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Tu1942 Collagen Proteolysis is Important in Neutrophilic Intestinal AutoInflammation Found in Inflammatory Bowel Disease via Proline-Glycine-Proline
ELISA. PGP neutralization was realized by using the PGP antagonist, argine-threoninearginine (RTR), and specific antibodies. Protease release from neutrophils isolated from IBD patients and healthy controls was measured and neutrophil-conditioned medium was incubated with collagen type I to study degradation into PGP. Results: In the intestine of IBD patients, MMP-8 and MMP-9 were elevated in inflamed areas, while PE and PGP were both expressed at similar levels in non-inflamed areas and/or control tissue. Neutrophils isolated from IBD patients showed increased release of both MMPs and were far more capable of generating PGP from collagen compared to healthy control neutrophils. In the inflamed intestine of DSS treated mice, similar results were found. Furthermore, PGP neutralization during DSS colitis lead to a significant reduction of intestinal inflammation, especially in the infiltration of neutrophils. Conclusion: The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of IBD patients and DSS treated mice. PGP neutralization in DSS treated mice showed that PGP guided neutrophilic infiltration plays an important role in intestinal inflammation, and opens new venues in treating the ongoing inflammation of IBD patients.
Cigarette Smoke Improves Selectively Colon and Not Small Bowel Inflammation Through NKT Cell Activation Muriel Montbarbon, Muriel Pichavant, Audrey Langlois, Edmone Erdual, Sebastien Dharancy, Laurent Dubuquoy, François Trottein, Pierre Desreumaux, Philippe Gosset, Benjamin Bertin INTRODUCTION Epidemiological studies strongly linked cigarette smoking and inflammatory bowel diseases (IBD). Smoking protects against ulcerative colitis but increases the risk of developing Crohn's disease. The mechanisms sustaining the dual effects of cigarette smoke (CS) in IBD remain unknown. AIM To compare the effect of CS on small bowel and colon inflammation in mice and to characterize the mechanisms of action at cellular and molecular levels. METHODS C57BL/6 mice and NKT deficient mice (Jα18KO and CD1dKO) were exposed during 3 weeks to CS of 5 cigarettes/days using the InExpose® exposure system (Scireq Inc). Colitis abd ileitis were induced respectively by administration of 2.5 % DSS in drinking water or indomethacin (100mg/kg s.c.). Intestinal inflammation was assessed by clinical scores, quantification of the recruitment of cells in the gut by flow cytometry analysis and evaluation of intestinal cytokines by quantitative RT-PCR and ELISA. RESULTS In wild-type mice, CS exposure improved selectively DSS-induced colitis and not indomethacininduced iletitis, leading to an improvement of body weight loss (n=30; p<0.0001), clinical score (p=0.01) and weight/length colon ratio (p<0.0001)). This colonic improvement was associated with a significant decrease in colonic TNF (p=0.0169), IFNγ (p<0.0001) and IL22 (p=0.0016) mRNA expression together with an increased expression of IL-10 mRNA (p= 0.0035). CS induced a recruitment of activated Natural Killer T cells (CD45+ TCRβ+ CD1d tetramer+) in the colon without any modification of neutrophil infiltration (CD45+ CD11cLy6G+) and activation (MPO activity). Demonstration of the roles of NKT cells in CSinduced colitis improvement was performed using 2 different strains of NKT deficient mice. Indeed, in Jα18KO and CD1dKO animals, CS exposure failed to induce significant regulation of DSS-induced colitis both at the clinical and molecular levels. Restoration of the CS regulation on DSS-induced colitis was obtained in Jα18KO animals after adoptive transfer of WT NKT cells exposed to CS. CONCLUSION The regulatory role of CS on intestinal inflammation is more pronounced in the colon than the small bowel in mice. This protective effect is mediated at least in part through the colonic recruitment and activation of NKT cells.
Tu1943 Regulation of Anergy-Related E3 Ubiquitin Ligase GRAIL by Mir-290-5p in Inflammatory Bowel Diseases Akira Mukai, Hideki Iijima, Eri Shiraishi, Satoshi Hiyama, Takahiro Inoue, Sachiko Nakajima, Shinichiro Shinzaki, Masahiko Tsujii, Tetsuo Takehara [Background]Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of Crohn's disease(CD). Anergy of CD4+ T cells is one of the main mechanisms of tolerance and is regulated by an E3 ubiquitin ligase, GRAIL (gene related to anergy in lymphocyte). It is unknown, however, if GRAIL expression is altered in CD patients and how GRAIL expression is regulated. [Aim]We aimed to investigate the expression of GRAIL in patients with CD and mouse models of colitis. We also investigated the regulation of GRAIL by microRNA. [Materials and methods]CD4+ T cells isolated from the peripheral blood mononuclear cells of 37 patients with CD and 22 healthy volunteers (HV) hospitalized in Osaka University Hospital. CD patients included 17 active patients whose mean Crohn's disease active index (CDAI) was 251.1. GRAIL protein expression in the colon was investigated by immunohistochemistry. Experimental colitis was examined in mice induced by dextran sodium sulfate and in IL-10 deficient mice. RNA interference microRNA array was performed using murine CD4+ T cells isolated from lamia propria and spleen of colitis mice. [Results]In peripheral blood CD4+ T cells, both mRNA and protein levels of GRAIL were lower in patients with CD than HV. In contrast, GRAIL protein expression was higher in inflamed colon of CD patients than in the colon of HV. Similarly, protein level of GRAIL was higher in the colonic lamina propria lymphocytes of colitic mice than non-colitic mice while mRNA expression of GRAIL was higher in colitic mice than non-colitic mice. In colitic mice, there was a discrepancy between mRNA and protein levels in the colon but not in other peripheral lymphoid organs. We performed miRNA array and revealed that miR-2905p which can potentially regulate GRAIL was decreased in lamia propria lymphocytes of colitic mice compare to those of wild-type mice. [Conclusion]GRAIL expression is suggested to be regulated by miRNA in the colon. Regulation of anergy-related molecule by miRNA may be a novel mechanism to control intestinal inflammation via alteration of immune status of CD4+ T cells in IBD.
Tu1941 Adoptive Transfer of Heme Oxygenase-1 Highly Expressed Macrophage Ameliorated TNBS-Induced Colitis in Mice Akihito Harusato, Yuji Naito, Tomohisa Takagi, Kazuhiko Uchiyama, Katsura Mizushima, Yasuko Hirai, Toshifumi Tsuji, Wataru Fukuda, Hiroyuki Yoriki, Munehiro Kugai, Ryusuke Horie, Akifumi Fukui, Takaya Iida, Kazuhiro Katada, Osamu Handa, Hiroshi Ichikawa, Akihiko Muto, Kazuhiko Igarashi, Toshikazu Yoshikawa Background and Aims: In the host innate immune system, macrophages play an important role in the regulation of inflammation in the gut. BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1), which plays a crucial role in the protection of cells/tissues in inflammation. Macrophages derived from Bach1 deficient mice have an enhanced expression of HO-1, and are considered to have an anti-inflammatory property. The aim of this study was to elucidate the role of Bach1 deficient HO-1 highly expressed macrophages in intestinal inflammation by using experimental colitis model. Methods: C57BL/6 (wild-type) and homozygous Bach1 deficient C57BL/6 mice were used in the present study. We isolated peritoneal macrophages from the respective mice and analysed these macrophages by using western blotting and real-time PCR. Adoptive transfer of macrophages was performed by injecting with these peritoneal macrophages into wild-type mice, and then acute colitis was induced by an enema administration of 2,4,6-trinitrobenzine sulfonic acid (TNBS). The extent of colitis was evaluated macroscopically, histologically, and biochemically. Results: The expression of HO-1 mRNA and protein in isolated peritoneal macrophages derived from Bach1-deficient mice were significantly increased as compared to macrophages derived from wild-type mice. The mRNA expression of M2 macrophage markers such as Arginase-1, Fizz1, Ym1 and MRC1 are up-regulated in the macrophages derived from Bach1 deficient mice. TNBS-induced colonic mucosal injuries were remarkably ameliorated by transferring the Bach1 deficient M2 macrophages into wild-type mice. Tissue MPO activity levels were also significantly decreased by transferring the Bach1 deficient macrophages. Administration of TNBS induced expression of cytokines including KC, TNFα and IFN-γ, which was suppressed by transferring the Bach1 deficient macrophages. Conclusion: Macrophages derived from Bach1 deficient mice were M2 macrophages which are considered to have an anti-inflammatory functions indicating that HO-1 highly expressed macrophages could be a candidate to treat Inflammatory Bowel Diseases.
Tu1944 TLR7 Ligand Amelioretes TNBS Colitis by Induction of CCR9 and Accumulation of Regulatory T Cells Kyoko Katakura, Ryoma Suzuki, Tatsuo Fujiwara, Naohiko Gunji, Hiromasa Ohira Background & Aims: We have previously demonstrated that activation of TLR9 by commensal bacteria has an essential role in maintaining colonic homeostasis. TLR7 agonist Imiquimod (IMQ) has been commercially tested, and same as TLR9 ligand, agonistic stimulation of TLR7 in plasmacytoid dendritic cells (pDCs) results in type-1 IFN production. Here, we investigated whether IMQ protects mice from colonic inflammation and what mechanisms would be lying in such immunoregulatory conditions. Methods: Murine colitis was induced to BALB/c mice by administration of trinitrobenzene sulfonic acid (TNBS) with or without daily intraperitoneal administration of IMQ. Colitis was evaluated by body-weight decreasing and histological score. Spleen and mesenteric lymph nodes (MLN) from IMQ treated mice were collected, and mRNA expressions were measured by RT-PCR and cytokine productions were measured by ELISA. Regulatory T cells (Tregs) and chemokine expression were analyzed in TNBS colon by immunohistochemistry. To confirm the induction of Tregs by type-1 IFN from pDC, we generated mouse bone marrow derived pDC and co-cultured with CD4+T cells isolated from mouse spleen with or without IMQ stimulation. Cytokine productions in the culture supernatant were measured by ELISA. Results: Administration of IMQ significantly suppressed colonic inflammation of TNBS-induced colitis. In colon of IMQ treated mice, mRNA expression of TNF-α was decreased and strong expressions of IL-6, IFN-β and TGFβ were detected. In MLN of IMQ treated mice, strong expressions of TLR7, IFN-β, TGF-β and Foxp3 mRNA were detected. In the inflamed colon, Tregs and CCR9 were detected. IL-10 production from MLN cells stimulated with αCD3/αCD28 antibody was increased in IMQ treated group. IL-10 and TGF-β production were increased in the co-cultured supernatant stimulated with IMQ, although we couldn't detect CD4+CD25+Foxp3+Tregs induction in IMQ stimulated co-cultured cells. Conclusion: These results suggest that IMQ protects mice from TNBS colitis through induction of CCR9 which regulated accumulation of Tregs to inflamed colon. IMQ would offer a novel tool for the treatment in inflammatory bowel disease.
Tu1942 Collagen Proteolysis is Important in Neutrophilic Intestinal AutoInflammation Found in Inflammatory Bowel Disease via Proline-GlycineProline Pim J. Koelink, Saskia A. Overbeek, Saskia Braber, Mary E. Morgan, Paul A. Henricks, Hein W. Verspaget, Simone C. Wolfkamp, Anje A. Te Velde, Patricia L. Jackson, J. Edwin Blalock, Johan Garssen, Gert Folkerts, Aletta D. Kraneveld Background & Aims: Recently, a collagen derived fragment, Proline-Glycine-Proline (PGP), was shown to have chemotactic properties on neutrophils via CXCR2 in several lung diseases. PGP is formed from collagen by the combined action of matrix metalloproteinase (MMP)8 and/or MMP-9 and prolyl endopeptidase (PE). We aimed to investigate the role of PGP in inflammatory bowel disease (IBD). Methods: MMP-8, MMP-9 and PE were detected in intestinal tissue of human IBD patients, colorectal cancer controls, and mice undergoing Dextran Sodium Sulfate (DSS)-induced colitis by ELISA, immunoblot and immunohistochemistry. PGP levels were measured by mass spectrometry in intestinal tissue homogenates and neutrophil infiltration was assessed by immunohistochemistry and myeloperoxidase
S-883
AGA Abstracts
AGA Abstracts
Tu1940
Cigarette Smoke Improves Selectively Colon and Not Small Bowel Inflammation Through NKT Cell Activation Muriel Montbarbon, Muriel Pichavant, Audrey Langlois, Edmone Erdual, Sebastien Dharancy, Laurent Dubuquoy, François Trottein, Pierre Desreumaux, Philippe Gosset, Benjamin Bertin INTRODUCTION Epidemiological studies strongly linked cigarette smoking and inflammatory bowel diseases (IBD). Smoking protects against ulcerative colitis but increases the risk of developing Crohn's disease. The mechanisms sustaining the dual effects of cigarette smoke (CS) in IBD remain unknown. AIM To compare the effect of CS on small bowel and colon inflammation in mice and to characterize the mechanisms of action at cellular and molecular levels. METHODS C57BL/6 mice and NKT deficient mice (Jα18KO and CD1dKO) were exposed during 3 weeks to CS of 5 cigarettes/days using the InExpose® exposure system (Scireq Inc). Colitis abd ileitis were induced respectively by administration of 2.5 % DSS in drinking water or indomethacin (100mg/kg s.c.). Intestinal inflammation was assessed by clinical scores, quantification of the recruitment of cells in the gut by flow cytometry analysis and evaluation of intestinal cytokines by quantitative RT-PCR and ELISA. RESULTS In wild-type mice, CS exposure improved selectively DSS-induced colitis and not indomethacininduced iletitis, leading to an improvement of body weight loss (n=30; p<0.0001), clinical score (p=0.01) and weight/length colon ratio (p<0.0001)). This colonic improvement was associated with a significant decrease in colonic TNF (p=0.0169), IFNγ (p<0.0001) and IL22 (p=0.0016) mRNA expression together with an increased expression of IL-10 mRNA (p= 0.0035). CS induced a recruitment of activated Natural Killer T cells (CD45+ TCRβ+ CD1d tetramer+) in the colon without any modification of neutrophil infiltration (CD45+ CD11cLy6G+) and activation (MPO activity). Demonstration of the roles of NKT cells in CSinduced colitis improvement was performed using 2 different strains of NKT deficient mice. Indeed, in Jα18KO and CD1dKO animals, CS exposure failed to induce significant regulation of DSS-induced colitis both at the clinical and molecular levels. Restoration of the CS regulation on DSS-induced colitis was obtained in Jα18KO animals after adoptive transfer of WT NKT cells exposed to CS. CONCLUSION The regulatory role of CS on intestinal inflammation is more pronounced in the colon than the small bowel in mice. This protective effect is mediated at least in part through the colonic recruitment and activation of NKT cells.
Tu1943 Regulation of Anergy-Related E3 Ubiquitin Ligase GRAIL by Mir-290-5p in Inflammatory Bowel Diseases Akira Mukai, Hideki Iijima, Eri Shiraishi, Satoshi Hiyama, Takahiro Inoue, Sachiko Nakajima, Shinichiro Shinzaki, Masahiko Tsujii, Tetsuo Takehara [Background]Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of Crohn's disease(CD). Anergy of CD4+ T cells is one of the main mechanisms of tolerance and is regulated by an E3 ubiquitin ligase, GRAIL (gene related to anergy in lymphocyte). It is unknown, however, if GRAIL expression is altered in CD patients and how GRAIL expression is regulated. [Aim]We aimed to investigate the expression of GRAIL in patients with CD and mouse models of colitis. We also investigated the regulation of GRAIL by microRNA. [Materials and methods]CD4+ T cells isolated from the peripheral blood mononuclear cells of 37 patients with CD and 22 healthy volunteers (HV) hospitalized in Osaka University Hospital. CD patients included 17 active patients whose mean Crohn's disease active index (CDAI) was 251.1. GRAIL protein expression in the colon was investigated by immunohistochemistry. Experimental colitis was examined in mice induced by dextran sodium sulfate and in IL-10 deficient mice. RNA interference microRNA array was performed using murine CD4+ T cells isolated from lamia propria and spleen of colitis mice. [Results]In peripheral blood CD4+ T cells, both mRNA and protein levels of GRAIL were lower in patients with CD than HV. In contrast, GRAIL protein expression was higher in inflamed colon of CD patients than in the colon of HV. Similarly, protein level of GRAIL was higher in the colonic lamina propria lymphocytes of colitic mice than non-colitic mice while mRNA expression of GRAIL was higher in colitic mice than non-colitic mice. In colitic mice, there was a discrepancy between mRNA and protein levels in the colon but not in other peripheral lymphoid organs. We performed miRNA array and revealed that miR-2905p which can potentially regulate GRAIL was decreased in lamia propria lymphocytes of colitic mice compare to those of wild-type mice. [Conclusion]GRAIL expression is suggested to be regulated by miRNA in the colon. Regulation of anergy-related molecule by miRNA may be a novel mechanism to control intestinal inflammation via alteration of immune status of CD4+ T cells in IBD.
Tu1941 Adoptive Transfer of Heme Oxygenase-1 Highly Expressed Macrophage Ameliorated TNBS-Induced Colitis in Mice Akihito Harusato, Yuji Naito, Tomohisa Takagi, Kazuhiko Uchiyama, Katsura Mizushima, Yasuko Hirai, Toshifumi Tsuji, Wataru Fukuda, Hiroyuki Yoriki, Munehiro Kugai, Ryusuke Horie, Akifumi Fukui, Takaya Iida, Kazuhiro Katada, Osamu Handa, Hiroshi Ichikawa, Akihiko Muto, Kazuhiko Igarashi, Toshikazu Yoshikawa Background and Aims: In the host innate immune system, macrophages play an important role in the regulation of inflammation in the gut. BTB and CNC homolog 1 (Bach1) is a transcriptional repressor of heme oxygenase-1 (HO-1), which plays a crucial role in the protection of cells/tissues in inflammation. Macrophages derived from Bach1 deficient mice have an enhanced expression of HO-1, and are considered to have an anti-inflammatory property. The aim of this study was to elucidate the role of Bach1 deficient HO-1 highly expressed macrophages in intestinal inflammation by using experimental colitis model. Methods: C57BL/6 (wild-type) and homozygous Bach1 deficient C57BL/6 mice were used in the present study. We isolated peritoneal macrophages from the respective mice and analysed these macrophages by using western blotting and real-time PCR. Adoptive transfer of macrophages was performed by injecting with these peritoneal macrophages into wild-type mice, and then acute colitis was induced by an enema administration of 2,4,6-trinitrobenzine sulfonic acid (TNBS). The extent of colitis was evaluated macroscopically, histologically, and biochemically. Results: The expression of HO-1 mRNA and protein in isolated peritoneal macrophages derived from Bach1-deficient mice were significantly increased as compared to macrophages derived from wild-type mice. The mRNA expression of M2 macrophage markers such as Arginase-1, Fizz1, Ym1 and MRC1 are up-regulated in the macrophages derived from Bach1 deficient mice. TNBS-induced colonic mucosal injuries were remarkably ameliorated by transferring the Bach1 deficient M2 macrophages into wild-type mice. Tissue MPO activity levels were also significantly decreased by transferring the Bach1 deficient macrophages. Administration of TNBS induced expression of cytokines including KC, TNFα and IFN-γ, which was suppressed by transferring the Bach1 deficient macrophages. Conclusion: Macrophages derived from Bach1 deficient mice were M2 macrophages which are considered to have an anti-inflammatory functions indicating that HO-1 highly expressed macrophages could be a candidate to treat Inflammatory Bowel Diseases.
Tu1944 TLR7 Ligand Amelioretes TNBS Colitis by Induction of CCR9 and Accumulation of Regulatory T Cells Kyoko Katakura, Ryoma Suzuki, Tatsuo Fujiwara, Naohiko Gunji, Hiromasa Ohira Background & Aims: We have previously demonstrated that activation of TLR9 by commensal bacteria has an essential role in maintaining colonic homeostasis. TLR7 agonist Imiquimod (IMQ) has been commercially tested, and same as TLR9 ligand, agonistic stimulation of TLR7 in plasmacytoid dendritic cells (pDCs) results in type-1 IFN production. Here, we investigated whether IMQ protects mice from colonic inflammation and what mechanisms would be lying in such immunoregulatory conditions. Methods: Murine colitis was induced to BALB/c mice by administration of trinitrobenzene sulfonic acid (TNBS) with or without daily intraperitoneal administration of IMQ. Colitis was evaluated by body-weight decreasing and histological score. Spleen and mesenteric lymph nodes (MLN) from IMQ treated mice were collected, and mRNA expressions were measured by RT-PCR and cytokine productions were measured by ELISA. Regulatory T cells (Tregs) and chemokine expression were analyzed in TNBS colon by immunohistochemistry. To confirm the induction of Tregs by type-1 IFN from pDC, we generated mouse bone marrow derived pDC and co-cultured with CD4+T cells isolated from mouse spleen with or without IMQ stimulation. Cytokine productions in the culture supernatant were measured by ELISA. Results: Administration of IMQ significantly suppressed colonic inflammation of TNBS-induced colitis. In colon of IMQ treated mice, mRNA expression of TNF-α was decreased and strong expressions of IL-6, IFN-β and TGFβ were detected. In MLN of IMQ treated mice, strong expressions of TLR7, IFN-β, TGF-β and Foxp3 mRNA were detected. In the inflamed colon, Tregs and CCR9 were detected. IL-10 production from MLN cells stimulated with αCD3/αCD28 antibody was increased in IMQ treated group. IL-10 and TGF-β production were increased in the co-cultured supernatant stimulated with IMQ, although we couldn't detect CD4+CD25+Foxp3+Tregs induction in IMQ stimulated co-cultured cells. Conclusion: These results suggest that IMQ protects mice from TNBS colitis through induction of CCR9 which regulated accumulation of Tregs to inflamed colon. IMQ would offer a novel tool for the treatment in inflammatory bowel disease.
Tu1942 Collagen Proteolysis is Important in Neutrophilic Intestinal AutoInflammation Found in Inflammatory Bowel Disease via Proline-GlycineProline Pim J. Koelink, Saskia A. Overbeek, Saskia Braber, Mary E. Morgan, Paul A. Henricks, Hein W. Verspaget, Simone C. Wolfkamp, Anje A. Te Velde, Patricia L. Jackson, J. Edwin Blalock, Johan Garssen, Gert Folkerts, Aletta D. Kraneveld Background & Aims: Recently, a collagen derived fragment, Proline-Glycine-Proline (PGP), was shown to have chemotactic properties on neutrophils via CXCR2 in several lung diseases. PGP is formed from collagen by the combined action of matrix metalloproteinase (MMP)8 and/or MMP-9 and prolyl endopeptidase (PE). We aimed to investigate the role of PGP in inflammatory bowel disease (IBD). Methods: MMP-8, MMP-9 and PE were detected in intestinal tissue of human IBD patients, colorectal cancer controls, and mice undergoing Dextran Sodium Sulfate (DSS)-induced colitis by ELISA, immunoblot and immunohistochemistry. PGP levels were measured by mass spectrometry in intestinal tissue homogenates and neutrophil infiltration was assessed by immunohistochemistry and myeloperoxidase
S-883
AGA Abstracts
AGA Abstracts
Tu1940