Persistence of neutrophil abnormalities inCOVID-19 convalescence

Background/AimsPatients infected with severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) may develop acute respiratory inflammation, due toan exaggerated immune response and some develop chroniccomplications. Neutrophils play a major role in the pathology ofinflammatory diseases and have been shown to contribute to lung andvascular damage in COVID-19. Our aim was to establish a relationshipbetween neutrophil phenotype and disease severity and to determinewhether neutrophil abnormalities persist in convalescent patients.MethodsPeripheral blood samples were obtained from acute COVID-19patients (n = 74), follow-up (FU) patients discharged following inpatientadmission (n = 56), a median of 87 days after discharge, and healthycontrols (HCs, n = 22). Patients were stratified by disease severitybased on inspired oxygen (FiO2) and admission to intensive care (ICU).Neutrophils were isolated from whole blood by negative selection forphenotyping and functional analysis. PBMC Isolation Tubes were usedto quantify and phenotype low density neutrophils (LDNs) within thePBMC fraction. For quantification of reactive oxygen species (ROS)production, isolated neutrophils were incubated with a ROS reactivedye, DHR-123 and stimulated with PMA. All samples were stained andfixed prior to analysis by flow cytometry.ResultsThere was a marked increase in neutrophils expressing the activationand degranulation markers, CD64 (P < 0.0001) and CD63 (P < 0.0001)and a reduction in neutrophils expressing the maturity markers, CD10(P < 0.0005) and CD101 (P < 0.0005) in patients with acute COVID-19compared to HCs. Increased frequency of neutrophils expressingCD64 (P < 0.005), CD63 (P < 0.01) and expressing decreased CD101(P < 0.0001) were also detected in FU patients compared to HCs.Notably, 42.3 4.4% of neutrophils were CD101lo in FU patients, compared to 29.0 3.7% in acute patients and 9.6 4.1% in HCs.These changes were most apparent in FU patients recovering fromsevere COVID-19 compared to mild or moderate disease. Thefrequency of LDNs in PBMCs from acute patients was significantlyhigher than HCs (P < 0.0001), and correlated with disease severity.Similarly, the frequency of LDNs in FU patients was significantly higherthan in HCs (P < 0.0005). We found a trend towards higher basal ROSproduction in acute and FU patients, but a blunted response to PMAstimulated ROS production in neutrophils from acute patients versusHCs (P < 0.0001). Impaired ROS production persisted in FU patientscompared to HCs (P < 0.01).ConclusionCirculating neutrophils in acute COVID-19 have an altered phenotypeand comprise immature and activated cells. This altered phenotypepersisted in convalescence and may contribute to the persistence ofsymptoms and an increased susceptibility to subsequent infections.Future work will aim to investigate the functional implications of thesefindings.