Laboratory of Pulmonary Immuno-toxicology, Environmental Toxicology Department, Southern University and A&M College, Baton Rouge, LA, 70813, USA.
Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USA.
Arch Toxicol. 2018 May;92(5):1767-1783. doi: 10.1007/s00204-018-2185-0. Epub 2018 Apr 6.
Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death and disability worldwide by 2030; with cigarette smoking (active or passive) being one of the chief cause of its occurrence. Cigarette smoke exposure has been found to result in excessive inflammation and tissue injury, which might lead to COPD, although the exact pathophysiology of the disease remains elusive. While previous studies have demonstrated the role of membrane-bound Toll-like receptors (TLRs) in cigarette smoke (CS)-induced inflammation, scant information is available about the role of cytosolic NOD-like receptors (NLRs) in regulating CS-mediated inflammatory responses. Thus, we investigated the role of NLRP10 and NLRP12 in regulating inflammatory responses in human alveolar type II epithelial cells (A549) and human monocytic cells (THP-1) in response to a challenge with cigarette smoke extract (CSE). We observed CSE-mediated increase in caspase-1 activity; production of IL-1β and IL-18; and expression of NLRP10 and NLRP12 in A549 and THP-1 cells. Interestingly, immunofluorescence imaging results demonstrated an increase in the membrane recruitment of NLRP10 and NLRP12 proteins in CSE-challenged A549 cells. We also observed an increase in the expression of lipid raft proteins (caveolin-1, caveolin-2, and flotillin-1) and an induction of lipid raft assembly following CSE-exposure in A549 cells. Lipid rafts are cholesterol-rich membrane microdomains well known to act as harbours for signalling molecules. Here we demonstrate the recruitment of NLRP10 and NLRP12 in lipid raft entities as well as the interaction of NLRP12 with the lipid raft protein caveolin-1 in CSE-challenged A549 cells. Furthermore, enrichment of lipid raft entities with poly-unsaturated fatty acids (PUFA) rescued A549 cells from CSE-mediated membrane recruitment of NLRP10 and NLRP12, and also from inflammatory responses and inflammasome activation. Enrichment of membrane microdomains with PUFA was able to reverse filipin (chemical agent used for disrupting lipid rafts)-mediated enhanced inflammation in CSE-challenged A549 cells. Overall, our findings unveil a novel mechanism by identifying an important role of membrane microdomains (lipid rafts) in regulating CSE-induced inflammation and NLRP10/NLRP12-dependent signalling in A549 cells.
慢性阻塞性肺疾病（COPD）预计将成为 2030 年全球第三大死亡和残疾原因；吸烟（主动或被动）是其主要发生原因之一。已经发现，吸烟会导致过度炎症和组织损伤，从而导致 COPD，尽管该疾病的确切病理生理学仍不清楚。虽然先前的研究已经表明，膜结合的 Toll 样受体（TLR）在香烟烟雾（CS）诱导的炎症中起作用，但关于细胞溶质核苷酸结合寡聚化结构域样受体（NLR）在调节 CS 介导的炎症反应中的作用的信息很少。因此，我们研究了 NLRP10 和 NLRP12 在调节人肺泡 II 型上皮细胞（A549）和人单核细胞（THP-1）对香烟烟雾提取物（CSE）的炎症反应中的作用。我们观察到 CSE 介导的半胱天冬酶-1 活性增加；IL-1β和 IL-18 的产生；以及 A549 和 THP-1 细胞中 NLRP10 和 NLRP12 的表达。有趣的是，免疫荧光成像结果表明，在 CSE 挑战的 A549 细胞中，NLRP10 和 NLRP12 蛋白的膜募集增加。我们还观察到，在 A549 细胞中暴露于 CSE 后，脂筏蛋白（窖蛋白-1、窖蛋白-2 和 flotillin-1）的表达增加，并诱导脂筏组装。脂筏是富含胆固醇的膜微区，已知作为信号分子的停泊点。在这里，我们证明了 NLRP10 和 NLRP12 在脂筏实体中的募集以及 NLRP12 与脂筏蛋白窖蛋白-1 在 CSE 挑战的 A549 细胞中的相互作用。此外，富含多不饱和脂肪酸（PUFA）的脂筏实体可使 A549 细胞免受 CSE 介导的 NLRP10 和 NLRP12 的膜募集，并免受炎症反应和炎性体激活的影响。用 PUFA 富集膜微区能够逆转 filipin（用于破坏脂筏的化学剂）介导的 CSE 挑战的 A549 细胞中增强的炎症。总的来说，我们的研究结果揭示了一种新的机制，即确定了膜微区（脂筏）在调节 CSE 诱导的炎症和 A549 细胞中 NLRP10/NLRP12 依赖性信号中的重要作用。
