- Email: [email protected]
M1820 Activated Toll-Like Receptor 4 (TLR4) Directs Differentiation of Goblet and Paneth Cells
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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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.
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in ileal mucosa was lower in villin-TLR4 than in WT mice. Lysozyme production was also lower in villin-TLR4 Paneth cells than that in WT. In contrast, the percentage of goblet cells per total IECs in each crypt unit was significantly higher in villin-TLR4 mice than in WT mice (13.11±0.36 vs 11.20±0.32%, p<0.01). The number of bacterial colonies growing from stool of WT mice was significantly higher than that in stool from villin-TLR4 in both aerobic and anaerobic cultures. In addition, considerable differences were observed in types of bacteria isolated from stool cultures between villin-TLR4 and WT mice. Conclusions: Epithelial TLR4 signaling positively regulates differentiation of goblet cells but negatively that of Paneth cells. Although Paneth cells are fewer, epithelial expression of TLR4 suppresses fecal bacterial load. These results indicate that TLRs can alter differentiation of cells as they emerge from the stem cell niche.
M1823 Genetic and Functional Analysis of Intestinal Organic Cation/L-Carnitine Transporter (OCTN) in Crohn's Disease Marc Girardin, Philippe Goyette, John D. Rioux, Serge Dionne, Patrick Charlebois, Ernest G. Seidman Background: The IBD5 locus has been repeatedly implicated as a genetic risk factor for Crohn's Disease (CD). This locus codes for the organic cation/carnitine transporters (OCTN1 & 2) that transport cations as well as carnitine, an essential cofactor for long-chain fatty acid oxidation. Two variants in the OCTN cluster have been reported, forming a haplotype associated with susceptibility to CD. They were shown to be associated with modified transporter functions of OCTN1 In Vitro. OCTN1 is a bidirectional transporter, with a low affinity for carnitine compared to OCTN2. The aim of this study is to investigate the functional aspects of intestinal OCTNs in inflammatory bowel disease (IBD) in relation to genetic polymorphisms. Patients & Methods: Intestinal tissue was obtained from endoscopic biopsies and surgical resections in consenting IBD patients (n =33 & 14, resp.) as well from normal controls (n =22 & 14, resp.). Western Blot analysis was used to measure OCTN protein levels in homogenates of intestinal biopsies, using actin as a control. Functional analyses were performed utilizing brush border membrane vesicles isolated from intestinal resections. Carnitine transport was measured across the apical membrane using H3 radiolabeled substrate. Genetic analyses (common OCTN1 & 2 polymorphisms) were carried out using leukocytes. Results: We documented the presence of intestinal OCTN1 & 2 (65 KDa) in both IBD and control patients' tissue by Western blot. OCTN1 protein levels were significantly higher in ileal compared to colonic tissue (2.95% ± 0.4 vs 0.66% ± 0.2, resp.; p<0.0002). OCTN1 expression was found to be higher in CD patients with homo or heterozygous mutations (0.6% ± 0.1 vs 3% ± 0.8, resp., p<0.02). Functional studies showed a very rapid, Na+ dependent transport of carnitine across the apical intestinal border (10 sec). No difference in carnitine transport was found comparing CD and control groups (0.45 ± 0.12 vs 0.51 ± 0.12 nM carnitine/mg prot/min, resp.). Carnitine transport tended to be higher in tissue from patients with homo or heterozygous OCTN1 mutations (0.19 ± 0.03 vs 0.59 ± 0.12, resp.). However, this difference did not reach statistical significance, due to the limited number of cases. Conclusions: The present data reveal the presence of higher OCTN protein levels in intestinal tissue from IBD patients. Our results suggest that ileal carnitine transport is similar in CD and control groups. However, there was a trend towards higher carnitine transport in patients with OCTN1 mutations. Further analyses are underway in a larger cohort to confirm these findings. Supported by a Crohn's and Colitis Foundation of Canada grant.
M1821 Prostaglandin E2 Inhibits Migration of Colonic Lamina Propria Fibroblasts Florian Rieder, Martina Georgieva, Anja Schirbel, Monika Artinger, Anita Zuegner, Martin Blank, Julia Brenmoehl, Jurgen Scholmerich, Gerhard Rogler BACKGROUND: Migration of colonic lamina propria fibroblasts (CLPF) is an important mechanism during wound healing in inflammatory bowel disease (IBD). The concentration of Prostaglandin E2 (PGE2) is increased in the intestinal mucosa of IBD patients. We therefore investigated the role of PGE2 in CLPF migration. METHODS: Primary cultures of CLPF were isolated from healthy controls and Crohn's disease patients. Migration assays were performed in the Boyden chamber and wound scratch assays. Expression of EP-receptors on CLPF, secretion of PGE2, levels of intracellular cAMP, expression and distribution of Factin, α-smooth muscle actin (SMA) and myosin light chain (MLC) were determined by immunoblotting, immunocytochemistry and ELISA, respectively. RESULTS: All four EPreceptor subtypes were present on all tested CLPF. PGE2 and agonists to the EP2- and EP4receptor reduced the migration of CLPF, whereas ligation of the EP1/EP3-receptors did not alter CLPF migration. Blockade of the EP2- and the EP4-receptor inhibited the effect of PGE2 on CLPF migration. Increase in intracellular cAMP by forskolin or phosphodiesterase inhibitors reduced CLPF migration. PGE2 increased the levels of cAMP in CLPF, with abrogation after addition of EP2- and EP4-receptor antagonists. PGE2 and forskolin decreased the expression of α-SMA and F-actin and reduced cell polarization and lamellipodium formation in a wound scratch assay. In addition, forskolin reduced the phoshorylation of MLC (pMLC) and led to lack of accumulation of pMLC in the leading edge of CLPF. Finally CLPF secreted increased amounts of PGE2 after activation with IL-1β. CONCLUSIONS: PGE2 reduced the migration of CLPF via its EP2- and EP4-receptors and elevation of intracellular cAMP. Potential mechanisms are changes in expression of cytoskeletal proteins, failure of CLPF to polarize and a decreased amount of pMLC. CLPF secrete enhanced amounts of PGE2 under inflammatory conditions. This might be one possible reason for the impairment of intestinal wound healing in IBD.
M1824 Inhibitor of Apoptosis Protein 2 (cIAP2) Overexpression in Cytokine Stimulated Colonocytes Modifies Inflammation Driven Propagation of DNADamaged Cells Jakob B. Seidelin, Ole H. Nielsen
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Introduction: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer, especially in poorly controlled, long-standing, and extensive cases. DNA-instability is an early prerequisite for cancer development. Inhibition of apoptosis pathways lead to survival of DNA-damaged cells and might be of importance for the development of cancer in UC. DNA-strand breaks result in phosphorylation of histone H2A.X which is a part of the cellular DNA-damage surveillance system, and accordingly this phosphorylation can be measured as a surrogate marker of DNA damage. We have previously described that cIAP2 is upregulated in regenerating colonocytes of active UC, but not in quiescent UC [1], and colonocytes have an increased H2A.X phosphorylation in active stages of UC [2]. Aim: The aim was to investigate if cytokines stimulate cIAP2 upregulation in Caco2 cells and lead to increased H2A.X phosphorylation as a sign of DNA-damage. Further, the aim was to investigate if an increased cIAP2 expression prolongs DNA-damage in colonocytes. Methods: Caco2 cells were stimulated with either a combination of cytokines (IL-1β, TNF-α, and INF-γ, all at 1 μM) or doxorubicin (10 μM) for 4 hours. Expression of cIAP2, H2A.X and phospho-H2A.X was determined by immunoblotting at different time points. cIAP2 was inhibited by siRNA interference. Cell death was measured by the LDH-release assay. Results: After 24 hours H2A.X phosphorylation was increased > 2 fold in cells stimulated with cytokines compared to unstimulated controls (p<0.03); the increment was similar to that of doxorubicin treated cells. H2A.X phoshorylation remained high after 72 hours post-stimulation in both cytokine as well as doxorubicin stimulated cells. cIAP2 expression was increased both at mRNA and protein levels in cytokine stimulated cells as compared to controls (p<0.02). Inhibition of cIAP2 expression with siRNA interference caused normalization of H2A.X phosphorylation within 48 hours. cIAP2 inhibition also caused increased cell death in cytokine stimulated colonocytes (p<0.01). Conclusions: Cytokine stimulation of colonocytes in UC promotes DNA-damage at similar levels as doxorubicin, indicating a direct association between proinflammatory mediators and DNA-damage. This DNA-damage is prolonged by a concomitant increased cIAP2 expression, and inhibition of cIAP2 decreases DNA-damage. Thus, cIAP2 might be of importance for DNA-damage propagation and thereby for carcinogenesis observed in UC. References: 1. Seidelin JB et al. Virchows Arch 2007;451:1031 2. Risques RA et al. Gastroenterology 2008;135:410
Lack of Epigenetic Changes Associated With Tolerized Genes During Sustained Inflammatory Gene Expression by Human Intestinal Fibroblasts (HIF) Melania Scarpa, Franco Scaldaferri, Tammy Sadler, Claudio Fiocchi, Eleni Stylianou Background: Tolerance is indispensable to maintenance of intestinal immune homeostasis. Lack of tolerance results in abnormal immune responses eventually leading to prolonged inflammation in the gut, like in IBD. Mucosal tolerance is mediated by immune cells, however, other cells in the mucosa, such as HIF, may not be tolerizable. Our previous studies have shown that HIF fail to become tolerant to bacterial products and pro-inflammatory cytokines. Epigenetic changes, e.g., histone modifications of chromatin have been implicated in regulating tolerance in immune cells by repressing pro-inflammatory gene expression in a cell-, stimulus- and gene-specific manner. Whether the same or other modifications control the expression of genes in HIF is unknown. Aims: To investigate the histone modifications associated with lack of tolerance in HIF. Materials and Methods: THP-1 monocytes and HIF were either exposed to a single dose of the cytokine TNF (responsive cells) or submitted to a classical tolerization protocol in which initial pretreatment of THP-1 or HIF with TNF for 16h was followed by a further exposure for 1 to 6 hours (tolerized cells). IL6 and IL8 mRNA expression levels (qRT-PCR) were assessed. Histone modifications associated with activation (H3S10Ph, H3K4me3, H3Ac) or repression (H3K9me2, H3K27me3) were measured by Chromatin Immunoprecipitation. Results: IL6 and IL8 mRNA levels were upregulated in responsive THP-1 (after a single exposure to TNF), peaking and decreasing rapidly by 2 hours. Repeated exposure to TNF (tolerized THP-1) abrogated induction of the same cytokine mRNA. Conversely, IL6 and IL8 mRNA levels in “tolerized” HIF increased above those of responsive HIF. In THP-1, levels of H3S10Ph, a mark of transcriptionally active genes, paralleled IL6 gene expression, increasing in responsive THP-1 and selectively decreasing in tolerized THP-1. In contrast, H3S10Ph levels were not increased with cytokine gene expression in responsive or “tolerized” HIF. Moreover, decreased H3K9me2 at the IL8 promoter, associated with gene silencing, was detected in responsive but not in tolerized THP-1. Of the other modifications detected in HIF, H3Ac and K3K27me3 were not associated with changes in IL6 gene expression. Conclusions: These preliminary results indicate that whereas decreased H3S10Ph mediates tolerization of inflammatory genes such as IL6 in THP-1 this is not the case in “tolerized” HIF indicating that these cells sustain inflammatory gene expression and potentially gut inflammation in a distinct cell and gene-specific manner. The other types of epigenetic modification that may be involved are currently being investigated.
M1825 Repeated Enemas of Recombinant Human Hepatocyte Growth Factor Ameliorate Rat Experimental Colitis Without Increasing Serum HGF Levels Hitoshi Setoyama, Akio Ido, Toshio Sakiyama, Fumisato Sasaki, Shuji Kanmura, Naohisa Yamaji, Masatsugu Numata, Akihiro Moriuchi, Hirohito Tsubouchi Background and aims: Inflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn's disease, is an idiopathic and refractory disorder with an unknown etiology. Immune-modulating agents have been used as conventional therapies for IBD. Recently, therapies that regenerate damaged epithelium have been developed as a potential therapeutic modality for IBD. Hepatocyte growth factor (HGF) is a major agent that promotes hepatocyte
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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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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.
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in ileal mucosa was lower in villin-TLR4 than in WT mice. Lysozyme production was also lower in villin-TLR4 Paneth cells than that in WT. In contrast, the percentage of goblet cells per total IECs in each crypt unit was significantly higher in villin-TLR4 mice than in WT mice (13.11±0.36 vs 11.20±0.32%, p<0.01). The number of bacterial colonies growing from stool of WT mice was significantly higher than that in stool from villin-TLR4 in both aerobic and anaerobic cultures. In addition, considerable differences were observed in types of bacteria isolated from stool cultures between villin-TLR4 and WT mice. Conclusions: Epithelial TLR4 signaling positively regulates differentiation of goblet cells but negatively that of Paneth cells. Although Paneth cells are fewer, epithelial expression of TLR4 suppresses fecal bacterial load. These results indicate that TLRs can alter differentiation of cells as they emerge from the stem cell niche.
M1823 Genetic and Functional Analysis of Intestinal Organic Cation/L-Carnitine Transporter (OCTN) in Crohn's Disease Marc Girardin, Philippe Goyette, John D. Rioux, Serge Dionne, Patrick Charlebois, Ernest G. Seidman Background: The IBD5 locus has been repeatedly implicated as a genetic risk factor for Crohn's Disease (CD). This locus codes for the organic cation/carnitine transporters (OCTN1 & 2) that transport cations as well as carnitine, an essential cofactor for long-chain fatty acid oxidation. Two variants in the OCTN cluster have been reported, forming a haplotype associated with susceptibility to CD. They were shown to be associated with modified transporter functions of OCTN1 In Vitro. OCTN1 is a bidirectional transporter, with a low affinity for carnitine compared to OCTN2. The aim of this study is to investigate the functional aspects of intestinal OCTNs in inflammatory bowel disease (IBD) in relation to genetic polymorphisms. Patients & Methods: Intestinal tissue was obtained from endoscopic biopsies and surgical resections in consenting IBD patients (n =33 & 14, resp.) as well from normal controls (n =22 & 14, resp.). Western Blot analysis was used to measure OCTN protein levels in homogenates of intestinal biopsies, using actin as a control. Functional analyses were performed utilizing brush border membrane vesicles isolated from intestinal resections. Carnitine transport was measured across the apical membrane using H3 radiolabeled substrate. Genetic analyses (common OCTN1 & 2 polymorphisms) were carried out using leukocytes. Results: We documented the presence of intestinal OCTN1 & 2 (65 KDa) in both IBD and control patients' tissue by Western blot. OCTN1 protein levels were significantly higher in ileal compared to colonic tissue (2.95% ± 0.4 vs 0.66% ± 0.2, resp.; p<0.0002). OCTN1 expression was found to be higher in CD patients with homo or heterozygous mutations (0.6% ± 0.1 vs 3% ± 0.8, resp., p<0.02). Functional studies showed a very rapid, Na+ dependent transport of carnitine across the apical intestinal border (10 sec). No difference in carnitine transport was found comparing CD and control groups (0.45 ± 0.12 vs 0.51 ± 0.12 nM carnitine/mg prot/min, resp.). Carnitine transport tended to be higher in tissue from patients with homo or heterozygous OCTN1 mutations (0.19 ± 0.03 vs 0.59 ± 0.12, resp.). However, this difference did not reach statistical significance, due to the limited number of cases. Conclusions: The present data reveal the presence of higher OCTN protein levels in intestinal tissue from IBD patients. Our results suggest that ileal carnitine transport is similar in CD and control groups. However, there was a trend towards higher carnitine transport in patients with OCTN1 mutations. Further analyses are underway in a larger cohort to confirm these findings. Supported by a Crohn's and Colitis Foundation of Canada grant.
M1821 Prostaglandin E2 Inhibits Migration of Colonic Lamina Propria Fibroblasts Florian Rieder, Martina Georgieva, Anja Schirbel, Monika Artinger, Anita Zuegner, Martin Blank, Julia Brenmoehl, Jurgen Scholmerich, Gerhard Rogler BACKGROUND: Migration of colonic lamina propria fibroblasts (CLPF) is an important mechanism during wound healing in inflammatory bowel disease (IBD). The concentration of Prostaglandin E2 (PGE2) is increased in the intestinal mucosa of IBD patients. We therefore investigated the role of PGE2 in CLPF migration. METHODS: Primary cultures of CLPF were isolated from healthy controls and Crohn's disease patients. Migration assays were performed in the Boyden chamber and wound scratch assays. Expression of EP-receptors on CLPF, secretion of PGE2, levels of intracellular cAMP, expression and distribution of Factin, α-smooth muscle actin (SMA) and myosin light chain (MLC) were determined by immunoblotting, immunocytochemistry and ELISA, respectively. RESULTS: All four EPreceptor subtypes were present on all tested CLPF. PGE2 and agonists to the EP2- and EP4receptor reduced the migration of CLPF, whereas ligation of the EP1/EP3-receptors did not alter CLPF migration. Blockade of the EP2- and the EP4-receptor inhibited the effect of PGE2 on CLPF migration. Increase in intracellular cAMP by forskolin or phosphodiesterase inhibitors reduced CLPF migration. PGE2 increased the levels of cAMP in CLPF, with abrogation after addition of EP2- and EP4-receptor antagonists. PGE2 and forskolin decreased the expression of α-SMA and F-actin and reduced cell polarization and lamellipodium formation in a wound scratch assay. In addition, forskolin reduced the phoshorylation of MLC (pMLC) and led to lack of accumulation of pMLC in the leading edge of CLPF. Finally CLPF secreted increased amounts of PGE2 after activation with IL-1β. CONCLUSIONS: PGE2 reduced the migration of CLPF via its EP2- and EP4-receptors and elevation of intracellular cAMP. Potential mechanisms are changes in expression of cytoskeletal proteins, failure of CLPF to polarize and a decreased amount of pMLC. CLPF secrete enhanced amounts of PGE2 under inflammatory conditions. This might be one possible reason for the impairment of intestinal wound healing in IBD.
M1824 Inhibitor of Apoptosis Protein 2 (cIAP2) Overexpression in Cytokine Stimulated Colonocytes Modifies Inflammation Driven Propagation of DNADamaged Cells Jakob B. Seidelin, Ole H. Nielsen
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Introduction: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer, especially in poorly controlled, long-standing, and extensive cases. DNA-instability is an early prerequisite for cancer development. Inhibition of apoptosis pathways lead to survival of DNA-damaged cells and might be of importance for the development of cancer in UC. DNA-strand breaks result in phosphorylation of histone H2A.X which is a part of the cellular DNA-damage surveillance system, and accordingly this phosphorylation can be measured as a surrogate marker of DNA damage. We have previously described that cIAP2 is upregulated in regenerating colonocytes of active UC, but not in quiescent UC [1], and colonocytes have an increased H2A.X phosphorylation in active stages of UC [2]. Aim: The aim was to investigate if cytokines stimulate cIAP2 upregulation in Caco2 cells and lead to increased H2A.X phosphorylation as a sign of DNA-damage. Further, the aim was to investigate if an increased cIAP2 expression prolongs DNA-damage in colonocytes. Methods: Caco2 cells were stimulated with either a combination of cytokines (IL-1β, TNF-α, and INF-γ, all at 1 μM) or doxorubicin (10 μM) for 4 hours. Expression of cIAP2, H2A.X and phospho-H2A.X was determined by immunoblotting at different time points. cIAP2 was inhibited by siRNA interference. Cell death was measured by the LDH-release assay. Results: After 24 hours H2A.X phosphorylation was increased > 2 fold in cells stimulated with cytokines compared to unstimulated controls (p<0.03); the increment was similar to that of doxorubicin treated cells. H2A.X phoshorylation remained high after 72 hours post-stimulation in both cytokine as well as doxorubicin stimulated cells. cIAP2 expression was increased both at mRNA and protein levels in cytokine stimulated cells as compared to controls (p<0.02). Inhibition of cIAP2 expression with siRNA interference caused normalization of H2A.X phosphorylation within 48 hours. cIAP2 inhibition also caused increased cell death in cytokine stimulated colonocytes (p<0.01). Conclusions: Cytokine stimulation of colonocytes in UC promotes DNA-damage at similar levels as doxorubicin, indicating a direct association between proinflammatory mediators and DNA-damage. This DNA-damage is prolonged by a concomitant increased cIAP2 expression, and inhibition of cIAP2 decreases DNA-damage. Thus, cIAP2 might be of importance for DNA-damage propagation and thereby for carcinogenesis observed in UC. References: 1. Seidelin JB et al. Virchows Arch 2007;451:1031 2. Risques RA et al. Gastroenterology 2008;135:410
Lack of Epigenetic Changes Associated With Tolerized Genes During Sustained Inflammatory Gene Expression by Human Intestinal Fibroblasts (HIF) Melania Scarpa, Franco Scaldaferri, Tammy Sadler, Claudio Fiocchi, Eleni Stylianou Background: Tolerance is indispensable to maintenance of intestinal immune homeostasis. Lack of tolerance results in abnormal immune responses eventually leading to prolonged inflammation in the gut, like in IBD. Mucosal tolerance is mediated by immune cells, however, other cells in the mucosa, such as HIF, may not be tolerizable. Our previous studies have shown that HIF fail to become tolerant to bacterial products and pro-inflammatory cytokines. Epigenetic changes, e.g., histone modifications of chromatin have been implicated in regulating tolerance in immune cells by repressing pro-inflammatory gene expression in a cell-, stimulus- and gene-specific manner. Whether the same or other modifications control the expression of genes in HIF is unknown. Aims: To investigate the histone modifications associated with lack of tolerance in HIF. Materials and Methods: THP-1 monocytes and HIF were either exposed to a single dose of the cytokine TNF (responsive cells) or submitted to a classical tolerization protocol in which initial pretreatment of THP-1 or HIF with TNF for 16h was followed by a further exposure for 1 to 6 hours (tolerized cells). IL6 and IL8 mRNA expression levels (qRT-PCR) were assessed. Histone modifications associated with activation (H3S10Ph, H3K4me3, H3Ac) or repression (H3K9me2, H3K27me3) were measured by Chromatin Immunoprecipitation. Results: IL6 and IL8 mRNA levels were upregulated in responsive THP-1 (after a single exposure to TNF), peaking and decreasing rapidly by 2 hours. Repeated exposure to TNF (tolerized THP-1) abrogated induction of the same cytokine mRNA. Conversely, IL6 and IL8 mRNA levels in “tolerized” HIF increased above those of responsive HIF. In THP-1, levels of H3S10Ph, a mark of transcriptionally active genes, paralleled IL6 gene expression, increasing in responsive THP-1 and selectively decreasing in tolerized THP-1. In contrast, H3S10Ph levels were not increased with cytokine gene expression in responsive or “tolerized” HIF. Moreover, decreased H3K9me2 at the IL8 promoter, associated with gene silencing, was detected in responsive but not in tolerized THP-1. Of the other modifications detected in HIF, H3Ac and K3K27me3 were not associated with changes in IL6 gene expression. Conclusions: These preliminary results indicate that whereas decreased H3S10Ph mediates tolerization of inflammatory genes such as IL6 in THP-1 this is not the case in “tolerized” HIF indicating that these cells sustain inflammatory gene expression and potentially gut inflammation in a distinct cell and gene-specific manner. The other types of epigenetic modification that may be involved are currently being investigated.
M1825 Repeated Enemas of Recombinant Human Hepatocyte Growth Factor Ameliorate Rat Experimental Colitis Without Increasing Serum HGF Levels Hitoshi Setoyama, Akio Ido, Toshio Sakiyama, Fumisato Sasaki, Shuji Kanmura, Naohisa Yamaji, Masatsugu Numata, Akihiro Moriuchi, Hirohito Tsubouchi Background and aims: Inflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn's disease, is an idiopathic and refractory disorder with an unknown etiology. Immune-modulating agents have been used as conventional therapies for IBD. Recently, therapies that regenerate damaged epithelium have been developed as a potential therapeutic modality for IBD. Hepatocyte growth factor (HGF) is a major agent that promotes hepatocyte
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