Exosome-shuttled miRNAs contribute to the modulation of the neuroinflammatory microglia phenotype by mesenchymal stem cells. Implication for amyotrophic lateral sclerosis

Background Mesenchymal stromal/stem cells (MSCs) are characterized by neuroprotective, immunomodulatory, and neuroregenerative properties, which support their therapeutic potential for neurodegenerative diseases driven by microglia-associated inflammation, such as amyotrophic lateral sclerosis (ALS). One mode of action through which MSCs exert their immunomodulatory effects is the release of extracellular vesicles, including exosomes, that carry proteins, mRNAs, and microRNAs (miRNAs), which, once transferred, modify the function of target cells. We have investigated the role of miRNAs present in exosomes derived from IFN-γ-primed mouse MSCs in the modulation of microglia activation, and analysed their effect on target genes and signaling pathways. Methods We compared miRNA expression in IFN-γ-primed vs unprimed mouse MSCs by microarray and measured the levels of relevant miRNAs in their respective exosomes through RT-PCR. To assess the effect of dysregulated MSC-derived miRNAs, we transfected lipopolysaccharide-activated N9 microglial line cells and primary microglia from late-symptomatic SOD1G93A ALS mice with their specific mimics and analysed the mRNA expression of pro/anti-inflammatory genes in the cells. We used mirWalk and Panther and KEGG Pathway databases to predict target genes of specific miRNAs and possible pathways they regulate. Data were compared using Student’s t-test. Results We identified nine miRNAs that were significantly dysregulated in IFN-γ-primed MSCs, but present at different levels in their derived exosomes. Transfection with three of the four miRNAs significantly upregulated in IFN-γ-primed MSC-derived exosomes, namely miR-467f, miR-466q and miR-466m-5p, could modulate the pro-inflammatory phenotype of N9 microglia by downregulating Tnf and/or Il1b expression, and/or upregulating Cx3cr1 expression. We obtained similar results in primary microglia from SOD1G93A mice transfected with miR-467f and miR-466q. Further analysis of the mode of action of miR-467f and miR-466q indicated that they dampen the pro-inflammatory phenotype of microglia by modulating the p38 MAPK signaling pathway via inhibition of the expression of their target genes, Map3k8 and Mk2. Conclusion These results suggest that exosome-mediated transfer of functional miRNAs could be one mode of action through which MSCs exert their therapeutic effect in ALS by downregulating neuroinflammatory microglia, and identify miR-467f and miR-466q as immunomodulatory miRNAs involved in this process.