title: Second-hand cigarette smoke impairs bacterial phagocytosis in macrophages by modulating CFTR dependent lipid-rafts.
abstract: BACKGROUND
  First/Second-hand cigarette-smoke (FHS/SHS) exposure weakens immune defenses inducing chronic obstructive pulmonary disease (COPD) but the underlying mechanisms are not fully understood. Hence, we evaluated if SHS induced changes in membrane/lipid-raft (m-/r)-CFTR (cystic fibrosis transmembrane conductance regulator) expression/activity is a potential mechanism for impaired bacterial phagocytosis in COPD.
  
  
  METHODS
  RAW264.7 murine macrophages were exposed to freshly prepared CS-extract (CSE) containing culture media and/or Pseudomonas-aeruginosa-PA01-GFP for phagocytosis (fluorescence-microscopy), bacterial survival (colony-forming-units-CFU), and immunoblotting assays. The CFTR-expression/activity and lipid-rafts were modulated by transient-transfection or inhibitors/inducers. Next, mice were exposed to acute/sub-chronic-SHS or room-air (5-days/3-weeks) and infected with PA01-GFP, followed by quantification of bacterial survival by CFU-assay.
  
  
  RESULTS
  We investigated the effect of CSE treatment on RAW264.7 cells infected by PA01-GFP and observed that CSE treatment significantly (p<0.01) inhibits PA01-GFP phagocytosis as compared to the controls. We also verified this in murine model, exposed to acute/sub-chronic-SHS and found significant (p<0.05, p<0.02) increase in bacterial survival in the SHS-exposed lungs as compared to the room-air controls. Next, we examined the effect of impaired CFTR ion-channel-activity on PA01-GFP infection of RAW264.7 cells using CFTR172-inhibitor and found no significant change in phagocytosis. We also similarly evaluated the effect of a CFTR corrector-potentiator compound, VRT-532, and observed no significant rescue of CSE impaired PA01-GFP phagocytosis although it significantly (p<0.05) decreases CSE induced bacterial survival. Moreover, induction of CFTR expression in macrophages significantly (p<0.03) improves CSE impaired PA01-GFP phagocytosis as compared to the control. Next, we verified the link between m-/r-CFTR expression and phagocytosis using methyl-β-cyclodextran (CD), as it is known to deplete CFTR from membrane lipid-rafts. We observed that CD treatment significantly (p<0.01) inhibits bacterial phagocytosis in RAW264.7 cells and adding CSE further impairs phagocytosis suggesting synergistic effect on CFTR dependent lipid-rafts.
  
  
  CONCLUSIONS
  Our data suggest that SHS impairs bacterial phagocytosis by modulating CFTR dependent lipid-rafts.
authors: Department of Pediatric Respiratory Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors: Inzer
authors: I
authors: Ni
authors: Department of Pediatric Respiratory Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors: Changhoon
authors: C
authors: Ji
authors: Department of Pediatric Respiratory Science, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America; Department of Foundational Sciences, College of Medicine, Central Michigan University, Mount Pleasant, Michigan, United States of America.
authors: Neeraj
authors: N
authors: Vij
mesh_terms: D000818:Animals; D002470:Cell Survival; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000382:microbiology*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D010587:Phagocytosis / Q000187:drug effects*; D000067996:RAW 264.7 Cells; D014028:Tobacco Smoke Pollution / Q000009:adverse effects*; D014162:Transfection
chemical_list: D014028:Tobacco Smoke Pollution; D019005:Cystic Fibrosis Transmembrane Conductance Regulator