Abstract
IntroductionOur previous works demonstrated that systemic orbital fat-derived stem cell (OFSC) transplantation was effective in ameliorating lipopolysaccharide (LPS)-induced extensive acute lung injury (ALI) in vivo mainly through paracrine regulation of macrophage-mediated cytokine-storm. In this study, we explore the molecular mechanism(s) of OFSCs regulating macrophage activity in a cytokine-inducible fashion.MethodsLPS (100 ng/ml)-activated macrophages were treated by conditioned medium from OFSCs (OFSCs-CM) or non-contact cultured with OFSCs for 6 hours. The potency of OFSCs on macrophage proliferation and pro-inflammation ability were determined. Expression levels of pro-inflammatory cytokines in macrophages, inducible immuno-modulatory factors in OFSCs, were investigated. Deep sequencing analysis as well as interaction between microRNA (miRNA) and genes of immuno-modulators in OFSCs induced by activated macrophages was predicted by miRTar. Transfection of miRNA inhibitor into OFSCs was performed. Real-time RT-PCR and transplantation of OFSCs into mice with LPS-induced ALI confirmed the in vitro and in vivo mechanism.ResultsThe paracrine effect of OFSCs on inhibition of macrophage pro-inflammatory cytokine release was more potent than induction of macrophage G0/G1 cell cycle arrest. OFSCs-CM suppressed LPS-induced inducible nitric oxide synthetase and the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 alpha, and IL-1 beta expression in macrophages. Under non-contact culture, LPS-activated macrophages effectively triggered the expression of soluble immuno-modulating factors in OFSCs, i.e., IL-10, IL-1 receptor antagonist (IL-1 RA), indoleamine 2,3-dioxygenase, and soluble TNF receptor type II (sTNF RII). Under miRTar prediction, miR-671-5p was identified as a critical microRNA in regulation of multiple immune-modulating factors in OFSCs response to macrophages. The baseline level of miR-671-5p was high in OFSCs, and down-regulation of miR-671-5p upon co-culture with activated macrophages was observed. MiR-671-5p inhibitor transfection into OFSCs selectively enhanced the IL-1 RA and sTNF RII expressions. In addition, inhibition of miR-671-5p in OFSCs enhanced the anti-inflammatory ability against LPS-induced ALI.ConclusionThe paracrine effect of OFSCs inhibits the pro-inflammatory ability and proliferation of macrophages. The immune-modulation capacity of OFSCs can be triggered by activated macrophages, and down-regulation of miR-671-5p enhances OFSC immuno-modulation ability by up-regulating IL-1 RA and sTNF RII expression.
Highlights
Our previous works demonstrated that systemic orbital fat-derived stem cell (OFSC) transplantation was effective in ameliorating lipopolysaccharide (LPS)-induced extensive acute lung injury (ALI) in vivo mainly through paracrine regulation of macrophage-mediated cytokine-storm
LPS dose-dependently increased the expression of CD68, inducible nitric oxide synthase (iNOS), and tumor necrosis factor alpha (TNFα) in macrophages (Figure 1A, left panel), and 100 ng/ml LPS and above significantly triggered the CD68 expression and iNOS production in macrophages (Figure 1A, right panel)
To determine the paracrine potency of OFSCs inhibiting iNOS production from LPS-activated macrophages, macrophages were treated for 6 hours with condition medium collected from various numbers of OFSCs depending on the ratio of OFSCs versus macrophages (OFSC/macrophage ratio from 0.5 to 4)
Summary
Our previous works demonstrated that systemic orbital fat-derived stem cell (OFSC) transplantation was effective in ameliorating lipopolysaccharide (LPS)-induced extensive acute lung injury (ALI) in vivo mainly through paracrine regulation of macrophage-mediated cytokine-storm. Cytokine storm-mediated extensive lung injury is the ultimate pathomechanism of acute respiratory distress syndrome and severe acute respiratory syndrome [2,3]. Except for differentiation ability [9], the MSC as an immunomodulator is a powerful therapeutic strategy in graft versus host disease, autoimmune neurological disease, systemic lupus nephritis, acute lung tissue injury and diabetes [6,10]. MSCs achieve immunomodulation effects on both innate and adaptive immunities by secreting critical soluble factors and/or direct contact regulation of immune cells [6,7], and procytokines such as interferon gamma (IFNγ), interleukin (IL)-1β or tumor necrosis factor alpha (TNFα) stimulate the immunomodulatory ability of MSCs [11,12]. Transforming growth factor beta (TGFβ), hepatocyte growth factor, IL-10, indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 are thought to be inducible immunomodulating factors secreted from MSCs upon procytokine stimulation for targeting T cells, B cells and natural killer cells [13,14,15,16]
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