Neutrophil‐Derived Microvesicles Enhance Pulmonary Vascular Inflammation via a Toll‐like Receptor 4 Signaling‐Dependent Mechanism

Circulating neutrophil-derived microvesicles (NMVs) are markedly elevated during sepsis and therefore could have a role in the development of indirect acute lung injury (ALI). We recently found that NMV-enriched CD11b+ MVs, immunoaffinity isolated from lipopolysaccharide (LPS)-stimulated healthy volunteer blood, have potent pro-inflammatory activity in a human peripheral blood mononuclear cell (PBMC) and lung microvascular endothelial cell (HLMEC) coculture model of pulmonary vascular inflammation (1). By contrast, immunoaffinity isolated platelet-MVs (CD61+ ) produced negligible responses in these assays, suggesting specificity of the NMV-enriched CD11b+ MV, activity. Here, we investigated the signaling mechanisms responsible for NMV-mediated activation of monocytes and HLMECs in this model.Heparinized blood from healthy donors was treated with LPS (100 ng/ml, 3 h) and NMVs (CD66b+ ) were isolated by positive immunoaffinity selection. NMVs were then incubated in PBMC-HLMEC cocultures for 4 h. NMV-induced responses were determined by flow cytometric quantification of cell surface activation markers (ICAM-1 for HLMECs, ICAM-1 and tissue factor for monocytes) and pro-inflammatory cytokine release by ELISA (TNFα, IL-8, IL-6, MCP-1).NMV-induced HLMEC activation required the presence of monocytes and was completely attenuated by anti-TNFα neutralizing antibody (ICAM-1: isotype control 302±103 vs anti-TNFα 66±29; p<0.01). Pharmacological inhibition of mitogen-activated protein kinases p38, MEK1-MEK2 and phosphoinositide-3 kinase ablated monocyte and HLMEC activation. Monocyte activation was prevented in the presence of the Toll-like receptor 4 (TLR4) inhibitor TAK-242 (e.g., TNFα release: vehicle 1108±257 vs TAK-242 16±12; p<0.001) and partially inhibited by an anti-TLR4 neutralizing antibody. However, the LPS inhibitor polymyxin-B had no effect on these responses, indicating that LPS contamination was unlikely. Furthermore, NMV activity was found to be sensitive to proteinase K and heat-treatment (55 °C, 15 min) (e.g., TNFα release: untreated 474±267 vs proteinase K 12±15; p<0.0001 vs heat-treatment 200±84; p<0.01).We have demonstrated that NMVs produced within LPS-stimulated blood are potent mediators of lung microvascular endothelial cells via monocyte-derived TNFα signaling. NMV-induced monocyte activation involves TLR4 signaling and is protease sensitive, suggesting that MVs serve as vehicles of damage-associated molecular pattern (DAMP) activity, mediated by surface proteins. Together these findings support a major role for NMVs as novel long-range mediators for systemic propagation of inflammation, contributing to the pathogenesis of sepsis-induced indirect acute lung injury. (1) Tsiridou et al. European Respiratory Journal. 2020;56:4468.