In-vitro NET-osis induced by COVID-19 sera is associated to severe clinical course in not vaccinated patients and immune-dysregulation in breakthrough infection

: Since neutrophil extracellular traps formation (NET-osis) can be assessed indirectly by treating healthy neutrophils with blood-derived fluids from patients and then measuring the NETs response, we designed a pilot study to convey high-dimensional cytometry of peripheral blood immune cells and cytokines, combined with clinical features, to understand if NET-osis assessment could be included in the immune risk profiling to early prediction of clinical patterns, disease severity, and viral clearance at 28 days in COVID-19 patients. Immune cells composition of peripheral blood, cytokines concentration and in-vitro NETosis were detected in peripheral blood of 41 consecutive COVID-19 inpatients, including 21 mild breakthrough infections compared to 20 healthy donors, matched for sex and age. Major immune dysregulation in peripheral blood in not-vaccinated COVID-19 patients compared to healthy subjects included: a significant reduction of percentage of unswitched memory B-cells and transitional B-cells; loss of naïve CD3+CD4+CD45RA+ and CD3+CD8+CD45RA+ cells, increase of IL-1β, IL-17A and IFN-γ. Myeloid compartment was affected as well, due to the increase of classical (CD14++CD16-) and intermediate (CD14++CD16+) monocytes, overexpressing the activation marker CD64, negatively associated to the absolute counts of CD8+ CD45R0+ cells, IFN-γ and IL-6, and expansion of monocytic-like myeloid derived suppressor cells. In not-vaccinated patients who achieved viral clearance by 28 days we found at hospital admission lower absolute counts of effector cells, namely CD8+T cells, CD4+ T-cells and CD4+CD45RO+ T cells. Percentage of in-vitro NET-osis induced by patients' sera and NET-osis density were progressively higher in moderate and severe COVID-19 patients than in mild disease and controls. The percentage of in-vitro induced NET-osis was positively associated to circulating cytokines IL-1β, IFN-γ and IL-6. In breakthrough COVID-19 infections, characterized by mild clinical course, we observed increased percentage of in-vitro NET-osis, higher CD4+ CD45RO+ and CD8+ CD45RO+ T cells healthy or mild-COVID-19 not-vaccinated patients, reduced by 24 h of treatment with ACE inhibitor ramipril. Taken together our data highlight the role of NETs in orchestrating the complex immune response to SARS-COV-2, that should be considered in a multi-target approach for COVID-19 treatment.