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Role of PARP-1 in the modulation of neutrophil function: Relevance for Inflammatory Bowel Disease C.B. Larmonier 1, K. W. Shehab 1, R-M. T. McFadden,1,3,

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Presentation on theme: "Role of PARP-1 in the modulation of neutrophil function: Relevance for Inflammatory Bowel Disease C.B. Larmonier 1, K. W. Shehab 1, R-M. T. McFadden,1,3,"— Presentation transcript:

1 Role of PARP-1 in the modulation of neutrophil function: Relevance for Inflammatory Bowel Disease C.B. Larmonier 1, K. W. Shehab 1, R-M. T. McFadden,1,3, F.K. Ghishan 1 and P.R. Kiela 1, 2 1 Departments of Pediatrics, Steele Children’s Research Center, 2 Department of Immunobiology, University of Arizona Health Sciences Center, Tucson, Arizona, 3 School of Dentistry, Oral Biology Program, University of North Carolina, Chapel Hill, North Carolina - USA Abstract Aberrant immune response has been proposed as the underlying cause of inflammatory bowel disease. Disease progression and relapse are attributed to a hyperactive adaptive immune response to the bowel luminal contents accompanied by disproportionate and persistent neutrophil (PMN) influx. Poly(ADP- ribose) polymerases (PARPs), including its most abundant isoform PARP1, catalyzes the transfer of ADP-ribose units from NAD + to a number of target proteins. In vivo studies have demonstrated that genetic ablation or pharmacological inhibition of PARP1 ameliorates the pathophysiologic changes of experimental colitis. Considering the essential role of PARP1 in the regulation of inflammation, we propose to evaluate the effect of Parp1 deletion on neutrophil function in a model of dextran sulfate sodium (DSS)-induced colitis. In comparison to WT mice, Parp1 -/- were protected from the effects of DSS, with significantly lower weight loss, and reduced proinflammatory mediators, including metalloproteinases and chemoattractants. The regulation of neutrophil-related genes in the colon was also studied by microarray. Similarly, Parp1 -/- neutrophils demonstrated impaired chemotaxis in vitro and reduced motility in vivo in a model of aseptic peritonitis. TER (%) Figure 2: (A) Neutrophil-specific immunohistochemistry staining with MCA771GA antibody recognizing a polymorphic 40-kDa antigen expressed by murine polymorphonuclear (PMN) cells (brown dots). Distal colon of WT or Parp1 -/- mice on control H 2 O or 4% DSS for 7 days. (B) Quantitative assessment of PMN infiltration by real-time RT-PCR analysis of MMP8, a neutrophil collagenase, MMP9, MIP2 and CXCL5 chemokine. Statistical analysis was performed with ANOVA followed by Fisher PLSD post hoc test. Differences were considered statistically significant at P ≤ 0.05 Conclusion In light of these current findings, we postulate that the pathogenic role of PARP1 may be mediated in part by regulation of neutrophil function. Funding: NIH Grant 1K01DK (C.Larmonier) Impaired chemotaxis on Parp1 deficient neutrophil Figure 5: Chemotaxis assay with calcein orange stained bone marrow-derived mouse neutrophils against recombinant MIP-2 (20ng/ml) or conditional medium obtained from interperitoneale macrophages stimulated for 24h with cecal antigen (CA) (10ng/ml) using ChemoTx® (Neutroprobe – 5  m). Ly-6G positive neutrophils were purified from the total population of bone marrow derived cells using automated magnetic separation (autoMACS TM, Miltenyi Biotec). Cell purity was assessed by flow cytometry using FITC conjugated rat anti-mouse Ly-6G and Ly-6C monoclonal antibody (BD, Pharmingen). Statistical analysis was performed by Student t-test. Differences were considered statistically significant at P ≤ 0.05 ControlMIP-2 20ng/ml Control sup. Non- treated intraperitoneal macrophage Sup from Ip macrophage treated with cecal antigen WTParp1 -/- 500  l - 3% Na- thioglycolate -3 hours Fold change * * Reduced neutrophil recruitment in DSS-treated Parp1 deficient Reduced neutrophil recruitment in DSS-treated Parp1 deficient mice Distal Colon WTParp1 -/- WTParp1 -/- Control H 2 O4% DSS A. Ip lavage / giemmsa staining Control 3% thioglycollate A. Parp1 -/- WT H2OH2ODSSH2OH2O Parp1 -/- WT H2OH2ODSSH2OH2O Parp1 -/- WT H2OH2ODSSH2OH2O Parp1 -/- WT H2OH2ODSSH2OH2O Regulation of Chemotaxis- related genes Figure 3: Heat map illustrating chemotaxis-related gene expression in the colon of WT and Parp1-/- mice on control H2O and 4% DSS. Genes up- or down-regulated >1.5-fold in DSS-treated WT mice (p ≤ 0.05; Moderated T-test with Storey Multiple testing Correction) were analyzed with Gene Ontology Tool (GeneSpring, Agilent) and genes in neutrophil chemotaxis category were selected. This 16-gene list was reiterated against all four genotypes/treatment conditions with expression values normalized to the median of control samples (control WT mice) and presented in log2 scale. Reduced neutrophil recruitment in Parp1 -/- mice in a model of aseptic peritonitis. B. C. WTParp1 -/- WT Parp1 -/- Figure 6: Neutrophil recruitment in thioglycollate induced aseptic peritonitis. (A-B) Experimental Design. Peritonitis was induced by i.p.injection of 500  l of 3% thioglycollate (wt/vol;Sigma) in WT or Parp1 KO mice. 3 hours later,intra-peritoneal cells were lavaged with 5mL of cold RPMI 1640 and the number of cells was determined using The Vi-Cell XR automatic cell counter and viability analyzer (Beckman Coulter). (C) Quantitative assessment of PMN activation by real-time PCR analysis of MMP8, a neutrophil collagenase Statistical analysis was performed with ANOVA followed by Fisher PLSD post hoc test. Differences were considered statistically significant at P ≤0.05. Fluorescence units * * * Parp1 deficiency is protective in DSS-induced colitis A. B. IFN  pg/50mg of colonic tissue C. TNF  pg/50mg of colonic tissue * WT DSS WT DSS KO DSS KO DSS D. WT + DSS KO + DSS WT H 2 O WT DSS Parp1 -/- H 2 O Parp1 -/- DSS Cxcr2Fcgr3 (CD16)Fcer1g Itgb2 (CD18; Integrin beta 2) IFN  Ccl2 (MCP1) Ccl3 (MIP-1  ) Prkca (protein kinase c, alpha) Nckap1 (NCK-associate protein 1) Cxadr IL-1  Csf3rCxcl2 (MIP-2)Itgam (Mac-1)Cklf (Chemokine-like factor) Amica 1 Figure 1: (A) Representative H&E staining from the proximal and distal colon of WT and Parp1 -/- mice after 5 day DSS treatment (4%). (B) Survival of WT and Parp1 -/- mice in this model of chronic colitis (cyclical DSS administration at 4%). (C) Real-time RT-PCR analysis of colonic cytokine expression also showed significant decrease in expression of IFN , TNF , IL12p40 and IL-17 in DSS-treated Parp1 -/- mice compared to DSS-treated WT mice. (D) IFN  and TNF  concentration in the colonic explant culture of WT and Parp1 -/- mice (ELISA). Statistical analysis was performed with ANOVA followed by Fisher PLSD post hoc test. Differences were considered statistically significant at P ≤ 0.05 * * * * * *


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