Abstract
Purpose :
Microglial activation is known to play an important role in retinal inflammatory and degenerative diseases. This study aims to investigate the signature miRNAs carried by exosomes from activated microglia and their potential roles in retinal pathologies.
Methods :
Murine microglia cell line BV-2 was cultured in DMEM with 2% exosome-depleted FBS, and stimulated with LPS (100ng/ml) + INF-γ (20ng/ml) or IL-4 (20ng/ml) + IL-13 (20ng/ml) for 24hrs to induce M1 and M2 polarization. The supernatant was harvested and sequentially centrifuged (300g/200g/16000g) to remove dead cells and large debris. The supernatant was then filtered through 0.22um filter to remove large vesicles and remaining debris. Exosome was purified from the supernatant using QIAGEN Exoeasy kit, and exosomal RNA was isolated using the RNeasy micro kit. The concentration, size distribution and structures of isolated exosome were determined by Nanoparticle Tracking Analysis (NTA) and transmission electron microscope (TEM). Exosome markers were evaluated by western blotting. Three independent replicate exosomal RNA samples of each group (M0, M1, M2) were subjected to small RNA-seq. Sequencing bias was eliminated by unique molecular identifiers (UMI) tagging during sequencing library preparation.
Results :
The median size of BV-2 derived exosome was 134.6nm. TEM showed clear vesicle structure and typical cup shape. Among thousands of miRNAs identified by sequencing, 477 were up-regulated in exosomes from M1 microglia and 303 were up-regulated in exosomes from M2 microglia; 300 and 315 miRNAs were down-regulated in exosomes from M1 and M2 microglia respectively. Among exosomal miRNAs that were changed after activation, the largest group was metabolic pathways related (10.1%). miR-146a-5p was dominant among miRNAs identified in exosomes, and was markedly increased following M1 activation. Microglia-derived miR-146a-5p was known to induce proinflammatory responses, and participate in neuron synapsis control. miR-21a-5p was dominant following M2 activation and is known to be involved in fibrosis.
Conclusions :
Exosomes from polarized microglia carry distinct miRNAs, which may participate in cell-to-cell communication and regulation of retinal inflammation under disease conditions.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.