title: The ability to suppress macrophage-mediated inflammation in orbital fat stem cells is controlled by miR-671-5p.
abstract: BACKGROUND
  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. In this study, we explore the molecular mechanism(s) of OFSCs regulating macrophage activity in a cytokine-inducible fashion.
  
  
  METHODS
  LPS (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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Gi-Shih
authors.initials: GS
authors.lastname: Lien
authors.forename: Jen-Fang
authors.initials: JF
authors.lastname: Liu
authors.forename: Ming-Hsien
authors.initials: MH
authors.lastname: Chien
authors.forename: Wei-Tse
authors.initials: WT
authors.lastname: Hsu
authors.forename: Tzu-Hao
authors.initials: TH
authors.lastname: Chang
authors.forename: Chia-Chi
authors.initials: CC
authors.lastname: Ku
authors.forename: Andrea Tung-Qian
authors.initials: AT
authors.lastname: Ji
authors.forename: Peng
authors.initials: P
authors.lastname: Tan
authors.forename: Ting-Lieh
authors.initials: TL
authors.lastname: Hsieh
authors.forename: Liang-Ming
authors.initials: LM
authors.lastname: Lee
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: Ho
mesh_terms: D000273:Adipose Tissue / Q000166:cytology; D000818:Animals; D018920:Coculture Techniques; D015536:Down-Regulation; D059014:High-Throughput Nucleotide Sequencing; D053582:Interleukin-1alpha / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides; D008264:Macrophages / Q000378:metabolism / Q000502:physiology*; D045164:Mesenchymal Stem Cell Transplantation; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009915:Orbit / Q000166:cytology; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; C000597535:MIRN671 microRNA, mouse; D035683:MicroRNAs; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II