Karpurapu Manjula, Nie Yunjuan, Chung Sangwoon, Yan Jiasheng, Dougherty Patrick, Pannu Sonal, Wisler Jon, Harkless Ryan, Parinandi Narasimham, Berdyshev Evgeny, Pei Dehua, Christman John W
Division of Pulmonary, Critical Care and Sleep Medicine, Ohio State University Wexner Medical Center, Davis Heart and Lung Research Institute, Columbus, OH, United States.
Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu, China.
Front Physiol. 2024 May 15;15:1378565. doi: 10.3389/fphys.2024.1378565. eCollection 2024.
Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.
细胞外囊泡通过运输生物活性大分子介导细胞间通讯。我们之前的研究表明，活化T细胞核因子细胞质成员3（NFATc3）在小鼠肺巨噬细胞中因脂多糖（LPS）而被激活。新型细胞可渗透的钙调神经磷酸酶肽抑制剂CNI103对NFATc3激活的抑制减轻了LPS处理小鼠急性肺损伤（ALI）的发展。尽管促炎脂质介质是已知的肺部炎症和损伤的促成因素，但钙调神经磷酸酶-NFAT途径是否调节细胞外囊泡（EV）脂质含量以及该含量是否促成ALI发病机制仍不清楚。在本研究中，通过液相色谱结合质谱（LC-MS/MS）分析了来自小鼠支气管肺泡灌洗液（BALF）的EVs的脂质介质。我们的数据表明，来自LPS处理小鼠的EVs含有显著更高水平的花生四烯酸（AA）代谢物，而预先用CNI103处理则发现其水平较低。ALI期间肺组织细胞质磷脂酶A2（cPLA2）的催化活性增加，这与EVs中花生四烯酸（AA）量的增加相关。此外，ALI与肺组织中cPLA2、环氧化酶2（COX2）和脂氧合酶（5-LOX、12-LOX和15-LOX）表达增加有关，并且用CNI103预处理可抑制cPLA2的催化活性以及cPLA2、COX和LOX转录本的表达。此外，小鼠肺微血管内皮细胞（PMVEC）单层与来自LPS处理小鼠的BALF EVs共培养的NFAT-荧光素酶报告巨噬细胞增加了肺微血管内皮细胞（PMVEC）单层屏障通透性和巨噬细胞中的荧光素酶活性。然而，来自CNI103处理小鼠的EVs对PMVEC单层屏障完整性没有负面影响。总之，来自LPS处理小鼠的BALF EVs携带具有生物活性的NFATc依赖性、源自AA的脂质，这些脂质在调节PMVEC单层屏障功能中起作用。
