Introduction 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. matrix proteins that form a biofilm involved in pathogenesis of chronic lung disease [13 27 30 31 32 Furthermore these chronic bacterial exacerbations are the major cause of mortality in CF/COPD subjects [22 24 Smoke exposure has been shown to deplete CFTR from cell membrane (m-) and lipid-rafts (r-) while increasing its internalization and endoplasmic reticulum (ER) accumulation as a result of impaired peripheral- and ER- associated degradation [7 12 28 33 Moreover recent studies verify that dysfunctional CFTR (mutation/acquired) impairs bacterial clearance by macrophages [18 34 35 that increase susceptibility to bacterial infections [12 36 37 38 39 40 The CFTR deficiency has also been linked to defective reactive oxidative species (ROS) and acid sphingomyelinase (ASMase) activation which are required for bacterial killing by macrophages [41 42 Despite the intriguing evidence around the pathogenic role of smoke exposure in increasing the risk of bacterial infection [5 10 15 18 the underlying mechanism(s) by which SHS exposure impairs CFTR dependent bacterial phagocytosis in macrophages is not fully understood. Hence we evaluated if SHS induced m-/r- CFTR dysfunction in macrophages is usually PIM-1 Inhibitor 2 a critical mechanism for impaired bacterial phagocytosis in COPD. We first verified the pathogenic role of SHS in diminishing bacterial phagocytosis that can initiate chronic obstructive lung disease. We also found that SHS exposure can modulate CFTR dependent lipid-rafts as a potential mechanism for impaired bacterial phagocytosis. Materials and Methods Reagents and treatments The murine macrophage cell line RAW264.7 was cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM-F-12; Corning Cellgro Manassas VA) media. The media was supplemented with 10% Fetal Bovine Serum (FBS; Corning Cellgro) and 1% Penicillin Streptomycin Amphotericin B (PSA; Corning Cellgro). For second hand smoke (SHS) exposure experiments cigarette smoke extract PIM-1 Inhibitor 2 (CSE) was directly collected in culture media and used at a PIM-1 Inhibitor 2 concentration of 10% for 150 minutes (min) exposure as described below. To inhibit cystic fibrosis transmembrane conductance regulator (CFTR) channel activity in RAW264.7 cells CFTR(inh)-172 (Sigma-Aldrich St. Louis MO) was used at a concentration of 10μM for overnight treatments. Similarly overnight treatment with flavonoid Rutin Hydrate (10μM; Sigma-Aldrich) or Quercetin (10μM; Sigma) was used to modulate CFTR channel activity. To test the effect of increased CFTR expression/activity on phagocytosis RAW264.7 cells were treated overnight with VRT-532 (10μM; CF Foundation Bethesda MD) [43 44 While effect of CFTR-lipid-raft disruption was evaluated using the CFTR/cholesterol depleting reagent methyl-β-cyclodextran (CD; Sigma) at a concentration of 5mM for 16 hours (hrs). RAW264.7 cells were also transiently transfected for 48 hrs with the vacant vector pcDNA3.1 or pcDNA3.1-WTCFTR plasmid using the Lipofectamine 2000 reagent (Invitrogen Carlsbad CA) following the manufacturer’s instructions. For contamination experiments ((SHS) exposure model PIM-1 Inhibitor 2 by adding CSE containing culture media at indicated time points . Immunoblotting Total protein extract was isolated using 1X Radio-Immunoprecipitation Assay buffer (RIPA; Sigma) supplemented with ethylene-di-amine-tetra-acetic-acid (1%; EDTA Sigma) and protease inhibitor cocktail (1%; Sigma). Total protein concentration was quantified using PIM-1 Inhibitor 2 Protein Assay Reagent (Bio-Rad Hercules CA) and VersaMax absorbance microplate reader (595nm Molecular Devices). The 80μg of total protein was loaded in each well. CTSL1 Changes in protein expression were quantified by immunoblotting for CFTR (596-ab; CF Foundation) NF-κB (Santa Cruz Biotechnology Santa Cruz CA) or β-actin (Sigma) antibodies. The secondary antibodies were anti-mouse (Amersham Amersham UK) and anti-rabbit (Amersham). Fluorescence microscopy for functional quantification of phagocytosis RAW264.7 cells were seeded onto 24 well plates and treated overnight with CFTR(inh)-172 (10μM) rutin hydrate (10μM) quercetin (10μM) VRT-532 PIM-1 Inhibitor 2 (10μM) or CD (5mM) or transiently transfected for 48 hours with control pcDNA3.1 vector or pcDNA3.1-WTCFTR plasmid construct. These.