Resident University of Wisconsin - Madison Madison, Wisconsin, United States
Background: An unmet need in the advancement of clinical tools for assessing early risks of neurodevelopmental disorders (NDDs) is a lack of validated brain biomarkers or biomarker signatures. Emerging evidence showed that macromolecules (RNAs, proteins) carried by the circulating brain-derived exosomes can be utilized as biomarkers. Assessment of cytokines from brain-derived exosomes may uncover the state of brain inflammation. Leveraging our discovery of the link between maternal obesity and neonatal systemic inflammation using cord blood plasma, this study aims to establish biomarker signatures demonstrating a link between neonatal systemic and central nervous system (CNS) inflammation, a known risk factor for NDDs. Objective: Identify brain-exosomal cytokine profiles and demonstrate a link between systemic and CNS inflammation using cytokine signatures of brain exosomes in cord blood Design/Methods: Cord blood plasma were collected and banked from healthy term newborns, gestational age ≥36 weeks, born by scheduled, uncomplicated cesarean delivery at a single center to limit the possibility of subclinical chorioamnionitis and variability of umbilical cord cytokines based on delivery mode. After collection, heparinized cord blood was processed within 12 hrs. Plasma was banked at -80C. Exosomes (n=40) were isolated from plasma (100µl) using the Exo-Spin 96 kit (Cell Guidance Systems). Brain exosomes were then enriched by immunoprecipitation using antibodies targeting contactin-2 (CNTN2), a brain-specific cell surface protein (1µg, Invitrogen) and validated using a nanoparticle tracker (NanoSight) and dot blot. 10^12 particles were first screened for cytokines (47 analytes, Milliplex) and then assayed for identified cytokines (CRP, Eotaxin, MCP-1, RANTES). Data were analyzed using linear regression (Graphpad). Results: Brain exosomes were confirmed by size (50-120 nm) and CNTN2+ dot plot. Positive correlations were found between plasma CRP and brain exosomal Eotaxin (R^2=0.33, P=0.03), MCP-1 (R^2=0.40, P=0.02), RANTES (R^2=0.53, P=0.005), and a trend with CRP (R^2=0.21, P=0.08).
Conclusion(s): To our knowledge, this is a first report that demonstrates a link between peripheral and CNS inflammation in newborns due to maternal obesity. The correlations of inflammatory markers between the CNS and periphery support the utility of brain exosomes as carriers of biomarkers relevant to brain health. This study paves the way for further analyses to establish brain exosomal Eotaxin and MCP-1 as biomarker signatures for clinical assessment of the risks of NDDs due to active neuroinflammation in newborns.
