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S1731 Tolerance of Toll-Like Receptor 4 Mediated Signaling Pathway Protects Mice from Experimental Colitis
was taken for assessment of gross inflammatory scores, histological scores and myeloperoxidase (MPO) assay. Serum was obtained for multiple cytokine assessment using a Luminex assay. Results: Both WT and Tollip-/- mice developed colitis following TNBS administration. Utilization of the agarose gel promoted TNBS retention and minimized variability within treatment groups. WT mice exhibited more severe weight loss compared to Tollip-/- animals (15.1 vs. 11.7% weight loss by day 2, p < 0.01). More severe gross scores were noted in the WT group (Mean ± SEM: Day 3 - 8.1 ± 0.4 vs 6.2 ± 0.4 , p < 0.05; Day 7 - 9.1 ± 0.7 vs 5.7 ± 0.6, p < 0.001) with increased MPO activity in the colon as well (29.4 ± 4.6 vs. 19.7 ± 2.4 MPO units/mg tissue on day 7, p < 0.05). Serum levels of IL-2 increased in WT mice on day 7 compared to Tollip-/- animals (20.9 ± 6.3 vs 5.5 ± 1.9 pg/ml); serum concentrations of IL-1β on day 7 remained elevated in WT mice as well (20.0 ± 7.3 vs. 3.1 ± 1.6 pg/ml, p < 0.05). Conclusion: Tollip-/- mice show diminished clinical severity and cytokine production compared to WT animals during TNBS colitis. This highlights a unique role for Tollip in the early events during intestinal inflammatory responses. Ultimately, a better understanding of the role Tollip plays in the intestinal tract may provide insight into the pathogenesis of inflammatory bowel disease in susceptible individuals.
Tolerance of Toll-Like Receptor 4 Mediated Signaling Pathway Protects Mice from Experimental Colitis Keietsu Saito, Kyoko Katakura, Kaori Kanno, Ryoma Suzuki, Hiromasa Ohira Background:The basis of inflammatory bowel disease (IBD) is multi-factorial involving susceptibility genes, and immune and environmental factors. There has been a rapid increase in the prevalence of IBD in industrialized countries. The hygiene hypothesis proposes that the present clean surroundings in less exposure to bacteria are involved in development of this disease. Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), has a phenomenon termed LPS tolerance such as an initial exposure to LPS induce a transient state of hyporesponsiveness to a subsequent challenge with LPS. Based on In Vitro studies, the mechanisms of this phenomenon are gradually becoming clear, although that has not been formally proven in inflammatory disease models In Vivo. Therefore we hypothesized that repeated LPS administration could protect colonic inflammation of mouse experimental colitis model. Methods:Murine colitis was induced to Balb/c mice by oral administration of 7% dextran sulphate sodium (DSS) with or without daily intraperitoneal administration of LPS. Colitis was evaluated by a macroscopic disease activity index, myeloperoxidase (MPO) activity in colon and histological score. Cytokine mRNA expressions in colon were also measured by RT-PCR. To confirm the phenomenon of LPS tolerance, we generated mouse conventional bone marrow derived dendritic cells (BMDC), and these were preincubated with or without LPS and restimulated with LPS after 24 hours from first stimulation. Cytokine productions in the culture supernatant were measured by ELISA, and mRNA expressions of the cells were evaluated by RT-PCR. Furthermore, we investigated negative regulators of LPS tolerance and expression of them in model of experimental colitis. Results:Administration of LPS significantly suppressed colonic inflammation of DSS-induced colitis, although there were no difference in mRNA expression of IL-12p40, IL-6, TNF-α, IL-10, and TLR4. TLR4 in BMDC was activated with LPS and induced TNF-α production. After second stimulation with LPS, TNF-α and IL-6 production were reduced which followed previous reports. IRAKM mRNA expression, which had participation in LPS tolerance, was upregulated in the LPS treated BMDC and mice colon in comparison to the control mice. Conclusion:LPS tolerance could protect mice from DSS-induced colitis, and IRAK-M had participation in LPS tolerance of TLR4 signaling pathway. Taken together, these observations suggested that loss of exposure to LPS may be one of the pathogenesis of IBD.
S1734 Apolipoprotein C3 Is a Novel Binding Partner of CD1D and Regulates CD1DRestricted Immunopathologies Arthur Kaser, Zhangguo Chen, T Jake Liang, Hyun S. Kim, David E. Cohen, Richard S. Blumberg Introduction: Lipid antigens recognized by the MHC class I homolog CD1d regulate a wide array of immune functions that range from hepatitis to inflammatory bowel disease by virtue of CD1d-restricted natural killer T (NKT) cells. We posited that extracellular binding partners might regulate CD1d function. Methods: The extracellular domain of human CD1d was used as bait in a yeast two-hybrid experiment with a liver cDNA library as prey. Immunoprecipitation was used to biochemically confirm the interaction, and a gene-targeted knock-out mouse was used to functionally explore the interaction between CD1d and its novel binding partner. Results: Final screening of initially 2x10(6) colonies revealed a sequence homologous to apolipoprotein C3 (apoC3) cDNA. Binding was confirmed by immunoprecipitation of human plasma-purified apoC3 with an extracellular CD1d expressed as a GST fusion protein. ApoC3 is an abundant plasma protein present in triglyceride-rich plasma lipoproteins (VLDL, chylomicrons and HDL). Upon injection of the CD1d-presented model glycolipid α-galactosyl-ceramide (aGalCer), apoC3-/- mice exhibited increased intracellular IFNγ and IL-4 expression in CD1d-restricted invariant NKT cells. ApoC3-/- mice were more susceptible to aGalCer hepatitis, a CD1d-restricted immunopathology. This was accompanied by increased IFNγ and IL-4 serum levels. ApoC3-/- mice also exhibited increased ear swelling in the oxazolone delayed type hypersensitivity (DTH) model, which is dependent on CD1d function. Conclusions: We identified apoC3 as a binding partner of the extracellular CD1d domain, and show that apoC3 deficiency results in increased CD1d immune function, including enhanced immunopathologies in two independent CD1d-restricted mouse models. Hence, our data suggest that the apoC3 - CD1d interaction might represent an unexpected interaction between metabolism and the immune system.
S1732 Elafin Is Protective Against the Development of Colitis in IBD Murine Models Jean-Michel Sallenave, Camila S. Dale, Viviane Balloy, Michel Chignard, Nathalie Vergnolle Elafin is a mucosal endogenous inhibitor of serine proteases, with a dual activity of inhibiting neutrophil-derived proteases and killing bacterial pathogens. While Elafin is not expressed in mice, its expression is increased in tissues from Crohn's disease and Ulcerative Colitis patients, compared to control patients, suggesting that elafin may play a role in the course of those diseases. Since proteases have been shown to have deleterious pro-inflammatory properties in the gut, a shifted balance between proteases and their inhibitors may play a central role in the development of chronic intestinal inflammation. We made the hypothesis that elafin could be protective against the development of colitis. METHODS: We have generated transgenic mice (eTg) that express elafin either under the cytomegalovirus (CMV) or the endothelin (ENDO) promoter (both on a C57Bl6 background), and we have compared the inflammatory response of wild-type (non-expressors) and eTg mice in two models of colitis. The latter was induced by adding dextran sodium sulfate (DSS: 3 %) to the drinking water of mice or by intrarectal administration of trinitrobenzene sulfuric acid (TNBS: 2 mg in 50 % Ethanol). Macroscopic damage scores, myeloperoxydase (MPO) activity, trypsinlike activity, and elastase activity were measured in tissues or lumenal washes of colons of mice 7 days after the induction of colitis. RESULTS: Macroscopic damage scores and increased MPO activity observed after DSS colitis were significantly reduced in eTg-CMV mice, but only macroscopic damage score was reduced in eTg-ENDO mice compared to wild-types. Elastase and trypsin-like activities were significantly increased by DSS colitis in wild-type mice both in tissues and lumenal washes, and this increase was abolished in the two eTg strains. After TNBS colitis, all inflammatory signs, trypsin-like and elastase activities were also significantly reduced in both eTg strains compared to wild-types. CONCLUSION: Expression of elafin protects mice against the development of colitis. Proteolytic activity (elastase and trypsin-like) follows the pattern of inflammatory parameters, further suggesting that the balance between proteases and anti-protease activity might be critical in the development of chronic intestinal inflammatory diseases. Elafin could be viewed as a potential mediator for the treatment of inflammatory bowel diseases.
S1735 Oral Administration of Amniotic Fluid Reduces Necrotizing Enterocolitis in Preterm Pigs Jayda L. Siggers, Richard H. Siggers, Kerstin Skovgaard, Mette Schmidt, Hanne K. Moeller, Mette Boye, Per T. Sangild Preterm neonates are susceptible to gastrointestinal (GIT) disorders such as necrotizing enterocolitis (NEC). Maternal milk protects against NEC via growth promoting, immunomodulatory and antimicrobial factors, but often maternal milk is limited following preterm birth. Amniotic fluid (AF), the fetal enteral “diet”, contains similar bioactive components, but it remains unknown if AF is beneficial for the immature intestine after preterm birth. Our preliminary studies in murine dendritic cells showed that porcine AF decreased inflammatory cytokine (IL-6, IL-12 and TNF) release in response to bacterial stimuli, suggesting an immunomodulatory function. This study aimed to determine the impact of AF on inflammation and function of the preterm intestine using a pig model of NEC. Thirty preterm pigs (92% gestation) were delivered via caesarean section and maintained on total parental nutrition (TPN) for 48 h and then fed colostrum (COLOS, n=7), formula (FORM, n=13) or formula+porcine AF (AF, n=10). AF pigs were fed amniotic fluid (10-12 mL/kg/3h) throughout the TPN and enteral feeding periods. The GIT was evaluated for NEC lesions and scores ≥ 2 (range: 0-4) in any GIT region was defined as NEC. Fifty-four percent (7/ 13) of FORM pigs developed NEC compared with 10% (1/10) of AF and 0% (0/10) of COLOS pigs (both P<0.05). There was a significant increase in brush border disaccharidase and amninopeptidase (Ap) activities in the COLOS pigs, compared with FORM and AF pigs (lactase: +42-44%, maltase: +59-63%, DPPIV: +13-25%, ApN: +25-28%, all P<0.05). Mean villus height did not differ across treatment groups. AF pigs had significantly higher weight gain (+48-76%), lower kidney (-22-29%) and spleen weights (-26-27%), compared with COLOS and FORM pigs (all P<0.05). Microarray analysis targeting over 200 immune related genes was performed on distal intestinal tissue. Twenty-six immune genes were differentially regulated among the three groups. Only nine differentially expressed genes (5 up, 4 down) were detected between AF and COLOS pigs, while 25 differentially expressed genes (10 up, 15 down) were detected between AF and FORM pigs. Thus, AF and COLOS tissues appeared more immunologically similar than AF and FORM tissues. Many of the differentially regulated genes were identified as being involved in the innate immune response (e.g. IgM, IL-1α, mucin 5AC, NFκB, prepro-β-defensin, TNF, CCR5, lysozyme). In conclusion, amniotic fluid administration immediately after preterm birth modulates the gut innate immune system and may reduce the incidence of NEC.
S1733 Toll-Interacting Protein (Tollip) Increases the Severity of Acute Colitis Christopher Waterhouse, Donna-Marie McCafferty, Paul Kubes Background: The normal bacterial flora plays an important role for intestinal homeostasis and healing following injury. In predisposed animal and human hosts, however, dysregulated responses to intestinal bacteria lead to inflammatory bowel disease (IBD). Recognition of bacteria within the intestine is mediated, in part, through Toll-like receptors (TLRs). Tollinteracting protein (Tollip) is an intracellular protein known to modulate signaling via members of the interleukin (IL)-1 receptor superfamily including several TLRs. Most evidence indicates that Tollip acts as an inhibitor of myeloid differentiation primary response gene (MyD)88-dependent signaling following receptor activation. Signaling through MyD88 has been shown by others to be protective during experimental colitis. To date, no role for Tollip has been established in the intestinal inflammatory response In Vivo. We hypothesized that Tollip deficiency would result in less severe inflammatory responses during acute colitis in mice. Experimental design: Wild type (WT) or Tollip deficient (-/-) mice 8-12 weeks of age were treated with 2 mg trinitrobenzene sulfonic acid (TNBS) in 30% ethanol and 0.2% agarose gel per rectum for induction of colitis and followed for 7 days. Colonic tissue
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AGA Abstracts
AGA Abstracts
S1731
Tolerance of Toll-Like Receptor 4 Mediated Signaling Pathway Protects Mice from Experimental Colitis Keietsu Saito, Kyoko Katakura, Kaori Kanno, Ryoma Suzuki, Hiromasa Ohira Background:The basis of inflammatory bowel disease (IBD) is multi-factorial involving susceptibility genes, and immune and environmental factors. There has been a rapid increase in the prevalence of IBD in industrialized countries. The hygiene hypothesis proposes that the present clean surroundings in less exposure to bacteria are involved in development of this disease. Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), has a phenomenon termed LPS tolerance such as an initial exposure to LPS induce a transient state of hyporesponsiveness to a subsequent challenge with LPS. Based on In Vitro studies, the mechanisms of this phenomenon are gradually becoming clear, although that has not been formally proven in inflammatory disease models In Vivo. Therefore we hypothesized that repeated LPS administration could protect colonic inflammation of mouse experimental colitis model. Methods:Murine colitis was induced to Balb/c mice by oral administration of 7% dextran sulphate sodium (DSS) with or without daily intraperitoneal administration of LPS. Colitis was evaluated by a macroscopic disease activity index, myeloperoxidase (MPO) activity in colon and histological score. Cytokine mRNA expressions in colon were also measured by RT-PCR. To confirm the phenomenon of LPS tolerance, we generated mouse conventional bone marrow derived dendritic cells (BMDC), and these were preincubated with or without LPS and restimulated with LPS after 24 hours from first stimulation. Cytokine productions in the culture supernatant were measured by ELISA, and mRNA expressions of the cells were evaluated by RT-PCR. Furthermore, we investigated negative regulators of LPS tolerance and expression of them in model of experimental colitis. Results:Administration of LPS significantly suppressed colonic inflammation of DSS-induced colitis, although there were no difference in mRNA expression of IL-12p40, IL-6, TNF-α, IL-10, and TLR4. TLR4 in BMDC was activated with LPS and induced TNF-α production. After second stimulation with LPS, TNF-α and IL-6 production were reduced which followed previous reports. IRAKM mRNA expression, which had participation in LPS tolerance, was upregulated in the LPS treated BMDC and mice colon in comparison to the control mice. Conclusion:LPS tolerance could protect mice from DSS-induced colitis, and IRAK-M had participation in LPS tolerance of TLR4 signaling pathway. Taken together, these observations suggested that loss of exposure to LPS may be one of the pathogenesis of IBD.
S1734 Apolipoprotein C3 Is a Novel Binding Partner of CD1D and Regulates CD1DRestricted Immunopathologies Arthur Kaser, Zhangguo Chen, T Jake Liang, Hyun S. Kim, David E. Cohen, Richard S. Blumberg Introduction: Lipid antigens recognized by the MHC class I homolog CD1d regulate a wide array of immune functions that range from hepatitis to inflammatory bowel disease by virtue of CD1d-restricted natural killer T (NKT) cells. We posited that extracellular binding partners might regulate CD1d function. Methods: The extracellular domain of human CD1d was used as bait in a yeast two-hybrid experiment with a liver cDNA library as prey. Immunoprecipitation was used to biochemically confirm the interaction, and a gene-targeted knock-out mouse was used to functionally explore the interaction between CD1d and its novel binding partner. Results: Final screening of initially 2x10(6) colonies revealed a sequence homologous to apolipoprotein C3 (apoC3) cDNA. Binding was confirmed by immunoprecipitation of human plasma-purified apoC3 with an extracellular CD1d expressed as a GST fusion protein. ApoC3 is an abundant plasma protein present in triglyceride-rich plasma lipoproteins (VLDL, chylomicrons and HDL). Upon injection of the CD1d-presented model glycolipid α-galactosyl-ceramide (aGalCer), apoC3-/- mice exhibited increased intracellular IFNγ and IL-4 expression in CD1d-restricted invariant NKT cells. ApoC3-/- mice were more susceptible to aGalCer hepatitis, a CD1d-restricted immunopathology. This was accompanied by increased IFNγ and IL-4 serum levels. ApoC3-/- mice also exhibited increased ear swelling in the oxazolone delayed type hypersensitivity (DTH) model, which is dependent on CD1d function. Conclusions: We identified apoC3 as a binding partner of the extracellular CD1d domain, and show that apoC3 deficiency results in increased CD1d immune function, including enhanced immunopathologies in two independent CD1d-restricted mouse models. Hence, our data suggest that the apoC3 - CD1d interaction might represent an unexpected interaction between metabolism and the immune system.
S1732 Elafin Is Protective Against the Development of Colitis in IBD Murine Models Jean-Michel Sallenave, Camila S. Dale, Viviane Balloy, Michel Chignard, Nathalie Vergnolle Elafin is a mucosal endogenous inhibitor of serine proteases, with a dual activity of inhibiting neutrophil-derived proteases and killing bacterial pathogens. While Elafin is not expressed in mice, its expression is increased in tissues from Crohn's disease and Ulcerative Colitis patients, compared to control patients, suggesting that elafin may play a role in the course of those diseases. Since proteases have been shown to have deleterious pro-inflammatory properties in the gut, a shifted balance between proteases and their inhibitors may play a central role in the development of chronic intestinal inflammation. We made the hypothesis that elafin could be protective against the development of colitis. METHODS: We have generated transgenic mice (eTg) that express elafin either under the cytomegalovirus (CMV) or the endothelin (ENDO) promoter (both on a C57Bl6 background), and we have compared the inflammatory response of wild-type (non-expressors) and eTg mice in two models of colitis. The latter was induced by adding dextran sodium sulfate (DSS: 3 %) to the drinking water of mice or by intrarectal administration of trinitrobenzene sulfuric acid (TNBS: 2 mg in 50 % Ethanol). Macroscopic damage scores, myeloperoxydase (MPO) activity, trypsinlike activity, and elastase activity were measured in tissues or lumenal washes of colons of mice 7 days after the induction of colitis. RESULTS: Macroscopic damage scores and increased MPO activity observed after DSS colitis were significantly reduced in eTg-CMV mice, but only macroscopic damage score was reduced in eTg-ENDO mice compared to wild-types. Elastase and trypsin-like activities were significantly increased by DSS colitis in wild-type mice both in tissues and lumenal washes, and this increase was abolished in the two eTg strains. After TNBS colitis, all inflammatory signs, trypsin-like and elastase activities were also significantly reduced in both eTg strains compared to wild-types. CONCLUSION: Expression of elafin protects mice against the development of colitis. Proteolytic activity (elastase and trypsin-like) follows the pattern of inflammatory parameters, further suggesting that the balance between proteases and anti-protease activity might be critical in the development of chronic intestinal inflammatory diseases. Elafin could be viewed as a potential mediator for the treatment of inflammatory bowel diseases.
S1735 Oral Administration of Amniotic Fluid Reduces Necrotizing Enterocolitis in Preterm Pigs Jayda L. Siggers, Richard H. Siggers, Kerstin Skovgaard, Mette Schmidt, Hanne K. Moeller, Mette Boye, Per T. Sangild Preterm neonates are susceptible to gastrointestinal (GIT) disorders such as necrotizing enterocolitis (NEC). Maternal milk protects against NEC via growth promoting, immunomodulatory and antimicrobial factors, but often maternal milk is limited following preterm birth. Amniotic fluid (AF), the fetal enteral “diet”, contains similar bioactive components, but it remains unknown if AF is beneficial for the immature intestine after preterm birth. Our preliminary studies in murine dendritic cells showed that porcine AF decreased inflammatory cytokine (IL-6, IL-12 and TNF) release in response to bacterial stimuli, suggesting an immunomodulatory function. This study aimed to determine the impact of AF on inflammation and function of the preterm intestine using a pig model of NEC. Thirty preterm pigs (92% gestation) were delivered via caesarean section and maintained on total parental nutrition (TPN) for 48 h and then fed colostrum (COLOS, n=7), formula (FORM, n=13) or formula+porcine AF (AF, n=10). AF pigs were fed amniotic fluid (10-12 mL/kg/3h) throughout the TPN and enteral feeding periods. The GIT was evaluated for NEC lesions and scores ≥ 2 (range: 0-4) in any GIT region was defined as NEC. Fifty-four percent (7/ 13) of FORM pigs developed NEC compared with 10% (1/10) of AF and 0% (0/10) of COLOS pigs (both P<0.05). There was a significant increase in brush border disaccharidase and amninopeptidase (Ap) activities in the COLOS pigs, compared with FORM and AF pigs (lactase: +42-44%, maltase: +59-63%, DPPIV: +13-25%, ApN: +25-28%, all P<0.05). Mean villus height did not differ across treatment groups. AF pigs had significantly higher weight gain (+48-76%), lower kidney (-22-29%) and spleen weights (-26-27%), compared with COLOS and FORM pigs (all P<0.05). Microarray analysis targeting over 200 immune related genes was performed on distal intestinal tissue. Twenty-six immune genes were differentially regulated among the three groups. Only nine differentially expressed genes (5 up, 4 down) were detected between AF and COLOS pigs, while 25 differentially expressed genes (10 up, 15 down) were detected between AF and FORM pigs. Thus, AF and COLOS tissues appeared more immunologically similar than AF and FORM tissues. Many of the differentially regulated genes were identified as being involved in the innate immune response (e.g. IgM, IL-1α, mucin 5AC, NFκB, prepro-β-defensin, TNF, CCR5, lysozyme). In conclusion, amniotic fluid administration immediately after preterm birth modulates the gut innate immune system and may reduce the incidence of NEC.
S1733 Toll-Interacting Protein (Tollip) Increases the Severity of Acute Colitis Christopher Waterhouse, Donna-Marie McCafferty, Paul Kubes Background: The normal bacterial flora plays an important role for intestinal homeostasis and healing following injury. In predisposed animal and human hosts, however, dysregulated responses to intestinal bacteria lead to inflammatory bowel disease (IBD). Recognition of bacteria within the intestine is mediated, in part, through Toll-like receptors (TLRs). Tollinteracting protein (Tollip) is an intracellular protein known to modulate signaling via members of the interleukin (IL)-1 receptor superfamily including several TLRs. Most evidence indicates that Tollip acts as an inhibitor of myeloid differentiation primary response gene (MyD)88-dependent signaling following receptor activation. Signaling through MyD88 has been shown by others to be protective during experimental colitis. To date, no role for Tollip has been established in the intestinal inflammatory response In Vivo. We hypothesized that Tollip deficiency would result in less severe inflammatory responses during acute colitis in mice. Experimental design: Wild type (WT) or Tollip deficient (-/-) mice 8-12 weeks of age were treated with 2 mg trinitrobenzene sulfonic acid (TNBS) in 30% ethanol and 0.2% agarose gel per rectum for induction of colitis and followed for 7 days. Colonic tissue
A-259
AGA Abstracts
AGA Abstracts
S1731