Ni Inzer, Ji Changhoon, Vij Neeraj
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.
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.
PLoS One. 2015 Mar 20;10(3):e0121200. doi: 10.1371/journal.pone.0121200. eCollection 2015.
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.
一手/二手香烟烟雾（FHS/SHS）暴露会削弱免疫防御，诱发慢性阻塞性肺疾病（COPD），但其潜在机制尚未完全明确。因此，我们评估了SHS诱导的膜/脂筏（m-/r）-CFTR（囊性纤维化跨膜传导调节因子）表达/活性变化是否是COPD中细菌吞噬功能受损的潜在机制。
将RAW264.7小鼠巨噬细胞暴露于新鲜制备的含香烟提取物（CSE）的培养基和/或铜绿假单胞菌-PA01-GFP中，进行吞噬作用（荧光显微镜检查）、细菌存活（菌落形成单位-CFU）和免疫印迹分析。通过瞬时转染或抑制剂/诱导剂调节CFTR表达/活性和脂筏。接下来，将小鼠暴露于急性/亚慢性SHS或室内空气（5天/3周）中，并用PA01-GFP感染，然后通过CFU测定法对细菌存活进行定量。
我们研究了CSE处理对被PA01-GFP感染的RAW264.7细胞的影响，发现与对照组相比，CSE处理显著（p<0.01）抑制PA01-GFP的吞噬作用。我们还在暴露于急性/亚慢性SHS的小鼠模型中验证了这一点，发现与室内空气对照组相比，暴露于SHS的肺部细菌存活率显著（p<0.05，p<0.02）增加。接下来，我们使用CFTR172抑制剂研究了CFTR离子通道活性受损对RAW264.7细胞PA01-GFP感染的影响，发现吞噬作用没有显著变化。我们同样评估了CFTR校正增强剂化合物VRT-532的作用，尽管它显著（p<0.05）降低了CSE诱导的细菌存活率，但未观察到对CSE受损的PA01-GFP吞噬作用有显著的挽救作用。此外，与对照组相比，巨噬细胞中CFTR表达的诱导显著（p<0.03）改善了CSE受损的PA01-GFP吞噬作用。接下来，我们使用甲基-β-环糊精（CD）验证了m-/r-CFTR表达与吞噬作用之间的联系，因为已知它会从膜脂筏中耗尽CFTR。我们观察到CD处理显著（p<0.01）抑制RAW264.7细胞中的细菌吞噬作用，添加CSE会进一步损害吞噬作用，表明对CFTR依赖性脂筏有协同作用。
我们的数据表明，SHS通过调节CFTR依赖性脂筏损害细菌吞噬作用。
