Second-hand cigarette smoke impairs bacterial phagocytosis in macrophages by modulating CFTR dependent lipid-rafts

• Published in: PloS one Vol. 10; no. 3; p. e0121200
• Main Authors: Ni, Inzer, Ji, Changhoon, Vij, Neeraj
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20-03-2015; Public Library of Science (PLoS)
• Subjects: Animal models; Animals; Bacteria; Bacterial infections; Biomedical engineering; Cell culture; Cell Survival; Chronic obstructive lung disease; Chronic obstructive pulmonary disease; Cigarette smoke; Cigarettes; Conductance; Culture media; Cystic fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Emphysema; Experiments; Exposure; Fibrosis; Fluorescence; Health aspects; Immunoblotting; Inducers; Infection; Infections; Lipid rafts; Lipids; Lung diseases; Lungs; Macrophages; Macrophages - cytology; Macrophages - drug effects; Macrophages - microbiology; Media (culture); Medicine; Membrane Microdomains - metabolism; Mice; Microscopy; Mutation; Obstructive lung disease; Pathogenesis; Pediatrics; Phagocytosis; Phagocytosis - drug effects; Pseudomonas aeruginosa; Rafts; RAW 264.7 Cells; Resistance; Rodents; Smoke; Smoking; Survival; Synergistic effect; Tobacco Smoke Pollution - adverse effects; Transfection; United States > US; Maryland; Baltimore Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0121200

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.
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.
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.
Our data suggest that SHS impairs bacterial phagocytosis by modulating CFTR dependent lipid-rafts.