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86 IL-7-Producing Mesenchymal Stem Cells Sustain Colitogenic Memory CD4+ T Cells for the Persistence of Chronic Colitis
chronic inflammation in the jejunum (score of 2.3 vs. 1.7) and ileum (score of 3.0 vs. 1.1). In myeloid Tg mice, increased chronic inflammation were observed only in the ileum compared to WT (2.3 vs. 1.1). We did not observe any acute or chronic inflammation in the colon of Tg mice. We found increased expression of IFNγ, TNFα, IL17A, and IL17F in the lymphoid Tg compared to WT mice at 2 and 10 months of age (3, 3, 15 fold increase in small bowel respectively at both time points). In contrast, myeloid TL1A Tg mice exhibited increased IFNγ, TNFα, and IL13 expression (approximately 7 fold increase for all cytokines) at 10 months; No elevation in these pro-inflammatory cytokines were observed at 2 months of age. Conclusion: Mild small bowel inflammation is induced by constitutive TL1A expression in either myeloid or lymphoid cells. The more extensive small bowel inflammation in lymphoid TL1A Tg mice may be due to induction of pro-inflammatory cytokine at an earlier age in these mice. 86 IL-7-Producing Mesenchymal Stem Cells Sustain Colitogenic Memory CD4+ T Cells for the Persistence of Chronic Colitis Yasuhiro Nemoto, Takanori Kanai, Teruji Totsuka, Tetsuya Nakamura, Ryuichi Okamoto, Kiichiro Tsuchiya, Naoya Sakamoto, Mamoru Watanabe Background: Inflammatory bowel disease (IBD) is the chronic and persistent disease with recurrences for a long time. We hypothesize that the disease is remitted by the reactivation of colitogenic memory CD4+ T cells, which respond to corresponding intestinal bacterial antigens. We have previously demonstrated not only that IL-7 is essential for the persistence of chronic colitis as a survival factor of colitogenic memory CD4+ T cells in a murine model of IBD, but also that bone marrow (BM), which constitutively produces IL-7, but lacks bacterial antigens, sustains them even in IL-7-decreasing condition of inflamed mucosa due to goblet cells disappearance. Aim & Methods: To confirm a role of IL-7-producing BMderived cells for the perpetuation of colitis, IL-7-/- x RAG1-/- mice were transplanted BM cells of IL-7+/+ x RAG1-/- or IL-7-/- x RAG1-/- mice, and were transferred with CD4+CD45RBhigh T cells 8 wks after transplantation. To further identify IL-7-producing cells in BM, we isolated BM mesenchymal stem cells (MSC) and examined their IL-7 expression and an ability to sustain colitogenic CD4+ memory T cells In Vitro and In Vivo. Results: IL-7 mRNA and protein were detected in BM, but not in colon, of in IL-7-/- x RAG1-/- mice transplanted with BM cells from IL-7+/+ x RAG1-/- mice (IL-7+/+→IL-7-/-), but not from IL-7-/- x RAG1-/mice (IL-7-/-→IL-7-/-). Surprisingly, IL-7+/+→IL-7-/-, but not IL-7-/-→IL-7-/- recipients, thereafter transferred with CD4+CD45RBhigh T cells, developed a wasting disease and colitis with the increased Th1/Th17 cytokine production. Of note, the absolute number of CD3+CD4+ T cells recovered from BM of IL-7+/+→IL-7-/- recipients was significantly higher than that of IL-7-/-→IL-7-/- recipients. MSC isolated from BM of IL-7+/+ x RAG1-/- mice (IL-7+ MSC), but not from BM of IL-7-/- x RAG1-/- mice (IL-7- MSC), expressed higher levels of IL-7 mRNA and protein than those of freshly isolated IL-7+ BM cells. Co-culture with IL-7+, but not IL-7-, MSC induced extensive proliferation of CFSE-labeled colitogenic CD4+ T cells In Vitro. Furthermore, CD4+CD45RBhigh T cell-transferred IL-7-/- x RAG1-/mice previously transplanted with IL-7+MSC, but not IL-7-MSC, developed Th1/Th17meidated colitis. Conclusion: BM MSC-derived IL-7, but not intestinal IL-7, is involved in the persistence of chronic colitis through the maintenance of colitogenic CD4+ memory T cells, suggesting their systemic control for the cure of IBD.
84 Mitochondrial Stress Induction in the Epithelium Fuels Endoplasmic Reticulum Unfolded Protein Responses via Double-Stranded RNA-Activated Protein Kinase (PKR) Under Conditions of Chronic Intestinal Inflammation Eva Rath, Emanuel Berger, Anja Messlik, Tobias Fromme, Martin Klingenspor, Regina Ensenauer, Nicholas J. Hoogenraad, Bo Liu, Sandra C. Kim, R. Balfour Sartor, Dirk Haller Background & Aim. Recently it has been shown that Endoplasmic reticulum (ER) stress responses in intestinal epithelial cells (IEC) contribute to the initiation and perpetuation of chronic intestinal inflammation. The aim of this study was to characterize the role of mitochondrial stress responses in the intestinal epithelium of patients with ulcerative colitis (UC) and two murine models of chronic, T cell-mediated colitis. Methods & Results. Primary IEC were isolated from an adoptive transfer model of colitis, recombination activating gene deficient (RAG2-/-) and RAG2-/- x Interleukin 10 deficient (IL-10-/-) mice reconstituted with CD4+ T cells, as well as gnotobiotic IL-10-/- mice mono- or dual associated with colitogenic E. coli and E. faecalis. Proteome analysis of primary IEC using 2D-SDS PAGE and MALDI-TOF mass spectrometry identified 160 differentially regulated proteins clustered around the mitochondrial stress chaperonin 60 (CPN60) linking ER and mitochondrial unfolded protein response (UPR) under conditions of chronic inflammation. To study the specific role of mitochondrial stress signaling we stimulated the IEC line Mode-K with the selective mt stress inducer truncated ornithine transcarbamylase (OTCΔ). Additionally, cells were treated with Tunicamycin (Tm) to induce ER-associated UPR. Under mt stress, the double-stranded RNA-activated protein kinase (PKR) was identified to be selectively induced at the mRNA- as well as protein level. Subsequent chromatin immunoprecipitation (ChIP) analysis revealed recruitment of the transcription factor (TF) AP-1 to the PKR promoter. In addition and most importantly for the understanding of mt stress integration into the ER signaling pathway, PKR co-immunoprecipitated with eIF2α pointing towards a direct role of PKR in eIF2α phosphorylation after selective mt stress induction. Furthermore, both mtand ER UPR converged in the recruitment of TF CHOP to the Cpn60 promoter. Interestingly, mt respiration and β-oxidation were reduced under ER- as well as mt stress, suggesting metabolic changes in IEC under chronic inflammation. Finally, Western blot analysis of primary IEC from UC patients and murine models of colitis confirmed that increased expression levels of GRP78 and CPN60 were associated with an induction of PKR. Conclusion. Primary IEC from both human UC patients and mouse models of colitis show signs of activated mt stress signaling. Our data demonstrate that mt stress responses enhance ERassociated UPR through PKR induction, eIF2α phosphorylation and CHOP recruitment to the Cpn60 promoter. In conclusion, mt stress fuels pathology-relevant ER stress mechanisms under chronic inflammatory conditions.
87 Conditional Loss of Intestinal Hedgehog Signaling Results in a Regenerative Response and Subsequently in Loss of Villi and the Spontaneous Development of Chronic Enteritis Willemijn A. van Dop, Elisabeth A. Wassenberg, Beate Lanske, Alan R. Clarke, Johan Offerhaus, Daniel W. Hommes, James C. Hardwick, Izak Biemond, Gijs R. van den Brink Indian hedgehog (Ihh) is expressed by differentiated intestinal epithelial cells and acts on the subepithelial myofibroblasts. We previously found that this epithelial mesenchymal interaction is part of a negative feedback loop from the differentiated cells to the intestinal epithelial precursor cells. Genetic variants in the hedgehog pathway have recently been linked to the development of inflammatory bowel disease. Here we studied the effect of loss of Hh signaling on the small intestinal epithelium. We conditionally deleted Ihh by injecting naphtoflavone in Cyp1a1-Cre*Ihhfl/fl mice. Deletion of Ihh was confirmed by immunohistochemistry for Ihh and quantitative RT-PCR for targets of Hh signaling. The phenotype of Ihh mutant mice was examined using routine histological and immunohistochemical techniques. We found that loss of Hedgehog signaling initially caused a regenerative response of the intestinal epithelium with deepening and fissioning of the crypts and lengthening of the villi. Profound morphological changes were found in the intestinal subepithelial myofibroblasts and smooth muscle cells in the villus core which lost their elongated appearance and rolled up into little balls. Later time points were characterized by complete loss of myofibroblasts and smooth muscle cells from the villus core, loss of small intestinal villi and the development of a chronic inflammatory infiltrate. In chronic intestinal inflammation epithelial regeneration is generally believed to result from epithelial damage by the inflammatory process. Our results show that epithelial regeneration and chronic intestinal inflammation may be separate processes and suggest that we should reappraise our thinking of cause and consequence in chronic intestinal inflammation and epithelial regeneration. Our data provide further evidence for a potential link between aberrant Hh signaling and the development of inflammatory bowel disease.
85 Constitutive In Vivo Expression of TNFSF15 in Myeloid or Lymphoid Cells Induce Mild Small Bowel Inflammation in Mice David Q. Shih, Robert Barrett, Xiaolan Zhang, Brian Ko, Michelle H. Wong, Piangwarin Phaosawasdi, Nicole Yeager, Gislaine Martins, Hidetoshi Takedatsu, Kathrin S. Michelsen, Stephan R. Targan Background: TL1A is the product of the TNFSF15 gene and is expressed by both lymphoid and myeloid derived cells. A role of TL1A in gut mucosal inflammation is highlighted by the finding that neutralizing TL1A antibody prevented and treated chronic colitis in mice. The contribution of either lymphoid or myeloid derived TL1A to the development of gut inflammation is not known. Aim: To investigate the In Vivo role of TL1A in myeloid and lymphoid cells. Methods: Transgenic (Tg) mice with constitutive TL1A expression in myeloid or lymphoid cells were generated using either FMS or LCK-CD2 promoter, respectively. TL1A expressing cells are marked by GFP using an IRES element in the Tg construct. Histological analyses of H&E stained gut sections were analyzed for inflammation in a blinded fashion. mRNA was measured by qtPCR and protein expression was measured by flow cytometry. Results: Progeny from both myeloid and lymphoid Tg mice were born healthy but at lower than expected Mendelian frequency when Tg were crossed to WT mice (myeloid 26/63, 41%; lymphoid 16/44, 36%; expected 50%). Specificity of TL1A overexpression in myeloid or lymphoid Tg mice is confirmed by both expression analysis and green florescence in myeloid or lymphoid cells, respectively. Lymphoid, but not myeloid, Tg mice are approximately 10% lighter in weight compared to WT littermates. Overexpression of TL1A in lymphoid or myeloid cells did not alter the proportion of macrophages, dendritic cells, CD4+ or CD8+ T cells. Compared to WT mice, lymphoid Tg mice showed increased
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AGA Abstracts
regulatory T-cells (Tregs) or Tregs obtained from the spleens of DKO mice to evaluate the ability of these Tregs to suppress the development of chronic gut inflammation. We found, using blinded histopathological analysis, that adoptive transfer of WT T-cells induced moderate-to-severe colitis in RAG-/- recipients at 8 wks post transfer whereas transfer of DKO Tcells induced little or no disease. The inability to induce disease by the DKO T-cells was not due to a delay in the onset of disease as we continued to observe significantly less disease even at 25 wks post transfer. Furthermore, failure to induce disease correlated well with large and significant decreases in the numbers of T-cells within the mesenteric lymph nodes (MLNs) and colonic lamina propria (cLP) suggesting that T-cell activation and/or trafficking may be defective in the DKO T-cells. In order to differentiate between these two possibilities we evaluated T-cell activation and trafficking In Vivo using adoptive transfer of congenic CSFE-labeled WT (CD45.1) and DKO (CD45.2) T-cells (107 cells for each) into RAG-/- mice. At 7 days following transfer we observed 12- and 4-fold fewer T-cells within the MLNs and cLP, respectively of the DKO→RAG-/- mice vs. WT→RAG-/- animals. In addition, we observed that both WT and DKO T-cells in the cLP generated similar amounts of IFN-γ and IL-17 and lost equivalent amounts of CFSE fluorescence. These data suggested that CD62L and β7 are not required for T-cell activation and polarization In Vivo. In the second series of studies, we found that DKO Tregs were substantially less effective than WT Tregs at suppressing the development of chronic colitis when co-transferred with WT CD4+CD4RBhigh T-cells. Taken together, our data suggest that both L-selectin and β7 are required for the T-cell-dependent induction and suppression of chronic colitis (NIHPO1 47385).
84 Mitochondrial Stress Induction in the Epithelium Fuels Endoplasmic Reticulum Unfolded Protein Responses via Double-Stranded RNA-Activated Protein Kinase (PKR) Under Conditions of Chronic Intestinal Inflammation Eva Rath, Emanuel Berger, Anja Messlik, Tobias Fromme, Martin Klingenspor, Regina Ensenauer, Nicholas J. Hoogenraad, Bo Liu, Sandra C. Kim, R. Balfour Sartor, Dirk Haller Background & Aim. Recently it has been shown that Endoplasmic reticulum (ER) stress responses in intestinal epithelial cells (IEC) contribute to the initiation and perpetuation of chronic intestinal inflammation. The aim of this study was to characterize the role of mitochondrial stress responses in the intestinal epithelium of patients with ulcerative colitis (UC) and two murine models of chronic, T cell-mediated colitis. Methods & Results. Primary IEC were isolated from an adoptive transfer model of colitis, recombination activating gene deficient (RAG2-/-) and RAG2-/- x Interleukin 10 deficient (IL-10-/-) mice reconstituted with CD4+ T cells, as well as gnotobiotic IL-10-/- mice mono- or dual associated with colitogenic E. coli and E. faecalis. Proteome analysis of primary IEC using 2D-SDS PAGE and MALDI-TOF mass spectrometry identified 160 differentially regulated proteins clustered around the mitochondrial stress chaperonin 60 (CPN60) linking ER and mitochondrial unfolded protein response (UPR) under conditions of chronic inflammation. To study the specific role of mitochondrial stress signaling we stimulated the IEC line Mode-K with the selective mt stress inducer truncated ornithine transcarbamylase (OTCΔ). Additionally, cells were treated with Tunicamycin (Tm) to induce ER-associated UPR. Under mt stress, the double-stranded RNA-activated protein kinase (PKR) was identified to be selectively induced at the mRNA- as well as protein level. Subsequent chromatin immunoprecipitation (ChIP) analysis revealed recruitment of the transcription factor (TF) AP-1 to the PKR promoter. In addition and most importantly for the understanding of mt stress integration into the ER signaling pathway, PKR co-immunoprecipitated with eIF2α pointing towards a direct role of PKR in eIF2α phosphorylation after selective mt stress induction. Furthermore, both mtand ER UPR converged in the recruitment of TF CHOP to the Cpn60 promoter. Interestingly, mt respiration and β-oxidation were reduced under ER- as well as mt stress, suggesting metabolic changes in IEC under chronic inflammation. Finally, Western blot analysis of primary IEC from UC patients and murine models of colitis confirmed that increased expression levels of GRP78 and CPN60 were associated with an induction of PKR. Conclusion. Primary IEC from both human UC patients and mouse models of colitis show signs of activated mt stress signaling. Our data demonstrate that mt stress responses enhance ERassociated UPR through PKR induction, eIF2α phosphorylation and CHOP recruitment to the Cpn60 promoter. In conclusion, mt stress fuels pathology-relevant ER stress mechanisms under chronic inflammatory conditions.
87 Conditional Loss of Intestinal Hedgehog Signaling Results in a Regenerative Response and Subsequently in Loss of Villi and the Spontaneous Development of Chronic Enteritis Willemijn A. van Dop, Elisabeth A. Wassenberg, Beate Lanske, Alan R. Clarke, Johan Offerhaus, Daniel W. Hommes, James C. Hardwick, Izak Biemond, Gijs R. van den Brink Indian hedgehog (Ihh) is expressed by differentiated intestinal epithelial cells and acts on the subepithelial myofibroblasts. We previously found that this epithelial mesenchymal interaction is part of a negative feedback loop from the differentiated cells to the intestinal epithelial precursor cells. Genetic variants in the hedgehog pathway have recently been linked to the development of inflammatory bowel disease. Here we studied the effect of loss of Hh signaling on the small intestinal epithelium. We conditionally deleted Ihh by injecting naphtoflavone in Cyp1a1-Cre*Ihhfl/fl mice. Deletion of Ihh was confirmed by immunohistochemistry for Ihh and quantitative RT-PCR for targets of Hh signaling. The phenotype of Ihh mutant mice was examined using routine histological and immunohistochemical techniques. We found that loss of Hedgehog signaling initially caused a regenerative response of the intestinal epithelium with deepening and fissioning of the crypts and lengthening of the villi. Profound morphological changes were found in the intestinal subepithelial myofibroblasts and smooth muscle cells in the villus core which lost their elongated appearance and rolled up into little balls. Later time points were characterized by complete loss of myofibroblasts and smooth muscle cells from the villus core, loss of small intestinal villi and the development of a chronic inflammatory infiltrate. In chronic intestinal inflammation epithelial regeneration is generally believed to result from epithelial damage by the inflammatory process. Our results show that epithelial regeneration and chronic intestinal inflammation may be separate processes and suggest that we should reappraise our thinking of cause and consequence in chronic intestinal inflammation and epithelial regeneration. Our data provide further evidence for a potential link between aberrant Hh signaling and the development of inflammatory bowel disease.
85 Constitutive In Vivo Expression of TNFSF15 in Myeloid or Lymphoid Cells Induce Mild Small Bowel Inflammation in Mice David Q. Shih, Robert Barrett, Xiaolan Zhang, Brian Ko, Michelle H. Wong, Piangwarin Phaosawasdi, Nicole Yeager, Gislaine Martins, Hidetoshi Takedatsu, Kathrin S. Michelsen, Stephan R. Targan Background: TL1A is the product of the TNFSF15 gene and is expressed by both lymphoid and myeloid derived cells. A role of TL1A in gut mucosal inflammation is highlighted by the finding that neutralizing TL1A antibody prevented and treated chronic colitis in mice. The contribution of either lymphoid or myeloid derived TL1A to the development of gut inflammation is not known. Aim: To investigate the In Vivo role of TL1A in myeloid and lymphoid cells. Methods: Transgenic (Tg) mice with constitutive TL1A expression in myeloid or lymphoid cells were generated using either FMS or LCK-CD2 promoter, respectively. TL1A expressing cells are marked by GFP using an IRES element in the Tg construct. Histological analyses of H&E stained gut sections were analyzed for inflammation in a blinded fashion. mRNA was measured by qtPCR and protein expression was measured by flow cytometry. Results: Progeny from both myeloid and lymphoid Tg mice were born healthy but at lower than expected Mendelian frequency when Tg were crossed to WT mice (myeloid 26/63, 41%; lymphoid 16/44, 36%; expected 50%). Specificity of TL1A overexpression in myeloid or lymphoid Tg mice is confirmed by both expression analysis and green florescence in myeloid or lymphoid cells, respectively. Lymphoid, but not myeloid, Tg mice are approximately 10% lighter in weight compared to WT littermates. Overexpression of TL1A in lymphoid or myeloid cells did not alter the proportion of macrophages, dendritic cells, CD4+ or CD8+ T cells. Compared to WT mice, lymphoid Tg mice showed increased
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AGA Abstracts
AGA Abstracts
regulatory T-cells (Tregs) or Tregs obtained from the spleens of DKO mice to evaluate the ability of these Tregs to suppress the development of chronic gut inflammation. We found, using blinded histopathological analysis, that adoptive transfer of WT T-cells induced moderate-to-severe colitis in RAG-/- recipients at 8 wks post transfer whereas transfer of DKO Tcells induced little or no disease. The inability to induce disease by the DKO T-cells was not due to a delay in the onset of disease as we continued to observe significantly less disease even at 25 wks post transfer. Furthermore, failure to induce disease correlated well with large and significant decreases in the numbers of T-cells within the mesenteric lymph nodes (MLNs) and colonic lamina propria (cLP) suggesting that T-cell activation and/or trafficking may be defective in the DKO T-cells. In order to differentiate between these two possibilities we evaluated T-cell activation and trafficking In Vivo using adoptive transfer of congenic CSFE-labeled WT (CD45.1) and DKO (CD45.2) T-cells (107 cells for each) into RAG-/- mice. At 7 days following transfer we observed 12- and 4-fold fewer T-cells within the MLNs and cLP, respectively of the DKO→RAG-/- mice vs. WT→RAG-/- animals. In addition, we observed that both WT and DKO T-cells in the cLP generated similar amounts of IFN-γ and IL-17 and lost equivalent amounts of CFSE fluorescence. These data suggested that CD62L and β7 are not required for T-cell activation and polarization In Vivo. In the second series of studies, we found that DKO Tregs were substantially less effective than WT Tregs at suppressing the development of chronic colitis when co-transferred with WT CD4+CD4RBhigh T-cells. Taken together, our data suggest that both L-selectin and β7 are required for the T-cell-dependent induction and suppression of chronic colitis (NIHPO1 47385).