Abstract: BACKGROUND: One of the main mechanisms for stem cell transplantation to treat diseases is regulating inflammatory response in a manner of paracrine or distant secretion. Transplanted stem cells secrete a large number of biologically molecules, such as transforming growth factor beta, prostaglandin E2, interleukin 10, and tumor necrosis factor α stimulating gene-6 protein (TSG-6), which affect the conversion of monocyte/macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, and coordinating the balance of pro-inflammatory and anti-inflammatory factors. The mechanism by which stem cell secreting factors promote the conversion of monocyte/macrophages M1/M2 subtypes is not clear.  
OBJECTIVE: To investigate the effects of biologically active molecule secreted by human umbilical cord blood mesenchymal stem cells TSG-6 on the conversions of phenotype and inflammatory factors of mice bone marrow-derived macrophages, and research into the mechanism of TSG-6 protein-mediated M2 polarization in macrophages.
METHODS:  (1) Bone marrow derived monocytes aseptically extracted from 6-8-week-old Balb/c mice were differentiated into M1 macrophages with lipopolysaccharide and interferon-γ. M1 macrophages were divided into four groups: M1(M1), MT(M1+rhTSG-6), MM(M1+MSCs), and MMsi(M1+MSCs+TSG-6 siRNA). After co-culture for 3 days, cells and supernatant fluid were collected. The macrophages M1/M2 ratio were measured by flow cytometry. The concentrations of inflammatory factors interleukin 1β, interleukin 12, tumor necrosis factor α, transforming growth factor β1, interleukin 10, and TSG-6 were measured by ELISA. Inside the macrophage, p/t-STAT1/3/6 and TSG-6 levels were measured by western blot assay. mRNA expression levels of arginase 1, interleukin 10, inducible nitric oxide synthase, and tumor necrosis factor alpha were measured by RT-PCR. (2) After blocking CD44, M1 macrophages were divided into five groups: M1(M1), MT(M1+rhTSG-6), bMT(M1+TSG-6+blkCD44), MM(M1+MSCs), and bMM(M1+MSC+blkCD44). After co-culture for 3 days, cells were collected to perform western blot assay for the concentrations of CD44 and p/t-STAT1/3/6. RT-PCR was conducted for the expression of CD44 and STAT1/3/6 mRNA and target genes. TSG-6 adhered M1 macrophages were measured by flow cytometry.  
RESULTS AND CONCLUSION: Mouse M1 macrophages co-cultured with rhTSG-6 or human cord blood mesenchymal stem cells showed: (1) M1 cells decreased, M2 cells increased, and the ratio of M1/M2 decreased (P < 0.05). (2) The pro-inflammatory factors tumor necrosis factor alpha, interleukin 1beta and interleukin 12 decreased (P < 0.05), and the levels of anti-inflammatory factors interleukin 10, transforming growth factor beta1 and TSG-6 increased (P < 0.05). (3) p/t-STAT1 levels decreased and p/t-STAT3/6 level increased (P < 0.05). (4) Tumor necrosis factor alpha and inducible nitric oxide synthase mRNA expression decreased, and interleukin 10 mRNA and arginase 1 mRNA expression increased (P < 0.05). After blocking CD44: (1) TSG-6 adhered macrophages decreased (P < 0.05). (2) CD44 protein and mRNA expression decreased (P < 0.05). (3) The mRNA expression of p/t-STAT1, tumor necrosis factor alpha, and inducible nitric oxide synthase significantly increased (P < 0.05), and the mRNA expression of p/t-STAT3/6, interleukin 10, and arginase 1 (P < 0.01). Therefore, human umbilical cord blood mesenchymal stem cells secreting TSG-6 mediates M1 to M2 conversion and inflammatory regulation in macrophages by affecting STAT 1/3/6 signaling pathway in a CD44-dependent manner.

Key words: inflammation, human umbilical cord blood mesenchymal stem cells, mononcyte/macrophages, M1/M2, TSG-6, CD44, STAT