SYK Kinase As a Master Regulator of Netosis

Background NETosis is a key neutrophil response to tissue damage and pathogens. It is a form of programmed cell death resulting in chromatin decondensation, histone citrullination and controlled extracellular release of chromosomal DNA. This DNA complexed to a variety of noxious proteins such as elastase, myeloperoxidase and citrullinated histones is called neutrophil extracellular traps (NETs). NETs are largely responsible for anti-viral and anti-microbial effects of neutrophils during infection. NETs can cause significant tissue damage in various pathological conditions such as severe infections (COVID-19 and others) and multiple autoimmune and autoinflammatory diseases characterized by hyperactivation of innate and adaptive immune systems. Together with concurrent platelet and coagulation cascade activation, NETs formation is known to drive coagulopathy and thrombosis often associated with sterile inflammation in autoimmune and autoinflammatory diseases and in severe COVID-19 (Y. Zhou et al.). Strong correlation between NETosis biomarkers and thrombosis has been established in patients with many thrombogenic diseases (Mołek et al.; Martos et al.). Inhibition of NETs using DNAse treatment, protein arginine deiminase 4 (PAD4) inhibitors, or PAD4 knockout results in decrease in thrombi formation in vitro and in vivo (Franck et al.; Martinod et al.; Yan et al.). NETs release induced by plasma from severe COVID-19 patients in normal human neutrophils can be blocked in the presence of the highly selective SYK inhibitor R406, an active metabolite of the immune thrombocytopenia drug fostamatinib (Strich et al.). The inhibitor is thought to block antibody- and viral particle-mediated neutrophil activation through FcgRIIA and C-type lectin receptors (CLRs) controlled by SYK.

Methods/Results To further elucidate the role of SYK kinase in NETosis, we have analyzed healthy human neutrophil responses to various stimuli in the presence of multiple commercially available SYK inhibitors using the Incucyte Live-Cell Analysis System. As expected, R406 potently inhibited immune complex induced NETosis mediated by FcgRIIA. To our surprise, every SYK inhibitor we've tested selectively blocked seemingly unrelated LPS-mediated TLR4-dependent NETs release. The effect was clearly specific as SYK inhibition had no effect on SYK-independent LPS-induced cytokine secretion or on cell proliferation at concentrations far exceeding those fully blocking NETosis. In contrast to SYK inhibitors, neither a selective JAK inhibitor nor dexamethasone had a significant effect on NETs release under the same assay conditions.

Moreover, R406 potently inhibited NETs production in response to hydrogen peroxide and monosodium urate (MSU) crystal treatment of healthy neutrophils. The mechanisms behind the ability of SYK inhibitors to block NETs formation in response to such a plethora of unrelated stimuli will be further discussed.

Conclusions The ability of SYK inhibitors to block both NETosis and platelet activation (in case of platelets, via CLEC2 and GPVI - two receptors involved in thrombosis yet dispensable for hemostasis) is consistent with possibility that, in addition to anti-inflammatory properties, SYK inhibition has potential anti-thrombotic effects (Harbi et al.). Indeed, the latter might be behind the lower than expected rates of thrombotic events in ITP patients observed in clinical trials of fostamatinib (Cooper et al.).

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Laura Martos et al. Int J Mol Sci. 2020 Aug 6;21(16):5651.

Patrycja Mołek et al. Thromb Res. 2022 May;213:1-7.

Grégory Franck et al. Circ Res. 2018 June 22;123(1): 33-42.

Kimberly Martinod et al. Proc Natl Acad Sci U S A. 2013 May 21;110(21):8674-8679.

Yanyan Yan et al. Aging (Albany NY). 2019 Sep 15;11(17):6951-6959.

Jeffrey R Strich et al. J Infect Dis. 2021 Mar 15;223(6):981-984.

Maan H. Harbi et al. Int J Mol Sci. 2022 Jul;23(13):6982.

Nichola Cooper et al. Ther Adv Hematol. 2021 Apr 30;12:20406207211010875

Markovtsov:Rigel Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Yi:Rigel Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Masuda:Rigel Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company.