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497 ER Stress Transcription Factor XBP1 Regulates NfκB Activation in Intestinal Epithelial Cells
magnitude of these changes correlates with the grade of disease activity. However, only patients with longstanding disease were evaluated, making it impossible to distinguish between primary and secondary changes. The aim of this study was to determine of if genetic ablation of MLCK can prevent the development of experimental IBD In Vivo. METHODS: MLCK-/-RAG1-/- mice and RAG1-/- control mice received CD4+CD45RBhi cells by adoptive transfer followed by regular analysis of clinical status and body weight. Mice were sacrificed at indicated time points, and epithelial MLCK expression, MLC phosphorylation, and tight junction protein status were assessed by SDS-PAGE immunoblot and immunofluorescence microscopy. Barrier function was determined by serum recovery of fluorescent-dextran delivered by gavage. RESULTS: CD4+CD45RBhi T cell-induced colitis in RAG1-/- recipients was associated with increased epithelial MLCK expression, MLC phosphorylation, and tight junction reorganization. Weight loss occurred within 17.3±2.5 days, and serum dextran recovery, was 18.3±1.6 μg/ml at 17 days. MLCK-/-RAG1-/- recipients showed significantly less colonic epithelial MLC phosphorylation and more limited tight junction reorganization. MLCK-/-RAG1-/- recipients did not develop weight loss until 26.6±3.2 days after adoptive transfer (p<0.05), had significantly lower clinical scores, and had significantly lower serum dextran recovery at 17 days (13.8±0.3 μg/ml, p<0.05) than RAG1-/- mice. Histological analysis confirmed reduced colitis severity in MLCK-/-RAG1-/- mice 17 days after adoptive transfer, although mucosal damage was present in all mice. At later times weight loss was similar in both groups, but histological damage remained less severe in MLCK-/-RAG1-/recipients. CONCLUSION: Intestinal epithelial MLCK activity and expression are increased in human and experimental IBD. Genetic ablation of MLCK limits epithelial MLC phosphorylation, tight junction disruption, and intestinal barrier dysfunction. These events contribute to IBD progression, as disease onset is delayed and severity attenuated in experimental IBD. These data suggest that suppression of MLCK expression or activity may be useful therapeutic strategies in human IBD.
ER Stress Transcription Factor XBP1 Regulates NfκB Activation in Intestinal Epithelial Cells Lukas Niederreiter, Sebastian Zeissig, Herbert Tilg, Richard S. Blumberg, Arthur Kaser Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium of mice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/- epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBα mRNA, a downstream target of NFκB was substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship
500 MTG16 Contributes to Epithelial Integrity in Response to Colonic Injury Christopher S. Williams, Rupesh Chaturvedi, Mary Kay Washington, Amanda D. Williams, Melissa A. Steapleton, Michael E. Engel, Keith T. Wilson, Scott W. Hiebert Myeloid Translocation Gene (MTGs) proteins are transcriptional co-repressors, two of which were first identified disrupted by the t(8;21) chromosomal translocations associated with acute myeloid leukemia (AML). Both Mtg8-/- and Mtgr1-/- mice show developmental and/or differentiation abnormalities in the intestine. A role for MTG16, the remaining MTG family member, in the intestine is unclear; therefore, we generated Mtg16-/- mice to explore its biologic function. Unlike the Mtgr1-/- mice, the small intestine and colon are normal in appearance and all intestinal lineages are present in expected ratios. However, similar to the Mtgr1-/- animals, there was increased colonic epithelial proliferation with an expansion of the proliferative zone (2.5x increased BrdU positive cells/HPF, p<0.001). To unmask colonic stress-induced phenotypes we treated Mtg16-/- mice with 3% low-molecular weight DSS. In comparison to wild-type animals, the Mtg16-/- mice developed accelerated weight loss, severe diarrhea, and increased mortality with severe acute colitis (severity index 43.4 vs 17.1, p<0.006). To explore the mechanistic underpinnings of this phenotype we surveyed for differences in cytokine production in the intestine using TaqMan probes. We found elevated Interferon-γ (IFN-γ) mRNA and protein levels in Mtg16-/- colons, consistent with a Th1 response. Colonic immunophenotyping using flow cytometry against cell surface markers for PMNs (Gr1), T cells (CD3) and T helper cells (CD4) cells indicated increased Gr1+ (10-fold, p<0.01) and CD4+ (11-fold, p<0.05) cells in the Mtg16-/- colons after DSS treatment. Compared to wild-type mice, Mtg16-/- mice exhibited a 17.6-fold and a 7.4-fold increase in colonic CD4+/IFN-γ+ cells in vehicle treated and DSS-treated mice, respectively, as assessed by flow cytometry (p < 0.05 for both). Adoptive transfer of WT marrow into the Mtg16-/- recipients followed by DSS treatment did not rescue the Mtg16-/- DSS phenotype, suggesting that this phenotype was not the result of a hematopoietic cell-intrinsic Mtg16-/defect, but rather implicating an epithelial or stromal based defect. To determine if there were abnormal epithelial inflammatory responses we isolated colonic epithelial cells from DSS treated WT or Mtg16-/- mice and measured cytokine mRNA levels. KC (Cxcl1) is a chemokine responsible for recruiting PMNs; we found that KC mRNA levels were increased 8.1 ± 1.7-fold in the Mtg16-/- DSS colitis tissues when compared to WT mice. These novel observations suggest that MTG16 is critical for survival and regeneration in response to intestinal injury and provides evidence that MTGs regulate inflammatory recruitment in response to injury.
498 Autocrine Intestinal Epithelial Cell GM-CSF Signaling Promotes Homeostatic Responses to Gut Injury Xiaonan Han, Shila Gilbert, Ingrid Jurickova, Charles M. Samson, Jonathan Gully, Tara Willson, Lee Denson Background: Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) may relieve symptoms in Crohn Disease (CD) by enhancing mucosal barrier function. We have reported that gm-csf deficiency exacerbates NSAID-induced ileitis in mice. Our recent study exhibited that loss of the gm-csf receptor β chain (gm-csfrβc) on intestinal epithelial cells (IEC) results in defective barrier function. We hypothesized that GM-CSF maintains gut homeostasis through an autocrine loop which involves GM-CSF production derived by intestinal epithelial cells (IEC). Methods: We utilized wild type (WT), gm-csf deficient mice (gm-csf-/-), and bone marrow (BM) chimeras containing WT BM and either WT or gm-csf-/- intestinal epithelium (WTBM, EP-GMKO). Reconstitution of GM-CSF expression on intestinal laminar propria mononuclear cells (LPMC) was confirmed by FACS analysis. Mice were placed on chow containing the NSAID piroxicam (200 ppm) for two weeks, and ileal histology and bacterial translocation to mesenteric lymph nodes (MLN) were determined. Ileal IEC were then isolated with EDTA, GM-CSF production by IEC was assessed using ELISA and immunohistochemistry (IHC). IEC survival and proliferation were determined by cleaved caspase3, p21 and Cyclin D1 western blot and BrdU labeling. RNA interference was used to knockdown gm-csfrβc expression in Caco-2 monolayer and sub-confluent Caco-2 cells, and survival, proliferation and monolayer permeability following TNFα (100 ng/ml) and Indomethacin (NSAID, IN) exposure were determined. Results: GM-CSFRβc was highly expressed on ileal IEC. GM-CSF production by isolated ileal IEC measured by ELISA was equal to 2.6 ± 0.5 pg/ml. IHC confirmed that GM-CSF was expressed on both crypt and villus cells. Compared to WTBM controls, EP-GMKO mice developed severe ileitis following NSAID exposure; this was associated with significantly increased bacterial translocation to MLN. Under these conditions, ileal IEC apoptosis as measured by cleaved caspase 3 staining was increased, while the IEC proliferative response as measured by cyclin D and p21 abundance was attenuated. GM-CSF at 1 ng/ml normalized proliferation of caco-2 cells exposed to IN and TNFα. Following knock down of gm-csfrβc expression in Caco-2 cells, cell proliferation was reduced and TNFα-induced increase in monolayer permeability was enhanced. TNFα and IN suppression of cellular proliferation was also significantly increased by gm-csfrβc knock down, suggesting an autocrine effect. Conclusion: Autocrine GM-CSF signaling on IEC contributes to ileal homeostasis and cellular function; furthermore, targeting GM-CSF derived by IEC may provide the new therapeutic strategy for CD.
501 Hypoxia Inducible Factor (HIF-1α) Plays a Critical Innate Protective Role in Clostridium difficile (Cdif) Toxin-Induced Intestinal Injury and Inflammation Simon A. Hirota, Jeff Ng, Eikichi Ihara, Yan Li, Daniel Muruve, Glen D. Armstrong, Sean P. Colgan, Daniel Chung, Paul L. Beck, Justin A. MacDonald Cdif-associated diarrhea (CDAD) is the leading cause of nosocomial diarrhea. The spread of virulent antibiotic-resistant, strains has resulted in increased mortality so that CDAD causes more deaths in the USA than HIV. Since Cdif toxin-mediated events occur rapidly and the adaptive immune response has not been found to provide significant protection, we have focused our studies on innate responses. We hypothesized that HIF-1α plays a protective role in CDAD since our preliminary studies showed that patients with CDAD had altered expression of the transcription factor HIF-1α and recent studies suggest that HIF-1α plays important roles in maintenance of intestinal epithelial barrier and early innate immune responses. Aims: 1) Assess the impact of Cdif toxins on HIF-1α transcription, translation and DNA binding events, 2) Assess the role of HIF-1α in Cdif mediated injury/ inflammation In Vitro and In Vivo, 3) Define the mechanisms involved in HIF-1α-mediated protective events in response to Cdif toxin. Methods: In Vitro studies assessed HIF-1α mRNA, protein levels and DNA binding events in Caco-2 cells exposed to Cdif toxin. In Vivo studies employed the murine ileal loop model of Cdif toxin-induced intestinal injury. Animals were exposed to toxin, sacrificed and assessed for inflammatory markers, tissue damage, mRNA and protein levels. Cell lines with siRNA knock down of HIF-1α and a murine line with targeted deletion of HIF-1α in the intestinal epithelium were used to assess the impact of
499 Genetic Ablation of Myosin Light Chain Kinase (MLCK) Limits Epithelial Barrier Dysfunction and Attenuates Experimental Inflammatory Bowel Disease (IBD) Liping Su, Sam C. Nalle, Erika A. Sullivan, Yang-Xin Fu, Jerrold R. Turner Previous studies using In Vitro and acute In Vivo models have shown that MLCK-mediated myosin II regulatory light chain (MLC) phosphorylation is central to TNF-induced epithelial tight junction disruption and barrier dysfunction. In mice, epithelial expression of constitutively-active MLCK accelerates onset and enhances severity of experimental IBD. In human IBD, intestinal epithelial MLCK expression and MLC phosphorylation are increased and the
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ER Stress Transcription Factor XBP1 Regulates NfκB Activation in Intestinal Epithelial Cells Lukas Niederreiter, Sebastian Zeissig, Herbert Tilg, Richard S. Blumberg, Arthur Kaser Background: The unfolded protein response (UPR) is a mechanism that allows cells to cope with endoplasmic reticulum (ER) stress due to the accumulation of misfolded proteins. Among the three proximal effectors of the UPR, the IRE1/XBP1 pathway represents the most conserved pathway. We have recently reported that conditional deletion of Xbp1 specifically in the intestinal epithelium of mice results in spontaneous small intestinal enteritis reminiscent of human inflammatory bowel disease (IBD), and that polymorphisms in the XBP1 gene are associated with both, Crohn's disease (CD) and ulcerative colitis (UC). We have shown that Xbp1-/- epithelia deplete Paneth cells due to apoptosis, and exhibit a pro-inflammatory phenotype as evidenced by JNK phosphorylation. We hypothesized that XBP1 knock-down might increase NFκB signaling as a consequence of IRE1 overactivation. Methods: XBP1 expression was silenced in the intestinal epithelial cell (IEC) line MODE-K via a retroviral shRNA vector, and cells stimulated with TLR ligands or TNFα. Phosphorylation status of IKKs, IκBα, and NFκB were analyzed by phospho-specific antibodies. DNA binding activity of nuclear extracts to the NFκB consensus sequence was assessed, and expression of a classical target gene of the canonical NFκB pathway, IκBα, measured by qPCR. Results: TNFα stimulation resulted in a marked increase in the extent and duration of IKK phosphorylation in XBP1-silenced MODE-Ks. IkBα phosphorylation was increased after TNFα stimulation at early time-points in Xbp1-silenced relative to control cells, as was nuclear NFκB phosphorylation. NFκB protein was retained in nuclei for a prolonged period of time after TNFα stimulation of Xbp1-silenced cells compared to control-silenced MODE-Ks. NFκB p65 DNA binding activity in nuclear extracts of XBP1-silenced MODE-Ks was also increased after TNFα stimulation. Expression of IκBα mRNA, a downstream target of NFκB was substantially increased in TNFα and TLR3 ligand-stimulated MODE-K cells. Discussion: We show that unresolved ER stress in IECs as modeled by XBP1 knock-down results in marked overactivation of the NFκB pathway. Similar to increased JNK phosphorylation as we have previously reported, this is most likely due to marked overactivation of IRE1α in XBP1-deficient IECs since phosphorylated IRE1α has previously been shown to physically interact with the adapter proteins TRAF2 and IKK2 under conditions of ER stress. These data show that the pro-inflammatory consequences of hypomorphic XBP1 function includes dysregulation of a key pro-inflammatory signal transduction pathway, NFκB. RSB and AK share senior authorship
500 MTG16 Contributes to Epithelial Integrity in Response to Colonic Injury Christopher S. Williams, Rupesh Chaturvedi, Mary Kay Washington, Amanda D. Williams, Melissa A. Steapleton, Michael E. Engel, Keith T. Wilson, Scott W. Hiebert Myeloid Translocation Gene (MTGs) proteins are transcriptional co-repressors, two of which were first identified disrupted by the t(8;21) chromosomal translocations associated with acute myeloid leukemia (AML). Both Mtg8-/- and Mtgr1-/- mice show developmental and/or differentiation abnormalities in the intestine. A role for MTG16, the remaining MTG family member, in the intestine is unclear; therefore, we generated Mtg16-/- mice to explore its biologic function. Unlike the Mtgr1-/- mice, the small intestine and colon are normal in appearance and all intestinal lineages are present in expected ratios. However, similar to the Mtgr1-/- animals, there was increased colonic epithelial proliferation with an expansion of the proliferative zone (2.5x increased BrdU positive cells/HPF, p<0.001). To unmask colonic stress-induced phenotypes we treated Mtg16-/- mice with 3% low-molecular weight DSS. In comparison to wild-type animals, the Mtg16-/- mice developed accelerated weight loss, severe diarrhea, and increased mortality with severe acute colitis (severity index 43.4 vs 17.1, p<0.006). To explore the mechanistic underpinnings of this phenotype we surveyed for differences in cytokine production in the intestine using TaqMan probes. We found elevated Interferon-γ (IFN-γ) mRNA and protein levels in Mtg16-/- colons, consistent with a Th1 response. Colonic immunophenotyping using flow cytometry against cell surface markers for PMNs (Gr1), T cells (CD3) and T helper cells (CD4) cells indicated increased Gr1+ (10-fold, p<0.01) and CD4+ (11-fold, p<0.05) cells in the Mtg16-/- colons after DSS treatment. Compared to wild-type mice, Mtg16-/- mice exhibited a 17.6-fold and a 7.4-fold increase in colonic CD4+/IFN-γ+ cells in vehicle treated and DSS-treated mice, respectively, as assessed by flow cytometry (p < 0.05 for both). Adoptive transfer of WT marrow into the Mtg16-/- recipients followed by DSS treatment did not rescue the Mtg16-/- DSS phenotype, suggesting that this phenotype was not the result of a hematopoietic cell-intrinsic Mtg16-/defect, but rather implicating an epithelial or stromal based defect. To determine if there were abnormal epithelial inflammatory responses we isolated colonic epithelial cells from DSS treated WT or Mtg16-/- mice and measured cytokine mRNA levels. KC (Cxcl1) is a chemokine responsible for recruiting PMNs; we found that KC mRNA levels were increased 8.1 ± 1.7-fold in the Mtg16-/- DSS colitis tissues when compared to WT mice. These novel observations suggest that MTG16 is critical for survival and regeneration in response to intestinal injury and provides evidence that MTGs regulate inflammatory recruitment in response to injury.
498 Autocrine Intestinal Epithelial Cell GM-CSF Signaling Promotes Homeostatic Responses to Gut Injury Xiaonan Han, Shila Gilbert, Ingrid Jurickova, Charles M. Samson, Jonathan Gully, Tara Willson, Lee Denson Background: Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) may relieve symptoms in Crohn Disease (CD) by enhancing mucosal barrier function. We have reported that gm-csf deficiency exacerbates NSAID-induced ileitis in mice. Our recent study exhibited that loss of the gm-csf receptor β chain (gm-csfrβc) on intestinal epithelial cells (IEC) results in defective barrier function. We hypothesized that GM-CSF maintains gut homeostasis through an autocrine loop which involves GM-CSF production derived by intestinal epithelial cells (IEC). Methods: We utilized wild type (WT), gm-csf deficient mice (gm-csf-/-), and bone marrow (BM) chimeras containing WT BM and either WT or gm-csf-/- intestinal epithelium (WTBM, EP-GMKO). Reconstitution of GM-CSF expression on intestinal laminar propria mononuclear cells (LPMC) was confirmed by FACS analysis. Mice were placed on chow containing the NSAID piroxicam (200 ppm) for two weeks, and ileal histology and bacterial translocation to mesenteric lymph nodes (MLN) were determined. Ileal IEC were then isolated with EDTA, GM-CSF production by IEC was assessed using ELISA and immunohistochemistry (IHC). IEC survival and proliferation were determined by cleaved caspase3, p21 and Cyclin D1 western blot and BrdU labeling. RNA interference was used to knockdown gm-csfrβc expression in Caco-2 monolayer and sub-confluent Caco-2 cells, and survival, proliferation and monolayer permeability following TNFα (100 ng/ml) and Indomethacin (NSAID, IN) exposure were determined. Results: GM-CSFRβc was highly expressed on ileal IEC. GM-CSF production by isolated ileal IEC measured by ELISA was equal to 2.6 ± 0.5 pg/ml. IHC confirmed that GM-CSF was expressed on both crypt and villus cells. Compared to WTBM controls, EP-GMKO mice developed severe ileitis following NSAID exposure; this was associated with significantly increased bacterial translocation to MLN. Under these conditions, ileal IEC apoptosis as measured by cleaved caspase 3 staining was increased, while the IEC proliferative response as measured by cyclin D and p21 abundance was attenuated. GM-CSF at 1 ng/ml normalized proliferation of caco-2 cells exposed to IN and TNFα. Following knock down of gm-csfrβc expression in Caco-2 cells, cell proliferation was reduced and TNFα-induced increase in monolayer permeability was enhanced. TNFα and IN suppression of cellular proliferation was also significantly increased by gm-csfrβc knock down, suggesting an autocrine effect. Conclusion: Autocrine GM-CSF signaling on IEC contributes to ileal homeostasis and cellular function; furthermore, targeting GM-CSF derived by IEC may provide the new therapeutic strategy for CD.
501 Hypoxia Inducible Factor (HIF-1α) Plays a Critical Innate Protective Role in Clostridium difficile (Cdif) Toxin-Induced Intestinal Injury and Inflammation Simon A. Hirota, Jeff Ng, Eikichi Ihara, Yan Li, Daniel Muruve, Glen D. Armstrong, Sean P. Colgan, Daniel Chung, Paul L. Beck, Justin A. MacDonald Cdif-associated diarrhea (CDAD) is the leading cause of nosocomial diarrhea. The spread of virulent antibiotic-resistant, strains has resulted in increased mortality so that CDAD causes more deaths in the USA than HIV. Since Cdif toxin-mediated events occur rapidly and the adaptive immune response has not been found to provide significant protection, we have focused our studies on innate responses. We hypothesized that HIF-1α plays a protective role in CDAD since our preliminary studies showed that patients with CDAD had altered expression of the transcription factor HIF-1α and recent studies suggest that HIF-1α plays important roles in maintenance of intestinal epithelial barrier and early innate immune responses. Aims: 1) Assess the impact of Cdif toxins on HIF-1α transcription, translation and DNA binding events, 2) Assess the role of HIF-1α in Cdif mediated injury/ inflammation In Vitro and In Vivo, 3) Define the mechanisms involved in HIF-1α-mediated protective events in response to Cdif toxin. Methods: In Vitro studies assessed HIF-1α mRNA, protein levels and DNA binding events in Caco-2 cells exposed to Cdif toxin. In Vivo studies employed the murine ileal loop model of Cdif toxin-induced intestinal injury. Animals were exposed to toxin, sacrificed and assessed for inflammatory markers, tissue damage, mRNA and protein levels. Cell lines with siRNA knock down of HIF-1α and a murine line with targeted deletion of HIF-1α in the intestinal epithelium were used to assess the impact of
499 Genetic Ablation of Myosin Light Chain Kinase (MLCK) Limits Epithelial Barrier Dysfunction and Attenuates Experimental Inflammatory Bowel Disease (IBD) Liping Su, Sam C. Nalle, Erika A. Sullivan, Yang-Xin Fu, Jerrold R. Turner Previous studies using In Vitro and acute In Vivo models have shown that MLCK-mediated myosin II regulatory light chain (MLC) phosphorylation is central to TNF-induced epithelial tight junction disruption and barrier dysfunction. In mice, epithelial expression of constitutively-active MLCK accelerates onset and enhances severity of experimental IBD. In human IBD, intestinal epithelial MLCK expression and MLC phosphorylation are increased and the
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