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Mo1786 ADAMDEC1: A Novel Molecule in Inflammation and Bowel Disease
Mo1788
ADAMDEC1: A Novel Molecule in Inflammation and Bowel Disease Nuala R. O'Shea, Thean Soon Chew, Gavin W. Sewell, Stuart L. Bloom, Andrew M. Smith, Anthony W. Segal
HIF Expression in the Lamina Propria Correlates With Histological Damage in Patients With Inflammatory Bowel Disease Jesus Cosin-Roger, Dolores Ortiz-Masia, Mario Manresa, Joaquin Hinojosa, Rafael Alos, Maria Jesus Nicolau-Ribera, Dolores Barrachina
Background: Innate immunity is attenuated in patients with Crohn's disease (CD) with impaired neutrophil recruitment to skin and bowel, delayed clearance of E. coli from the skin, and impaired secretion of pro-inflammatory cytokines from macrophages (Marks et al, 2006; Smith et al, 2009). The primary defect of acute inflammation results in failure to eradicate bacterial flora entering the bowel wall leading to the chronic granulomatous inflammation characteristic of CD. Microarray analysis of monocyte derived macrophage mRNA expression, confirmed by qPCR, revealed that ADAMDEC1 (ADAM like Decysin1) was under-expressed in 10% (6/60) of CD patients. ADAMDEC1, a Metalloprotease and Decysin, is part of a family of proteins involved in wound healing and tissue repair, and is almost exclusively expressed in macrophages, dendritic cells and the gastrointestinal tract. To determine the role of this protein we examined E. coli induced inflammation and Dextran Sodium Sulphate (DSS) colitis in Adamdec1 knockout (KO) mice. Method: In an acute colitis model, Adamdec1 KO mice were exposed to 2% DSS for 7 days. Controls, wild type (WT) litter mates, were age, weight and sex matched (n=11 per group). Clinical colitis scores (weight loss, PR blood, loose stool) were recorded daily. Histology was obtained from small and large bowels. For bacterial inflammation, 5x108 heat killed E. coli (HkEc) were injected subcutaneously (SC) into two sites on the backs of KO and WT mice (n=8 per group). Mice were weighed, injection sites inspected for ulceration and subcutaneous inflammatory nodules measured daily. Injection sites were excised at different times for histology and identification of infiltrating cells by FACS. Results: Adamdec1 KO mice were more susceptible to DSS colitis. They demonstrated higher clinical colitis scores with an earlier and more dramatic weight loss (p<0.001). A more florid inflammatory response was seen on histology. In response to a subcutaneous injection of E. coli, Adamdec1 KO mice had smaller inflammatory nodules and less ulceration at the injection sites after 48-72 hours, than WT mice (p<0.001). Conclusion: Mice lacking Adamdec1 develop a phenotype that closely mirrors that observed in patients with CD, an attenuated and delayed E.coli induced acute inflammatory response and an increased susceptibility to bowel inflammation. These results suggest ADAMDEC1 plays an important role in the acute inflammatory response to bacteria and has a protective role within the intestine; reduced levels may have a pivotal role in the development and persistence of CD. Studies are currently underway to further investigate the impaired cellular recruitment and potentially defective bacterial clearance at these early stages of inflammation in our model which could predispose to a more exuberant secondary response and chronic inflammation.
Introduction: Epithelial barrier function is impaired in the inflammatory bowel disease (IBD). Hypoxia and cytokines, key features of inflammation, modulate the activity of hypoxia inducible factor (HIF), a transcription factor related to the induction of genes involved in mucosal healing. We aim to determine the pattern of HIF expression in the intestinal mucosa of IBD patients. Patients and Methods: Both damaged and non-damaged mucosa from patients with ulcerative colitis (UC) and Crohn`s disease (CD) were obtained. Paraffin embedded tissues were used for histological analysis (score 1-4) and ki67 immunostaining (cellular proliferation, score 1-4). Presence of macrophages CD206+ (M2 phenotype), HIF1α and HIF-2α were analyzed by immunohistochemistry. Quantification of macrophages CD206+, HIF-1α and HIF-2α positive cells was performed counting positive cells in a total area of 0,152 mm2 (4 photographs 40X) using an inverted microscope. Results: Cellular proliferation was increased in damaged mucosa of UC (2.8±0.3) and CD (1.7±0.3) compared with non-damaged mucosa (1.3±0.3 and 1±0.05, respectively). A correlation was observed between mucosal damage and the number of macrophages in the lamina propria; the Spearman correlation coefficient was r=0.6181 (P=0.0185, n=14) for UC and r=0.6392 (P= 0.0187, n=13) for CD. HIF-1α immunostaining was observed in few epithelial cells and in some cells of the lamina propria. In contrast, an increased HIF-2α immunostaining was observed only in cells of the lamina propria of the damaged area of UC and CD. A quantitative analysis in the lamina propria showed higher expression of HIF-2α than HIF-1α both in UC (7.11±1.45 vs. 2.33±0.72 respectively) and in CD (6.50±1.38 vs. 2.00±0.54 respectively). Moreover, a correlation was observed between macrophages CD206+ and HIF-1α of the lamina propria (r=0.5636 P=0.023, n=16) and between macrophages CD206+ and HIF-2α (r=0.7864, P=0.0002, n=17) in patients with IBD. The number of HIF-1α positive cells of the lamina propria positively correlated with damage in UC; the Spearman correlation coefficient was r=0.9478 (P<0.0001, n=13). However, the number of HIF-2α positive cells correlated with mucosal damage in both, UC and CD; the Spearman correlation coefficient was r=0.7593 (P=0.0016, n=14) for UC and r=0.7412 (P=0.0058, n=12) for CD. Conclusion: The number of HIF-1α and HIF-2α positive cells in the lamina propria, which positively correlates with M2 phenotype of macrophages, increases with the severity of disease in the intestinal mucosa of patients with IBD. Mo1789
Mo1787
Immunoglobulin a Plasma Cell Depletion in the Small Bowel Mucosa of Common Variable Immune Deficiency (CVID) and Asplenic Patients With Decreased Circulating Memory B Cells Antonio Di Sabatino, Maria Manuela Rosado, Rita Carsetti, Simona Cascioli, Ezio Giorda, Marco Scarsella, Stefania Petrini, Cinzia Milito, Alessandra Pasini, Isabella Quinti, Gino R. Corazza
Nicotinamide Ameliorates the Course of Citrobacter rodentium-Induced Colitis Through Enhanced Bacterial Killing Dominik Bettenworth, Tobias M. Nowacki, Pierre Kyme, Matthias Ross, Nils H. Thoennissen, Jan Heidemann Background and Aims: The myeloid-specific transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) is crucial for the terminal differentiation and functional maturation of neutrophils and activation of downstream antimicrobial targets. Recently, we demonstrated that the Histon deacetylase inhibitor nicotinamide (NAM; vitamin B3) increases the transcriptional activity of C/EBPε by modulating the acetylation of its lysine residues which in turn can lead to enhanced killing of bacteria by the innate immune system (Thoennissen et al., abstract #925, ASH 2010). Aim of the present study was to assess the effect of NAM in Citrobacter rodentium-induced colitis and to elucidate its impact on bacterial killing In Vivo and ex vivo. Methods: C57BL/6 WT mice were orally gavaged with 5×108 colony-forming units of C. rodentium. From day 2 post infection onward, animals (n=9/group) were treated either with NAM (250 mg/kg i.p. daily), or with an equivalent volume of PBS as control. Fecal excretion of C. rodentium and changes of body weight were monitored routinely during the course of disease. Hematopoietic changes were serially analyzed by FACS analyses of peripheral blood. At day 12 post infection, animals were euthanized and the systemic spreading of C. rodentium was assessed by incubation of tissue homogenates (mesenterial lymph nodes, spleen). Moreover, inflammatory changes of the colon were evaluated histologically. In addition, killing of C. rodentium in NAM-stimulated murine and human peripheral whole blood was assessed using safe NAM doses ex vivo. Results: From day 10 until the end of experiment, the fecal excretion of C. rodentium in NAM-treated mice was markedly decreased by up to 4 log10 compared to PBS-treated animals (day 12: 2.1x1014 ± 1.3x101 vs. 3.1x109 ± 1.2x101; p=0.008). Accordingly, systemic invasion of C. rodentium as measured by post mortem analysis of mesenterial lymph nodes and spleens was significantly reduced in NAM-treated mice (61.1% vs. 94.4% in controls; respectively; p=0.02). Histological damage of colonic mucosa reflected by lengthening of crypts was clearly reduced in NAMtreated animals (15.3AU ± 1.1 vs 11.8AU ± 0.8; p=0.03). FACS analyses of peripheral blood revealed no statistical difference in neutrophil counts comparing NAM vs. control group over the course of infection. Ex vivo, the pre-stimulation of whole blood from healthy WT mice with NAM for 20 hrs led to a significantly increased killing of C. rodentium by up to 2.6 log10 (p=0.04). Conclusions: We have demonstrated that pharmacological application of NAM critically impacts the host's ability to fight C. rodentium infections. In face of the currently increasing antibiotic resistance, NAM might serve as a complementary antimicrobial agent to fight intestinal bacterial infections.
Background & Aims. Memory B cells preserve and recall previous antigenic experience in order to prevent or limit re-infection. The common immunological disorder observed in patients with a reduced frequency of memory B cells is the increased susceptibility to bacterial infections at parenchimal and mucosal sites. Secretory IgA (sIgA) is the tool used by mucosal B cells to protect the gut. Patients & Methods. We studied two groups of patients with reduced number of memory B cells: 34 patients with CVID and 7 splenectomized patients. Diagnosis of CVID was done according to the ESID/PAGID criteria: marked decrease in serum IgG and IgA, onset age>2 years, poor response to vaccines and exclusion of other causes of hypogammaglobulinaemia. All CVID patients were on intravenous or subcutaneous immunoglobulins. 50 healthy donors were enrolled as controls for blood values, and 15 for intestinal biopsies. Peripheral blood mononuclear cells were isolated and stained with combination of monoclonal antibodies to recognize memory B cells. Duodenal biopsies were collected and frozen in liquid nitroge. Multiple cryostat sections were incubated with Phalloidin-TRITC, anti-human-IgA, anti-human Ig kappa light chain, goat anti-human IgM, anti-human secretory component and anti-human J-chain. Duodenal sections were analysed at confocal microscope to analyze mucosal IgA plasma cells and sIgA. Results. Reduction of memory B cells in peripheral blood of splenectomized patients was associated to a significant diminution of IgA plasma cells and the disruption of the film of sIgA on the luminal side of epithelial cells. In patients with CVID, the absence of sIgA in the gut and IgA in the serum were associated to a significant reduction of memory B cells in the peripheral blood. The subset of CVID patients with sIgA at mucosal sites showed a normal number of circulating memory B cells, detectable serum IgA and have a low risk of respiratory infections. Conclusions. Memory B cells are indispensable for the production of sIgA at mucosal sites. The disruption of the sIgA film in the gut impairs the local defense against invading pathogens. New tools should be developed in order to replace the function of sIgA in immune-deficient and splenectomized patients. Mo1790 NOD2 Induced Peyers Patches Dysfunction: Respective Roles of Immune and Epithelial Cells in Mouse Ziad Alnabhani, Nicolas Montcuquet, Camille Jung, Patricia Lepage, Maryline Roy, Monique Dussaillant, Bertrand Meresse, Nadine Cerf-Bensussan, Jean-Pierre Hugot, Frédérick Barreau Background: Crohn's disease (CD) is a chronic inflammatory bowel disease characterized by an excessive immune reaction in response to abnormal microbiota in genetically predisposed individuals. Although, NOD2 mutations have been associated with susceptibility to CD, their role in the genesis of the disease remains unknown. A key role of Peyer's patches (PP) in CD has been supported by a spatiotemporal relationship between the CD lesions and PP. PP are characterized by a dialogue between immune cells and epithelial cells, regulated by the bacterial recognition receptors, including Nod2. We have shown that Nod2 played
S-685
AGA Abstracts
AGA Abstracts
Mo1786
ADAMDEC1: A Novel Molecule in Inflammation and Bowel Disease Nuala R. O'Shea, Thean Soon Chew, Gavin W. Sewell, Stuart L. Bloom, Andrew M. Smith, Anthony W. Segal
HIF Expression in the Lamina Propria Correlates With Histological Damage in Patients With Inflammatory Bowel Disease Jesus Cosin-Roger, Dolores Ortiz-Masia, Mario Manresa, Joaquin Hinojosa, Rafael Alos, Maria Jesus Nicolau-Ribera, Dolores Barrachina
Background: Innate immunity is attenuated in patients with Crohn's disease (CD) with impaired neutrophil recruitment to skin and bowel, delayed clearance of E. coli from the skin, and impaired secretion of pro-inflammatory cytokines from macrophages (Marks et al, 2006; Smith et al, 2009). The primary defect of acute inflammation results in failure to eradicate bacterial flora entering the bowel wall leading to the chronic granulomatous inflammation characteristic of CD. Microarray analysis of monocyte derived macrophage mRNA expression, confirmed by qPCR, revealed that ADAMDEC1 (ADAM like Decysin1) was under-expressed in 10% (6/60) of CD patients. ADAMDEC1, a Metalloprotease and Decysin, is part of a family of proteins involved in wound healing and tissue repair, and is almost exclusively expressed in macrophages, dendritic cells and the gastrointestinal tract. To determine the role of this protein we examined E. coli induced inflammation and Dextran Sodium Sulphate (DSS) colitis in Adamdec1 knockout (KO) mice. Method: In an acute colitis model, Adamdec1 KO mice were exposed to 2% DSS for 7 days. Controls, wild type (WT) litter mates, were age, weight and sex matched (n=11 per group). Clinical colitis scores (weight loss, PR blood, loose stool) were recorded daily. Histology was obtained from small and large bowels. For bacterial inflammation, 5x108 heat killed E. coli (HkEc) were injected subcutaneously (SC) into two sites on the backs of KO and WT mice (n=8 per group). Mice were weighed, injection sites inspected for ulceration and subcutaneous inflammatory nodules measured daily. Injection sites were excised at different times for histology and identification of infiltrating cells by FACS. Results: Adamdec1 KO mice were more susceptible to DSS colitis. They demonstrated higher clinical colitis scores with an earlier and more dramatic weight loss (p<0.001). A more florid inflammatory response was seen on histology. In response to a subcutaneous injection of E. coli, Adamdec1 KO mice had smaller inflammatory nodules and less ulceration at the injection sites after 48-72 hours, than WT mice (p<0.001). Conclusion: Mice lacking Adamdec1 develop a phenotype that closely mirrors that observed in patients with CD, an attenuated and delayed E.coli induced acute inflammatory response and an increased susceptibility to bowel inflammation. These results suggest ADAMDEC1 plays an important role in the acute inflammatory response to bacteria and has a protective role within the intestine; reduced levels may have a pivotal role in the development and persistence of CD. Studies are currently underway to further investigate the impaired cellular recruitment and potentially defective bacterial clearance at these early stages of inflammation in our model which could predispose to a more exuberant secondary response and chronic inflammation.
Introduction: Epithelial barrier function is impaired in the inflammatory bowel disease (IBD). Hypoxia and cytokines, key features of inflammation, modulate the activity of hypoxia inducible factor (HIF), a transcription factor related to the induction of genes involved in mucosal healing. We aim to determine the pattern of HIF expression in the intestinal mucosa of IBD patients. Patients and Methods: Both damaged and non-damaged mucosa from patients with ulcerative colitis (UC) and Crohn`s disease (CD) were obtained. Paraffin embedded tissues were used for histological analysis (score 1-4) and ki67 immunostaining (cellular proliferation, score 1-4). Presence of macrophages CD206+ (M2 phenotype), HIF1α and HIF-2α were analyzed by immunohistochemistry. Quantification of macrophages CD206+, HIF-1α and HIF-2α positive cells was performed counting positive cells in a total area of 0,152 mm2 (4 photographs 40X) using an inverted microscope. Results: Cellular proliferation was increased in damaged mucosa of UC (2.8±0.3) and CD (1.7±0.3) compared with non-damaged mucosa (1.3±0.3 and 1±0.05, respectively). A correlation was observed between mucosal damage and the number of macrophages in the lamina propria; the Spearman correlation coefficient was r=0.6181 (P=0.0185, n=14) for UC and r=0.6392 (P= 0.0187, n=13) for CD. HIF-1α immunostaining was observed in few epithelial cells and in some cells of the lamina propria. In contrast, an increased HIF-2α immunostaining was observed only in cells of the lamina propria of the damaged area of UC and CD. A quantitative analysis in the lamina propria showed higher expression of HIF-2α than HIF-1α both in UC (7.11±1.45 vs. 2.33±0.72 respectively) and in CD (6.50±1.38 vs. 2.00±0.54 respectively). Moreover, a correlation was observed between macrophages CD206+ and HIF-1α of the lamina propria (r=0.5636 P=0.023, n=16) and between macrophages CD206+ and HIF-2α (r=0.7864, P=0.0002, n=17) in patients with IBD. The number of HIF-1α positive cells of the lamina propria positively correlated with damage in UC; the Spearman correlation coefficient was r=0.9478 (P<0.0001, n=13). However, the number of HIF-2α positive cells correlated with mucosal damage in both, UC and CD; the Spearman correlation coefficient was r=0.7593 (P=0.0016, n=14) for UC and r=0.7412 (P=0.0058, n=12) for CD. Conclusion: The number of HIF-1α and HIF-2α positive cells in the lamina propria, which positively correlates with M2 phenotype of macrophages, increases with the severity of disease in the intestinal mucosa of patients with IBD. Mo1789
Mo1787
Immunoglobulin a Plasma Cell Depletion in the Small Bowel Mucosa of Common Variable Immune Deficiency (CVID) and Asplenic Patients With Decreased Circulating Memory B Cells Antonio Di Sabatino, Maria Manuela Rosado, Rita Carsetti, Simona Cascioli, Ezio Giorda, Marco Scarsella, Stefania Petrini, Cinzia Milito, Alessandra Pasini, Isabella Quinti, Gino R. Corazza
Nicotinamide Ameliorates the Course of Citrobacter rodentium-Induced Colitis Through Enhanced Bacterial Killing Dominik Bettenworth, Tobias M. Nowacki, Pierre Kyme, Matthias Ross, Nils H. Thoennissen, Jan Heidemann Background and Aims: The myeloid-specific transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) is crucial for the terminal differentiation and functional maturation of neutrophils and activation of downstream antimicrobial targets. Recently, we demonstrated that the Histon deacetylase inhibitor nicotinamide (NAM; vitamin B3) increases the transcriptional activity of C/EBPε by modulating the acetylation of its lysine residues which in turn can lead to enhanced killing of bacteria by the innate immune system (Thoennissen et al., abstract #925, ASH 2010). Aim of the present study was to assess the effect of NAM in Citrobacter rodentium-induced colitis and to elucidate its impact on bacterial killing In Vivo and ex vivo. Methods: C57BL/6 WT mice were orally gavaged with 5×108 colony-forming units of C. rodentium. From day 2 post infection onward, animals (n=9/group) were treated either with NAM (250 mg/kg i.p. daily), or with an equivalent volume of PBS as control. Fecal excretion of C. rodentium and changes of body weight were monitored routinely during the course of disease. Hematopoietic changes were serially analyzed by FACS analyses of peripheral blood. At day 12 post infection, animals were euthanized and the systemic spreading of C. rodentium was assessed by incubation of tissue homogenates (mesenterial lymph nodes, spleen). Moreover, inflammatory changes of the colon were evaluated histologically. In addition, killing of C. rodentium in NAM-stimulated murine and human peripheral whole blood was assessed using safe NAM doses ex vivo. Results: From day 10 until the end of experiment, the fecal excretion of C. rodentium in NAM-treated mice was markedly decreased by up to 4 log10 compared to PBS-treated animals (day 12: 2.1x1014 ± 1.3x101 vs. 3.1x109 ± 1.2x101; p=0.008). Accordingly, systemic invasion of C. rodentium as measured by post mortem analysis of mesenterial lymph nodes and spleens was significantly reduced in NAM-treated mice (61.1% vs. 94.4% in controls; respectively; p=0.02). Histological damage of colonic mucosa reflected by lengthening of crypts was clearly reduced in NAMtreated animals (15.3AU ± 1.1 vs 11.8AU ± 0.8; p=0.03). FACS analyses of peripheral blood revealed no statistical difference in neutrophil counts comparing NAM vs. control group over the course of infection. Ex vivo, the pre-stimulation of whole blood from healthy WT mice with NAM for 20 hrs led to a significantly increased killing of C. rodentium by up to 2.6 log10 (p=0.04). Conclusions: We have demonstrated that pharmacological application of NAM critically impacts the host's ability to fight C. rodentium infections. In face of the currently increasing antibiotic resistance, NAM might serve as a complementary antimicrobial agent to fight intestinal bacterial infections.
Background & Aims. Memory B cells preserve and recall previous antigenic experience in order to prevent or limit re-infection. The common immunological disorder observed in patients with a reduced frequency of memory B cells is the increased susceptibility to bacterial infections at parenchimal and mucosal sites. Secretory IgA (sIgA) is the tool used by mucosal B cells to protect the gut. Patients & Methods. We studied two groups of patients with reduced number of memory B cells: 34 patients with CVID and 7 splenectomized patients. Diagnosis of CVID was done according to the ESID/PAGID criteria: marked decrease in serum IgG and IgA, onset age>2 years, poor response to vaccines and exclusion of other causes of hypogammaglobulinaemia. All CVID patients were on intravenous or subcutaneous immunoglobulins. 50 healthy donors were enrolled as controls for blood values, and 15 for intestinal biopsies. Peripheral blood mononuclear cells were isolated and stained with combination of monoclonal antibodies to recognize memory B cells. Duodenal biopsies were collected and frozen in liquid nitroge. Multiple cryostat sections were incubated with Phalloidin-TRITC, anti-human-IgA, anti-human Ig kappa light chain, goat anti-human IgM, anti-human secretory component and anti-human J-chain. Duodenal sections were analysed at confocal microscope to analyze mucosal IgA plasma cells and sIgA. Results. Reduction of memory B cells in peripheral blood of splenectomized patients was associated to a significant diminution of IgA plasma cells and the disruption of the film of sIgA on the luminal side of epithelial cells. In patients with CVID, the absence of sIgA in the gut and IgA in the serum were associated to a significant reduction of memory B cells in the peripheral blood. The subset of CVID patients with sIgA at mucosal sites showed a normal number of circulating memory B cells, detectable serum IgA and have a low risk of respiratory infections. Conclusions. Memory B cells are indispensable for the production of sIgA at mucosal sites. The disruption of the sIgA film in the gut impairs the local defense against invading pathogens. New tools should be developed in order to replace the function of sIgA in immune-deficient and splenectomized patients. Mo1790 NOD2 Induced Peyers Patches Dysfunction: Respective Roles of Immune and Epithelial Cells in Mouse Ziad Alnabhani, Nicolas Montcuquet, Camille Jung, Patricia Lepage, Maryline Roy, Monique Dussaillant, Bertrand Meresse, Nadine Cerf-Bensussan, Jean-Pierre Hugot, Frédérick Barreau Background: Crohn's disease (CD) is a chronic inflammatory bowel disease characterized by an excessive immune reaction in response to abnormal microbiota in genetically predisposed individuals. Although, NOD2 mutations have been associated with susceptibility to CD, their role in the genesis of the disease remains unknown. A key role of Peyer's patches (PP) in CD has been supported by a spatiotemporal relationship between the CD lesions and PP. PP are characterized by a dialogue between immune cells and epithelial cells, regulated by the bacterial recognition receptors, including Nod2. We have shown that Nod2 played
S-685
AGA Abstracts
AGA Abstracts
Mo1786