The ability to suppress macrophage-mediated inflammation in orbital fat stem cells is controlled by miR-671-5p

• DOI: 10.1186/scrt486
• Publication Date: 2014-08-13T21:03:45Z
• Authors: Gi-Shih Lien; Jen-Fang Liu; Ming-Hsien Chien; Wei-Tse Hsu; Tzu-Hao Chang; Chia-Chi Ku; Andrea Tung-Qian Ji; Peng Tan; Ting-Lieh Hsieh; Liang-Ming Lee; Jennifer H Ho
• Keywords: Lipopolysaccharides; 0301 basic medicine; Mice, Inbred BALB C; Tumor Necrosis Factor-alpha; Research; Macrophages; Interleukin-1beta; Down-Regulation; High-Throughput Nucleotide Sequencing; Nitric Oxide Synthase Type II; Mesenchymal Stem Cells; Mesenchymal Stem Cell Transplantation; Coculture Techniques; Receptors, Tumor Necrosis Factor; Up-Regulation; 3. Good health; MicroRNAs; 03 medical and health sciences; Adipose Tissue; Interleukin-1alpha; Animals; Orbit

Abstract Introduction 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.

Conclusion 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.