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Effecing Lesion-Forming Pathogen Citrobacter rodentium
AGA Abstracts
improvement in histology from the scores in the 8 day experiment, whereas mutant mice did not demonstrate significant restitution (<1% improvement). Conclusions: Our results support the hypothesis that Atoh1∆intestine mice are more susceptible to experimental colitis than WT mice, although only in the DSS model. Since DSS is thought to induce colitis in mice by direct gut epithelial injury secondary to its irritant properties whereas TNBS is thought to induce colitis through Th-1 mediated pathways after an initial sensitizing exposure, our findings raise the possibility that compromised gut epithelial barrier function may be an important underlying mechanism for this increased susceptibility. We are pursuing additional studies assessing proliferation, apoptosis, as well as gut permeability to define a mechanism of barrier function compromise that may be characteristic of Atoh1∆intestine mice.
chronic stress(IS). Indomethacin treatment induces an enteritis characterized by a cyclical alternation of active and reactive phases of inflammation (Porras et al, 2004). Chronic stress was induced during the reactive phase of the second inflammatory cycle(low blood leukocytes) by wrap restraint or water avoidance stress 1h/day on alternate days for 5 days. Body weight, pellets output and corticosterone levels were monitored as a control of the stress effect. 1 hour after the last stress session, mesenteric lymph nodes were cultured to determine bacterial translocation. Mucosal mast cell (MMC) number and myeloperoxidase activity (MPO) were determined in ileon. Both constitutive and inducible NOS and COX isoforms mRNA expression were measured by RT-PCR. Results:Control and indomethacin treated animals, studied during the reactive phase, showed similar results in all parameters evaluated. The exposure to chronic stress induced a significant reduction in blood leukocytes number and an increase in defecation frequency, in both CS and IS groups. There were no differences in nNOS, COX-1 and COX-2 in rats subjected to stress. However, chronic stress induced an increase in ileon mucosal mast cell number (8,9± 0,65 vs 5,9± 0,36 MMC/villi; p<0.01 vs CC), an important bacterial translocation (50% of animals showed positive cultures to Enterobacteria and 65% positive cultures to gram positive flora), a tend to increase in MPO activity and a significant reduction in iNOS mRNA expression (0,073±0,049 vs 0,30±0,098; p<0.05 vsIC) in IS group, but not in CS animals. Conclusion: These findings suggest that chronic stress could be a risk factor of intestinal inflammatory reactivation as shown by the increase of bacterial translocation related with an increase in mucosal mast cell number. However, this initial reactivation can be masked by the decrease of several inflammatory parameters (blood leukocytes number and iNOS expression), induced by chronic stress.
S1726 Cxcr2-Targeted Chemokines Are Critical Regulators of Host Defense Against the Attaching/Effecing Lesion-Forming Pathogen Citrobacter rodentium Martina E. Spehlmann, Sara M. Dann, Elaine Hanson, Petr Hruz, Lars Eckmann INTRODUCTION: Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and causes crypt hyperplasia and mucosal inflammation. Although several defense mechanisms have been described, critical aspects of the infectious pathogenesis remain unknown. METHODS: Genome-wide microarrays were employed to monitor changes in global gene expression in the colon during C. rodentium infection in a murine model. Microarray data were confirmed by real-time PCR. KO mice were used to determine the physiologic functions of specific target genes. RESULTS: Of the >30,000 unique probes on the microarrays, significant (>4-fold) increases above background were observed for ~700 genes after 1, 2, or 3 weeks after infection, with the majority occurring at 1 week. Of the top 25 genes with increased expression, five (20%) were the chemokines CXCL1/2/5 and CXCL9/10, which are ligands for the chemokine receptors, CXCR2 and CXRC3, respectively. Infection of mice lacking CXCR2 revealed that the receptor was important for effective host defense, since clearance did not occur until week 5 after infection compared to 2-3 weeks in wild-type mice. Histologic analysis of the colonic mucosa in Cxcr2-/- mice showed a lack of the normally observed mucosal hyperplasia and immune cell infiltration, yet serum IgG against C. rodentium was significantly higher in Cxcr2-/- mice compared to controls. CONCLUSIONS: The global expression analysis of the colonic response to C. rodentium infection revealed a limited set of significantly induced genes, an important fraction of which are chemokine ligands for CXCR2 and CXCR3. Deficiency for CXCR2 compromises bacterial clearance, indicating that this receptor and its cognate ligands play an important role in host defense against the attaching/effacing lesion-forming pathogen C. rodentium.
S1729 Upregulation of HIF-1α Is Associated with Augmented Rho-Kinase-Mediated Contraction of Ileal Smooth Muscle Following Exposure to Clostridium difficile Toxins in a Mouse Ileal Loop Model Eikichi Ihara, Simon A. Hirota, Paul L. Beck, Justin A. MacDonald Introduction: Clostridium difficile (C. diff.) infections cause colitis with associated diarrhea that can progress to toxic megacolon and sepsis, often leading to death. Upon clinical examination, C. diff.-associated colitis often resembles that which is caused by ischemia. C. diff. toxin A (TcdA) and toxin B (TcdB) can trigger immune responses that generate a cytokine milieu that may be responsible for the ischemia-like epithelial response and the altered motility associated with C. diff. infections. Thus we hypothesized that C. diff. toxin-induced inflammation activates protective hypoxic signaling pathways that control the immune response and intestinal motility during C. diff. infections. Methods: To study the effect of C. diff. toxins on intestinal epithelial cells Caco-2 cells were exposed to C.diff. toxin and the expression of HIF-1α was determined with real-time RT-PCR and Western blotting. HIF-1α-DNA binding was examined using EMSA to evaluate the putative induction of transcription events. To investigate the role of HIF-1α in the regulation of intestinal motility upon exposure to C. diff. toxins, ileal loops created in wild-type and epitheliumtargeted HIF-1α null mice (HIF-1α(Ep-/-)) were treated with crude C. diff. toxin (containing both TcdA and TcdB) for 4 hr. Ileal smooth muscle was then removed and contractions evoked by carbachol and KCl were examined in the presence of inhibitors of various contractile pathways. Results: HIF-1α mRNA levels were significantly elevated upon exposure to crude C. diff. toxin (0.1 - 50 µg/mL). Furthermore, HIF-1α protein levels and HIF-1αDNA binding were increased in response to crude C. diff. toxin (100 µg/mL). Injection of the crude toxin into ileal loops of both wild-type and HIF-1α (Ep-/-) mice led to an augmentation of KCl- and carbachol-induced contractions. However, the toxin-induced hyperresponsiveness to carbachol observed in the wild-type ileum was significantly attenuated in the presence of Y-27632 (10 µM) a selective inhibitor of Rho-kinase, an effect not observed in the HIF-1α (Ep-/-). Conclusions: C. diff. toxins upregulate epithelial HIF-1α signalling, through increased transcription, protein stabilization and HIF-1α-DNA binding. This effect may be responsible for the augmented agonist-induced Rho-kinase-mediated Ca2+-sensitization observed in ileal smooth muscle isolated from C. diff. toxin-treated ileal loops from wild-type, but not HIF-1α (Ep-/-) mice.
S1727 DC Subtypes in the Colon and Their Role in TNBS Colitis Colin de Haar, Pieter van Lierop, Dicky Lindenbergh-Kortleve, Mirjam Kool, Leonie S. van Rijt, Bart Lambrecht, Edward Nieuwenhuis, Janneke Samsom Introduction Mucosal dendritic cells (DC) play an important role in both tolerance induction to harmless antigens and protection from pathogenic infection. Hapten-induced inflammatory colitis models are considered T-cell dependent, although the characteristics of colonic DC subtypes are poorly defined. To determine the subsets of DC that are predominantly involved in antigen presentation during colonic inflammation we have characterized them during hapten induced colitis. Methods Changes in myeloid (CD11b+ CD8a-), lymphoid (CD11bCD8a+), double negative (CD11b- CD8a-) and plasmacytoid (CD11b- 120G8+ B220+) DC, were studied in the colon lamina propria (LP) and the mucosa draining lymph nodes (LN) of mice during homeostasis and during trinitro-benzene sulphonic acid (TNBS)-induced colitis. The role of DC in antigen presentation to T cells during colitis was further investigated by transiently depleting CD11c+ cells using diphtheria toxin injection in transgenic CD11cDTR mice. Results During homeostasis CD11c+ cells in colon LP mainly consisted of the myeloid phenotype while lymphoid and plasmacytoid DC subtypes were scarce. Upon induction of TNBS colitis a strong influx of monocytes into the colon LP occurred within 24h of hapten injection which coincided with a decrease in CD11c+ DC. At 72h of inflammation an increase in myeloid DC numbers in the mucosa draining LN was observed suggesting that DC from colon LP have migrated to the LN. However, despite a reduction in CD4+ T cells in the mucosa draining lymph node, depletion of DC did not reduce weightloss or histological damage in the colon. Upon characterization of mesenteric lymph node cells in CD11cdepleted mice, we observed a dominant population of Ly6c+ DC at 72h of colitis induction. This may indicate that other than classical CD11c+ DC may be involved in driving mucosal T cell activation in this model. Conclusion Colonic inflammation strongly affects myeloid DC in both the lamina propria of the colon and the mucosa draining LN. Depletion of DC during colitis reduces the number of CD4+T cells in the mucosa draining LN. Interestingly, this reduction of T cells did not reduce the severity of colitis. The presence of Ly6c+ DC may point to alternative ways of antigen presentation in TNBS colitis.
S1730 Display of Self Antigens By Lymph Node Stromal Cells Contributes to CD8 T Cell Tolerance Khashayarsha Khazaie, Fay Magnusson To evaluate the contribution of antigen specific CD8 and CD4 T-cells to the breakdown of the enteric glial (EGC) network and autoimmune pathology in the intestine, we used transgenic mice that express the influenza hemagglutinin(HA) in EGCs (GFAP-HA), and HA specific T cell receptor (TCR) transgenic mice. The mice were either crossed or HA specific T cells were adoptively transferred. Both CD8 and CD4 T-cell subtypes were activated In Vivo in an antigen-specific manner. However, they differed substantially in their ability to expand in the mesenteric lymph nodes, trigger pro-inflammatory cytokines, and induce autoimmune damage in the intestine. Direct presentation of antigen, provided by lymph node stromal cells, caused the activation and deletion of CD8 T-cells. This mechanism of T-cell tolerance did not affect CD4 T-cells, which produced antigen-specific lethal autoimmunity in the intestine. Breaking CD8 tolerance led to multi-organ autoimmunity, but the intestine remained protected. Our observations support a recently identified mechanism of peripheral T-cell tolerance that protects against CD8 T cell autoimmunity directed to the intestine. Furthermore, we show that in our model conventional CD4 T-cell are not affected by this mechanism of tolerance and their targeting of EGCs produces lethal autoimmune inflammatory pathology in the intestine.
S1728 Bacterial Translocation Induced By Chronic Stress During the Remission Phase of Rat Model of Enteritis: A Possible Risk of Inflammatory Reactivation? Esther Jorge, Patri Vergara, Maite Martin Inflammatory bowel diseases are characterized by chronic relapsing and remitting course.Psychological stress and normal intestinal resident bacteria are environmental factors that play a key role in the pathogenesis of the disease. Aims:The aims of this study were to evaluate bacterial translocation and COX and NOS isoforms mRNA expression in control animals subjected to stress, and to determine whether stress could influence the course of inflammation in a rat model of enteritis. Methods:Experimental groups: control rats(CC), control rats submitted to chronic stress(CS), rats treated with two subcutaneous administration of 7.5 mg/Kg indomethacin 48 hours apart(IC) and rats treated with indomethacin subjected to
AGA Abstracts
A-258
improvement in histology from the scores in the 8 day experiment, whereas mutant mice did not demonstrate significant restitution (<1% improvement). Conclusions: Our results support the hypothesis that Atoh1∆intestine mice are more susceptible to experimental colitis than WT mice, although only in the DSS model. Since DSS is thought to induce colitis in mice by direct gut epithelial injury secondary to its irritant properties whereas TNBS is thought to induce colitis through Th-1 mediated pathways after an initial sensitizing exposure, our findings raise the possibility that compromised gut epithelial barrier function may be an important underlying mechanism for this increased susceptibility. We are pursuing additional studies assessing proliferation, apoptosis, as well as gut permeability to define a mechanism of barrier function compromise that may be characteristic of Atoh1∆intestine mice.
chronic stress(IS). Indomethacin treatment induces an enteritis characterized by a cyclical alternation of active and reactive phases of inflammation (Porras et al, 2004). Chronic stress was induced during the reactive phase of the second inflammatory cycle(low blood leukocytes) by wrap restraint or water avoidance stress 1h/day on alternate days for 5 days. Body weight, pellets output and corticosterone levels were monitored as a control of the stress effect. 1 hour after the last stress session, mesenteric lymph nodes were cultured to determine bacterial translocation. Mucosal mast cell (MMC) number and myeloperoxidase activity (MPO) were determined in ileon. Both constitutive and inducible NOS and COX isoforms mRNA expression were measured by RT-PCR. Results:Control and indomethacin treated animals, studied during the reactive phase, showed similar results in all parameters evaluated. The exposure to chronic stress induced a significant reduction in blood leukocytes number and an increase in defecation frequency, in both CS and IS groups. There were no differences in nNOS, COX-1 and COX-2 in rats subjected to stress. However, chronic stress induced an increase in ileon mucosal mast cell number (8,9± 0,65 vs 5,9± 0,36 MMC/villi; p<0.01 vs CC), an important bacterial translocation (50% of animals showed positive cultures to Enterobacteria and 65% positive cultures to gram positive flora), a tend to increase in MPO activity and a significant reduction in iNOS mRNA expression (0,073±0,049 vs 0,30±0,098; p<0.05 vsIC) in IS group, but not in CS animals. Conclusion: These findings suggest that chronic stress could be a risk factor of intestinal inflammatory reactivation as shown by the increase of bacterial translocation related with an increase in mucosal mast cell number. However, this initial reactivation can be masked by the decrease of several inflammatory parameters (blood leukocytes number and iNOS expression), induced by chronic stress.
S1726 Cxcr2-Targeted Chemokines Are Critical Regulators of Host Defense Against the Attaching/Effecing Lesion-Forming Pathogen Citrobacter rodentium Martina E. Spehlmann, Sara M. Dann, Elaine Hanson, Petr Hruz, Lars Eckmann INTRODUCTION: Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and causes crypt hyperplasia and mucosal inflammation. Although several defense mechanisms have been described, critical aspects of the infectious pathogenesis remain unknown. METHODS: Genome-wide microarrays were employed to monitor changes in global gene expression in the colon during C. rodentium infection in a murine model. Microarray data were confirmed by real-time PCR. KO mice were used to determine the physiologic functions of specific target genes. RESULTS: Of the >30,000 unique probes on the microarrays, significant (>4-fold) increases above background were observed for ~700 genes after 1, 2, or 3 weeks after infection, with the majority occurring at 1 week. Of the top 25 genes with increased expression, five (20%) were the chemokines CXCL1/2/5 and CXCL9/10, which are ligands for the chemokine receptors, CXCR2 and CXRC3, respectively. Infection of mice lacking CXCR2 revealed that the receptor was important for effective host defense, since clearance did not occur until week 5 after infection compared to 2-3 weeks in wild-type mice. Histologic analysis of the colonic mucosa in Cxcr2-/- mice showed a lack of the normally observed mucosal hyperplasia and immune cell infiltration, yet serum IgG against C. rodentium was significantly higher in Cxcr2-/- mice compared to controls. CONCLUSIONS: The global expression analysis of the colonic response to C. rodentium infection revealed a limited set of significantly induced genes, an important fraction of which are chemokine ligands for CXCR2 and CXCR3. Deficiency for CXCR2 compromises bacterial clearance, indicating that this receptor and its cognate ligands play an important role in host defense against the attaching/effacing lesion-forming pathogen C. rodentium.
S1729 Upregulation of HIF-1α Is Associated with Augmented Rho-Kinase-Mediated Contraction of Ileal Smooth Muscle Following Exposure to Clostridium difficile Toxins in a Mouse Ileal Loop Model Eikichi Ihara, Simon A. Hirota, Paul L. Beck, Justin A. MacDonald Introduction: Clostridium difficile (C. diff.) infections cause colitis with associated diarrhea that can progress to toxic megacolon and sepsis, often leading to death. Upon clinical examination, C. diff.-associated colitis often resembles that which is caused by ischemia. C. diff. toxin A (TcdA) and toxin B (TcdB) can trigger immune responses that generate a cytokine milieu that may be responsible for the ischemia-like epithelial response and the altered motility associated with C. diff. infections. Thus we hypothesized that C. diff. toxin-induced inflammation activates protective hypoxic signaling pathways that control the immune response and intestinal motility during C. diff. infections. Methods: To study the effect of C. diff. toxins on intestinal epithelial cells Caco-2 cells were exposed to C.diff. toxin and the expression of HIF-1α was determined with real-time RT-PCR and Western blotting. HIF-1α-DNA binding was examined using EMSA to evaluate the putative induction of transcription events. To investigate the role of HIF-1α in the regulation of intestinal motility upon exposure to C. diff. toxins, ileal loops created in wild-type and epitheliumtargeted HIF-1α null mice (HIF-1α(Ep-/-)) were treated with crude C. diff. toxin (containing both TcdA and TcdB) for 4 hr. Ileal smooth muscle was then removed and contractions evoked by carbachol and KCl were examined in the presence of inhibitors of various contractile pathways. Results: HIF-1α mRNA levels were significantly elevated upon exposure to crude C. diff. toxin (0.1 - 50 µg/mL). Furthermore, HIF-1α protein levels and HIF-1αDNA binding were increased in response to crude C. diff. toxin (100 µg/mL). Injection of the crude toxin into ileal loops of both wild-type and HIF-1α (Ep-/-) mice led to an augmentation of KCl- and carbachol-induced contractions. However, the toxin-induced hyperresponsiveness to carbachol observed in the wild-type ileum was significantly attenuated in the presence of Y-27632 (10 µM) a selective inhibitor of Rho-kinase, an effect not observed in the HIF-1α (Ep-/-). Conclusions: C. diff. toxins upregulate epithelial HIF-1α signalling, through increased transcription, protein stabilization and HIF-1α-DNA binding. This effect may be responsible for the augmented agonist-induced Rho-kinase-mediated Ca2+-sensitization observed in ileal smooth muscle isolated from C. diff. toxin-treated ileal loops from wild-type, but not HIF-1α (Ep-/-) mice.
S1727 DC Subtypes in the Colon and Their Role in TNBS Colitis Colin de Haar, Pieter van Lierop, Dicky Lindenbergh-Kortleve, Mirjam Kool, Leonie S. van Rijt, Bart Lambrecht, Edward Nieuwenhuis, Janneke Samsom Introduction Mucosal dendritic cells (DC) play an important role in both tolerance induction to harmless antigens and protection from pathogenic infection. Hapten-induced inflammatory colitis models are considered T-cell dependent, although the characteristics of colonic DC subtypes are poorly defined. To determine the subsets of DC that are predominantly involved in antigen presentation during colonic inflammation we have characterized them during hapten induced colitis. Methods Changes in myeloid (CD11b+ CD8a-), lymphoid (CD11bCD8a+), double negative (CD11b- CD8a-) and plasmacytoid (CD11b- 120G8+ B220+) DC, were studied in the colon lamina propria (LP) and the mucosa draining lymph nodes (LN) of mice during homeostasis and during trinitro-benzene sulphonic acid (TNBS)-induced colitis. The role of DC in antigen presentation to T cells during colitis was further investigated by transiently depleting CD11c+ cells using diphtheria toxin injection in transgenic CD11cDTR mice. Results During homeostasis CD11c+ cells in colon LP mainly consisted of the myeloid phenotype while lymphoid and plasmacytoid DC subtypes were scarce. Upon induction of TNBS colitis a strong influx of monocytes into the colon LP occurred within 24h of hapten injection which coincided with a decrease in CD11c+ DC. At 72h of inflammation an increase in myeloid DC numbers in the mucosa draining LN was observed suggesting that DC from colon LP have migrated to the LN. However, despite a reduction in CD4+ T cells in the mucosa draining lymph node, depletion of DC did not reduce weightloss or histological damage in the colon. Upon characterization of mesenteric lymph node cells in CD11cdepleted mice, we observed a dominant population of Ly6c+ DC at 72h of colitis induction. This may indicate that other than classical CD11c+ DC may be involved in driving mucosal T cell activation in this model. Conclusion Colonic inflammation strongly affects myeloid DC in both the lamina propria of the colon and the mucosa draining LN. Depletion of DC during colitis reduces the number of CD4+T cells in the mucosa draining LN. Interestingly, this reduction of T cells did not reduce the severity of colitis. The presence of Ly6c+ DC may point to alternative ways of antigen presentation in TNBS colitis.
S1730 Display of Self Antigens By Lymph Node Stromal Cells Contributes to CD8 T Cell Tolerance Khashayarsha Khazaie, Fay Magnusson To evaluate the contribution of antigen specific CD8 and CD4 T-cells to the breakdown of the enteric glial (EGC) network and autoimmune pathology in the intestine, we used transgenic mice that express the influenza hemagglutinin(HA) in EGCs (GFAP-HA), and HA specific T cell receptor (TCR) transgenic mice. The mice were either crossed or HA specific T cells were adoptively transferred. Both CD8 and CD4 T-cell subtypes were activated In Vivo in an antigen-specific manner. However, they differed substantially in their ability to expand in the mesenteric lymph nodes, trigger pro-inflammatory cytokines, and induce autoimmune damage in the intestine. Direct presentation of antigen, provided by lymph node stromal cells, caused the activation and deletion of CD8 T-cells. This mechanism of T-cell tolerance did not affect CD4 T-cells, which produced antigen-specific lethal autoimmunity in the intestine. Breaking CD8 tolerance led to multi-organ autoimmunity, but the intestine remained protected. Our observations support a recently identified mechanism of peripheral T-cell tolerance that protects against CD8 T cell autoimmunity directed to the intestine. Furthermore, we show that in our model conventional CD4 T-cell are not affected by this mechanism of tolerance and their targeting of EGCs produces lethal autoimmune inflammatory pathology in the intestine.
S1728 Bacterial Translocation Induced By Chronic Stress During the Remission Phase of Rat Model of Enteritis: A Possible Risk of Inflammatory Reactivation? Esther Jorge, Patri Vergara, Maite Martin Inflammatory bowel diseases are characterized by chronic relapsing and remitting course.Psychological stress and normal intestinal resident bacteria are environmental factors that play a key role in the pathogenesis of the disease. Aims:The aims of this study were to evaluate bacterial translocation and COX and NOS isoforms mRNA expression in control animals subjected to stress, and to determine whether stress could influence the course of inflammation in a rat model of enteritis. Methods:Experimental groups: control rats(CC), control rats submitted to chronic stress(CS), rats treated with two subcutaneous administration of 7.5 mg/Kg indomethacin 48 hours apart(IC) and rats treated with indomethacin subjected to
AGA Abstracts
A-258