Altered neutrophil phenotype and function in non-ICU hospitalised COVID-19 patients correlated with disease severity

Abstract

Rational: Infection 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 tractable therapeutic targets. Objectives: Examine neutrophil phenotype and functional capacity in COVID-19 patients versus age-matched controls (AMC) Methods: Isolated neutrophils from 41 hospitalised, non-ICU COVID-19 patients and 23 AMC underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NET formation (NETosis) and cell surface receptor expression. DNAse 1 activity was measured, alongside circulating levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI. All measurements were correlated to clinical outcome. Serial sampling on day 3-5 post hospitalisation were also measured. Results: Compared 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. COVID-19 patients showed increased systemic markers of NETosis including increased cfDNA (p=0.0153) and impaired DNAse activity (p<0.0.001). MPO (p<0.0001), VEGF (p<0.0001), TNFRI (p<0.0001) and IL-6 (p=0.009) were elevated in COVID-19, which positively correlated with disease severity by 4C score. Conclusion: COVID-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.

Neutrophil subsets and their differential roles in viral respiratory diseases

“…Studies on immature neutrophils during inflammatory conditions suggest a high ROS function and migration capacity, but low NETs and phagocytosis function 259,260 . This was also shown with in vitro human studies, demonstrating an increased immature neutrophil migration through CXCL8 signaling, but a higher propensity for NET formation correlating with severe COVID‐19 disease 261 …”

“…259,260 This was also shown with in vitro human studies, demonstrating an increased immature neutrophil migration through CXCL8 signaling, but a higher propensity for NET formation correlating with severe COVID-19 disease. 261 The third layer of heterogeneity is the neutrophil's activation status. Reports of LDNs in VRDs have suggested a degranulated form of neutrophils capable of immunosuppression.…”