Increased IL-8, Neutrophil Activation Phenotypes and NETosis in Critically Ill COVID-19 Patients

Background:  Increased inflammation is a hallmark of COVID-19, with pulmonary and systemic inflammation identified in multiple cohorts of patients. Definitive cellular and molecular pathways driving severe forms of this disease remain uncertain. Neutrophils, the most numerous leukocytes in blood circulation, can contribute to immunopathology in infections, inflammatory diseases and acute respiratory distress syndrome (ARDS), a primary cause of morbidity and mortality in COVID-19. Neutrophilia, elevated neutrophil:lymphocyte ratios, and elevated neutrophil-associated cytokines are present in COVID-19, but changes in neutrophil functions have not been characterized. Here we analyzed the functional state of circulating neutrophils in COVID-19. Methods:  Blood was obtained from critically ill COVID-19 patients over two weeks and healthy controls across multiple timepoints. Plasma cytokine profiles were assessed by bead array. Neutrophils were isolated and tested ex vivo for oxidative burst, neutrophil extracellular trap formation (NETosis) and phagocytosis. Lung tissue was obtained immediately post-mortem from COVID-19 patients for immunostaining. Results:  Elevations in neutrophil-associated cytokines IL-8 and IL-6 were identified in COVID-19 plasma both at the first measurement and across their hospitalization (p < 0.0001). Elevations in cytokines IP-10, GM-CSF, IL-1b, IL-10 and TNF were also present at the first measurement and across hospital stays. Functionally, circulating neutrophils from COVID-19 patients had exaggerated oxidative burst (p < 0.0001), NETosis (p < 0.0001) and phagocytosis (p < 0.0001) relative to controls. Increased NETosis was found to be correlated with both leukocytosis and neutrophilia in COVID-19 patients. Neutrophils and NETs were identified within airways and alveoli in lung parenchyma. While elevations in IL-8 and ANC correlated to COVID-19 disease severity, plasma IL-8 levels alone correlated with death. Conclusions:  Circulating neutrophils in COVID-19 exhibit an activated phenotype with increased oxidative burst, NETosis and phagocytosis. Readily accessible and dynamic, plasma IL-8 and circulating neutrophil function can be explored as potential COVID-19 disease biomarkers. Funding Statement: This work was supported by the Department of Veterans Affairs (salary support and VA Merit Award, PI Crotty Alexander) and NIH NHLBI (PI Crotty Alexander). Declaration of Interests: The authors report no conflicts of interest. Ethics Approval Statement: The research protocol was approved by the UCSD, VASDHS and Rady Children’s Hospital institutional review boards (IRBs) and all participants or designated family member gave written informed consent.