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Mo1744 Pregnancy-Specific Glycoprotein 1 (PSG1) Activates TGF-β and Prevents Dextran Sodium Sulfate (DSS)-Induced Colitis in Mice
Mo1745
AGA Abstracts
All-Trans-Retinoic Acid (ATRA) Counteracts Inhibition of DRA Function and Expression in Intestinal Inflammation Shubha Priyamvada, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Tarunmeet Gujral, Alip Borthakur, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja DRA (Down Regulated in Adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl- absorption in intestinal epithelial cells (IECs). Reduction in DRA function and expression has been implicated in diarrhea associated with inflammatory bowel diseases (IBD). Previous studies have shown a decrease in DRA expression by proinflammatory cytokines such as IL-1β and IFN-γ. Upregulation of DRA, therefore, appears to be a novel approach to treat IBD associated diarrhea. In this regard, ATRA, a key metabolite of vitamin A is known to have anti-inflammatory and immunomodulatory properties. We have earlier demonstrated that ATRA, increased DRA function, expression and promoter activity through RAR-β via the involvement of transcription factor HNF-1β. Whether, ATRA could modulate DRA function and expression under inflammatory conditions is not known. The objective of the current studies was to evaluate the efficacy of ATRA in attenuating the inhibitory effects of IFN-γ on DRA utilizing Caco-2 cells as an in vitro model and Dextran Sodium Sulfate (DSS)-induced colitis as an in vivo mouse model (3% DSS in drinking water for 7 days). Caco-2 cells grown on filter inserts were co-treated with IFN-γ (30 ng/ml) and ATRA for 24h. Results demonstrated that ATRA abrogated IFN-γ induced decrease in DRA function as measured by DIDS-sensitive 125I uptake. Parallel to the functional studies, IFN-γ-induced decrease in DRA mRNA (50%, p<0.05) and protein expression (40%, p<0.05) was markedly alleviated by ATRA co-treatment. Further, ATRA significantly blocked the inhibitory effects of IFN-γ on DRA promoter activity (Control, 100; ATRA, 262 ± 22; IFN-γ, 48 ± 1; ATRA+IFNγ; 154 ± 3, expressed as % of control, p<0.05). To evaluate if ATRA exerted these effects through modulation of IFN-γ induced signaling cascade, signal transducer and activator of transcription factor-1 (STAT-1) phosphorylation levels were analyzed. IFN-γ treatment induced the activation of STAT-1 at 6 h and this was sustained until 24h. ATRA alone had no effect on STAT-1 phosphorylation; however, ATRA co-treatment significantly diminished IFN-γ induced STAT-1 phosphorylation both at 6h and 24h time points. To complement the in vitro studies, effects of ATRA on DRA expression and inflammatory markers was analyzed in mice with DSS-induced colitis. ATRA (1 mg/kg body wt., i.p. for 7 days) partly blocked the reduced expression of DRA mRNA and protein levels in distal colon of DSStreated mice. Further, the enhanced expression of inflammatory cytokines IL-1β (~10 fold) and CXCl1 (~18 fold) induced by DSS was also alleviated by ATRA treatment. These data indicate that ATRA increases DRA function and expression under inflammatory conditions and this could be exploited as a novel therapeutic approach in management of IBD associated diarrhea (Supported by NIDDK and Dept. of Veteran Affairs).
Mo1743 CXCR3 Deficiency Enhances Intestinal Inflammation in Mice Geethanjali Pickert, Irmgard Tegeder, Nerea Ferreiròs, Detlef Schuppan Background and aims: The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are characterized by a chronic self-destructive inflammation of the gastrointestinal tract. In both UC and CD, leukocyte infiltration into the intestine is appears to precede clinical disease development. Chemokines and their receptors are central orchestrators of tissue-specific and cell type-selective trafficking of leukocytes. CXCR3 signaling is considered critical for the chemoattraction of activated Th1 cells, CTLs, NK cells, macrophages and DCs to inflammatory sites. Although CXCR3 and its ligands (CXCL9, CXCL10 and CXCL11) are highly expressed in IBD, the role of the CXCR3 axis in intestinal inflammation of IBD is insufficiently understood. Methods and Results: We studied the role of CXCR3 by using the CXCR3 deficient mice in dextran sodium sulphate (DSS)-induced colitis (2.5% DSS for 10 days in the drinking water). CXCR3-/- mice displayed significantly enhanced weight loss and increased epithelial erosions compared to their wildtype controls. CXCR3 deficiency dramatically increased the infiltration of polymorphonuclear leukocytes in the colonic mucosa, which was confirmed by vivo luciferase imaging of myeloperoxidase activity. FACSanalysis revealed an increased infiltration of CD11b+ F4/80+ inflammatory myeloid in the colonic mucosa and the intraepithelial compartment of CXCR3-/- mice. Furthermore, inflammation in CXCR3-/- mice was associated with elevated levels of proinflammatory lipid mediators like C16-ceramide, lysophosphatidic acid and prostaglandins in the colon and in the plasma. Conclusions: 1) The chemokine receptor und leukocyte chemoattractant CXCR3 plays an important role in inflammatory bowel disease. 2) Unexpectedly, deficiency of CXCR3 enhances intestinal inflammation in murine DSS-induced colitis. 3) The mechanisms of the immunoprotection by CXCR3 and its ligands are currently under investigation.
Mo1746 TNF Inhibits SLC26A3 Expression via Activation of NF-κB Pathway Anoop Kumar, Tarunmeet Gujral, Ravinder K. Gill, Hayley Coffing, Arivarasu Natarajan Anbazhagan, Alip Borthakur, Waddah A. Alrefai, Pradeep K. Dudeja SLC26A3 or DRA (Down Regulated in Adenoma) plays a major role in mediating Clabsorption in the mammalian intestine. Disturbances in DRA function and/or expression have been implicated in intestinal disorders such as infectious diarrhea and inflammation. while, a decrease in DRA expression is associated with intestinal inflammation; the direct effect of mediators that trigger intestinal inflammatory mediators on DRA has not been fully investigated. Our previous studies showed that IFN-γ decreases DRA expression via transcriptional mechanisms. In this regard, tumor necrosis factor (TNF) is also a central mediator of gut inflammation in IBD. Whether, TNF directly modulates DRA expression at the transcriptional level is not known. We hypothesized that TNF inhibits DRA expression in intestinal epithelial cells via NF-kB mediated pathway. To examine the effects of TNF, human intestinal HT-29 and LS174T cells were used as in vitro models. Promoter activity was measured by luciferase assays normalized to β-galactosidase activity. Protein and mRNA expression was measured by immunoblotting and real time PCR, respectively. Cellular localization of DRA and NF-κB subunit (p65) were detected by confocal microscopy. Our results showed that DRA protein expression was significantly decreased in response to treatment of HT-29 cells with 20 and 50 ng/ml TNF for 24h (~ 50-60%, p<0.05). Consistent with DRA protein levels, TNF (50 ng/ml) decreased DRA mRNA levels at 6-24h (60-70%, p<0.05). These effects occurred at the transcriptional level as TNF decreased DRA promoter activity (-1183/+114bp) in HT-29 cells (~60%, p<0.05) at 6,12 & 24h. A similar decrease in DRA mRNA expression in response to TNF was also observed in LS174T cells indicating that the effects are not cell line specific. In addition, TNF treatment did not alter the mRNA levels of another apical anion exchanger, SLC26A6 (PAT-1) in LS174T cells indicating that the observed effects were specific to DRA. Elucidation of molecular mechanisms underlying TNF effects on DRA showed direct involvement of NF-κB in decreasing DRA expression as: i) the decrease in DRA mRNA expression by TNF was blocked in the presence of specific NF-κB inhibitor CAPE (50 μM); ii) TNF increased nuclear localization of NF-κB subunit p65 in HT-29 cells (~2.5 fold, p<0.05) and p50 (~3 fold, p<0.05); iii) TNF increased reporter activity of NF-κB in intestinal epithelial cells (~3-4 fold, p<0.05); iv) Activation of NF-κB pathway achieved by siRNA knock down of IKB-α increased nuclear translocation of p65 and resulted in decreased DRA mRNA and protein expression as assessed by qRT-PCR and confocal microscopy, respectively. Conclusion: These results demonstrate that TNF-mediated activation of NF-κB directly suppresses DRA transcription in IBD and may contribute to associated diarrhea (Supported by NIDDK and Dept. of Veterans Affairs).
Mo1744 Pregnancy-Specific Glycoprotein 1 (PSG1) Activates TGF-β and Prevents Dextran Sodium Sulfate (DSS)-Induced Colitis in Mice Sandra M. Blois, Ivan J. Fuss, Warren Strober, Gabriela Dveksler Background: Transforming growth factor-βs (TGF-βs) are secreted from cells as latent complexes and the activity of TGF-βs is controlled predominantly through activation of these complexes. Tolerance to the fetal allograft is essential for pregnancy success; TGF-β1 and TGF-β2 play important roles in regulating these processes. Pregnancy-specific β-glycoproteins (PSGs) are present in the maternal circulation at a high concentration throughout pregnancy and have been proposed to have anti-inflammatory functions. Methods: ELISA determined PSG1 from human maternal circulation. Recombinant PSG1 was generated in Mouse Embryonic Fibroblast TGF-β1-null cells which served for detecting the presence of active TGF-β To test whether PSG1 could activate latent TGF-β1, a stable TGF-β reporter cell line by transducing HEK-293T cells with lentivirus particles containing the firefly luciferase gene under a CMV promoter. DSS colitis was generated by the administration of 3.5 % DSS to C57BL/6 mice with or without concomitant treatment with r-PSG1. Mice were sacrificed on Day 10 and LPMC examined for cytokine secretion and FoxP3 regulatory T cell formation. Results: We found that PSG1 is secreted in increased concentrations by the syncytiotrophoblast layer of the placenta to the maternal circulation and is most closely related to the carcinoembryonic (CEA) gene family Ig superfamily. PSG1 is associated with LAP and active TGF-β1 (and TGF-β2) within the circulation. PSG1 can activate latent TGFβ and its induction can be blocked with Anti-TGF-β or ALK5 inhibitor. Recombinant and native PSG1 activated TGF-β1 and TGF-β2 in vitro. Consistent with these findings, administration of PSG1 protected mice from dextran sodium sulfate (DSS)-induced colitis, reduced the secretion of pro-inflammatory cytokines, and increased the number of mucosal FoxP3 T regulatory cells. The PSG1-mediated protection was greatly inhibited by the coadministration of neutralizing anti-TGF-β antibody. Conclusion: Our results indicate that proteins secreted by the placenta directly contribute to the generation of active TGF-β and formation of FoxP3 T regulatory cells and plays a significant role in mucosal homeostasis.
AGA Abstracts
S-650
AGA Abstracts
All-Trans-Retinoic Acid (ATRA) Counteracts Inhibition of DRA Function and Expression in Intestinal Inflammation Shubha Priyamvada, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Tarunmeet Gujral, Alip Borthakur, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja DRA (Down Regulated in Adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl- absorption in intestinal epithelial cells (IECs). Reduction in DRA function and expression has been implicated in diarrhea associated with inflammatory bowel diseases (IBD). Previous studies have shown a decrease in DRA expression by proinflammatory cytokines such as IL-1β and IFN-γ. Upregulation of DRA, therefore, appears to be a novel approach to treat IBD associated diarrhea. In this regard, ATRA, a key metabolite of vitamin A is known to have anti-inflammatory and immunomodulatory properties. We have earlier demonstrated that ATRA, increased DRA function, expression and promoter activity through RAR-β via the involvement of transcription factor HNF-1β. Whether, ATRA could modulate DRA function and expression under inflammatory conditions is not known. The objective of the current studies was to evaluate the efficacy of ATRA in attenuating the inhibitory effects of IFN-γ on DRA utilizing Caco-2 cells as an in vitro model and Dextran Sodium Sulfate (DSS)-induced colitis as an in vivo mouse model (3% DSS in drinking water for 7 days). Caco-2 cells grown on filter inserts were co-treated with IFN-γ (30 ng/ml) and ATRA for 24h. Results demonstrated that ATRA abrogated IFN-γ induced decrease in DRA function as measured by DIDS-sensitive 125I uptake. Parallel to the functional studies, IFN-γ-induced decrease in DRA mRNA (50%, p<0.05) and protein expression (40%, p<0.05) was markedly alleviated by ATRA co-treatment. Further, ATRA significantly blocked the inhibitory effects of IFN-γ on DRA promoter activity (Control, 100; ATRA, 262 ± 22; IFN-γ, 48 ± 1; ATRA+IFNγ; 154 ± 3, expressed as % of control, p<0.05). To evaluate if ATRA exerted these effects through modulation of IFN-γ induced signaling cascade, signal transducer and activator of transcription factor-1 (STAT-1) phosphorylation levels were analyzed. IFN-γ treatment induced the activation of STAT-1 at 6 h and this was sustained until 24h. ATRA alone had no effect on STAT-1 phosphorylation; however, ATRA co-treatment significantly diminished IFN-γ induced STAT-1 phosphorylation both at 6h and 24h time points. To complement the in vitro studies, effects of ATRA on DRA expression and inflammatory markers was analyzed in mice with DSS-induced colitis. ATRA (1 mg/kg body wt., i.p. for 7 days) partly blocked the reduced expression of DRA mRNA and protein levels in distal colon of DSStreated mice. Further, the enhanced expression of inflammatory cytokines IL-1β (~10 fold) and CXCl1 (~18 fold) induced by DSS was also alleviated by ATRA treatment. These data indicate that ATRA increases DRA function and expression under inflammatory conditions and this could be exploited as a novel therapeutic approach in management of IBD associated diarrhea (Supported by NIDDK and Dept. of Veteran Affairs).
Mo1743 CXCR3 Deficiency Enhances Intestinal Inflammation in Mice Geethanjali Pickert, Irmgard Tegeder, Nerea Ferreiròs, Detlef Schuppan Background and aims: The inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis are characterized by a chronic self-destructive inflammation of the gastrointestinal tract. In both UC and CD, leukocyte infiltration into the intestine is appears to precede clinical disease development. Chemokines and their receptors are central orchestrators of tissue-specific and cell type-selective trafficking of leukocytes. CXCR3 signaling is considered critical for the chemoattraction of activated Th1 cells, CTLs, NK cells, macrophages and DCs to inflammatory sites. Although CXCR3 and its ligands (CXCL9, CXCL10 and CXCL11) are highly expressed in IBD, the role of the CXCR3 axis in intestinal inflammation of IBD is insufficiently understood. Methods and Results: We studied the role of CXCR3 by using the CXCR3 deficient mice in dextran sodium sulphate (DSS)-induced colitis (2.5% DSS for 10 days in the drinking water). CXCR3-/- mice displayed significantly enhanced weight loss and increased epithelial erosions compared to their wildtype controls. CXCR3 deficiency dramatically increased the infiltration of polymorphonuclear leukocytes in the colonic mucosa, which was confirmed by vivo luciferase imaging of myeloperoxidase activity. FACSanalysis revealed an increased infiltration of CD11b+ F4/80+ inflammatory myeloid in the colonic mucosa and the intraepithelial compartment of CXCR3-/- mice. Furthermore, inflammation in CXCR3-/- mice was associated with elevated levels of proinflammatory lipid mediators like C16-ceramide, lysophosphatidic acid and prostaglandins in the colon and in the plasma. Conclusions: 1) The chemokine receptor und leukocyte chemoattractant CXCR3 plays an important role in inflammatory bowel disease. 2) Unexpectedly, deficiency of CXCR3 enhances intestinal inflammation in murine DSS-induced colitis. 3) The mechanisms of the immunoprotection by CXCR3 and its ligands are currently under investigation.
Mo1746 TNF Inhibits SLC26A3 Expression via Activation of NF-κB Pathway Anoop Kumar, Tarunmeet Gujral, Ravinder K. Gill, Hayley Coffing, Arivarasu Natarajan Anbazhagan, Alip Borthakur, Waddah A. Alrefai, Pradeep K. Dudeja SLC26A3 or DRA (Down Regulated in Adenoma) plays a major role in mediating Clabsorption in the mammalian intestine. Disturbances in DRA function and/or expression have been implicated in intestinal disorders such as infectious diarrhea and inflammation. while, a decrease in DRA expression is associated with intestinal inflammation; the direct effect of mediators that trigger intestinal inflammatory mediators on DRA has not been fully investigated. Our previous studies showed that IFN-γ decreases DRA expression via transcriptional mechanisms. In this regard, tumor necrosis factor (TNF) is also a central mediator of gut inflammation in IBD. Whether, TNF directly modulates DRA expression at the transcriptional level is not known. We hypothesized that TNF inhibits DRA expression in intestinal epithelial cells via NF-kB mediated pathway. To examine the effects of TNF, human intestinal HT-29 and LS174T cells were used as in vitro models. Promoter activity was measured by luciferase assays normalized to β-galactosidase activity. Protein and mRNA expression was measured by immunoblotting and real time PCR, respectively. Cellular localization of DRA and NF-κB subunit (p65) were detected by confocal microscopy. Our results showed that DRA protein expression was significantly decreased in response to treatment of HT-29 cells with 20 and 50 ng/ml TNF for 24h (~ 50-60%, p<0.05). Consistent with DRA protein levels, TNF (50 ng/ml) decreased DRA mRNA levels at 6-24h (60-70%, p<0.05). These effects occurred at the transcriptional level as TNF decreased DRA promoter activity (-1183/+114bp) in HT-29 cells (~60%, p<0.05) at 6,12 & 24h. A similar decrease in DRA mRNA expression in response to TNF was also observed in LS174T cells indicating that the effects are not cell line specific. In addition, TNF treatment did not alter the mRNA levels of another apical anion exchanger, SLC26A6 (PAT-1) in LS174T cells indicating that the observed effects were specific to DRA. Elucidation of molecular mechanisms underlying TNF effects on DRA showed direct involvement of NF-κB in decreasing DRA expression as: i) the decrease in DRA mRNA expression by TNF was blocked in the presence of specific NF-κB inhibitor CAPE (50 μM); ii) TNF increased nuclear localization of NF-κB subunit p65 in HT-29 cells (~2.5 fold, p<0.05) and p50 (~3 fold, p<0.05); iii) TNF increased reporter activity of NF-κB in intestinal epithelial cells (~3-4 fold, p<0.05); iv) Activation of NF-κB pathway achieved by siRNA knock down of IKB-α increased nuclear translocation of p65 and resulted in decreased DRA mRNA and protein expression as assessed by qRT-PCR and confocal microscopy, respectively. Conclusion: These results demonstrate that TNF-mediated activation of NF-κB directly suppresses DRA transcription in IBD and may contribute to associated diarrhea (Supported by NIDDK and Dept. of Veterans Affairs).
Mo1744 Pregnancy-Specific Glycoprotein 1 (PSG1) Activates TGF-β and Prevents Dextran Sodium Sulfate (DSS)-Induced Colitis in Mice Sandra M. Blois, Ivan J. Fuss, Warren Strober, Gabriela Dveksler Background: Transforming growth factor-βs (TGF-βs) are secreted from cells as latent complexes and the activity of TGF-βs is controlled predominantly through activation of these complexes. Tolerance to the fetal allograft is essential for pregnancy success; TGF-β1 and TGF-β2 play important roles in regulating these processes. Pregnancy-specific β-glycoproteins (PSGs) are present in the maternal circulation at a high concentration throughout pregnancy and have been proposed to have anti-inflammatory functions. Methods: ELISA determined PSG1 from human maternal circulation. Recombinant PSG1 was generated in Mouse Embryonic Fibroblast TGF-β1-null cells which served for detecting the presence of active TGF-β To test whether PSG1 could activate latent TGF-β1, a stable TGF-β reporter cell line by transducing HEK-293T cells with lentivirus particles containing the firefly luciferase gene under a CMV promoter. DSS colitis was generated by the administration of 3.5 % DSS to C57BL/6 mice with or without concomitant treatment with r-PSG1. Mice were sacrificed on Day 10 and LPMC examined for cytokine secretion and FoxP3 regulatory T cell formation. Results: We found that PSG1 is secreted in increased concentrations by the syncytiotrophoblast layer of the placenta to the maternal circulation and is most closely related to the carcinoembryonic (CEA) gene family Ig superfamily. PSG1 is associated with LAP and active TGF-β1 (and TGF-β2) within the circulation. PSG1 can activate latent TGFβ and its induction can be blocked with Anti-TGF-β or ALK5 inhibitor. Recombinant and native PSG1 activated TGF-β1 and TGF-β2 in vitro. Consistent with these findings, administration of PSG1 protected mice from dextran sodium sulfate (DSS)-induced colitis, reduced the secretion of pro-inflammatory cytokines, and increased the number of mucosal FoxP3 T regulatory cells. The PSG1-mediated protection was greatly inhibited by the coadministration of neutralizing anti-TGF-β antibody. Conclusion: Our results indicate that proteins secreted by the placenta directly contribute to the generation of active TGF-β and formation of FoxP3 T regulatory cells and plays a significant role in mucosal homeostasis.
AGA Abstracts
S-650