Circulating Neutrophils from Critically Ill COVID-19 Patients Have Increased NETosis Via Canonical and Non-Canonical Pathways

Abstract

Introduction. The pathophysiology of infection with SARS-CoV-2 involves the lower airways and host-launched aggressive inflammatory responses leading to exacerbated lung damage in these vital tissues. Early clinical studies found that COVID-19 patients have higher levels of neutrophils in the circulation. Neutrophils are the most abundant leukocyte in circulation and are known to be highly proinflammatory due to production of neutrophil extracellular traps (NETosis). NETs are web-like chromatin structures coated with histones and proteases that both capture and kill invading pathogens. However, while being an effective countermeasure towards foreign microbes, this process also causes undesirable damage in host tissues. Therefore, we sought to characterize NETosis in circulating neutrophils from COVID-19 patients to determine whether this immunological response might be exacerbating or driving the disease state in COVID-19, rather than mitigating the virus. Methods.Blood was drawn daily from critically ill COVID-19 patients (n=16) after consent was obtained. Healthy controls (n=13) were screened for COVID-19 and gave blood once a week. Blood was drawn into lithium heparin tubes (BD Vacutainer). Neutrophils were isolated using PolymorphprepTM(PROGEN) per manufacturer's instructions. Cells were resuspended at 2x106 cells/ml for functional assays. Neutrophils were stimulated with increasing concentrations of PMA (Phorbol 12-myristate 13-acetate) of 2.5nM, 25nM and 250nM to stimulate NETosis via the canonical pathway, and nigericin at 15uM for the non-canonical pathway. NETosis was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen) and by NET visualization via myeloperoxidase and nuclear staining (using Polyclonal Rabbit Anti-Human Myeloperoxidase by Dako and Hoescht stain by Invitrogen). Results.Functional NETosis assays of circulating neutrophils from COVID-19 patients demonstrate overall increased NETosis determined by increased release of dsDNA. This enhanced NETosis occurred at baseline and after stimulation with PMA when compared to healthy controls (Figure 1A, p <0.0001). Fluorescent microscopy also demonstrated increased NETosis in neutrophils from COVID-19 patients (Figure 1B;MPO-green and nucleus-blue). NETosis via the non-canonical pathway (induction with nigericin) was also increased in COVID-19 patients versus controls (p=0.02). Conclusions.Circulating neutrophils from critically ill COVID-19 patients are more prone to produce NETs than circulating neutrophils from healthy individuals. This is likely to lead to NETmediated tissue injury once neutrophils enter inflamed tissue, where they can potentially drive acute lung injury and acute respiratory distress syndrome, common causes of mortality in COVID-19. The finding of increased production of NETs by both canonical and non-canonical pathways is consistent with an overall hyper-activated state in COVID-19.