Attending Pediatric Surgeon University of Toronto Toronto, Ontario, Canada
Background: Necrotizing enterocolitis (NEC) is a devastating neonatal disease that affects the intestine of premature babies and is associated with neurodevelopmental delay in 25% of survivors. The brain of babies with NEC undergoes neuroinflammation with activated microglia. We have previously shown that neuroinflammation is partially mediated by inflammatory molecules released in extracellular vesicles (M-EVs) by the activated microglia. EVs are nanoparticles that cross the blood-brain barrier, can be collected in peripheral blood, and can potentially be used as biomarkers of neuroinflammation. Objective: To characterize M-EVs in plasma derived from neonates with NEC. Design/Methods: Following ethics approval (IRB:1000058989), blood was collected from 6 NEC babies (32-34 weeks of gestation) and 6 age-matched controls. Bulk EVs were obtained from plasma using size-exclusion chromatography (qEV) with 70nm columns. Bulk EV characterization was conducted using nanoparticle tracking analysis for EV size and Western blotting for expression of EV canonical markers (TSG101 and HSP70) and absence of nuclear debris (H3K27me3). M-EVs were enriched by serial immunocapture of canonical microglia receptors with a biotin-streptavidin platform: - EVs were incubated with P2Y12 antibody and biotin-tagged secondary antibody - Streptavidin coated agarose beads were added - After washes, P2Y12+EVs were released by acid catalysis - Incubation steps were repeated using TMEM119 antibody, and P2Y12+/TMEM119+ EVs were separated. M-EVs were assessed for size (NTA), and protein expression of canonical EV (CD63, TSG101, Histone-H3) and inflammasome (NLRP3, IL1β) markers by Western blot. Results: Of the 20 fractions collected with qEV, fractions 12-18 contained EVs. Samples from controls and NEC patients and had similar EV size (controls: 104 +/-13 nm; NEC: 119 +/-2), maximal yield (control: 4.6x10^11 particles/ml; NEC: 4.5x10^11), and expression of canonical EV markers. M-EVs from NEC patients were enriched for the inflammasome mediator NLRP3.
Conclusion(s): This is the first study on the isolation and characterization of EVs from human plasma in preterm infants. M-EVs in NEC have a distinct inflammasome signature that can be detected in serum. Further studies are under way to establish serum derived M-EVs as potential biomarkers for assessing severity of NEC induced brain injury.
