Abstract 350: High-dimensional Analysis Of Circulating Blood Cells Reveals Distinct Granulocyte Activation And NETosis Profiles In COVID-19

Abstract

Background: Coronavirus disease 19 (COVID-19) represents a perfect storm for immunothrombosis leading to severe disease, with release of neutrophil extracellular traps (NETs) being an important contributor now frequently described both in blood and in tissues. NETs contribute to thrombosis by trapping platelets and activating the coagulation cascade, as well as endotheliopathy. NET formation is an active process initiated by factors such as activated platelets and involving histone modifications including citrullination. Aims: We aimed to characterize early activation toward NETosis in circulating granulocytes in the context of neutrophil activation, immunothrombosis, and the broader immune response in COVID-19, particularly in severe disease involving ICU stays. Methods: We designed a custom panel of 42 antibodies to stain peripheral blood collected from COVID-19 patients in ICU at UZ Leuven during the COVID-19 Delta-wave. This panel comprised cell-specific epitopes, surface activation markers, intracellular staining for NET-associated cell biological changes, and platelet markers to identify complexes. Samples were run using cytometry by time-of-flight (CyTOF) on a Helios mass cytometer and analyzed using FlowJo software with FlowSOM clustering and tSNE visualisation. Results: Nonbiased clustering analysis was performed and identified 18 distinct populations of granulocytes. Four of these populations were present only in COVID-19 patients, with high expression of CD11b, CD177, and CD16. A distinct population of citrullinated histone-positive eosinophils was also identified in COVID-19 patients, with higher expression than any of the neutrophil populations. Ex vivo stimulation of healthy donor granulocytes toward NETosis shifted the expression profile toward that of COVID-19 patient granulocytes. However, morphometric analysis using scatterbody targets failed to identify cell biological changes common to NET formation. Conclusions: High dimensional profiling of circulating blood cells identifies distinct granulocyte populations with activation profiles in COVID-19, highlighting the heterogeneity of neutrophil responses and involvement of eosinophils in extracellular trap release.