Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland.
Division of Pulmonary & Critical Care Medicine, Department of Medicine, State University of New York Downstate Medical Center, Brooklyn, NY, USA.
EBioMedicine. 2017 Sep;23:173-184. doi: 10.1016/j.ebiom.2017.08.013. Epub 2017 Aug 16.
Identification of mechanisms promoting neutrophil trafficking to the lungs of patients with cystic fibrosis (CF) is a challenge for next generation therapeutics. Cholesterol, a structural component of neutrophil plasma membranes influences cell adhesion, a key step in transmigration. The effect of chronic inflammation on neutrophil membrane cholesterol content in patients with CF (PWCF) remains unclear. To address this we examined neutrophils of PWCF to evaluate the cause and consequence of altered membrane cholesterol and identified the effects of lung transplantation and ion channel potentiator therapy on the cellular mechanisms responsible for perturbed membrane cholesterol and increased cell adhesion.
PWCF homozygous for the ΔF508 mutation or heterozygous for the G551D mutation were recruited (n=48). Membrane protein expression was investigated by mass spectrometry. The effect of lung transplantation or ivacaftor therapy was assessed by ELISAs, and calcium fluorometric and μ-calpain assays.
Membranes of CF neutrophils contain less cholesterol, yet increased integrin CD11b expression, and respond to inflammatory induced endoplasmic reticulum (ER) stress by activating μ-calpain. In vivo and in vitro, increased μ-calpain activity resulted in proteolysis of the membrane cholesterol trafficking protein caveolin-1. The critical role of caveolin-1 for adequate membrane cholesterol content was confirmed in caveolin-1 knock-out mice. Lung transplant therapy or treatment of PWCF with ivacaftor, reduced levels of circulating inflammatory mediators and actuated increased caveolin-1 and membrane cholesterol, with concurrent normalized neutrophil adhesion.
Results demonstrate an auxiliary benefit of lung transplant and potentiator therapy, evident by a reduction in circulating inflammation and controlled neutrophil adhesion.
确定促进囊性纤维化（CF）患者中性粒细胞向肺部迁移的机制是下一代治疗方法的一个挑战。胆固醇是中性粒细胞质膜的结构成分，影响细胞黏附，这是迁移的关键步骤。慢性炎症对 CF 患者（PWCF）中性粒细胞膜胆固醇含量的影响尚不清楚。为了解决这个问题，我们检查了 PWCF 的中性粒细胞，以评估改变的膜胆固醇的原因和后果，并确定了肺移植和离子通道增强剂治疗对负责扰乱膜胆固醇和增加细胞黏附的细胞机制的影响。
招募了纯合子ΔF508 突变或杂合子 G551D 突变的 PWCF（n=48）。通过质谱法研究膜蛋白表达。通过 ELISA、钙荧光和μ-钙蛋白酶测定评估肺移植或 ivacaftor 治疗的效果。
CF 中性粒细胞的膜胆固醇含量较低，但整合素 CD11b 表达增加，并且通过激活 μ-钙蛋白酶对炎症诱导的内质网（ER）应激做出反应。在体内和体外，增加的 μ-钙蛋白酶活性导致膜胆固醇转运蛋白 caveolin-1 的蛋白水解。在 caveolin-1 敲除小鼠中证实了 caveolin-1 对适当的膜胆固醇含量的关键作用。肺移植治疗或用 ivacaftor 治疗 PWCF 可降低循环炎症介质水平，并激活 caveolin-1 和膜胆固醇增加，同时使中性粒细胞黏附正常化。
结果表明，肺移植和增强剂治疗具有辅助益处，这表现在循环炎症减少和中性粒细胞黏附得到控制。
