Extracellular vesicle miRNA signatures in pediatric onset-multiple sclerosis and obesity-driven immune and metabolic dysregulation

Abstract

Background: Multiple sclerosis (MS) is a chronic, demyelinating autoimmune syndrome of the central nervous system (CNS). The cause of MS remains unknown; however, a dysregulated metabolome appears to play a contributing role. Up to 10% of all MS cases occur in childhood or adolescence. Childhood obesity is an independent risk factor for the development of MS. Pediatric-onset MS (POMS) provides a unique opportunity to study obesity-associated autoimmunity in the absence of age-related comorbidities. Circulating extracellular vesicle-encapsulated microRNAs (EV-miRNAs) have emerged as stable biomarkers and regulators of immune and metabolic pathways in neuroinflammatory disorders but have not been investigated in POMS.

Methods: We performed bulk and single EV analysis on platelet-poor plasma EVs from children with POMS and age-, sex-, and body mass index (BMI)-matched controls. EVs were isolated via tangential flow titration (TFF) and subjected to miRNA-Seq. Differential expression analyses were performed to identify EV-miRNAs unique to MS and obesity. Functional enrichment was employed to elucidate impacted biological pathways. Key findings were cross-referenced with known and published pathways. Single EV analysis using total internal reflection fluorescence microscopy (TIRFM) was employed to identify EV and obesity-specific markers at single-particle resolution.

Results: Bulk EV characterization confirmed the presence of small EVs with classic biophysical characteristics. Particle size was comparable across both groups and ranged between 70 to 91 nm and concentration range was 2 x 10 ¹⁰ to 1 x 10 ¹¹ / mL. EV-miRNA sequencing demonstrated marked global differential transcriptomics between POMS and controls, irrespective of BMI. Notably, we identified MS-enriched EV-miRNAs, such as miR-29b-3p, miR-4326, miR-671-3p, and miR-139-3p, that are involved in cholesterol homeostasis, inflammatory signaling, T-cell regulation and blood-brain-barrier (BBB) integrity. These MS-specific alterations implicate EV-miRNA cargo in modulating pathobiologically relevant pathways, such as immune regulation, neuroinflammation, and lipid metabolism. Obesity also had a pronounced, group-specific impact on EV-miRNAs. Subgroup analysis comparing controls to obese POMS revealed alterations in EV-miRNAs involved in regulating metabolism homeostasis, pro-adipogenesis and inflammation (e.g., miR-29a-3p, miR-142-5p, miR-29c-3p, miR-27a-3p). In POMS patients, obesity further amplified these trends with markedly increased pathogenic EV-miRNAs, including miR-142-5p, miR-29a-3p, and let-7d-5p, and significant suppression of protective EV-miRNAs, such as miR-30d-5p, let-7c-5p, and miR-151a-5p when comparing normal weight MS versus obese MS. Enrichment analysis revealed dysregulated EV-miRNAs in MS target pathways central to immune activation, neuroinflammation, and lipid metabolism, while obesity accentuated disturbances in pathways, for example AMPK signaling, insulin resistance, and axon guidance. Single EV analysis via TIRFM confirmed the presence of obesity-specific EVs within the circulating plasma EV pool of POMS.

Conclusion: EV-miRNA profiles differ significantly between POMS patients and controls, reflecting aberrant immune and metabolic regulation in disease. Furthermore, obesity resulted in an expanded MS-related, EV-miRNA dysregulated repertoire, underscoring a prospectively novel cellular mechanism underlying obesity-associated CNS autoimmunity. These findings highlight plasma EVs as promising minimally invasive biomarkers in POMS and provide novel therapeutic candidates for future validation in larger cohorts.