Abstract Rationale Following acute pulmonary infections, some individuals can experience persistent symptoms of fatigue and shortness of breath. While the etiology of these post-infectious symptoms may be multifactorial, following SARS-CoV-2 infection, persistent viral reservoirs have been shown to be associated with ongoing dysregulated immune responses. In the most severe post-infectious cases, multisystem inflammatory syndrome in children (MIS-C), we showed strong neutrophil activation correlates with disease. However, the role of neutrophil dysfunction has not yet been defined in the more indolent post-infectious syndrome, long COVID. Characterizing post-infectious neutrophil signals can offer key insight into pathology and therapeutic avenues for treatments. Methods In this study, whole blood was collected from 55 adolescents and young adults with long COVID, 19 children with MIS-C, and 65 pediatric controls to quantify levels of spike antigenemia, RNA expression, inflammatory cytokines, and cell-free DNA. Isolated neutrophils underwent single-cell RNA sequencing and microfluidic assays to assess live neutrophil functions, including neutrophil extracellular trap (NET) formation, chemotaxis, phagocytosis, and spontaneous migration. Finally, ex vivo studies were conducted to model the functional alterations of neutrophils in long COVID. Results Circulating SARS-CoV-2 spike protein was identified in 15% of long COVID participants, suggesting a subgroup with persistent viral reservoirs. Bulk and scRNA-seq revealed changes in long COVID neutrophil functions, including NETosis and chemotaxis, accompanied by activation of the NF-κB and AP-1 transcription factors. Significant increases in inflammatory cytokines were detected in long COVID children with spike antigen, including IFNγ, IL-4, IL-10, and IL-13 (p 0.05, respectively). Long COVID spike-positive and MIS-C neutrophils had significantly higher levels of NETosis (p 0.001), concentrations of cell-free DNA in circulation (p 0.05, p 0.001, respectively), and blunted neutrophil chemotaxis to a distinct chemoattractant (p 0.05) compared to healthy children. Incubation of healthy donor neutrophils and spike immune complexes was able to recapitulate the changes previously observed in a dose-dependent manner. Conclusion In post-infectious long COVID, neutrophil functions that support tissue repair, such as clearance of proinflammatory stimuli, debris phagocytosis, and angiogenesis, are reduced. Instead, persistent spike reservoirs promote NETosis and sustained immune dysfunction. Viral gastrointestinal viral reservoirs and zonulin-mediated intestinal permeability may promote spike antigen leak, which exacerbates this dysfunctional response. These findings suggest that post-acute viral reservoirs sustain neutrophil activation, contributing to ongoing symptoms. Therapeutic strategies that limit spike trafficking or mitigate dysregulated neutrophilic inflammation may help reduce antigen leak, restore neutrophil function, and improve clinical outcomes. This abstract is funded by: This abstract is funded by: NIH/NHLBI grant 1R01HL173059; PolyBio Research Foundation, NIGMS grant T32-GM144273
Alvarez-Carcamo et al. (Fri,) studied this question.