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Mo1931 Monocyte-Derived Macrophages Are Essential for the Resolution of Inflammation and Tissue Repair in Colitis
recently been identified to play an important role in immune mediated inflammatory diseases. AIM: The aim of this study was analyze a panel of innate immune response genes in the colonic mucosa from UC patients. PATIENTS AND METHODS: We studied a total of 60 patients with definitive diagnosis of UC (30 active and 30 in remission) and healthy control group (30 subjects) without endoscopic evidence of intestinal inflammation. In all groups, the gene expression of FCGR2A, TNFRSF14, DOK3, SNX20 and LAMP3 were measured by real-time polymerase chain reaction (RT-PCR) Expression of GAPDH a housekeeping gene was analyzed for normalization purposes and quality controls. For q-PCR assays quality control, determination of linearity and reproducibility was evaluated (VC<10%). The mRNA relative quantification of target genes was conducted using the LightCycler software 4.1, according to the 2-delta-delta Ct method. The statistical analysis was performed using SPSS version 17.0, using Kruskall Wallis non-parametric test. A P value <0.05 was considered statistically significant. RESULTS: The patients with active UC had significantly higher FCGR2A gene expression in mucosa compared to remission patients and controls (P= 0.035 and P=0.050). TNFRS14 mRNA expression was increased in patients with active UC compared to remission UC and normal controls (P=0.010 and P=0.000). The LAMP3 expression was increased in patients with active UC compared to UC patients in remission and the control group (P=0.020 and P =0.0005). Conversely, DOK3 gene expression was decreased in active and remission UC groups compared to normal controls (P=0.024 and P= 0.010). The SNX20 expression was decreased in patients with active UC compared to UC patients in remission and the normal control group (P=0.000 and P =0.00001). No statistical significant differences were found in association to FCGR2A, TNFRSF14, DOK3, SNX20 and LAMP3 gene expression and clinical characteristics such as age at diagnosis, extent of disease, extraintestinal manifestations, medical treatment and the need of surgery. CONCLUSION: This is the first depiction of the presence of FCGR2A, TNFRSF14, DOK3, SNX20 y LAMP3 genes in the colonic mucosa from patients with UC suggesting that these genes could be involved in the pathogenesis of UC.
Table 2. Acute experimental colitis
Mo1931 Monocyte-Derived Macrophages Are Essential for the Resolution of Inflammation and Tissue Repair in Colitis Michelle Stakenborg, Gera Goverse, Giovanna Farro, Pedro J. Gomez-Pinilla, Guy E. Boeckxstaens, Gianluca Matteoli Background: Monocyte recruitment in the gut wall via C-C chemokine receptor 2 (CCR2) is a major hallmark in the pathogenesis of inflammatory bowel disease (IBD). Classically, monocytes are considered as the main mediators of tissue damage during colitis. However, monocyte-derived macrophages may be essential for the resolution of inflammation. In the current study, we aim to identify the function of monocytes recruited during colitis and to define whether monocytes play a role in the induction of inflammation and/or are crucial for tissue repair. Methods: To study the contribution of monocytes, acute colitis was induced in wild type (WT, C57BL/6) and CCR2-/- mice by 2,5% dextran sodium sulfate (DSS) in drinking water for 5 days. Disease progression was assessed via a standardized disease activity index (DAI) including body weight loss, stool consistency and blood in the feces. To study the role of monocytes during chronic colitis, mice were subjected to 3 cycles of 2% DSS. Monocyte and neutrophil recruitment and macrophage differentiation in the colon were assessed by flow cytometry and cell sorting. Histological evaluation of colonic tissues was performed. Results: During acute DSS colitis (day 5), CCR2-/- mice displayed a reduced DAI and body weight loss as compared to WT mice indicating that inhibiting monocyte recruitment reduces tissue inflammation. On the contrary, during the recovery phase (day 10 to 14), CCR2-/- mice showed increased DAI and body weight loss compared to WT (24.5% vs 8.6%). Interestingly, the induction of chronic colitis confirmed that CCR2monocytes are crucial for the resolution of inflammation. During the 1st cycle of DSS, body weight loss in WT mice (6.8%) was 10-fold higher than in CCR2-/- animals (0.67%). Interestingly from day 10 onward, CCR2-/- mice continued to lose weight, showing altered stool consistency and blood in the feces. During the 2nd and 3rd subsequent DSS cycles, CCR2-/- mice failed to recover body weight and presented increased disease severity compared to WT. This phenotype in CCR2-/- mice correlated with increased colonic tissue alterations such as epithelial erosion, cell infiltration and fibrosis. Flow cytometry analysis showed increased accumulation of ROS-producing neutrophils in CCR2-/- mice (WT:9.5x10^5; CCR2-/-:37.6x10^5; p<0.05), while immune infiltrate in WT mice mainly consisted of differentiated MHCII+ macrophages expressing typical M2 markers such as Arg-1, IL-10, CD163, MRC1, Lyve1 and Stab1. Conclusion: Our data demonstrate a dual role of monocytes during colitis, inducing the inflammatory response during the first phase, and playing a crucial role in the resolution of inflammation and tissue repair at a later stage. Further understanding of the mechanisms leading to immune-regulation and mucosal repair is vitally important to improve treatment for patients suffering from IBD.
Mo1929 Critical Role of Chromogranin-A on Macrophage Intrinsic Apoptotic Pathway in Colitis: Human and Animal studies Nour Eissa, Mohammad F. Rabbi, Peris M. Munyaka, Azin Khafipour, Charles N. Bernstein, Jean-Eric Ghia Background: There is evidence in ulcerative colitis (UC) and murine models of colitis that Chromogranin-A (CgA), released by the enterochromaffin cells, is elevated, that macrophages are dysregulated and that an increase of apoptosis is detected within the mucosa. Apoptosis can result from an activation of the extrinsic and/or intrinsic pathways. We hypothesized that CgA is involved in the pathogenesis of colitis through the modulation of the macrophage apoptotic pathway.Aims: To determine the expression and correlation between CgA, classically activated (M1), alternatively activated (M2) macrophages and intrinsic, extrinsic proapoptotic markers using rectal biopsies from active UC, murine colitis and mouse peritoneal M1, M2 macrophages.Methods: mRNA levels of CgA, M1 markers (MCP-1, CD120b, TLR2, SLAMF7), M2 markers (C-MYC, MR, CD1b, IL-10), intrinsic (PUMA, BAD, BAX, BAK) and extrinsic (CASP8, TRAIL) apoptotic pathway and their correlations were determined in human rectal biopsies collected from healthy (n=9) and active UC patients (n=7). Colitis was induced in CgA-C57BL/6-deficient (CgA-/-) and wild type mice by dextran sulfate sodium (DSS 5%) for 5 days (n=12). Disease activity index (DAI), macro- and microscopic scores were determined. Myeloperoxidase (MPO) activity, CgA, TNF- a, IL-1b, M1 markers (iNOS, MCP-1, CCL3, CCL4), M2 markers (C-MYC, Arg-1, YM1, FIZZ1, IL-10), intrinsic and extrinsic apoptosis pathways were quantified (see above) in colon using ELISA/RT-qPCR. In vitro, CgA, M1, M2 and apoptotic pathways markers of polarized M1 & M2 peritoneal macrophages isolated from naïve CgA-/- and wild-type (WT) mice were determined.Results: In human active UC biopsies, there was a significant increase of CgA, M1 and intrinsic apoptotic markers, and a decrease of M2 markers and no changes in the extrinsic apoptosis pathway was detected. CgA showed a positive linear correlation with the M1 markers and the intrinsic apoptotic pathway, and a negative linear correlation with the M2 markers. In murine colitis (CgA-/-mice) DAI, macroscopic & histological damage scores, colonic MPO activity, TNF-a, IL-1b, M1 and intrinsic apoptosis markers were significantly decreased. Conversely, an up-regulation of IL-10 and M2 markers expression levels was detected. No changes in the extrinsic apoptotic markers were detected in mice. In non-colitic mice the CgA deficiency did not affect any inflammatory, M1, M2 or apoptotic markers. In vitro, when compared to WT, in colitic conditions IL-1 b, M1 and the intrinsic apoptotic markers were decreased, while M2 markers were increased in CgA-/-macrophages.Conclusions: CgA signaling is critical in the pathogenesis of inflammation in both the clinical and experimental setting through the modulation of the macrophage intrinsic apoptotic pathway. These findings may lead to novel therapeutic strategies in UC modulating CgA.
Mo1932 Titanium Dioxide Nanoparticles Promote Intestinal Inflammation: Role of the Inflammasome Pedro A. Ruiz, Belen Moron, Helen M. Becker, Silvia Lang, Kirstin Atrott, Marianne R. Spalinger, Michael Scharl, Kacper Wojtal, Anne Fischbeck-Terhalle, Isabelle Frey-Wagner, martin hausmann, Thomas Kraemer, Gerhard Rogler
Mo1930
Western life style and diet are major environmental factors playing a role in the onset of inflammatory bowel diseases (IBDs). Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations, and are consumed by millions of people on a daily basis. Mounting evidence suggest that TiO2 particles are implicated in a wide range of disease pathologies, including chronic inflammation. Since the NLRP3 inflammasome has been shown to mediate the deleterious effects of TiO2, we investigated the impact of orally administered TiO2 nanoparticles in wild type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis. Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2 administered mice. In vitro, TiO2 particles were taken up by intestinal epithelial cells (IECs) and macrophages, and triggered IL-1 bcleavage, caspase-1-ASC-NLRP3 assembly, and the release of NLRP3-associated interleukin (IL)-1 b
Transcriptome Analysis of Immune Innate Response Genes in the Colonic Mucosa from Patients With Ulcerative Colitis Gabriela Fonseca-Camarillo, Emilio Iturriaga-Goyon, Lucero A. Salazar-Salas, Jesus K. Yamamoto-Furusho INTRODUCTION: Ulcerative Colitis (UC) is caused by an aberrant immune response to environmental triggers in genetically susceptible individuals. The exact contribution of the adaptive immune system has not been elucidated. However, recent advances in discovery of new inflammatory mediators such as FCGR2A, TNFRSF14, DOK3, SNX20 y LAMP3 highlight the crucial role of the innate immune system in IBD. This panel of genes have
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AGA Abstracts
AGA Abstracts
Table 1. Chronic experimental colitis
Table 2. Acute experimental colitis
Mo1931 Monocyte-Derived Macrophages Are Essential for the Resolution of Inflammation and Tissue Repair in Colitis Michelle Stakenborg, Gera Goverse, Giovanna Farro, Pedro J. Gomez-Pinilla, Guy E. Boeckxstaens, Gianluca Matteoli Background: Monocyte recruitment in the gut wall via C-C chemokine receptor 2 (CCR2) is a major hallmark in the pathogenesis of inflammatory bowel disease (IBD). Classically, monocytes are considered as the main mediators of tissue damage during colitis. However, monocyte-derived macrophages may be essential for the resolution of inflammation. In the current study, we aim to identify the function of monocytes recruited during colitis and to define whether monocytes play a role in the induction of inflammation and/or are crucial for tissue repair. Methods: To study the contribution of monocytes, acute colitis was induced in wild type (WT, C57BL/6) and CCR2-/- mice by 2,5% dextran sodium sulfate (DSS) in drinking water for 5 days. Disease progression was assessed via a standardized disease activity index (DAI) including body weight loss, stool consistency and blood in the feces. To study the role of monocytes during chronic colitis, mice were subjected to 3 cycles of 2% DSS. Monocyte and neutrophil recruitment and macrophage differentiation in the colon were assessed by flow cytometry and cell sorting. Histological evaluation of colonic tissues was performed. Results: During acute DSS colitis (day 5), CCR2-/- mice displayed a reduced DAI and body weight loss as compared to WT mice indicating that inhibiting monocyte recruitment reduces tissue inflammation. On the contrary, during the recovery phase (day 10 to 14), CCR2-/- mice showed increased DAI and body weight loss compared to WT (24.5% vs 8.6%). Interestingly, the induction of chronic colitis confirmed that CCR2monocytes are crucial for the resolution of inflammation. During the 1st cycle of DSS, body weight loss in WT mice (6.8%) was 10-fold higher than in CCR2-/- animals (0.67%). Interestingly from day 10 onward, CCR2-/- mice continued to lose weight, showing altered stool consistency and blood in the feces. During the 2nd and 3rd subsequent DSS cycles, CCR2-/- mice failed to recover body weight and presented increased disease severity compared to WT. This phenotype in CCR2-/- mice correlated with increased colonic tissue alterations such as epithelial erosion, cell infiltration and fibrosis. Flow cytometry analysis showed increased accumulation of ROS-producing neutrophils in CCR2-/- mice (WT:9.5x10^5; CCR2-/-:37.6x10^5; p<0.05), while immune infiltrate in WT mice mainly consisted of differentiated MHCII+ macrophages expressing typical M2 markers such as Arg-1, IL-10, CD163, MRC1, Lyve1 and Stab1. Conclusion: Our data demonstrate a dual role of monocytes during colitis, inducing the inflammatory response during the first phase, and playing a crucial role in the resolution of inflammation and tissue repair at a later stage. Further understanding of the mechanisms leading to immune-regulation and mucosal repair is vitally important to improve treatment for patients suffering from IBD.
Mo1929 Critical Role of Chromogranin-A on Macrophage Intrinsic Apoptotic Pathway in Colitis: Human and Animal studies Nour Eissa, Mohammad F. Rabbi, Peris M. Munyaka, Azin Khafipour, Charles N. Bernstein, Jean-Eric Ghia Background: There is evidence in ulcerative colitis (UC) and murine models of colitis that Chromogranin-A (CgA), released by the enterochromaffin cells, is elevated, that macrophages are dysregulated and that an increase of apoptosis is detected within the mucosa. Apoptosis can result from an activation of the extrinsic and/or intrinsic pathways. We hypothesized that CgA is involved in the pathogenesis of colitis through the modulation of the macrophage apoptotic pathway.Aims: To determine the expression and correlation between CgA, classically activated (M1), alternatively activated (M2) macrophages and intrinsic, extrinsic proapoptotic markers using rectal biopsies from active UC, murine colitis and mouse peritoneal M1, M2 macrophages.Methods: mRNA levels of CgA, M1 markers (MCP-1, CD120b, TLR2, SLAMF7), M2 markers (C-MYC, MR, CD1b, IL-10), intrinsic (PUMA, BAD, BAX, BAK) and extrinsic (CASP8, TRAIL) apoptotic pathway and their correlations were determined in human rectal biopsies collected from healthy (n=9) and active UC patients (n=7). Colitis was induced in CgA-C57BL/6-deficient (CgA-/-) and wild type mice by dextran sulfate sodium (DSS 5%) for 5 days (n=12). Disease activity index (DAI), macro- and microscopic scores were determined. Myeloperoxidase (MPO) activity, CgA, TNF- a, IL-1b, M1 markers (iNOS, MCP-1, CCL3, CCL4), M2 markers (C-MYC, Arg-1, YM1, FIZZ1, IL-10), intrinsic and extrinsic apoptosis pathways were quantified (see above) in colon using ELISA/RT-qPCR. In vitro, CgA, M1, M2 and apoptotic pathways markers of polarized M1 & M2 peritoneal macrophages isolated from naïve CgA-/- and wild-type (WT) mice were determined.Results: In human active UC biopsies, there was a significant increase of CgA, M1 and intrinsic apoptotic markers, and a decrease of M2 markers and no changes in the extrinsic apoptosis pathway was detected. CgA showed a positive linear correlation with the M1 markers and the intrinsic apoptotic pathway, and a negative linear correlation with the M2 markers. In murine colitis (CgA-/-mice) DAI, macroscopic & histological damage scores, colonic MPO activity, TNF-a, IL-1b, M1 and intrinsic apoptosis markers were significantly decreased. Conversely, an up-regulation of IL-10 and M2 markers expression levels was detected. No changes in the extrinsic apoptotic markers were detected in mice. In non-colitic mice the CgA deficiency did not affect any inflammatory, M1, M2 or apoptotic markers. In vitro, when compared to WT, in colitic conditions IL-1 b, M1 and the intrinsic apoptotic markers were decreased, while M2 markers were increased in CgA-/-macrophages.Conclusions: CgA signaling is critical in the pathogenesis of inflammation in both the clinical and experimental setting through the modulation of the macrophage intrinsic apoptotic pathway. These findings may lead to novel therapeutic strategies in UC modulating CgA.
Mo1932 Titanium Dioxide Nanoparticles Promote Intestinal Inflammation: Role of the Inflammasome Pedro A. Ruiz, Belen Moron, Helen M. Becker, Silvia Lang, Kirstin Atrott, Marianne R. Spalinger, Michael Scharl, Kacper Wojtal, Anne Fischbeck-Terhalle, Isabelle Frey-Wagner, martin hausmann, Thomas Kraemer, Gerhard Rogler
Mo1930
Western life style and diet are major environmental factors playing a role in the onset of inflammatory bowel diseases (IBDs). Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations, and are consumed by millions of people on a daily basis. Mounting evidence suggest that TiO2 particles are implicated in a wide range of disease pathologies, including chronic inflammation. Since the NLRP3 inflammasome has been shown to mediate the deleterious effects of TiO2, we investigated the impact of orally administered TiO2 nanoparticles in wild type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis. Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2 administered mice. In vitro, TiO2 particles were taken up by intestinal epithelial cells (IECs) and macrophages, and triggered IL-1 bcleavage, caspase-1-ASC-NLRP3 assembly, and the release of NLRP3-associated interleukin (IL)-1 b
Transcriptome Analysis of Immune Innate Response Genes in the Colonic Mucosa from Patients With Ulcerative Colitis Gabriela Fonseca-Camarillo, Emilio Iturriaga-Goyon, Lucero A. Salazar-Salas, Jesus K. Yamamoto-Furusho INTRODUCTION: Ulcerative Colitis (UC) is caused by an aberrant immune response to environmental triggers in genetically susceptible individuals. The exact contribution of the adaptive immune system has not been elucidated. However, recent advances in discovery of new inflammatory mediators such as FCGR2A, TNFRSF14, DOK3, SNX20 y LAMP3 highlight the crucial role of the innate immune system in IBD. This panel of genes have
S-819
AGA Abstracts
AGA Abstracts
Table 1. Chronic experimental colitis