Disruption of neutrophil homeostasis is associated with functional alterations in mitochondria of critically ill COVID−19 patients

Abstract

Abstract Understanding the molecular mechanisms underlying neutrophil dynamics during COVID−19 disease progression is essential for managing severe inflammatory conditions. We investigated whether alterations in neutrophil mitochondrial function and calcium handling are associated with disrupted neutrophil homeostasis in critically ill COVID−19 patients. We analyzed neutrophil counts, phenotypes, and apoptotic profiles in critically ill COVID−19 survivors (ICU-S) and non-survivors (ICU-NS) compared with healthy controls. Flow cytometry, metabolic profiling, immunofluorescence imaging, and small RNA sequencing (miRNA-seq) were used to characterize neutrophil apoptosis-related pathways and mitochondrial function in freshly isolated neutrophils. Critically ill COVID−19 patients showed marked neutrophilia and a higher proportion of immature CD16low neutrophils relative to controls. Both ICU-S and ICU-NS groups exhibited reduced neutrophil apoptosis, as evidenced by fewer annexin V+ cells and lower cleaved caspase−3 signal compared with healthy controls. Although exploratory miRNA-seq in a small subset of ICU patients identified differentially expressed miRNAs with predicted enrichment in apoptosis- and calcium-related pathways, these mortality-associated miRNA signatures were not corroborated by functional apoptosis readouts (cleaved caspase−3 and annexin V) between ICU-S and ICU-NS. Neutrophils from ICU patients also demonstrated altered calcium handling, hyperpolarized mitochondrial membrane potential, increased complex II–linked respiration, and elevated mitochondrial ROS relative to controls. Neutrophils from critically ill COVID−19 patients display coordinated alterations in calcium handling, mitochondrial activity, and apoptosis consistent with impaired neutrophil clearance and disrupted homeostasis. These findings are observational and do not establish causality; the miRNA results should be interpreted as hypothesis-generating rather than validated mortality biomarkers.