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Background Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Methods We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. Results We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a−/−/tlr2−/− mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a−/−/tlr2−/− mice. Conclusions This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

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Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa-induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with lipopolysaccharides of P. aeruginosa, and is responsible for P. aeruginosa-induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1-dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa-induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa-induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilatorassociated lung injury and fibrosis in P. aeruginosa-induced pneumonia. Results CLEC5A mediates P. aeruginosa-induced NET formation via caspase-1Lipopolysaccharides (LPS) from various bacterial strains have been shown to induce NET formation (32). To understand the roles of CLEC5A in P. aeruginosa infection, we isolated neutrophils from wild type (WT), Clec5a -/-, Tlr2 -/-, and Tlr4 -/mice, and compared their responses to various LPS. NET formation was determined by the colocalization of histone, MPO, and DNA under a confocal microscope (Suppl. Fig. 1A & 1B). Surprisingly, the levels of E. coli LPS-induced NET formation were similar in WT, Clec5a -/-, Tlr2 -/-, and Tlr4 -/neutrophils (Fig. 1A). A similar observation was found in neutrophils incubated with LPS from Klebsiella pneumoniae (K.p; Fig. 1A).However, NET formation induced by LPS from P. aeruginosa PAO1 strain was attenuated in Clec5a -/neutrophils (Fig. 1A). These observations suggest that LPS from PAO1 induces NET formation via CLEC5A. In contrast, phagocytosis and killing of P. aeruginosa in neutrophils were unaffected by Clec5a deletion (Suppl. Fig. 1C). We further compared NET formation in neutrophils incubated with live and UV-inactivated P. aeruginosa; whilst both live and UV-inactivated P. aeruginosa induced similar levels of NET formation in WT and Tlr4 -/mice, NET formation was reduced dramatically in Clec5a -/and Tlr2 -/neutrophils (Fig. 1B). These observations suggest that P. aeruginosa-induced NET formation occurs via CLEC5A and TLR2, and is not dependent on toxic substances secreted from live bacteria. TLR2 does not bind P. aeruginosa LPS, therefore the less potent effect of TLR2 deletion compared to Clec5a deletion may be due to the co-activation of CLEC5A/TLR2 complex by P. ...