SARS-CoV-2 primed platelets-derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation

BackgroundHyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive.MethodsWe explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting.ResultsCOVID-19 patient-derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet-derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-kappa B activation and IL-1 beta/TNF alpha/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement.ConclusionsWe identified SARS-CoV-2 triggered platelets-derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19. COVID-19 poses a crucial threat to global health. Excessive NETs contribute to microthrombi through platelet-neutrophil interactions, resulting in acute respiratory distress syndrome in severe COVID-19 cases. EVs play a crucial role in intercellular communication during infection. Herein, we showed that the viral spike protein activates platelets directly, followed by intracellular miR21/let7b upregulation, which is loaded into pEVs. The pEV-carried miR-21/let-7b could interact with TLR7/8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while NF-kappa B promotes the expression of proinflammatory cytokines. The miR-21 inhibitor and TLR8 antagonist could efficiently suppress spike protein-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between platelets-derived miRNAs, NETs formation and SARS-CoV-2 infection. Host-directed therapies are a relatively new and promising approach to COVID-19 treatment. The miR-21-TLR8 axis may be a potential therapeutic target for severe COVID-19.-_k_965S5Tdr4uMAbg8aGRVideo Abstract

Automated summary

The study revealed that EVs from COVID-19 patients induced NETs formation through endocytosis uptake. Platelet-derived EVs played a crucial role in SARS-CoV-2 spike protein-triggered NETs formation, with miR-21/let-7b identified as important factors in enhancing NETs. These miRNAs could potentially serve as therapeutic targets for severe COVID-19.