Circulating extracellular vesicles drive innate immune dysregulation in MIS-C.

Abstract

Multisystem inflammatory syndrome in children (MIS-C) is a post-severe acute respiratory syndrome coronavirus 2 hyperinflammatory condition with multiorgan involvement. The contribution of circulating extracellular vesicles (EVs) to MIS-C immunopathology remains incompletely defined. We sought to compare humoral and cytokine responses and EV-associated viral/inflammatory signatures in MIS-C versus pediatric coronavirus disease 2019 (COVID-19) across the clinical spectrum. In a prospective cohort, children with PCR-confirmed severe acute respiratory syndrome coronavirus 2 infection were classified as asymptomatic, mild/moderate, or severe COVID-19 or MIS-C and compared with healthy controls. Serum anti-spike, anti-receptor-binding domain (RBD), and anti-nucleocapsid IgG/IgA/IgM were quantified by ELISA and followed longitudinally. Nineteen cytokines were measured using multiplex bead assays. Plasma EVs were isolated by size-exclusion chromatography, characterized by tunable resistive pulse sensing/flow cytometry, assessed for spike/RBD by immunoblotting, tested for nuclear factor κB (NF-κB)/interferon regulatory factor (IRF) activation in THP1 reporter cells, and profiled by LC-MS/MS proteomics. Patients with MIS-C had higher anti-spike/RBD IgG (and broader isotype responses) at admission and sustained titers over follow-up, consistent with prolonged antigenic exposure. MIS-C showed a distinct inflammatory profile with prominent IL-17A (and IL-22) elevation relative to severe COVID-19. Spike and RBD were detectable in EVs from severe COVID-19 and MIS-C. Functionally, severe COVID-19 EVs activated both NF-κB and IRF pathways, whereas MIS-C EVs preferentially activated IRF with minimal NF-κB signaling. Proteomics identified an MIS-C-specific EV cargo enriched for innate immune pathways, neutrophil degranulation, and adhesion/integrin-associated proteins. MIS-C is characterized by sustained humoral responses, T_H17-skewed cytokines, and antigen-bearing EVs with distinct IRF-biased immunostimulatory and proteomic signatures. EV profiling and implicated pathways (eg, IL-17 and adhesion/integrin networks) may inform biomarker development and targeted immunomodulatory strategies in MIS-C.