Abstract
Purpose :
Extracellular Vesicles (EVs) are critical mediators of cell-cell communication via transfer of molecular cargo including genetic material like miRNAs and regulate transcription in various physiological and pathological conditions. In this study, we have characterized the miRNA expression profile in EVs released during bacterial endophthalmitis in a mice model, for understanding its pathophysiology and uncover potential biomarkers for diagnosis and/or therapeutic targets.
Methods :
Endophthalmitis was induced by intravitreal injection of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) in C57BL/6 mice followed by enucleation at 24 hours. EVs were isolated using the exosome isolation kit (Invitrogen), and characterized by DLS and western blotting with tetraspannin markers, CD9 and CD81. miRNA enriched total RNA was isolated by miRNeasy Serum/Plasma Advanced Kit (Qiagen) and sequenced on the Illumina Nextseq 2K platform. The miRNet platform was used to elucidate potential interactions between exosomal miRNA and retinal mRNA. Additionally, the inflammation targetome of the top 10 EV-miRNAs were imported into Enrichr and analyzed for over-expressed pathways annotated in Wikipathways, and gene-disease associations listed in DisGeNet database
Results :
Transcriptomic profiling of EV-miRNAs revealed a total of 82 differentially expressed miRNAs (DEM) in PA endophthalmitis, and 106 DEMs in SA endophthalmitis, of which 52 DEMs were common to both infections. Major miRNAs such as miR-(223-3p, 467a-3p and 467d-3p) were upregulated while miR-(690 and 6240) were downregulated. The differentially regulated pathways observed in our earlier proteomic study in bacterial endophthalmitis correlated with transcriptomic profiling of EV-miRNAs, particularly miR-223-3p (also present in humans) which promotes apoptosis and regulates NF-κB and PI3K-AKT inflammatory pathways while also downregulating cytokines such as TNFα, IL-1β and IFNγ.
Conclusions :
EV-miRNA cargo in bacterial endophthalmitis play a major role in regulating host-immune response. While, miR-(467a-3p, 467d-3p and 690) has potential application as diagnostic and prognostic markers, miR-223-3p may be a novel therapeutic immune-target in endophthalmitis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.