum assisted folding system for misfolded substrates. EDEM1 itself is rapidly degraded in a process dependent on the autophagy pathway gene ATG5. Aims: To quantify EDEM1 and ATG5 mRNA in ulcerative colitis and Crohn's colitis by qRT-PCR. Methods: Colonic mucosal biopsies were taken from macroscopically and histologically non-inflamed or inflamed colonic sites of treatment-naïve patients with inflammatory bowel disease (19 Crohn's disease and 25 ulcerative colitis), and 12 age-matched healthy patients undergoing cancer screening. RNA was extracted and qRT-PCR performed with EDEM1, ATG5 and VIL1 specific primers. Analysis: Fold changes for EDEM1 and ATG5 relative to VIL1 (epithelial specific gene encoding villin) were normalized to the mean of the control group and analysis of variance assessed with Kruskal-Wallis testing. Results: EDEM1 was significantly upregulated in inflamed ulcerative colitis, p=0.0002, and in inflamed Crohn's colitis, p=0.002. ATG5 was also significantly upregulated in both inflamed ulcerative colitis, p=0.013 and inflamed Crohn's colitis, p=0.02. The ratio of the fold change of EDEM1 to ATG5 was significantly different between inflamed ulcerative colitis and inflamed Crohn's colitis when assessed by Mann-Whitney testing, with higher levels of EDEM1 to ATG5 mRNA in the ulcerative colitis group. There was no significant difference between either of these groups and the control group. Discussion: EDEM1 mRNA is upregulated in ulcerative colitis, but with an attenuated ATG5 mRNA response compared to Crohn's colitis. Less tightly regulated expression of EDEM1 in goblet cells could lead to diversion of MUC2 to a degradation pathway contributing to the goblet cell pathology and mucus barrier deficiency seen in ulcerative colitis.
M1817 Regulation of Intestinal Permeability and Epithelial Cell Tight Junctions by the Ubiquitin-Editing Enzyme TNFAIP3 James P. Lodolce, Lauren Kolodziej, Jonathan Chang, Jeffrey R. Schneider, Jeannette S. Messer, Jerrold R. Turner, David L. Boone Gut homeostasis depends on the regulation of intestinal epithelial cell (IEC) tight junctions (TJ) necessary for maintaining the semi-permeable barrier of the intestine. Patients with Crohn's disease (CD) have disruption of TJ and impaired barrier function. In addition, CD patients have elevated levels of the pro-inflammatory cytokine TNF and myosin light chain kinase (MLCK) that mediate IEC permeability by regulating TJ. The anti-inflammatory protein TNFAIP3 is required to negatively regulate TNF pro-inflammatory signaling. TNFAIP3 is both a deubiquitinating enzyme and an ubiquitin ligating enzyme. Although TNFAIP3 is expressed in IEC, its role in regulating tight junctions and intestinal permeability remains unknown. METHODS: To determine if TNFAIP3 is required to maintain barrier function In Vivo, we measured FITC-dextran flux in intestinal loops explanted from TNFAIP3 +/+ and -/- mice. To assess TNFAIP3's role in maintaining TJ In Vitro we measured trans-epithelial resistance (TER) and paracellular flux in TNF-treated WT and TNFAIP3 overexpressing IEC monolayers or cells expressing TNFAIP3 shRNA. We investigated MLCK involvement in the TNF-induced decrease in TER by treating cells with PIK, a potent inhibitor of MLCK. To assess MLCK activity, we probed for phosphorylated MLC in TNF-treated cells. RESULTS: TNFAIP3 -/- mice exhibited significantly increased intestinal permeability, indicating TNFAIP3 is required to maintain intestinal barrier integrity In Vivo. In Vitro, IEC overexpressing TNFAIP3 were less susceptible to TNF-induced increases in flux and decreases in TER. TNFAIP3 shRNA expressing cells displayed greater decreases in TER in response to TNF compared to WT (control shRNA) cells. The MLCK inhibitor PIK prevented TER decreases in both control and TNFAIP3 shRNA expressing cells, thus implicating TNFAIP3 in the MLCK pathway of TJ regulation. However, IEC overexpressing TNFAIP3 displayed no difference in the activation of MLCK by TNF. This suggests that TNFAIP3 acts in the MLCK pathway of TJ regulation at a point downstream of MLCK activation. CONCLUSIONS: TNFAIP3 is required for normal intestinal barrier function, and TNFAIP3 can act within
M1820 Activated Toll-Like Receptor 4 (TLR4) Directs Differentiation of Goblet and Paneth Cells Cristhine Pastorini, Rebeca Santaolalla, Masayuki Fukata, John P. Sotolongo, Cecilia Espana, Lory Hayes, Maria T. Abreu Background: Paneth and goblet cells are intestinal secretory cells which play an important role in maintenance of host-microbial homeostasis by regulating microbial density in the small intestine. TLR4 expression in the small intestine increases intestinal proliferation and villus height. We hypothesized that TLR4 signaling could alter differentiation of stem cells into secretory cells. Methods: We developed mice that express constitutively-active TLR4 in intestinal epithelial cells (IECs) under the villin promoter (villin-TLR4 mice). We compared Paneth cell and goblet cell numbers between villin-TLR4 mice and wild-type (WT) littermates using H&E and Alcian blue-Periodic acid-Schiff staining, respectively. Paneth cell maturation was assessed by immunofluorescent detection of lysozyme. Ileal expression of pan-alpha defensin was determined by real time-PCR. Total number of intestinal bacteria was counted by culturing stool pellets from villin-TLR4 and WT mice. Results: The villin-TLR4 mice had a significant reduction in the average number of Paneth cells detected per crypt when compared with WT mice (3.65±0.59 vs 6.92±0.26, p<0.01). Expression of pan-alpha defensin
S-425
AGA Abstracts
AGA Abstracts
UC-CMFs. Myeloid dendritic cells (DC) generated from peripheral blood mononuclear cells by IL-4/GM-CSF treatment were used as control APCs. T cell phenotype was determined by immunostaining followed by FACS analysis. CFSE-proliferation assays, real-time RT-PCR were used to evaluate activation of Treg cells co-cultured with CMFs. Results: Co-culturing of allogeneic TH0 cells with both N- and UC-CMFs leads to the upregulation of FoxP3 mRNA expression. FACS analysis demonstrated that N-CMF induced generation of FoxP3+ T cells from TH0 cells at a rate of 10.2±3.6%, comparable to those co-cultured with DCs. N-CMFs induced FoxP3+ T cells were CD25highCD127-, express IL-10 and TGFβ and possess suppressive activity. Co-culture of the TH0 cells with UC-derived CMFs induced FoxP3 expression in cells bearing CD127+CD25low T effector cell phenotype. Since FoxP3 expression in CD127+CD25low T effectors has been associated with anergy, our data suggest that in contrast to N-CMFs, UC-derived CMFs have a reduced capacity to induce an active Treg and may lead to the induction of anergic FoxP3+ CD4+ T effector cells. Conclusions: These results support the notion that in normal colonic mucosa CMFs have an anti-inflammatory role and contribute to tolerance by supporting the Treg cell function. Our data also suggest that disruption in the capacity of UC-CMFs to induce active Treg may contribute to the progression of UC. Supported by NIDDK, CCFA and AGA.