Neutrophils from the circulation of critically ill COVID-19 patients may promote inflammation via increased production of reactive oxygen species

ABSTRACT

Background: Neutrophils are an important part of the innate immune system and play a vital role in host defense. Neutrophils target invading pathogens through both intracellular and extracellular mechanisms. One of their primary antimicrobial mechanisms is the production of reactive oxygen species (ROS) within phagolysosomes during oxidative burst. ROS production is a marker of the inflammatory/activity state of neutrophils. Although neutrophils are crucial in the defense against microorganisms, recruitment and hyperactivation of neutrophils can also cause collateral damage to the host. Recent studies have shown that neutrophils may be playing a role in acute respiratory distress syndrome (ARDS) in COVID-19. We hypothesized that ROS production would be increased secondary to elevated activation of circulating neutrophils in COVID-19 patients and may be playing a role in immunopathology.Methods: Circulating neutrophils were isolated from the blood of 20 human subjects with COVID-19 and 12 healthy controls. Neutrophils at 2x106 cells/ml were incubated with 10μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for 20min at 37°C. Neutrophils were centrifuged at 500g, resuspended in HBSS-/-, plated at 2x105 in 96-well plates and exposed to 0, 2.5, 25 and 250nM phorbol myristate acetate (PMA;in triplicate). Fluorescence was measured (Infinite M200, TECAN) at 495nm520nm every 15min for 2hr. ROS production was analyzed using mixed-effects models with Geisser- Greenhouse correction and Sidak's multiple comparisons test.Results: ROS production was increased in neutrophils isolated from the circulation of critically ill COVID-19 patients as compared to healthy controls (2581nm versus 3790nm, respectively, at 120min, p<0.05). Upon stimulation with PMA, ROS production was also increased in COVID-19 patients relative to controls (2.5 PMA 16597 versus 10549, respectively, <0.001;25 PMA 20633 versus 11312, respectively, p<0.0001). At the final timepoint, 1.57, 1.82 and 1.85-fold difference in ROS production was noted at 2.5, 25 and 250nM respectively between patient populations. Conclusion: Increased ROS production at baseline and upon stimulation demonstrates that neutrophils in the circulation of critically ill COVID-19 patients with ARDS have an elevated activation state. These patients are known to have recruitment of neutrophils to the lung parenchyma, where the production and release of ROS may cause collateral damage. Further, neutrophils have been implicated in the vascular and systemic immunopathology of COVID-19, which may be driven by release of these damaging and toxic molecules. Neutrophil-associated pathways may serve as therapeutic targets and quantitative markers of disease in COVID-19 patients.