key: cord-0855774-9mx79mou
authors: Strich, Jeffrey R; Ramos-Benitez, Marcos J; Randazzo, Davide; Stein, Sydney R; Babyak, Ashley; Davey, Richard T; Suffredini, Anthony F; Childs, Richard W; Chertow, Daniel S
title: Fostamatinib Inhibits Neutrophils Extracellular Traps Induced by COVID-19 Patient Plasma: A Potential Therapeutic
date: 2020-12-24
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa789
sha: bf39a3a0580e61a3060928e9bf6eed0284e06cbf
doc_id: 855774
cord_uid: 9mx79mou

Abstract:Neutrophil extracellular traps (NETs) contribute to immunothrombosis and have been associated with mortality in Coronavirus Disease 2019 (COVID-19). We stimulated donor neutrophils with plasma from patients with COVID-19 and demonstrate that R406 can abrogate the release of NETs. These data provide evidence for how fostamatinib may mitigate neutrophil-associated mechanisms contributing to COVID-19 immunopathogenesis.

A c c e p t e d M a n u s c r i p t While steroid therapy is an approach to dampen the immune response and other complications of COVID-19 such as acute respiratory distress syndrome (ARDS), concern remains about unintended consequences from steroid therapy, including prolonged viral shedding and risk of secondary infection [3] . Therefore, interest remains in identifying host-targeted immunomodulators to improve outcomes of patients with COVID-19 with limited or no adverse effects.

During systemic bacterial or viral infection, neutrophils release neutrophil extracellular traps (NETs) in a process called NETosis. NETs are web-like structures composed of DNA-decorated citrullinated histones (cHIS) and antimicrobial granules that limit dissemination of pathogens in the bloodstream [4] . However, elevated levels of serum NETs contribute to endothelial dysfunction and microvascular thrombosis that complicate severe COVID-19 and are associated with multiorgan pathology and mortality [5, 6] . Both host and microbial components in COVID-19 patient plasma contribute to NETosis [6] . One such pathway is the stimulation of FcRIIA receptors on neutrophils by antigen-antibody complexes which is mediated via spleen tyrosine kinase (SYK) phosphorylation and downstream signaling. R406, the metabolically active component of fostamatinib, an FDAapproved drug, is a potent SYK inhibitor. R406 has been shown to limit inflammatory cytokine production of human macrophages stimulated by COVID-19 plasma by disrupting FcRIIA receptormediated SYK signaling [7] . We sought to determine if SYK pathway inhibition limits NETosis in human neutrophils. We observed that R406 potently inhibits NETosis of healthy donor neutrophils stimulated with COVID-19 patient plasma, raising the potential for therapeutic benefit of fostamatinib for severe COVID-19. Units (RFU) were used to quantify NETs formation over 6 hours with data acquisition beginning ~15 minutes after stimulation. For inhibition assays, R406 (Rigel Pharmaceuticals) was preincubated with healthy neutrophils at either 1M and 4M for 30 minutes prior to stimulation.

We stimulated healthy neutrophils with plasma from hospitalized patients with COVID-19 (n=7) obtained from disease days ranging between day 11 and 27 relative to symptoms onset, healthy controls (n=6), or 1M phorbol myristate ester (PMA) as a positive control. The kinetics in NETs formation showed a gradual increase in NETs release over 6 hours when stimulated by plasma from patients with COVID-19 or PMA ( figure 1A) . We analyzed the RFU values at 6 hours and A c c e p t e d M a n u s c r i p t demonstrated significant differences in total NETs release triggered by plasma from COVID-19 positive patients, when compared to healthy control plasma (7.7e 6 vs 8.1e 5 ; p=0.01) ( Figure 1B) .

Pre-incubation of neutrophils with R406 dramatically reduced NETs release triggered by COVID-19 plasma across all time points but did not alter NETosis induced by PMA ( figure 1A) . The inhibitory effect of R406 was dose-dependent with a decrease of 72% (2.2e 6 RFU, p=0.04) at 1M and 81% (1.4e 6 RFU, p=0.02) at 4M, when compared to neutrophils stimulated with plasma from COVID-19 patients (figure 1B).

This is the first report to identify that R406, a potent SYK inhibitor and the metabolically active component of fostamatinib, inhibits NETosis among healthy donor neutrophils stimulated with COVID-19 patient plasma. This finding was consistent across seven different COVID-19 patients and R406 inhibition occurred in a concentration-dependent manner that is in the range of steady state concentration (800nM to 1.6M) from an oral dose of 150 mg twice daily. Fostamatinib is currently FDA approved for adults with immune thrombocytopenia who have had insufficient response to previous treatment [8] . SYK is a cytoplasmic tyrosine kinase involved in intracellular signaling through Fc receptors or c-type lectin (CLEC) receptors on neutrophils, monocytes, macrophages, and platelets [9] . SYK signaling is therefore involved in a variety of cells types and pathways that have been implicated to contribute to the aberrant immune response and immunothrombosis worsening outcomes in patients with severe COVID-19.

A major contributing factor to the development of immunothrombosis, a feature observed in COVID-19 induced ARDS that may be associated with clinical outcomes and an area of much investigation, is the release of NETs [6] . A well-characterized mechanism for NETs production is stimulation of the FcRIIA receptor and downstream SYK signaling, which contributes to heparininduced thrombocytopenia [10] . Signaling through the FcRIIA receptor is primarily driven by antigen-antibody complexes. In COVID-19 robust antibodies responses have been associated with poor clinical outcomes raising the possibility that antigen-antibody complexes contribute to A c c e p t e d M a n u s c r i p t pathogenic NETosis [11, 12] . Our results support the hypothesis that antigen-antibody complexes circulating in COVID-19 patient plasma might contribute to NETosis, which is inhibited by R406. By contrast R406 did not inhibit NETosis from neutrophils simulated with PMA in our experiments.

This finding is likely attributable to the fact that PMA induces NETosis through redundant pathways, including pathways independent of SYK phosphorylation [13] (supplemental figure 1) .

While immune complex mediated NETosis may play a role in COVID-19 immunopathogenesis, a variety of microorganisms and endogenous stimuli including cytokines and damage associated molecular patterns (DAMPs) also have the ability to stimulate the release of NETs and defining their role in COVID-19 disease remains to be elucidated [4] . We show that SYK inhibition using R406 prevents NETosis induced by COVID-19 patient plasma and support a potential therapeutic role for fostamatinib in the treatment of COVID-19 for which clinical trials are underway (ClinicalTrials.gov; NCT04579393) [7, 14] . 

Remdesivir for the Treatment of Covid-19 -Final Report

Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report

Corticosteroids in COVID-19 ARDS: Evidence and Hope During the Pandemic

Neutrophil extracellular traps in immunity and disease

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Neutrophil extracellular traps in COVID-19

Anti-SARS-CoV-2 IgG from severely ill COVID-19 patients promotes macrophage hyper-inflammation responses

The SYK tyrosine kinase: a crucial player in diverse biological functions

Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia

Kinetics of viral load and antibody response in relation to COVID-19 severity

Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection

Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps

A High-Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury

A c c e p t e d M a n u s c r i p t