(P008) EXTRACELLULAR VESICLES FROM HUMAN HEART EXPLANT-DERIVED CELLS ATTENUATE ACTIVATION OF THE NLRP3 INFLAMMASOME IN MACROPHAGES

Background: Intramyocardial injection of heart explant-derived cells (EDCs) improves cardiac function in preclinical models of ischemic cardiomyopathy. This therapeutic benefit is partially attributable to the anti-inflammatory cargo (micro RNAs and proteins) enriched within the extracellular vesicles (EVs) released by EDCs. Recent work has shown that activation of the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in both immune and non-immune cells plays a critical role in promoting cardiac inflammation and adverse remodeling. Although EDC EVs are known to modulate inflammatory mediators, their effects on the NLRP3 inflammasome are not known. Therefore, we explored the ability of EDC EVs to attenuate NLRP3 inflammasome activation in macrophages, the major pro-inflammatory cell type recruited to myocardium after an ischemic insult.

METHODS AND RESULTS: EVs were isolated from EDC conditioned media (ultracentrifugation) and characterized (Nanosight & antibody array). Monocytes (THP-1) were differentiated into macrophages (PMA; 3 days) and treated with EVs (20 hours) before priming (LPS; 4 hours) and activating (nigericin; 1 hour) the NLRP3 inflammasome. Secreted caspase-1 in the culture supernatants was measured by using a bioluminescent assay (Promega). The miRNA and protein cargo within EVs was profiled using miRNA detection (Nanostring) and liquid chromatography-mass spectrometry, respectively. miRNA and protein data were analyzed using appropriate bioinformatics tools (Tam 2.0, miRWalk, and Uniprot).
EV size (160±2 nm) and markers (ICAM, ALIX, CD81, CD63, EPCAM, ANXAS, TSG101, FLOT-1) confirmed EV identity. Macrophages pretreated with EVs (4E+10 EVs/mL) showed a significant attenuation in NLRP3 inflammasome induced caspase-1 vs. LPS+nigericin-only treated cells (50% lower, n=4-6, p=0.02). EV cargo profiling revealed that EVs were enriched with 22 distinct anti-inflammatory miRNAs (Tam 2.0). Specifically, 3 miRNAs (miR-21, miR-100, miR-181a, n=3) and 5 proteins (Peroxiredoxin-1, Thioredoxin-1, Caveolin-1, Sequestosome-1, n=3) abundantly found within EVs were predicted to inhibit the NLRP3 inflammasome (miRWalk & Uniprot).

Conclusion: EDC EVs suppress activation of the NLRP3 inflammasome in macrophages via transfer of anti-inflammatory miRNAs and proteins.