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M1768 Junctional Adhesion Molecule – A is a New Key Regulator in Colitis Associated Colon Cancer
of murine colonic mucosa was evaluated by endoscopic and histological scores . The grades of histological mucosal lesion were identified as glandular intraepithelial neoplasia (GIN), indicating small clusters of dysplastic colonic crypts on the mucosal surface, low grade (LG) and high grade (HG) adenomas. Colonic murine sections were also stained for Ki67 and β-catenin expression, and apoptosis analysis was performed by TUNEL assay. JAM-A protein was found to decrease significantly in human colon cancer surrounding cancer lesions, while no differences were observed in corresponding non-neoplastic mucosa compared to normal tissues. Interestingly, JAM-A expression was very low in HCT116, HT29 and SW620 cell lines compared to Caco-2, with lowest expression in the metastatic colon cancer line SW620. JAM-A -/- mice were significantly more susceptible to CAC as assessed by body weight loss (p<0.001), DAI (p<0.001), endoscopic and histological inflammation (p<0.05). Furthermore, JAM-A -/- mice showed a higher (p<0.01) number of tumor incidence in the distal segment of the colon characterized by a high grade (HG) of differentiation in comparison to WT mice. Additionally, JAM-A-/- mice displayed a significantly higher number of Ki-67-positive epithelial cells and TUNEL activity with increased nuclear localization of β-catenin. These results demonstrate that the low levels of JAM-A correlate with increased incidence of tumor development and tumor aggressiveness. These findings suggest that epithelial JAM-A is a new regulator of colon cancerogenesis.
Adipose Tissue-Derived Mesenchymal Stromal Cells Ameliorate Experimental Colitis: Evidence for Immunomodulatory Paracrine Effect Igor Diomará P. Soares, Daiana V. Lopes, Morgana T. Castelo-Branco, Alberto Schanaider, Sergio Augusto L. de Souza, Bianca Gutfilen, Lea Mirian B. Fonseca, Celeste C. Elia, Maria Isabel D. Rossi, Heitor Souza Background & Aims: Inflammatory bowel diseases (IBD) are characterized by chronic intestinal inflammation due to the loss of immune tolerance against mucosal antigens. Mesenchymal stromal cells were recently shown to possess immunomodulatory action with beneficial therapeutic effects in immune mediated disorders. We investigated the potential therapeutic action of subcutaneous adipose tissue (AT-MSCs) or bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a model of IBD. Methods: Male Wistar rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis were treated with AT-MSCs or BM-MSCs through intraperitoneal injection after colonoscopic detection of inflammation, at day 4. Colonoscopic and histologic scores were evaluated. Inflammatory response was determined by measuring the levels of inflammatory cytokines in cultures of colon samples, by ELISA. Collagen fibers were stained with picric acid dye and the density of collagen deposition was evaluated using a computerized image analysis system. MSCs were labeled with Tc-99m and administered contralateral to the site of inflammation. Scans were taken 30 minutes and 2 hours after injection, and isolated colon scans were obtained after euthanasia. Results: Intra-peritoneal injection of AT-MSCs or BM-MSCs significantly reduced the endoscopic (p<0.01) and histopathologic (p<0.02) severity of colitis . The therapeutic effect was mediated by downregulating the levels of TNF-alpha (p<0.01), and interleukin-1 beta (p<0.03). The high baseline levels of VEGF-a in TNBS-colitis did not decrease significantly after MSCs-therapy. A significant reduction in collagen deposition was observed in MSC-treated animals (p<0.01). Effects observed with AT-MSCs were significantly greater than the ones obtained with BMMSCs. Scintigraphy showed 99mTc-MSCs uptake in TNBS-induced animals but no uptake in controls. Labeled MSCs clearly migrated towards the sites of inflammation, and the uptake increased from 30 min to 2h. Conclusions: Intra-peritoneally injected MSCs are effective in the treatment of experimental colitis, probably due to a local paracrine anti-inflammatory action. Because MSCs are easily and safely obtained from the abundant subcutaneous fat tissue, AT-MSCs emerge as a promising therapy for IBD.
M1769 Intestinal Inflammation Down-Regulates SIGIRR/TIR8 Expression in Epithelial Cells by Inhibiting SP1-Mediated Pathway Chikara Kadota, Shunji Ishihara, Md. M. Aziz, Yoshiyuki Mishima, Naoki Oshima, Aya Otani, Akihiko Oka, Ryusaku Kusunoki, Yasumasa Tada, Ichiro Moriyama, Takafumi Yuki, Yuji Amano, Yoshikazu Kinoshita (Background & Aim) Intestinal epithelial cells (IECs) express various pattern recognition receptors including toll-like receptors (TLRs), which recognize luminal microbial components. Several negative regulatory mechanisms control TLR-mediated inflammatory responses and restore immune balance, including single immunoglobulin (Ig) IL-1R-related molecule (SIGIRR), an Ig-like membrane protein critical for negative regulation of TLR4-mediated signaling. We investigated SIGIRR expression and its regulation mechanism in IECs during inflammation. (Materials and Methods) First, we examined SIGIRR expression in human colonic mucosa with and without inflammation. Endoscopic biopsy specimens were taken from active and inactive colonic mucosa of patients with ulcerative colitis (UC), and SIGIRR expression was examined by real-time PCR and immunohistochemistry (IH). Next, mice experimental colitis models were established by administrations of TNBS and DSS, and colonic expression time-course changes were examined with real-time PCR, IH, and flow cytometry. Also, the effects of TLR ligands (LPS, flagellin) and TNFα on SIGIRR expression were evaluated In Vitro using cultured IECs (SW480, HCT15). Further, we transiently silenced endogenous SIGIRR expression in IECs using siRNA, and assessed NF-κB activity and IL-8 production. To elucidate SIGIRR expression regulation in IECs, the binding ability of the potent transcription factor Sp1 at the proximal-end responsive element of the SIGIRR promoter was examined using gel-shift and luciferase assays. (Results) In human colonic samples, SIGIRR was mainly expressed in IECs at levels significantly higher in inactive as compared to active mucosa. In the mice models, SIGIRR colonic expression rapidly decreased after colitis development and gradually returned to basal levels. Experimental colitis-mediated down-regulation of SIGIRR in IECs was also clearly confirmed by results of IH and flow cytometry. In Vitro, gene knockdown using siRNA for SIGIRR accelerated IL-8 production in LPS-stimulated IECs via activation of NF-κB. Further, inflammatory conditions induced by TLR ligands and TNFα caused significant down-regulation of SIGIRR expression in IECs, which was dependent on decreased Sp1 binding at the responsive element of the SIGIRR promoter. (Conclusion) SIGIRR is expressed in IECs and serves as a negative regulator to maintain gut innate immunity, which is down-regulated during inflammation by inhibiting an Sp1-mediated pathway.
M1767 Regional Differences of Mucosa-Associated Microbiota are Essential for Longitudinal Expression of TLR2 and TLR4 in Murine Colon Yunwei Wang, Suzanne Devkota, Dionysios Antonopoulos, Kenneth Drabik, Mark W. Musch, Eugene B. Chang Background: Interaction between enteric microbiota and host is pivotal for maintaining the homeostasis of mammalian gastrointestinal (GI) tract. Regional differences in immune function and gene expression of the GI tract may correlate with the composition and diversity of colonic microbes, particularly the indigenous microbiota associated with the mucosa. We therefore explored this possibility by examining the composition of the microbiota associated with the normal proximal and distal colonic mucosa as well as expression of two molecules sensing selective bacterial pattern, toll-like receptor 2 (TLR2) and TLR4, in C57BL/6J mice housed in specific pathogen free (SPF) and germ free (GT) environments. Methods: Proximal colon, distal colon and fecal samples of SPF mice obtained from the Jackson laboratory and the University of Chicago were collected and analyzed by 16S rRNA gene-based clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis. Expressions of TLR2 and TLR4 in colonic mucosa of SPF and GF mice were examined by laser capture microdissection, real time PCR and western blot. Fecal transplantation in GF mice was also used to determine the findings in SPF mice. Results: Significant differences in the community structure of the mucosa-associated microbiota of the murine proximal and distal colon were found in each individual mouse although gut microbiota differs in mice between litters and facilities. Low diversity of bacterial population was also discovered in distal colon compared to proximal colon and fecal sample. TLR2 was highly expressed at proximal colon and TLR4 was strongly expressed at distal colon in SPF mice by both RT-PCR and western blot analysis. However, there was no detectable protein expression of TLRs in the GF mice which suggested that enteric microbial population is essential in maintaining the differential expression of TLRs in the colon. More importantly, after the fecal transplantation in GF mice, both regional colonization of mucosa-associated microbes and differential expression of TLRs in murine colon could be reconstituted. Conclusions: These results provide direct evidence about the interaction between the microbial population and GI tract of the host. It also indicates that region-specific host assembly rules which may be relevant to essential microbial functions that contribute to host physiology and possibly disease.
M1770 Interaction Between Sex Hormones/Sex Hormone Receptors With the Intestinal Epithelium Modulates Barrier Function and Subsequent Inflammation in Experimental IBD Benedetta Mattioli, Rekha R. Garg, Luca Pastorelli, Xiao Wang, Sharon B. Hoang, Massimo Campieri, Theresa T. Pizarro Background: It is well established that the incidence and severity of several autoimmune disorders is increased in the female population, including Crohn's disease (CD). Previously, we demonstrated that earlier onset and increased severity of disease occurs in F vs M SAMP1/ YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, and that epithelial barrier dysfunction precedes histological evidence of inflammation in this strain. As such, a critical factor likely contributing to SAMP ileitis is the interaction between sex hormones/sex hormone receptors and intestinal epithelial cells (IEC) that modulate downstream inflammatory/ immune responses. Aim: To characterize gender-specific epithelial barrier dysfunction in SAMP mice and to mechanistically evaluate the role of sex hormones and their receptors regulating chronic ileitis. Methods: In Vivo small intestinal permeability was evaluated in 34 wk old (pre-inflammation) F- and M-SAMP and age/gender-matched AKR (parental control) mice by fractional urine excretion of lactulose/mannitol 22h after gavage, and RT-PCR was performed on isolated IEC for tight junction (TJ) proteins. Ex vivo permeability was assessed on ileal tissues following stimulation with ER agonists by measuring transepithelial electrical resistance (TEER) after 2h in culture. Ileal inflammation was histologically evaluated using an established scoring system and epithelial permeability measured by TEER after sex hormone replacement on gonadectomized (GDX) 10 wk old SAMP with established disease. Results: Prior to the onset of inflammation, small intestinal permeability was increased in F- compared to M-SAMP, with no gender difference observed in AKR, which was significantly decreased compared to SAMP, confirming our previously published studies. A 2.2-fold increase (P<0.05) was present in the TJ protein, claudin-2 (on ChrX), in F- vs M-SAMP, which was significantly increased compared to both F- and M-AKR, with no difference between genders. Administration of estrogen (E2), but not 5a-DHT (end metabolite of testosterone), significantly reduced ileal inflammation in GDX M-SAMP (9.0±0.7 vs 17.5±0.5
M1768 Junctional Adhesion Molecule - A is a New Key Regulator in Colitis Associated Colon Cancer Stefania Vetrano, Emanuela Sala, Carmen Correale, Vincenzo Arena, Alberto Malesci, Elisabetta Dejana, Silvio Danese Junctional Adhesion Molecule-A (JAM-A) is a tight junction protein expressed by epithelial and endothelial cells. We have shown previously that JAM-A exerts a crucial role in controlling mucosal homeostasis by regulating the integrity and permeability of the intestinal epithelial barrier function. Emerging evidences have indicated an association between increased tight junction permeability of the colon epithelium and tumor development. The aim of this study was to investigate the involvement of JAM-A in colitis-associated cancer (CAC). JAMA expression was analyzed by confocal microscopy in human colon cancer and control tissues and in four human colon cancer cell lines with distinct metastatic potential: CaCo2, HT29, HCT116 and SW620. JAM-A -/- and WT C57BL/6 mice were treated with azoxymethane and three oral cycles of 1,5% dextran sodium sulphate (DSS) each characterized by 7 days DSS exposure followed by 14 days of water. Mice were monitored daily for weight loss, fecal blood and diarrhea and a disease activity index (DAI) was calculated. The damage
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AGA Abstracts
AGA Abstracts
M1766
Adipose Tissue-Derived Mesenchymal Stromal Cells Ameliorate Experimental Colitis: Evidence for Immunomodulatory Paracrine Effect Igor Diomará P. Soares, Daiana V. Lopes, Morgana T. Castelo-Branco, Alberto Schanaider, Sergio Augusto L. de Souza, Bianca Gutfilen, Lea Mirian B. Fonseca, Celeste C. Elia, Maria Isabel D. Rossi, Heitor Souza Background & Aims: Inflammatory bowel diseases (IBD) are characterized by chronic intestinal inflammation due to the loss of immune tolerance against mucosal antigens. Mesenchymal stromal cells were recently shown to possess immunomodulatory action with beneficial therapeutic effects in immune mediated disorders. We investigated the potential therapeutic action of subcutaneous adipose tissue (AT-MSCs) or bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a model of IBD. Methods: Male Wistar rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis were treated with AT-MSCs or BM-MSCs through intraperitoneal injection after colonoscopic detection of inflammation, at day 4. Colonoscopic and histologic scores were evaluated. Inflammatory response was determined by measuring the levels of inflammatory cytokines in cultures of colon samples, by ELISA. Collagen fibers were stained with picric acid dye and the density of collagen deposition was evaluated using a computerized image analysis system. MSCs were labeled with Tc-99m and administered contralateral to the site of inflammation. Scans were taken 30 minutes and 2 hours after injection, and isolated colon scans were obtained after euthanasia. Results: Intra-peritoneal injection of AT-MSCs or BM-MSCs significantly reduced the endoscopic (p<0.01) and histopathologic (p<0.02) severity of colitis . The therapeutic effect was mediated by downregulating the levels of TNF-alpha (p<0.01), and interleukin-1 beta (p<0.03). The high baseline levels of VEGF-a in TNBS-colitis did not decrease significantly after MSCs-therapy. A significant reduction in collagen deposition was observed in MSC-treated animals (p<0.01). Effects observed with AT-MSCs were significantly greater than the ones obtained with BMMSCs. Scintigraphy showed 99mTc-MSCs uptake in TNBS-induced animals but no uptake in controls. Labeled MSCs clearly migrated towards the sites of inflammation, and the uptake increased from 30 min to 2h. Conclusions: Intra-peritoneally injected MSCs are effective in the treatment of experimental colitis, probably due to a local paracrine anti-inflammatory action. Because MSCs are easily and safely obtained from the abundant subcutaneous fat tissue, AT-MSCs emerge as a promising therapy for IBD.
M1769 Intestinal Inflammation Down-Regulates SIGIRR/TIR8 Expression in Epithelial Cells by Inhibiting SP1-Mediated Pathway Chikara Kadota, Shunji Ishihara, Md. M. Aziz, Yoshiyuki Mishima, Naoki Oshima, Aya Otani, Akihiko Oka, Ryusaku Kusunoki, Yasumasa Tada, Ichiro Moriyama, Takafumi Yuki, Yuji Amano, Yoshikazu Kinoshita (Background & Aim) Intestinal epithelial cells (IECs) express various pattern recognition receptors including toll-like receptors (TLRs), which recognize luminal microbial components. Several negative regulatory mechanisms control TLR-mediated inflammatory responses and restore immune balance, including single immunoglobulin (Ig) IL-1R-related molecule (SIGIRR), an Ig-like membrane protein critical for negative regulation of TLR4-mediated signaling. We investigated SIGIRR expression and its regulation mechanism in IECs during inflammation. (Materials and Methods) First, we examined SIGIRR expression in human colonic mucosa with and without inflammation. Endoscopic biopsy specimens were taken from active and inactive colonic mucosa of patients with ulcerative colitis (UC), and SIGIRR expression was examined by real-time PCR and immunohistochemistry (IH). Next, mice experimental colitis models were established by administrations of TNBS and DSS, and colonic expression time-course changes were examined with real-time PCR, IH, and flow cytometry. Also, the effects of TLR ligands (LPS, flagellin) and TNFα on SIGIRR expression were evaluated In Vitro using cultured IECs (SW480, HCT15). Further, we transiently silenced endogenous SIGIRR expression in IECs using siRNA, and assessed NF-κB activity and IL-8 production. To elucidate SIGIRR expression regulation in IECs, the binding ability of the potent transcription factor Sp1 at the proximal-end responsive element of the SIGIRR promoter was examined using gel-shift and luciferase assays. (Results) In human colonic samples, SIGIRR was mainly expressed in IECs at levels significantly higher in inactive as compared to active mucosa. In the mice models, SIGIRR colonic expression rapidly decreased after colitis development and gradually returned to basal levels. Experimental colitis-mediated down-regulation of SIGIRR in IECs was also clearly confirmed by results of IH and flow cytometry. In Vitro, gene knockdown using siRNA for SIGIRR accelerated IL-8 production in LPS-stimulated IECs via activation of NF-κB. Further, inflammatory conditions induced by TLR ligands and TNFα caused significant down-regulation of SIGIRR expression in IECs, which was dependent on decreased Sp1 binding at the responsive element of the SIGIRR promoter. (Conclusion) SIGIRR is expressed in IECs and serves as a negative regulator to maintain gut innate immunity, which is down-regulated during inflammation by inhibiting an Sp1-mediated pathway.
M1767 Regional Differences of Mucosa-Associated Microbiota are Essential for Longitudinal Expression of TLR2 and TLR4 in Murine Colon Yunwei Wang, Suzanne Devkota, Dionysios Antonopoulos, Kenneth Drabik, Mark W. Musch, Eugene B. Chang Background: Interaction between enteric microbiota and host is pivotal for maintaining the homeostasis of mammalian gastrointestinal (GI) tract. Regional differences in immune function and gene expression of the GI tract may correlate with the composition and diversity of colonic microbes, particularly the indigenous microbiota associated with the mucosa. We therefore explored this possibility by examining the composition of the microbiota associated with the normal proximal and distal colonic mucosa as well as expression of two molecules sensing selective bacterial pattern, toll-like receptor 2 (TLR2) and TLR4, in C57BL/6J mice housed in specific pathogen free (SPF) and germ free (GT) environments. Methods: Proximal colon, distal colon and fecal samples of SPF mice obtained from the Jackson laboratory and the University of Chicago were collected and analyzed by 16S rRNA gene-based clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis. Expressions of TLR2 and TLR4 in colonic mucosa of SPF and GF mice were examined by laser capture microdissection, real time PCR and western blot. Fecal transplantation in GF mice was also used to determine the findings in SPF mice. Results: Significant differences in the community structure of the mucosa-associated microbiota of the murine proximal and distal colon were found in each individual mouse although gut microbiota differs in mice between litters and facilities. Low diversity of bacterial population was also discovered in distal colon compared to proximal colon and fecal sample. TLR2 was highly expressed at proximal colon and TLR4 was strongly expressed at distal colon in SPF mice by both RT-PCR and western blot analysis. However, there was no detectable protein expression of TLRs in the GF mice which suggested that enteric microbial population is essential in maintaining the differential expression of TLRs in the colon. More importantly, after the fecal transplantation in GF mice, both regional colonization of mucosa-associated microbes and differential expression of TLRs in murine colon could be reconstituted. Conclusions: These results provide direct evidence about the interaction between the microbial population and GI tract of the host. It also indicates that region-specific host assembly rules which may be relevant to essential microbial functions that contribute to host physiology and possibly disease.
M1770 Interaction Between Sex Hormones/Sex Hormone Receptors With the Intestinal Epithelium Modulates Barrier Function and Subsequent Inflammation in Experimental IBD Benedetta Mattioli, Rekha R. Garg, Luca Pastorelli, Xiao Wang, Sharon B. Hoang, Massimo Campieri, Theresa T. Pizarro Background: It is well established that the incidence and severity of several autoimmune disorders is increased in the female population, including Crohn's disease (CD). Previously, we demonstrated that earlier onset and increased severity of disease occurs in F vs M SAMP1/ YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, and that epithelial barrier dysfunction precedes histological evidence of inflammation in this strain. As such, a critical factor likely contributing to SAMP ileitis is the interaction between sex hormones/sex hormone receptors and intestinal epithelial cells (IEC) that modulate downstream inflammatory/ immune responses. Aim: To characterize gender-specific epithelial barrier dysfunction in SAMP mice and to mechanistically evaluate the role of sex hormones and their receptors regulating chronic ileitis. Methods: In Vivo small intestinal permeability was evaluated in 34 wk old (pre-inflammation) F- and M-SAMP and age/gender-matched AKR (parental control) mice by fractional urine excretion of lactulose/mannitol 22h after gavage, and RT-PCR was performed on isolated IEC for tight junction (TJ) proteins. Ex vivo permeability was assessed on ileal tissues following stimulation with ER agonists by measuring transepithelial electrical resistance (TEER) after 2h in culture. Ileal inflammation was histologically evaluated using an established scoring system and epithelial permeability measured by TEER after sex hormone replacement on gonadectomized (GDX) 10 wk old SAMP with established disease. Results: Prior to the onset of inflammation, small intestinal permeability was increased in F- compared to M-SAMP, with no gender difference observed in AKR, which was significantly decreased compared to SAMP, confirming our previously published studies. A 2.2-fold increase (P<0.05) was present in the TJ protein, claudin-2 (on ChrX), in F- vs M-SAMP, which was significantly increased compared to both F- and M-AKR, with no difference between genders. Administration of estrogen (E2), but not 5a-DHT (end metabolite of testosterone), significantly reduced ileal inflammation in GDX M-SAMP (9.0±0.7 vs 17.5±0.5
M1768 Junctional Adhesion Molecule - A is a New Key Regulator in Colitis Associated Colon Cancer Stefania Vetrano, Emanuela Sala, Carmen Correale, Vincenzo Arena, Alberto Malesci, Elisabetta Dejana, Silvio Danese Junctional Adhesion Molecule-A (JAM-A) is a tight junction protein expressed by epithelial and endothelial cells. We have shown previously that JAM-A exerts a crucial role in controlling mucosal homeostasis by regulating the integrity and permeability of the intestinal epithelial barrier function. Emerging evidences have indicated an association between increased tight junction permeability of the colon epithelium and tumor development. The aim of this study was to investigate the involvement of JAM-A in colitis-associated cancer (CAC). JAMA expression was analyzed by confocal microscopy in human colon cancer and control tissues and in four human colon cancer cell lines with distinct metastatic potential: CaCo2, HT29, HCT116 and SW620. JAM-A -/- and WT C57BL/6 mice were treated with azoxymethane and three oral cycles of 1,5% dextran sodium sulphate (DSS) each characterized by 7 days DSS exposure followed by 14 days of water. Mice were monitored daily for weight loss, fecal blood and diarrhea and a disease activity index (DAI) was calculated. The damage
S-415
AGA Abstracts
AGA Abstracts
M1766