Introduction: Several serine/threonine kinases are essential for inducing neutrophil extracellular trap (NET) formation (NETosis); however, the involvement of tyrosine kinases (TKs) in NETosis is not clearly established. We hypothesized that many TKs also regulate NETosis by modulating reactive oxygen species (ROS) production and transcription activation. The objective of the study was to elucidate the role of TKs in NETosis and device target drugs for the treatment of NET-associated pathologies.

Methods and Results: We used neutrophils isolated from healthy individuals and induced NADPH oxidase 2 (NOX)-dependent NETosis with PMA and E. coli LPS 0128 in the absence or presence of a clinically approved TKs inhibitor, bosutinib, that inhibits several tyrosine kinases at different inhibitory concentrations. We showed that bosutinib, at low concentrations (<1000nM), does not affect baseline or NOX-dependent NETosis. At mid concentration range (1-10 μM), bosutinib inhibited baseline as well as NOX-dependent NETosis. By contrast, bosutinib at high concentrations (20-40 μM) promoted baseline and NOX-dependent NETosis. These data suggested that kinases that require high concentrations of bosutinib (e.g., Ret, PKA, CK1 and CK2) are suppressors of NETosis whereas kinases that require low concentrations of bosutinib (e.g., Csk, Syk and Alk) are promoters of NETosis. ROS measurements showed that bosutinib at concentrations up to 10 μM, both NOX and mitochondrial ROS (mROS) were suppressed during baseline NETosis. Interestingly, bosutinib at similar concentrations suppressed ROS production during LPS, but not PMA-mediated NETosis. Therefore, bosutinib suppressed PMA-induced NETosis by regulating the subsequent steps of NETosis. High concentrations of bosutinib (40 μM) suppressed NOX-dependent ROS but enhanced NETosis by promoting mROS production. Tyrosine kinases with high IC₅₀ values (e.g., Ret, CK1, CK2 and PKA) are important for NOX-dependent ROS production, but they suppressed mROS production.

Conclusion: Thus, we propose that multiple tyrosine kinases regulate NETosis and targeting TKs hold promise in regulating NETosis associated clinical conditions.