Abstract P3158: Extracellular Vesicles From Ischemic Myocardium Mediate Regulatory T Cell Dysfunction Through MicroRNA-1-mediated Suppression Of VEGF-A

Abstract

Motivation: Myocardial infarction (MI) triggers an efflux of pro-inflammatory extracellular vesicles (EVs) from the heart into the circulation. Here, we test the hypothesis that circulating ischemic heart EVs (iEVs) attenuate regulatory T cell (Treg) proliferation and function post-MI. Methods: Hearts were explanted from mice and Langendorff-perfused with Tyrode buffer either continuously for two hours (nonischemic), or subjected to no-flow for 30 min, and reperfused for 1.5h. Heart effluent (~700ml) was collected for EV isolation by ultracentrifugation. EV size and concentration were measured by nanoparticle tracking analysis; EV markers were characterized by western blot. EVs from nonischemic heart effluent (nEVs) served as controls. RNA sequencing (RNAseq) followed by qPCR validation was used to identify microRNAs in EVs. Assessments of human Tregs included cell proliferation (Cell Counting Kit-8 assay), IL-10 production (qPCR of Tregs, and ELISA of Treg-conditioned media), and RNAseq after exposure to EVs. In vivo, an antagomir was administered retro-orbitally immediately post-MI, and splenic Tregs were measured by flow cytometry 7 days post-MI. Findings: MiR-1-3p was highly enriched in iEVs compared to nEVs. Exposure of human Tregs to iEVs or a miR-1 mimic resulted in decreased proliferation ( P <0.0001, N=8) and production of IL-10 (qPCR: P<0.0001, n=9; ELISA: P <0.0001, N=6) compared to nEVs. Antagomir-1 reversed iEV- and miR-1-induced inhibition of Treg proliferation. RNAseq on human Tregs revealed that VEGF-A, a miR-1 target gene, was suppressed after exposure to iEVs compared to nEVs. In vivo, the number of splenic Tregs decreased on day 7 post-MI, with a concomitant decrease in IL-10 + and VEGF-A + Tregs. Intriguingly, administration of antagomiR-1 restored numbers of splenic IL10 + and VEGF-A + Treg cells. Conclusions: iEVs cause Treg dysfunction by transferring miR-1. In turn, miR-1 suppresses VEGF-A, a known modulator of Treg function. Our findings reveal novel paracrine modulation of immune cells by ischemic myocardium. Targeting EV signaling post-MI represents a potential new strategy to augment Treg-mediated tissue repair.