Introduction: Our previous works demonstrated that systemic orbital fat-derived stem cell (OFSC) transplantation\nwas effective in ameliorating lipopolysaccharide (LPS)-induced extensive acute lung injury (ALI) in vivo mainly\nthrough paracrine regulation of macrophage-mediated cytokine-storm. In this study, we explore the molecular\nmechanism(s) of OFSCs regulating macrophage activity in a cytokine-inducible fashion.\nMethods: LPS (100 ng/ml)-activated macrophages were treated by conditioned medium from OFSCs (OFSCs-CM)\nor non-contact cultured with OFSCs for 6 hours. The potency of OFSCs on macrophage proliferation and\npro-inflammation ability were determined. Expression levels of pro-inflammatory cytokines in macrophages,\ninducible immuno-modulatory factors in OFSCs, were investigated. Deep sequencing analysis as well as interaction\nbetween microRNA (miRNA) and genes of immuno-modulators in OFSCs induced by activated macrophages was\npredicted by miRTar. Transfection of miRNA inhibitor into OFSCs was performed. Real-time RT-PCR and transplantation\nof OFSCs into mice with LPS-induced ALI confirmed the in vitro and in vivo mechanism.\nResults: The paracrine effect of OFSCs on inhibition of macrophage pro-inflammatory cytokine release was more\npotent than induction of macrophage G0/G1 cell cycle arrest. OFSCs-CM suppressed LPS-induced inducible nitric oxide\nsynthetase and the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-?), interleukin (IL)-1 alpha, and\nIL-1 beta expression in macrophages. Under non-contact culture, LPS-activated macrophages effectively triggered the\nexpression of soluble immuno-modulating factors in OFSCs, i.e., IL-10, IL-1 receptor antagonist (IL-1 RA), indoleamine\n2,3-dioxygenase, and soluble TNF receptor type II (sTNF RII). Under miRTar prediction, miR-671-5p was identified as a\ncritical microRNA in regulation of multiple immune-modulating factors in OFSCs response to macrophages. The\nbaseline level of miR-671-5p was high in OFSCs, and down-regulation of miR-671-5p upon co-culture with\nactivated macrophages was observed. MiR-671-5p inhibitor transfection into OFSCs selectively enhanced the IL-1\nRA and sTNF RII expressions. In addition, inhibition of miR-671-5p in OFSCs enhanced the anti-inflammatory ability\nagainst LPS-induced ALI.\nConclusion: The paracrine effect of OFSCs inhibits the pro-inflammatory ability and proliferation of macrophages.\nThe immune-modulation capacity of OFSCs can be triggered by activated macrophages, and down-regulation of\nmiR-671-5p enhances OFSC immuno-modulation ability by up-regulating IL-1 RA and sTNF RII expression.
Loading....