Mesenchymal Stem Cells Induce a Fibrolytic Phenotype By Regulating mmu-miR-6769b-5p Expression in Macrophages

Liver transplantation is the only radical treatment for decompensated cirrhosis, but its use is limited owing to a shortage of donors; hence, there is an urgent need for new treatments. Previously, we developed a liver-regeneration therapy using autologous bone marrow-derived mesenchymal stem cells (BMSCs), which is under clinical investigation. Cell-cell interactions between BMSCs and macrophages (Mφs) participate in the improvement of liver function and alleviation of liver fibrosis, although the associated mechanisms have not been elucidated. Therefore, in this study, we investigated phenotypic changes in Mφs caused by interactions with BMSCs, as well as the underlying mechanisms. Co-culturing lipopolysaccharide (LPS)-stimulated murine bone marrow-derived Mφs (BMDMs) with BMSCs substantially upregulated matrix metalloproteinase 9 (Mmp9), Mmp12, and Mmp13 expression, and downregulated tumor necrosis factor alpha (Tnfα) expression. To identify humoral factors involved in phenotypic changes occurring in Mφs, microarray analysis was performed with microRNAs (miRNAs) derived from extracellular vesicles in the supernatant of co-cultured BMSCs and LPS-stimulated BMDMs. We found that miR-6769b-5p was highly expressed and that transfecting miR-6769b-5p mimic upregulated MMP9 in LPS-stimulated BMDMs and downregulated Tnfα and interleukin-1 beta (Il-1β). MiR-6769b-5p expression in BMDMs was decreased by LPS stimulation but was increased by co-culture with BMSCs. Microarray and pathway analyses of gene expression in LPS-stimulated, miR-6769b-5p-transfected BMDMs revealed changes in the eukaryotic initiation factor 2-signaling pathway and decreased the expression of activating transcription factor 4 (Atf4). LPS-stimulated BMDMs exhibited increased MMP9 expression and decreased the expression of Tnfα and Il-1β by ATF4 knockdown. These findings indicate that upregulating miR-6769b-5p in BMDMs induced a fibrolytic phenotype, where MMP9 was highly expressed and inflammatory cytokine expression was decreased by the suppression of ATF4 expression. These findings imply that regulating miR-6769b-5p or ATF4 expression in BMDMs may be helpful for treating chronic liver disease.