Signaling Transduction Mechanisms Mediating Inflammatory Effects of Cd40l on Human Pulmonary Micro-Vascular Endothelial Cells

Background: Owing to its role in endothelial cell (EC) activation, CD40L figures prominently in the pathogenesis of acute lung injury (ALI). However, the precise mechanisms by which CD40L drives an inflammatory response in this disease remain unknown. <br><br>Methods: The expression, phosphorylation and sub-cellular localization of signaling proteins and cytokines studied were assessed by a combination of the PathScan Intracellular Signaling Array, Western Blotting, qRT-PCR and immunofluorescence analyses. Knockout mouse models were established by adeno-associated virus (AAV)-mediated delivery of sgRNAs into mice with EC-specific Cas9 expression. CD40L-induced pulmonary pathologic changes and cytokine secretion were assessed by the hematoxylin and eosin (H&amp;E) staining, Evans blue extravasation, wet-dry weight ratio and ELISA assays. <br><br>Findings: CD40L induced the transactivation of NF-κB and downstream inflammatory gene expression through a p38 MAPK-dependent mechanism in human pulmonary micro-vascular endothelial cells (HPMVECs). The effect of CD40L on ECs might also be correlated with a sequential activation of the IKK/IκB/NF-κB pathway and subsequent NF-κB nuclear translocation, being accompanied by dynamic actin cytoskeletal changes. Knockdown experiments demonstrated that CD40L mediated the activation of the p38 MAPK/NF-κB and IKK/IκB/NF-κB pathways, in large part, via GEF-H1/RhoA signaling. The critical role of GEF-H1/RhoA in CD40L-induced EC activation was further confirmed by in vivo findings that knockout of either GEF-H1 or RhoA blocked CD40L-triggered pulmonary pathological changes and secretion of NF-κB-dependent cytokines associated with ALI. <br><br>Interpretation: These findings may help to determine key pharmacological targets of intervention for preventing the progress of ALI.&nbsp; <br><br>Funding Information: This work was supported by the National Natural Science Foundation of China (Grant No. 81901959 and Grant No. 81971832), Natural Science Foundation of Shanghai (Grant No. 19ZR1432100 and Grant No. 21ZR1440600), and Shanghai Sailing Program (Grant No. 19YF1430500).<br> <br>Declaration of Interests: The authors declare that they have no competing interests.<br><br>Ethics Approval Statement: All experimental protocols were approved by the Research Ethics Review Committee of Shanghai Jiao Tong University. Methods used in the present study were carried out in accordance with approved guidelines and regulations. It conformed to the provisions of the Declaration of Helsinki.