COVID-19 neutrophils show elevated migration and NETosis, which is reduced by PI3K inhibition

Rational: Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics. Cell functions are interwoven pathways, so understanding the effect of COVID-19 across neutrophil function may identify therapeutic targets. We examined neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia (CAP) patients. Method(s): Isolated neutrophils underwent ex vivo analyses for migration, phagocytosis and NETosis, and the effect of PI3K inhibition. Circulating DNAse 1 activity and levels of cfDNA were measured. Result(s): Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397, A) and NETosis (p=0.0366, B), but impaired phagocytosis (p=0.0236, C) associated with impaired ROS generation (p<0.0001). COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001, D). Ex vivo inhibition of PI3K gamma and delta reduced NET release by COVID-19 neutrophils (p=0.0156). Conclusion(s): COVID-19 is associated with neutrophil dysfunction across all main effector functions, with elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function via PI3k may help modulate COVID-19 severity. (Figure Presented).

Dysfunctional neutrophil response in COVID-19 infection vary by subtype

S94 Figure 1Comparison of neutrophil effector functions between COVID-19 variants (alpha n=33, delta n=13, omicron n-14). A.% change in phagocytosis significantly increased between alpha and delta patients (p=0.0162). B. Fold change in cells migrated through a transwell pore to IL8 compared to vehicle control significantly reduced in omicron patients compared alpha and delta (vs alpha p=0.0018, vs delta p=0.0370). C. Neutrophil extracellular trap production after stimulation with PMA compared to vehicle control significantly reduced in omicron patients compared to alpha (p=0.0396)[Figure omitted. See PDF]DiscussionOur results showing changes in neutrophil „function and phenotype differ between variants of COVID-19 infection, potentially reflect viral evolution. This change in neutrophil function may contribute to the evolving clinical phenotype observed in patients. Our population of ward-based COVID-19 patients represents the majority of inpatient hospital burden where early intervention may prevent clinical deterioration. Targeting neutrophil function may be an effective way of improving infection outcome in the future.ReferenceBelchamber K, et al. Altered neutrophil phenotype and function in non-ICU hospitalised COVID-19 patients correlated with disease severity. medRxiv, 2021: p. 2021.06.08.21258535.

Elevated NETosis and migration but impaired anti-microbial responses in neutrophils from non-ICU, hospitalized COVID-19 patients

RationalInfection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based predominantly on transcriptomics or single functional assays. Cell functions are interwoven pathways, and so understanding the effect of COVID-19 across the spectrum of neutrophil function may identify therapeutic targets to treat disease.ObjectivesExamine neutrophil phenotype and functional capacity in COVID-19 patients versus age-matched controls (AMC).MethodsIsolated neutrophils from 41 non-ICU COVID-19 patients and 23 AMC underwent ex vivo analyses for migration, phagocytosis of Streptococcus pneumoniae, reactive oxygen species (ROS) generation, neutrophil extracellular trap formation (NETosis) and cell surface receptor expression. Serum DNAse 1 activity was measured, alongside circulating levels of cell-free (cf)DNA, myeloperoxidase (MPO), VEGF, IL-6 and sTNFRI. All measurements were correlated to clinical outcome. Serial sampling on day 3–5 post hospitalisation were also measured.ResultsCompared to AMC, COVID-19 neutrophils demonstrated elevated transmigration (p=0.0397) and NETosis (p=0.0366), but impaired phagocytosis (p=0.0236) associated with impaired ROS generation (p<0.0001). Surface expression of CD54 (p<0.0001) and CD11c (p=0.0008) was significantly increased and CD11b significantly decreased (p=0.0229) on COVID-19 patient neutrophils. On day 3–5 follow-up, levels of senescent neutrophils increased compared to day 1 (indicated by decreased CXCR2 and elevated CXCR4 expression (p=0.0332)). COVID-19 patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001). MPO, VEGF, sTNFRI, and IL-6 (p<0001) were elevated in COVID-19, which positively correlated with disease severity by 4C score.ConclusionCOVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration, impaired antimicrobial responses and elevated NETosis. These changes represent a clear mechanism for tissue damage and highlight that targeting neutrophil function may help modulate COVID-19 severity.Please refer to page A189 for declarations of interest related to this abstract.