Necroptosis and cellular stress characterize immune and endothelial dysfunction in long COVID

Long COVID, or post-acute sequelae of severe acute respiratory syndrome coronavirus 2 infection, affects a significant proportion of COVID-19 survivors and is associated with persistent fatigue, dysautonomia, and cardiovascular complications. The cellular mechanisms underlying these chronic symptoms remain incompletely understood.

We sought to investigate immune and endothelial cell dysfunction, with a focus on cell stress and death pathways, in individuals with long COVID compared with matched infection-recovered controls.

We conducted a cross-sectional study at the University of Miami Miller School of Medicine and the Miami VA Healthcare System enrolling adults who met the World Health Organization criteria for long COVID, along with age- and sex-matched controls with no history of long COVID symptoms. Clinical assessments included the modified COVID-19 Yorkshire Rehabilitation Scale, Composite Autonomic Symptoms Score, heart rate variability, and vascular reactivity index. Peripheral blood was analyzed by spectral flow cytometry to characterize immune cell and circulating endothelial cell (CEC) populations and their expression of markers related to necroptosis (phosphorylated mixed lineage kinase domain-like protein [pMLKL]), autophagy (microtubule-associated protein light chain 3), hypoxia (hypoxia-inducible factor 1-alpha), and neutrophil extracellular traps (myeloperoxidase, citrullinated histone H3, and neutrophil elastase).

Patients with long COVID (n = 73) showed significantly higher long COVID symptom scores compared with controls (n = 41), along with impaired heart rate variability and endothelial reactivity. Flow cytometry revealed increased expression of pMLKL and microtubule-associated protein light chain 3 (LC3) in classical and nonclassical monocytes, and LC3 in neutrophils, and eosinophils. CECs from participants with long COVID were substantially increased and demonstrated marked activation of necroptosis and autophagy pathways. These findings were accompanied by increased monocyte-platelet and CEC-platelet aggregates, consistent with a prothrombotic state. Elevated pMLKL expression in CECs strongly correlated with symptom severity and autonomic dysfunction.

Our findings demonstrate that long COVID is characterized by persistent inflammation and endothelial stress, involving necroptosis and autophagy pathways. These mechanisms may contribute to chronic vascular and autonomic dysfunction in patients with long COVID. Targeting these stress and death signaling pathways may offer novel therapeutic strategies to mitigate the long-term consequences of severe acute respiratory syndrome coronavirus 2 infection.