The Molecular Mechanism Of IFN-γ And TNF-α Synergistically Up-Regulating PD-L1 Expression To Enhance The Immunosuppressive Ability Of HUC-MSCs

Background:Mesenchymal stem cells(MSCs)are a group of pluripotent stem cells from a wide range of sources,which have the potential of terminal cell differentiation such as adipocytes,osteoblasts and chondroblasts.In addition to the ability of tissue regeneration and repair,MSCs also have a powerful immunosuppressive ability,so they are widely used in the treatment of various autoimmune and inflammatory diseases,such as rheumatoid arthritis,lupus nephritis,inflammatory bowel disease,and so on,and have achieved good results.The immunosuppressive ability of MSCs mainly derived from the direct interaction of MSCs with various immune cells and various immune factors secreted by MSCs.MSCs suppress the immune activity of T cells,B cells,DC cells or macrophages by secreting or expressing anti-inflammatory molecules such as PD-L1,IDO,TSG-6 and HGF,which reduce local or systemic inflammatory responses.These anti-inflammatory molecules,which are highly expressed in inflammatory environments,play a key role in the immunosuppressive capacity of MSCs.In order to improve the efficacy of MSCs transplantation therapy,it is of great significance to explore the internal mechanism of inducing and maintaining MSCs expression of anti-inflammatory molecules.PD-L1 is programmed cell death ligand 1,the ligand of the immunosuppressive molecule PD-1(programmed death receptor).PD-1 is widely distributed on the surface of activated T cells.And the binding of PD-L1 and PD-1 can significantly inhibit the activity and proliferation of T cells and other immune cells,which lowers the activity of the immune system.In tumors,the PD-L1/PD-1 axis is considered to be one of the important causes of tumor metastasis and immune tolerance,and biological agent antagonistic strategies targeting the PD-L1/PD-1 axis have been applied in various tumor therapies,and a large number of patients have benefited.On the other hand,PD-L1 is also one of the important mediators for MSCs to play an immunosuppressive role.The expression of PD-L1 enhanced the regulatory ability of MSCs on T cell proliferation,activation,anergy and apoptosis,thus improving the therapeutic effect of MSCs in rheumatoid arthritis,inflammatory bowel disease,multiple sclerosis and other T-cell hyperfunction diseases.At the same time,PD-L1 endows MSCs with the ability to balance the differentiation of various T cell subsets,which enables MSCs to have more diversified regulatory functions in adaptive immune regulation.Although it has been reported that IFN-γ,TNF-α or IL-1 alone can up-regulate the expression of PD-L1 in MSCs,the specific mechanism of the regulation of PD-L1 expression in MSCs,especially the synergistic up-regulation of PD-L1 expression by multiple factors,is still unclear.Further exploration of this mechanism can provide theoretical support for the further establishment of induction strategies for PD-L1 positive MSCs and the improvement of the efficacy of MSCs transplantation.Aim:1.To explore the molecular regulatory pathway mechanism of IFN-γ induced PD-L1 expression of hUC-MSCs;2.To explore the molecular regulatory mechanism of TNF-α synergistically enhancing the expression of PD-L1 in hUC-MSCs induced by IFN-γ;3.To establish the optimal in vitro induction system of IFN-γ and TNF-α to enhance the immunosuppressive function of hUC-MSCs,and to evaluate the immunosuppressive efficacy of the induced hUC-MSCs in ulcerative colitis model.Methods:1.We detected the changes of PD-L1 expression of hUC-MSCs after IFN-γ induction by RT-PCR,WB and flow cytometry,and then key pathway inhibitors were used to explore the specific pathway of IFN-γ-induced PD-L1 expression.Then we further investigated the binding region of IFN-γ-induced key transcription factor IRF1 to the PD-L1 gene promoter by luciferase reporter gene assay.2.Changes in PD-L1 expression of hUC-MSCs induced by IFN-γ alone or combined with TNF-α were detected by RT-PCR,WB and flow cytometry.And then the specific pathway of TNF-α synergistically enhancing IFN-γ-induced PD-L1 expression was explored by key pathway inhibitors.Furthermore,the mechanism of TNF-α enhancing IFN-γ-induced PD-L1 expression was investigated by luciferase reporter gene assay and overexpression assay.3.We evaluated the immunosuppressive enhancement and therapeutic effect of hUC-MSCs after synergistic pretreatment with TNF-α and IFN-γ in vitro lymphocyte proliferation inhibition test and DSS induced ulcerative colitis mice.And the therapeutic potential of hUC-MSCs in different treatment groups were analyzed according to the difference of lymphocyte proliferation rate and the disease status of colitis in mice.Specific scheme:The inhibitory effect of immunosuppressive enhanced hUC-MSCs on lymphocyte proliferation was evaluated at the cellular level according to the difference in lymphocyte proliferation rate;meanwhile,the therapeutic effect of immunosuppressive enhanced hUC-MSCs on inflammatory diseases was evaluated at the animal model level by transplantation of colitis mice.Results:1.Our results showed that IFN-γ activated the JAK/STAT1 pathway in hUC-MSCs,and then promoted the binding of IRF1 transcription factor to the PD-L1 gene promoter by upregulating the expression of IRF1,promoted the transcription of PD-L1 mRNA,and finally upregulated the expression levels of total protein and membrane protein of PD-L1.2.Our study found that TNF-α alone had no effect on the PD-L1 expression in hUC-MSCs,but TNF-α synergistically enhances IFN-γ-mediated activation of the JAK/STAT1/IRF1 pathway,and this effect is mediated by IFNGR1.Meanwhile,it was confirmed that TNF-α enhanced the binding of P65 transcription factor to IFNGR1 gene promoter through the activation of NF-κB pathway,and initiated IFNGR1 transcription expression in hUC-MSCs,thereby synergistically enhancing IFN-γ-mediated JAK/STAT1/IRF1 signaling activation.3.Our study found that hUC-MSCs pretreated with IFN-γ and TNF-α exhibited stronger inhibition of T lymphocyte proliferation than the untreated hUC-MSCs,and this capacity was eliminated by neutralizing antibody against PD-L1.At the same time,animal experiments confirm that hUC-MSCs pretreated with IFN-γ and TNF-α significantly alleviated weight loss and colon tissue injury of mice,decreased inflammatory cell infiltration and inflammatory cytokine levels in colon tissue.Conclusions:1.IFN-γ up-regulates the expression of IRF1 transcription factor by activating the JAK/STAT1 pathway,thereby mediating the up-regulation of PD-L1 mRNA transcription level.2.TNF-α promoted the expression of IFNGR1 mRNA and protein in hUC-MSCs by activating the NF-κB pathway,and synergistically enhanced IFN-γ-mediated JAK signaling,thereby enhanced PD-L1 expression.3.hUC-MSCs pretreated with IFN-γ and TNF-α have stronger immunosuppressive capacity and better therapeutic effect on ulcerative colitis,suggesting that IFN-γ and TNF-αcombined pretreatment can enhance the immunosuppressive capacity of hUC-MSCs and improve the therapeutic effect.