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

Abstract

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. 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.

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

Fig 1. Secondhand cigarette smoke exposure impairs bacterial phagocytosis and improves survival.

(A) RAW264.7 cells were seeded on a 24-well plate and infected with PA01-GFP (multiplicity of infection, MOI 10) and/or treated with cigarette smoke extract (CSE; 10%) for 150 min as a secondhand cigarette smoke (SHS) exposure model, followed by microscopy. Representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (B) CSE treatment as above, significantly (**p<0.01) impairs bacterial phagocytosis in RAW264.7 cells. (C) C57BL6 mice were exposed to either room-air (control) or SHS for 5 days. Mice were infected with intra-tracheal (i.t.) PA01-GFP (2x10⁶ at the indicated time points on the scale), followed by shredding of lungs for bacterial survival assay. (D) Acute-SHS exposed mice show significantly (*p<0.05) higher bacterial load (colony forming units, CFU) in the shredded lungs (low speed sonication; 1 pulse 10 sec) as compared to the room-air controls (n = 4, left: bar graph, right: scatter plot of same data). (E) The C57BL6 were exposed to either room-air (control) or SHS for 3 weeks (5 days/week) and were infected with intra-tracheal (i.t.) PA01-GFP (2x10⁶ at the indicated time points on the scale). Lungs were shredded and assessed for bacterial load by quantifying colony-forming units (CFU) at the end of the 3-week period. (F) The lungs of sub-chronic- SHS exposed mice show significantly (*p<0.02, > 2 fold) greater bacterial survival (n = 4, left: bar graph, right: scatter plot of same data) as compared to the room-air controls.

Fig 2. CFTR ion channel activity modulators cannot restore SHS impaired bacterial phagocytosis and limit survival.

(A) RAW264.7 cells were seeded on a 24-well plate and treated overnight with CFTR(inh)-172 (10 μM). In addition, cells were infected with PA01-GFP (MOI 10) for 150min followed by microscopy. Representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 6, white bar = 20μm). (B) Next we quantified (from 2A) the percentage of macrophages that were infected with PA01-GFP and found that inhibiting CFTR ion channel activity does not impair bacterial phagocytosis. (C) RAW264.7 cells were seeded on a 24-well plate and treated overnight with the flavonoids, Rutin Hydrate (RH, 10μM) or Quercetin (Q, 10μM). Cells were also infected with PA01-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150 min followed by microscopy. The representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 6, white bar = 20μm). (D) The quantification (from 2C) of bacterial phagocytosis shows significant (*p<0.05, **p<0.01) increase in Q or RH and CSE treated groups as compared to the CSE treated control group. But this change is not sufficient to restore SHS impaired bacterial phagocytosis to the levels seen in the control group. (E) In a separate experiment (similar to one described in 2C), media (100μl) was collected and spread on 2% LB agar plates. The plates were incubated overnight at 37°C and colony forming unit (CFU) counts were used to quantify bacterial survival. CSE treatment significantly (**p<0.01) improves bacterial survival, but RH or Q treatment of CSE group does not significantly affect bacterial counts.

Fig 3. Modulating CFTR expression may limit SHS induced bacterial survival.

(A) Immunoblotting of total protein extracts from RAW264.7 cells treated with VRT-532 (10μM; overnight, a known CFTR corrector and potentiator) or untreated group (control), show slightly higher protein levels of CFTR and reduced NF-κB levels in the treatment group as compared to the controls. β-actin was used as a loading control (n = 3). (B) CFTR and NF- κB protein expression (in 3A) was normalized to β—actin using an Image-J software. The densitometry analysis verifies that VRT-532 treatment can decrease NF-κB protein expression (*p<0.05) as compared to untreated control. (C) RAW264.7 cells were seeded on a 24-well plate and treated overnight with VRT-532 (10μM). Next, these cells were infected with PA01-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150mins. Representative bright field (top) and fluorescent microscopy images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (D) CSE treatment (in 3C) significantly (**p<0.01) inhibits bacterial phagocytosis, while VRT-532 is unable to restore SHS impaired phagocytosis. (E) In a separate experiment (as described in 3C), media (100μl) was collected, spread on agar plates, and then incubated overnight at 37°C. CFU counts of the extracellular bacteria (media) indicates that VRT-532 treatment can significantly (*p<0.05) decrease SHS induced bacterial survival. Note, this experiment was done in parallel with Fig. 2E (left panel, Rutin Hydrate), hence control and CSE samples used are common.

Fig 4. Transient transfection with WT-CFTR rescues SHS impaired bacterial phagocytosis and limits survival.

(A) RAW264.7 cells were seeded on a 24-well plate and transiently transfected (using Lipofectamine) with pcDNA3.1 control vector or pcDNA3.1-WTCFTR for 48 hours. Cells were infected with PA01-GFP (MOI 10) and/or treated with cigarette smoke extract (CSE; 10%; SHS model) for 150 min before microscopy. Representative bright field (top) and fluorescent images (bottom) are shown (magnification 40X, n = 4, white bar = 20μm). (B) Quantification of bacterial phagocytosis (from 4A) shows that transfection with WT-CFTR significantly (*p<0.03) rescues SHS impaired phagocytosis. (C) RAW264.7 cells were treated (as described in 4A) and media (100 μL) was collected and spread on 2% LB agar plates. The plates were incubated overnight at 37°C followed by quantification of bacterial survival by colony forming unit (CFU) counts. Transient transfection with WT-CFTR significantly (*p<0.02) limits bacterial survival in the CSE/SHS treated group, but bacterial survival remains higher than the untreated controls suggesting higher CFTR expression may be needed to limit bacterial burden.

Fig 5. Lipid-raft disruption impairs bacterial phagocytosis.

(A) RAW264.7 cells were seeded on a 24-well plate and treated overnight with methyl-β-cyclodextran (CD; 5mM), followed by PA01-GFP (MOI 10) and/or cigarette smoke extract (CSE; 10%; SHS model) treatment for 150 mins. Representative bright field (top) and fluorescent microscopy images (bottom) images are shown (magnification 40X, n = 4, white bar = 20μm). (B) Overnight CD treatment (in 5A) significantly (**p<0.01) impairs bacterial phagocytosis as compared to untreated controls. In addition, CD treatment further inhibits SHS impaired bacterial phagocytosis suggesting the role of CFTR dependent lipid-rafts in this process. (C) RAW264.7 cells were treated (as described in 5A), and media (100μl) was collected and spread on 2% LB agar plates, followed by overnight incubation at 37°C. CSE treatment significantly (**p<0.01) induces bacteria survival while CD treatment slightly elevates CSE induced bacterial survival.