- Email: [email protected]
1030-LBP
Abstracts / Human Immunology 75 (2014) 479–501
1029-LBP IMMUNE RESPONSES TO COLLAGEN IV AND FIBRONECTIN IN RENAL TRANSPLANT
RECIPIENTS WITH TRANSPLANT GLOMERULOPATHY. Muthukumar Gunasekaran 1,
Angaswamy nataraju 1, Klein Christina 2, Tiriveedhi Venkataswarup 1, Siddiq Anwar 2, Phelan Donna 3, Wellen Jason 1, Shenoy Surendra 1, Chapman William 1, Mohanakumar Thalachallour 1. 1 Department of Surgery, Washington University, Saint Louis, MO 63110, United States; 2 Department of Medicine, Washington University, Saint Louis, MO 63110, United States; 3 HLA Laboratory, Barnes-Jewish Hospital, Saint Louis, MO 63110, United States. Aim: Antibodies (Abs) to donor HLA (DSA) has been associated with transplant glomerulopathy (TG) following kidney transplantation (KTx). Immune responses to tissue restricted self-antigens (self-Ags) have been proposed to play a role in chronic rejection. In this study we determined the role of kidney restricted self tissue antigens collagen-IV (Col-IV) and fibronectin (FN) in the development of chronic rejection following KTx in TG patients. Methods: Biopsy proven 26 KTx TG patients, 10 stable KTx and 14 normal controls were enrolled in this study. Antibodies to self-Ags, Col-IV and FN were determined by ELISA. DSA was determined by solid phase assay and frequency of CD4+ T cells secreting IFN-c, IL-17 or IL-10 by ELISPOT. Results: Development of Abs to self-Ags following KTx increased the risk for TG with an odds ratio of 22 (p < 0.01). Abs to self-Ags were IgG and IgM isotypes. Pre-transplant Abs to self-Ags increased the risk of TG (22% vs 10%, p < 0.5). Abs to self-Ags were identified frequently in KTx with DSA. TG patients demonstrated increased Col-IV and FN specific CD4+ T cells secreting IFN-c (TG: 867 ± 241 pg/mL, stable: 407 ± 178 pg/mL, p = 0.02) and IL-17 (TG: 918 ± 197 pg/mL, stable: 327 ± 142 pg/mL, p = 0.009) with reduction in IL-10 (TG: 184 78 pg/mL, stable: 668 184 pg/mL, p = 0.001). Conclusions: In conclusion, the development of Abs to self-Ags is an independent risk factor and having both DSA and Abs to self-Ags increases the risk for TG. The increased frequency of self-Ag specific IFN-c and IL-17 cells with reduction in IL-10 demonstrate tolerance breakdown to self-Ags which we propose play a role in the pathogenesis of TG.
1030-LBP HEPATITIS C VIRUS INDUCED CHANGES IN MICRORNA-107 AND MICRORNA-449A MODULATE THE INFLAMMATORY CHEMOKINE CCL2 BY TARGETING IL6 RECEPTOR COMPLEX IN PATIENTS WITH HEPATIC FIBROSIS. Nayan Sarma 1, Venkataswarup
Tiriveedhi 1, Jeffrey Crippin 3, William Chapman 1, Thalachallour Mohanakumar 1,2. 1
Surgery, Washington Univ Sch Med, St. Louis, United States; 2 Pathology & Immunology, Washington Univ Sch Med, St. Louis, United States; 3 Medicine, Washington Univ Sch med, St. Louis, United States.
Aim: Hepatitis C virus (HCV) mediated liver diseases are one of the major health issues in United States and worldwide. This study is to identify the role of HCV induced changes in microRNAs (miRNA) that controls various cell surface receptors and gene regulatory complexes involved in patients with HCV mediated inflammation and fibrosis. Methods: Computational sequence analyses and reporter assays using the CCL2 promoter region were carried out to analyze transcription factors. Gene expression analyses were done using q-PCR. Gene overexpression and knockdowns in human hepatocytes were carried out using cDNA plasmids and siRNAs. Chromatin (ChIP) and protein (CoIP) immunoprecipitations were done to establish interactions between transcription factors and DNA. Results: We report here that down-regulation of miRNA-107 and miRNA-449a following HCV infection in patients modulate expression of the CCL2, an inflammatory chemokine up-regulated in patients with chronic liver diseases, by targeting components of the IL6R complex. Computational analysis for DNA bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBPa, spleen focus forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBPa, PU.1 and STAT3 interacted with each other physically to co-operatively bind to the promoter and activate CCL2 expression. Analysis of IL6R and JAK1 expression in HCV patients showed significant up regulation when there is impaired miRNA-107 and miRNA-449a expression along with up regulation of PU.1 and STAT3 but not CEBPa. miRNA449a and miRNA-107 target IL6R and JAK1 respectively, inhibit IL6 signaling and impair STAT3 activation. Conclusions: Our results demonstrate a novel gene regulatory mechanism wherein HCV induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL6 mediated signaling cascade in HCV patients which we propose will result in HCV mediated induction of inflammatory responses and fibrosis.
495
1029-LBP IMMUNE RESPONSES TO COLLAGEN IV AND FIBRONECTIN IN RENAL TRANSPLANT
RECIPIENTS WITH TRANSPLANT GLOMERULOPATHY. Muthukumar Gunasekaran 1,
Angaswamy nataraju 1, Klein Christina 2, Tiriveedhi Venkataswarup 1, Siddiq Anwar 2, Phelan Donna 3, Wellen Jason 1, Shenoy Surendra 1, Chapman William 1, Mohanakumar Thalachallour 1. 1 Department of Surgery, Washington University, Saint Louis, MO 63110, United States; 2 Department of Medicine, Washington University, Saint Louis, MO 63110, United States; 3 HLA Laboratory, Barnes-Jewish Hospital, Saint Louis, MO 63110, United States. Aim: Antibodies (Abs) to donor HLA (DSA) has been associated with transplant glomerulopathy (TG) following kidney transplantation (KTx). Immune responses to tissue restricted self-antigens (self-Ags) have been proposed to play a role in chronic rejection. In this study we determined the role of kidney restricted self tissue antigens collagen-IV (Col-IV) and fibronectin (FN) in the development of chronic rejection following KTx in TG patients. Methods: Biopsy proven 26 KTx TG patients, 10 stable KTx and 14 normal controls were enrolled in this study. Antibodies to self-Ags, Col-IV and FN were determined by ELISA. DSA was determined by solid phase assay and frequency of CD4+ T cells secreting IFN-c, IL-17 or IL-10 by ELISPOT. Results: Development of Abs to self-Ags following KTx increased the risk for TG with an odds ratio of 22 (p < 0.01). Abs to self-Ags were IgG and IgM isotypes. Pre-transplant Abs to self-Ags increased the risk of TG (22% vs 10%, p < 0.5). Abs to self-Ags were identified frequently in KTx with DSA. TG patients demonstrated increased Col-IV and FN specific CD4+ T cells secreting IFN-c (TG: 867 ± 241 pg/mL, stable: 407 ± 178 pg/mL, p = 0.02) and IL-17 (TG: 918 ± 197 pg/mL, stable: 327 ± 142 pg/mL, p = 0.009) with reduction in IL-10 (TG: 184 78 pg/mL, stable: 668 184 pg/mL, p = 0.001). Conclusions: In conclusion, the development of Abs to self-Ags is an independent risk factor and having both DSA and Abs to self-Ags increases the risk for TG. The increased frequency of self-Ag specific IFN-c and IL-17 cells with reduction in IL-10 demonstrate tolerance breakdown to self-Ags which we propose play a role in the pathogenesis of TG.
1030-LBP HEPATITIS C VIRUS INDUCED CHANGES IN MICRORNA-107 AND MICRORNA-449A MODULATE THE INFLAMMATORY CHEMOKINE CCL2 BY TARGETING IL6 RECEPTOR COMPLEX IN PATIENTS WITH HEPATIC FIBROSIS. Nayan Sarma 1, Venkataswarup
Tiriveedhi 1, Jeffrey Crippin 3, William Chapman 1, Thalachallour Mohanakumar 1,2. 1
Surgery, Washington Univ Sch Med, St. Louis, United States; 2 Pathology & Immunology, Washington Univ Sch Med, St. Louis, United States; 3 Medicine, Washington Univ Sch med, St. Louis, United States.
Aim: Hepatitis C virus (HCV) mediated liver diseases are one of the major health issues in United States and worldwide. This study is to identify the role of HCV induced changes in microRNAs (miRNA) that controls various cell surface receptors and gene regulatory complexes involved in patients with HCV mediated inflammation and fibrosis. Methods: Computational sequence analyses and reporter assays using the CCL2 promoter region were carried out to analyze transcription factors. Gene expression analyses were done using q-PCR. Gene overexpression and knockdowns in human hepatocytes were carried out using cDNA plasmids and siRNAs. Chromatin (ChIP) and protein (CoIP) immunoprecipitations were done to establish interactions between transcription factors and DNA. Results: We report here that down-regulation of miRNA-107 and miRNA-449a following HCV infection in patients modulate expression of the CCL2, an inflammatory chemokine up-regulated in patients with chronic liver diseases, by targeting components of the IL6R complex. Computational analysis for DNA bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBPa, spleen focus forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBPa, PU.1 and STAT3 interacted with each other physically to co-operatively bind to the promoter and activate CCL2 expression. Analysis of IL6R and JAK1 expression in HCV patients showed significant up regulation when there is impaired miRNA-107 and miRNA-449a expression along with up regulation of PU.1 and STAT3 but not CEBPa. miRNA449a and miRNA-107 target IL6R and JAK1 respectively, inhibit IL6 signaling and impair STAT3 activation. Conclusions: Our results demonstrate a novel gene regulatory mechanism wherein HCV induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL6 mediated signaling cascade in HCV patients which we propose will result in HCV mediated induction of inflammatory responses and fibrosis.
495