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

Abstract

BackgroundCirculating 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.MethodsHeparinized 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).ResultsNMV‐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).ConclusionsWe 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