Department of Immunology and Nano-Medicine, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States.
Respiratory Immunology Program, Lovelace Respiratory Research Institute, Albuquerque, NM, United States.
Front Immunol. 2022 Jun 14;13:803362. doi: 10.3389/fimmu.2022.803362. eCollection 2022.
Cigarette smoke (CS) exposure is strongly associated with chronic obstructive pulmonary disease (COPD). In respiratory airways, CS exposure disrupts airway barrier functions, mucous/phlegm production, and basic immune responses of airway epithelial cells. Based on our recent identification of a specific immunomodulatory long noncoding RNA (lncRNA), we investigated its role in CS-induced responses in bronchial airways of cynomolgus macaque model of CS-induced COPD and in former smokers with and without COPD. The lncRNA was significantly upregulated in CS-induced macaque airways and in COPD airways that exhibited higher mucus expression and goblet cell hyperplasia. Experimental models of cells derived from COPD subjects recapitulated the augmented inflammation and mucus expression following the smoke challenge. Blocking of lncRNA expression in cell culture setting suppressed the smoke-induced and COPD-associated dysregulated mucoinflammatory response suggesting that this airway specific immunomodulatory lncRNA may represent a novel target to mitigate the smoke-mediated inflammation and mucus hyperexpression.
In conducting airways, CS disrupts airway epithelial functions, mucociliary clearances, and innate immune responses that are primarily orchestrated by human bronchial epithelial cells (HBECs). Mucus hypersecretion and dysregulated immune response are the hallmarks of chronic bronchitis (CB) that is often exacerbated by CS. Notably, we recently identified a long noncoding RNA (lncRNA) antisense to ICAM-1 () that mediates airway epithelial responses.
To investigate the role of lncRNA in CS-induced airway inflammation and mucin hyperexpression in an animal model of COPD, and in HBECs and lung tissues from former smokers with and without COPD. To interrogate lncRNA role in CS-mediated airway mucoinflammatory responses by targeted gene editing.
Small airway tissue sections from cynomolgus macaques exposed to long-term mainstream CS, and those from former smokers with and without COPD were analyzed. The structured-illumination imaging, RNA fluorescence hybridization (FISH), and qRT-PCR were used to characterize lncRNA expression and the expression of inflammatory factors and airway mucins in a cell culture model of CS extract (CSE) exposure using HBECs from COPD (CHBEs) in comparison with cells from normal control (NHBEs) subjects. The protein levels of mucin MUC5AC, and inflammatory factors ICAM-1, and IL-6 were determined using specific ELISAs. RNA silencing was used to block lncRNA expression and lentivirus encoding lncRNA was used to achieve overexpression (LASI-OE).
Compared to controls, lncRNA was upregulated in CS-exposed macaques and in COPD smoker airways, correlating with mucus hyperexpression and mucus cell hyperplasia in severe COPD airways. At baseline, the unstimulated CHBEs showed increased lncRNA expression with higher expression of secretory mucin MUC5AC, and inflammatory factors, ICAM-1, and IL-6 compared to NHBEs. CSE exposure of CHBEs resulted in augmented inflammation and mucus expression compared to controls. While RNA silencing-mediated knockdown suppressed the mucoinflammatory response, cells overexpressing lncRNA showed elevated mRNA levels of inflammatory factors.
Altogether, lncRNA may represent a novel target to control the smoke-mediated dysregulation in airway responses and COPD exacerbations.
香烟烟雾（CS）暴露与慢性阻塞性肺疾病（COPD）密切相关。在呼吸道中，CS 暴露会破坏气道屏障功能、黏液/痰的产生以及气道上皮细胞的基本免疫反应。基于我们最近对一种特定的免疫调节长非编码 RNA（lncRNA）的鉴定，我们研究了其在 CS 诱导的 COPD 恒河猴模型支气管气道中的作用，以及在有和没有 COPD 的前吸烟者中的作用。lncRNA 在 CS 诱导的猕猴气道和 COPD 气道中显著上调，这些气道表现出更高的黏液表达和杯状细胞增生。来自 COPD 患者的细胞实验模型在烟雾暴露后表现出增强的炎症和黏液表达。在细胞培养环境中阻断 lncRNA 表达可抑制烟雾诱导和 COPD 相关的异常黏液炎症反应，这表明这种气道特异性免疫调节 lncRNA 可能是减轻烟雾介导的炎症和黏液高表达的新靶点。
在气道中，CS 会破坏气道上皮细胞的功能、黏液纤毛清除功能和主要由人支气管上皮细胞（HBECs）协调的固有免疫反应。黏液分泌过度和免疫反应失调是慢性支气管炎（CB）的特征，通常会因 CS 而加重。值得注意的是，我们最近鉴定了一个反义于 ICAM-1 的长非编码 RNA（lncRNA）（），它介导气道上皮细胞的反应。
在 COPD 动物模型中，研究 lncRNA 在 CS 诱导的气道炎症和黏液高表达中的作用，以及在有和没有 COPD 的前吸烟者的 HBECs 和肺组织中的作用。通过靶向基因编辑来研究 lncRNA 在 CS 介导的气道黏液炎症反应中的作用。
分析来自长期暴露于主流 CS 的恒河猴的小气道组织切片，以及来自有和没有 COPD 的前吸烟者的组织切片。使用结构光照明显微镜、RNA 荧光原位杂交（FISH）和 qRT-PCR 来表征 lncRNA 表达以及 CS 提取物（CSE）暴露后 HBECs 中炎症因子和气道黏液素的表达，与来自正常对照（NHBEs）的细胞进行比较。使用特定的 ELISA 测定黏蛋白 MUC5AC 和炎症因子 ICAM-1 和 IL-6 的蛋白水平。使用 RNA 沉默来阻断 lncRNA 表达，并用慢病毒编码 lncRNA 来实现过表达（LASI-OE）。
与对照组相比，lncRNA 在 CS 暴露的猕猴和 COPD 吸烟者的气道中上调，与严重 COPD 气道中的黏液过度表达和黏液细胞增生相关。在基线时，与 NHBEs 相比，未受刺激的 CHBEs 表现出更高的 lncRNA 表达，同时也表现出更高的分泌性黏蛋白 MUC5AC 和炎症因子 ICAM-1 和 IL-6 的表达。与对照组相比，CSE 暴露于 CHBEs 会导致炎症和黏液表达增强。虽然 RNA 沉默介导的 lncRNA 敲低抑制了黏液炎症反应，但过表达 lncRNA 的细胞表现出更高的炎症因子 mRNA 水平。
总之，lncRNA 可能是控制气道反应和 COPD 加重的烟雾介导失调的新靶点。
