Authors: Li Na, Nian Hong, Zhao Lu, Wei Yankai, Wu Yiyuan, Wei Ruihua
To investigate the effect of human umbilical cord mesenchymal stem cells derived exosomes (hUC-MSC-exo) on the phenotype of peripheral blood macrophages from rabbit autoimmune dry eye and the expression of related cytokines.
The hUC-MSCs were isolated and characterized.Exosomes derived from hUC-MSCs were extracted by ultracentrifugation and observed directly using electronic microscopy.Specific markers of exosomes were analyzed by Western blot.Six rabbits were randomly divided into the normal control group and the dry eye group by using the random number table method, 3 rabbits for each group.Rabbit model of autoimmune dry eye was established in the dry eye group, and the lacrimal glands were collected for quantitative real-time PCR (qRT-PCR) at the 8th week.In vitro, activated peripheral blood mononuclear cells (PBMCs) from rabbit autoimmune dry eye model were incubated with hUC-MSC-exo or phosphate buffered saline (PBS). After 48 hours, cells from the hUC-MSC-exo group and the PBS control group were collected.The mRNA expression levels of related cytokine genes and subpopulation-related marker genes in macrophages were quantified by qRT-PCR.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.This study protocol was approved by Ethic Committee of Tianjin Medical University Eye Hospital (No.TJYY20181217001). Written informed consent was obtained from each family before obtaining umbilical cord.
Exosomes derived from hUC-MSCs had typical morphology and specific markers.qRT-PCR results showed that, the relative expression quantity of M1 macrophages phenotypic molecular nitric oxide synthase 2 (NOS2) mRNA and inflammatory factor tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) mRNA in the lachrymal organization in the dry eye model group was significantly higher than those in the normal control group (3.06±1.00 vs. 1.00±0.03, 2.77±0.72 vs. 1.01±0.02 and 1.30±0.08 vs. 1.01±0.01, respectively), the relative expression quantity of M2 macrophages phenotypic molecular arginase 1 (Arg1), CD206 and IL-10 mRNA in the lachrymal organization in the dry eye model group was significantly lower than those in the normal control group (0.55±0.07 vs. 1.00±0.00, 0.60±0.13 vs.1.00±0.00, 0.65±0.14 vs. 1.01±0.01, respectively), with significant differences between them (all at P<0.05). In vitro, the relative expression quantity of M1 macrophages phenotypic molecular NOS2 mRNA and inflammatory factor TNF-α, IL-1β mRNA in PBMCs in the hUC-MSC-exo group was significantly lower than those in the PBS control group (0.59±0.08 vs.0.98±0.03, 0.56±0.07 vs. 1.03±0.11, 0.47±0.04 vs.1.00±0.08)(all at P<0.05); the relative expression quantity of M2 macrophages phenotypic molecular Arg1, CD206 mRNA and anti-inflammatory cytokine TGF-β, IL-10 and IL-4 mRNA in PBMCs in the hUC-MSC-exo group was significantly higher than those in the PBS control group (2.13±0.28 vs. 1.10±0.17, 1.32±0.03 vs. 1.01±0.06, 1.53±0.20 vs. 1.05±0.10, 1.47±0.08 vs.0.98±0.03, 1.51±0.16 vs. 1.01±0.03), with significant differences between them (all at P<0.05).
hUC-MSC-exo can polarize peripheral blood macrophages toward immune-suppressive M2-like phenotype, inhibit the production of pro-inflammatory cytokines TNF-α and IL-1β, and meanwhile increase the expression of anti-inflammatory factors IL-10 and TGF-β.