Sueblinvong Viranuj, Fan Xian, Hart Craishun, Molina Samuel, Koval Michael, Guidot David M
Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
FUJIFILM Irvine Scientific, Warminster, Pennsylvania, USA.
Alcohol Clin Exp Res (Hoboken). 2023 Oct;47(10):1839-1849. doi: 10.1111/acer.15174. Epub 2023 Aug 29.
Chronic alcohol ingestion predisposes to lung injury and disrepair during sepsis. Our previous studies outlined roles for transforming growth factor-beta 1 (TGFβ1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in epithelial barrier homeostasis and how alcohol perturbs their expression and signaling. Here we hypothesize that ethanol-exposed lung fibroblasts (LF) are a source of dysregulated TGFβ1 and GM-CSF and thereby alter airway epithelial barrier function.
Human or rat LF were cultured ± ethanol for 2 weeks and then co-cultured with human or rat airway epithelial cells (AEC) seeded on Transwell permeable supports. In selected groups, a TGFβ1 receptor type 1 (TGFβR1) inhibitor (SB431542) or a TGFβ1 neutralizing antibody was applied. Transepithelial electrical resistance (TER) was measured prior to co-culture and on day 5 of co-culture. AEC were then analyzed for the expression of selected tight junction and mesenchymal proteins, and transwell membranes were analyzed by immunofluorescence microscopy for ZO-1 expression and localization. TGFβ1 and GM-CSF levels in conditioned media from the co-cultures were quantified by ELISA.
AEC co-cultured with ethanol-exposed LF (ELF) showed a significant reduction in TER and corresponding decreases in ZO-1 expression, whereas collagen type 1A1 and α-smooth muscle actin protein expression were increased. In parallel, in conditioned media from the ELF + AEC co-cultures, activated TGFβ1 levels increased and GM-CSF levels decreased. Notably, all the effects of ELF on the AEC were prevented by blocking TGFβ1 activity.
Prior ethanol exposure to LF induces barrier dysfunction in naive AEC in a paracrine fashion through activation of TGFβ1 signaling and suppression of GM-CSF. These experimental findings provide a potential mechanism by which chronic alcohol ingestion impairs airway epithelial integrity and renders individuals susceptible to lung injury.
长期摄入酒精会使机体在脓毒症期间更易发生肺损伤且损伤后难以修复。我们之前的研究阐述了转化生长因子-β1（TGFβ1）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）在上皮屏障稳态中的作用，以及酒精如何扰乱它们的表达和信号传导。在此，我们假设乙醇处理过的肺成纤维细胞（LF）是TGFβ1和GM-CSF失调的来源，从而改变气道上皮屏障功能。
将人或大鼠的LF在有或无乙醇的条件下培养2周，然后与接种在Transwell可渗透支持物上的人或大鼠气道上皮细胞（AEC）共培养。在选定的组中，应用1型TGFβ1受体（TGFβR1）抑制剂（SB431542）或TGFβ1中和抗体。在共培养前和共培养第5天测量跨上皮电阻（TER）。然后分析AEC中选定的紧密连接和间充质蛋白的表达，并通过免疫荧光显微镜分析Transwell膜上ZO-1的表达和定位。通过酶联免疫吸附测定法（ELISA）对共培养条件培养基中的TGFβ1和GM-CSF水平进行定量。
与乙醇处理过的LF（ELF）共培养的AEC显示TER显著降低，ZO-1表达相应减少，而1A1型胶原蛋白和α-平滑肌肌动蛋白的蛋白表达增加。同时，在ELF + AEC共培养的条件培养基中，活化的TGFβ1水平升高，GM-CSF水平降低。值得注意的是，通过阻断TGFβ1活性可防止ELF对AEC的所有影响。
预先用乙醇处理LF会通过激活TGFβ1信号传导和抑制GM-CSF以旁分泌方式诱导未接触过乙醇的AEC出现屏障功能障碍。这些实验结果提供了一种潜在机制，通过该机制长期摄入酒精会损害气道上皮完整性并使个体易患肺损伤。
