Role Of PD-L1 In Human Adipose-derived Stem Cells Under Inflammatory Environments

Inflammatory disease has become a major disease that affects the health of people all over the world because of its complicated pathogenesis,different prognosis and wide range.In recent years,many studies have confirmed that MSCs has the effect of curing and alleviating inflammation,but the mechanism is not clear.And meanwhile,the interaction between MSCs and inflammatory environment is complex,the effects on homing rate and immune stability of MSCs need to be further studied.Programmed death ligand-1(PD-L1)is a type I transmembrane protein,which is expressed on many cell surfaces and maintains the tolerance of peripheral and central immune cells by binding to programmed death-1(PD-1).PD-L1 has been shown to be involved in the regulation of MSCs immune function.In this study,PD-L1 was used as a target to investigate the effect of inflammatory microenvironment on the migration and immune function of MSCs and the mechanism of PD-L1 expression in MSCs.The aim of this study is to provide a new way for the construction of gene-modified MSCs and to improve the therapeutic effect of MSCs on inflammatory diseases caused by immune imbalance.This study used human adipose-derived stem cells(hADSCs)as the research object,and stimulated cells with 10 mg/L LPS to construct an inflammatory model in vitro,the study included the following four aspects:(1)The expression of PD-L1 in hADSCs was detected by qRT-PCR,Western blot and IF methods.(2)To investigate the effect of PD-L1 expression in inflammatory microenvironment on the migration of hADSCs.Cell scratch and Transwell assay were used to detect the migration ability of hADSCs,and qRT-PCR was used to detect the expression of chemokines.(3)To investigate the role of PD-L1 expression in inflammatory microenvironment in the regulation of B cell immunity by hADSCs.The proliferation of B cells was detected by CCK-8 method,and the secretion of Ig G by B cells was detected by ELISA method.(4)To explore the regulatory mechanism of PD-L1 expression in hADSCs,and to detect the m RNA level changes of PD-L1 regulated genes in hADSCs by qRT-PCR.The results showed that:(1)After 10 mg/L LPS stimulation,the gene transcription level and protein expression of PD-L1 in hADSCs increased significantly.(2)After 10 mg/L LPS stimulation,the migration of hADSCs and the relative m RNA expression of chemotactic factors were significantly increased.However,after transfection with siRNA-PD-L1,the migration of hADSCs was inhibited and the relative m RNA expression of CXCR4 was significantly decreased.(3)In co-culture,hADSCs inhibited the proliferation and secretion of Ig G of B cells,and the inhibitory effect of hADSCs on B cells was partially reversed after transfection of siRNA-PD-L1.(4)The gene transcription level of PD-L1 and NF-κB in hADSCs increased significantly after 10 mg/L LPS stimulation,and the expression of PD-L1 and NF-κB in hADSCs decreased significantly after adding the inhibitor of NF-κB.In summary,we investigated the role of PD-L1 in MSCs therapy and inflammation mitigation using an inflammatory model in vitro of hADSCs,and found that high expression of PD-L1 in the inflammatory environment promoted the migration of hADSCs and the immunosuppression of B cells,meanwhile,transcription factor NF-κB is involved in the regulation of PD-L1 expression on hADSCs.The results show that MSCs could increase homing rate and immunosuppressive function by adding inflammatory factor pretreatment or gene overexpression of PD-L1,which provides a theoretical basis for improving the clinical application of MSCs in inflammatory diseases.