The Effects And Partial Mechanism Of STNFRⅡ-Fc Fusion Protein Modified Human Umbilical Cord-derived Mesenchymal Stem Cells On Collagen-induced Arthritis In Mice

Rheumatoid arthritis(RA)is an inflammatory,chronic,and systemic autoimmune disease with joint synovial inflammation as the main pathological manifestation.Its pathogenesis may be related to synergy among T cells,B cells,dendritic cells,macrophages and fibroblast-like synoviocytes(FLS)and inflammatory cytokines,and there is currently no satisfactory treatment.Tumor necrosis factor-α(TNF-α)plays an important role in the pathogenesis of RA.It binds to the tumor necrosis factor receptor(TNFR)on cells and induces RA inflammation.The production of mediators,infiltration of inflammatory cells,formation of synovial vasculature,and bone erosion play an important role in pathophysiological processes.In Clinical,TNF-α inhibitors represented by Etanercept,play a pivotal role in the treatment of severe and refractory RA.Etanercept is a fusion protein constructed by the human soluble tumor necrosis factor receptor Ⅱ(sTNFRⅡ)and Fc segment of human immunoglobulin(Ig)G1.As a pseudoreceptor,it competitively binding to TNF-α and blocks the transmission of TNF-α signal in immune cells,thereby inhibiting the activation of immune cells.However,because it acts on a single target,and cannot be regulated from multiple links to exert therapeutic effects.It also has the risk of inducing infection and tumor.Therefore,there is an urgent need for a safe,effective,and easily accessible vector to deliver drugs to the affected area to reduce the incidence of adverse reactions.Mesenchymal stem cells(MSC)are adult stem cells derived from mesoderm with high self-renewal ability and multi-directional differentiation potential.Because of its characteristics of low immunogenicity,tissue regeneration and damage repair ability,immune regulation ability,multi-directional differentiation ability,etc.,it provides new ideas for the treatment research of immune-related diseases.Currently,several clinical trials have confirmed the safety and effectiveness of using MSC to treat RA.However,studies have shown that the inflammatory environment is not conducive to the performance of MSC.Inflammatory cytokines(especially TNF-α)can induce autophagy and apoptosis of MSC,and increase the expression of HLA-DR in MSC,thereby accelerating the clearance of MSC by immune cells and affecting the therapeutic effect of MSC.RA is also an inflammatory and systemic autoimmune disease,with the increase of various inflammatory cytokines in the body.Would MSC undergo apoptosis and autophagy in the inflammatory microenvironment of RA,thereby affecting its efficacy? No reports yet.In summary,the application of MSC in the treatment of inflammatory diseases should consider reducing the induction of apoptosis and autophagy by the inflammatory microenvironment,so as to improve the therapeutic effect of MSC.With the development of tissue cell engineering and genetic engineering,biomedical targeted therapy of RA using MSC as a cell vector is feasible,and its prominent features may include:(1)MSC as a cell vector for gene therapy.(2)Genetically modified MSC still retain their immunoregulatory and differentiation properties,inhibit inflammation and repair damaged joint tissue.(3)MSC gene modification enhances the expression of a certain gene,and the genetically modified MSC can "homing" to the site of local inflammation damage,avoiding systemic adverse reactions caused by high-dose systemic administration.This project intends to construct a human umbilical cord MSC(UC-MSC)stable cell line(sTNFRⅡ-MSC)capable of stably expressing sTNFRⅡ-Fc.Using gene interference,gene transfection,transwell chamber,cell flow cytometry,Q-PCR and other technologies to study the therapeutic effect and characteristics of sTNFRⅡ-MSC in RA classical animal model(mouse CIA);In vitro,explore whether sTNFRⅡ-MSC could reduce TNF-α-induced apoptosis and autophagy by bindind and neutralizing TNF-α with secreted sTNFRⅡ-Fc,so as to better exert its therapeutic effects.It provides new therapeutic measures and experimental basis for clinical treatment of RA and other antigen-specific autoimmune diseases.Objective:To construct sTNFRⅡ-Fc fusion protein-modified human UC-MSC(sTNFRⅡ-MSC).To clarify the in vitro and in vivo biological characteristics of sTNFRⅡ-MSC,and the therapeutic effects of sTNFRⅡ-MSC on mice CIA,revealing the mechanism how did sTNFRⅡ-MSC work via multiple targets and multiple links to regulate the immune response.Provide new therapeutic strategies and experimental evidences for clinical treatment of RA and other autoimmune diseases.Methods:1.The sTNFRⅡ and IgG1-Fc gene fragments were constructed using a total synthetic method,and a flexible peptide(5’-GGAGGTGGAGGATCA-3 ’)was used to link the two genes to obtain the sTNFRⅡ-Fc fusion protein gene,which was ligated to the GV358 lentivirus plasmid.The sTNFRⅡ-Fc lentiviral vector was obtained through transformation,identification,packaging and purification.The sTNFRⅡ-Fc lentiviral vector was used to infect human UC-MSC,and a UC-MSC stable cell line sTNFRⅡ-MSC stably expressing the sTNFRⅡ-Fc fusion protein was constructed by a mixed cloning method and a limiting dilution method.2.Flow cytometry was used to detect sTNFRⅡ-MSC surface molecular markers(CD45,HLA-DR,CD34,CD90,CD73 and CD105),and sTNFRⅡ-MSC was used for osteogenic,chondrogenic and adipogenic differentiation.After TNF-α and IL-1β stimulation,Western blot and immunofluorescence were used to detect the expression of VCAM-1 and ICAM-1 in sTNFRⅡ-MSC.Transwell cells were used to co-culture sTNFRⅡ-MSC and fibroblast-like synoviocytes(FLS)from RA patients.After TNF-α and IL-1β stimulation,ELISA was used to detect the levels of RANKL and OPG in the co-culture system.NOD/SCID mice were pretreated with sTNFRⅡ-MSC and etanercept,and then acute inflammation was induced by intraperitoneal injection of LPS.Human sTNFRⅡ,TNF-α,IL-6 and IL-1β levels in serum were measured by ELISA at 0,1.5 and 6 h.3.Using chicken type Ⅱ collagen to establish a mouse CIA model,sTNFR Ⅱ-MSC or UC-MSC were injected into the tail vein;Etanercept is injected subcutaneously,and administered for four weeks.Observe the changes of overall indicators(body weight,arthritis index,and joint swelling number),changes of thymus and spleen index of each group of mice,score the pathology of spleen and ankle of each group,and measure the splenocytes and thymocytes proliferation induced by LPS or Con A and CC Ⅱ specific CD4+ T cell proliferation response with CCK-8.ELISA was used to detect IL-17,IL-4,IFN-γ,IL-10,IgG1,anti-CCⅡ,IgG2 a,levels in serum.Flow cytometry was used to detect changes in T and B cell subsets in spleen of CIA mice.Immunohistochemical method was used to detect the expression of ADAMTS-5,Collagen Ⅱ,MMP-13 and TIMP-1 in mouse ankle cartilage and synovial tissue,and immunofluorescence was used to detect the distribution of sTNFRⅡ-MSC in spleen and joint tissue of CIA mice.The expression of LC3B-Ⅱ and cleaved caspase 3 in UC-MSC remaining in the spleen of CIA mice was detected by immunofluorescence.4.Isolating CD4+ T cells in the spleen of CIA mice,and co-cultured with sTNFRⅡ-MSC.QPCR method was used to detect expression of transcription factorsT-bet,GATA-3,ROR-γt,Bcl6 and Foxp3 in CD4+ T cells.5.sTNFRⅡ-MSC were treated with TNF-α and CHX,and expression of Bax,Bcl2,Caspase 8,Caspase 3,Cleaved caspase 8,Cleaved caspase 3,LC3B-Ⅱ and TRIB3 protein in sTNFRⅡ-MSC was detect by western blot method.CCK-8 method was used to detect the effects of TNF-α-induced apoptosis and autophagy on the immunosuppressive ability of sTNFRⅡ-MSC.Annexin V/PI and TUNEL method was used to detect the apoptosis of sTNFRⅡ-MSC.sTNFRⅡ-MSC were stimulated with TNF-α,and the expressions of SOX-9 and Aggrecan proteins in sTNFRⅡ-MSC were detected by Western Blot method.Results: 1.Successful construction of sTNFRⅡ-MSC stable cell lineThe sTNFRⅡ-Fc fusion protein gene was obtained by a total synthesis method,which was ligated to the GV358 lentiviral plasmid,and the sTNFRⅡ-Fc lentiviral vector was successfully obtained through transformation,identification,packaging and purification.The sTNFRⅡ-Fc lentiviral vector was used to infect human UC-MSC,and the sTNFRⅡ-MSC stable cell line was obtained.The purity of the sTNFRⅡ-MSC stable cell line was above 98%.2.Biological characteristics of sTNFRⅡ-MSC in vitro and in vivoWe examined the levels of human sTNFRⅡ in sTNFRⅡ-MSC culture supernatants,and the results showed that the first and third generations of sTNFRⅡ-MSC were able to secrete higher levels of sTNFRⅡ-Fc after lentivirus transfection.Does UC-MSC after sTNFRⅡ-Fc lentivirus transfection still retain its inherent biological characteristics? We found that sTNFRⅡ-MSC hardly expresses CD45,CD34,HLA-DR,and their positive rates are below 2%;and sTNFRⅡ-MSC high expresses CD105,CD73,and CD90,and their positive rates are above 99%.At the same time,sTNFRⅡ-MSC still retained osteogenic,chondrogenic and adipogenic differentiation ability.These results suggest that sTNFRⅡ-MSC still retained the inherent biological characteristics of UC-MSC.Furthermore,we tested whether sTNFRⅡ-Fc secreted by sTNFRⅡ-MSC had biological effects in vitro.The results showed that sTNFRⅡ-MSC could resist the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α,but had no effect on the up-regulation of ICAM-1 and VCAM-1 induced by IL-1β,and sTNFRⅡ-MSC can resist TNF-α-induced up-regulation of RANKL and OPG in co-culture systems with FLS from RA patients,but it had no effect on IL-1β-induced up-regulation of RANKL and OPG in co-culture systems.These results suggested that sTNFRⅡ-Fc secreted by sTNFRⅡ-MSC could selectively neutralize TNF-α in vitro,thereby exerting its biological effects.In the biological characteristics of sTNFRⅡ-MSC,we found that the level of sTNFRⅡ in the serum of sTNFRⅡ-MSC group mice was significantly reduced 1.5 hours after LPS injection into NOD/SCID mice.After 1.5 hours of intraperitoneal injection of LPS in NOD/SCID mice,the serum TNF-α levels of mice in the EGFP-MSC group and Etanercept group increased sharply to about 2000 pg/ml,while the serum TNF-α levels in the sTNFRⅡ-MSC group were significantly lower than that in the EGFP-MSC group and Etanercept group.The serum TNF-α level of the mice in the sTNFRⅡ-MSC group was still significantly lower than that in the Etanercept group 6 hours after LPS injection.Six hours after intraperitoneal injection of LPS in NOD/SCID mice,serum IL-1β and IL-6 levels in mice in the EGFP-MSC and Etanercept groups began to increase,and serum IL-1β and IL-6 levels in sTNFRⅡ-MSC groups were significant lower than EGFP-MSC group and Etanercept group.The results suggested that sTNFRⅡ-MSC could secrete sTNFRⅡ-Fc,thereby blocking the biological function of TNF-α in mouse and reducing the levels of other inflammatory factors.3.Therapeutic effects of sTNFRⅡ-MSC on mouse CIATransplantation of sTNFRⅡ-MSC could obviously restore the weight of CIA mice,reduce the arthritis index and joint swelling score,reduce the spleen index of CIA mice,and reduce the pathological scores of spleen and ankle.sTNFRⅡ-MSC could significantly down-regulate the proliferation of spleen and thymocytes induced by LPS and Con A,and the CC Ⅱ specific CD4+ T cell proliferation response.sTNFRⅡ-MSC could significantly down-regulate the levels of IL-17,IFN-γ,anti-CC Ⅱ,and IgG2 a in the serum of CIA mice,and could significantly up-regulate IL-10 and IL-4.Immunohistochemical results showed that sTNFRⅡ-MSC transplantation could significantly down-regulate the expression of ADAMTS-5 and MMP-13 in ankle cartilage and synovial tissue of CIA mice,and significantly up-regulate the expression of Collagen Ⅱ in ankle cartilage,and TIMP-1 expression was not significantly affected in the tissues.Flow cytometry results showed that sTNFRⅡ-MSC transplantation could significantly reduce Th1(CD4+IFN-γ+),Th17(CD4+IL-17+),Tfh(CD4+CXCR5+ PD-1+)and plasma cells(CD19-CD138+)in spleen of CIA mice,and significantly increased the ratio of Th2(CD4+IL-4+),Treg(CD4+CD25+Foxp3+)and Breg(CD19+IL-10+).4.Effect of sTNFRⅡ-MSC on spleen CD4+ T cell proliferation and expression of related transcription factors in CIA miceQPCR results showed that sTNFRⅡ-MSC could significantly down-regulate the expression of transcription factorsT-bet,ROR-γt,and Bcl6,and significantly up-regulate the expression of Foxp3 and GATA-3 in spleen CD4+ T cells of CIA mice.5.sTNFRⅡ-MSC can migrate into spleen and ankle tissues of CIA mice,and sTNFRⅡ-Fc gene modification can reduce the occurrence of apoptosis and autophagy in UC-MSCImmunofluorescence results showed that after sTNFRⅡ-MSC injection by tail vein,EGFP+CD105+ sTNFRⅡ-MSC could be detected on d 1 to d 14 in spleen and ankle tissue of CIA mice.We tested the expression of LC3B-Ⅱ and cleaved caspase 3 in sTNFRⅡ-MSC in spleen tissue of CIA mice on d7 after sTNFRⅡ-MSC injection by tail vein.The results showed that compared with EGFP-MSC,the expression of LC3B-Ⅱ and cleaved caspase 3 in sTNFRⅡ-MSC was significantly reduced.6.TNF-α can induce apoptosis and autophagy of UC-MSC,reducing its immunosuppressive function,sTNFRⅡ-MSC can resist this ability of TNF-αWestern Blot results showed that after treatment with TNF-α/CHX,the expressions of Bax,Cleaved caspase-3,Cleaved caspase-8,and LC3B-Ⅱ proteins were significantly inncreased,and Bcl2,caspase-3,caspase-8,and TRIB3 proteins were significantly decreased in UC-MSC.Western Blot results showed that the expressions of Bax,Cleaved caspase-3,Cleaved caspase-8,and LC3B-Ⅱ protein in sTNFRⅡ-MSC group were significantly lower than those in UC-MSC group,but Bcl2,caspase-3,caspase-8 and TRIB3 protein expression was significantly increased after TNF-α/CHX treatment.CCK-8 results showed that TNF-α-induced autophagy and apoptosis reduce the immunosuppressive ability of UC-MSC,while sTNFRⅡ-MSC could resist this function of TNF-α.7.Effects of TNF-α on the chondrogenic differentiation of sTNFRⅡ-MSCWestern Blot results showed that the expression of SOX-9 and Aggrecan proteins in UC-MSC was significantly reduced after TNF-α treatment,while the expression of SOX-9 and Aggrecan proteins in sTNFRⅡ-MSC was significantly increased compared with EGFP-MSC.Alcian blue staining results showed that after TNF-α treatment,UC-MSC in chondrogenic medium cannot aggregate into spheres,and alcian blue is lightly stained;sTNFRⅡ-MSC grows in spheroid,and Alcian blue is darkly stained.Conclusion: 1.In this project,UC-MSC was used as the vector for constructing a UC-MSC stable cell line(sTNFRⅡ-MSC)stably expressing the sTNFRⅡ-Fc fusion protein using lentiviral transfection for the first time,and sTNFRⅡ-MSC still retained the inherent biology characteristics of MSC.2.The sTNFRⅡ-Fc fusion protein secreted by sTNFRⅡ-MSC has biological effects both in vivo and in vitro,and acts by neutralizing TNF-α in the culture supernatant and serum.3.Compared with UC-MSC,sTNFRⅡ-MSC had a better therapeutic effect on CIA mice.sTNFRⅡ-MSC could migrate into the spleen and ankle joint tissues of CIA mice,and exert its therapeutic effects by regulating T and B cell activation and differentiation,and reducing inflammation and promote tissue repair.4.The inflammatory environment is not conducive for UC-MSC to exert its therapeutic effects.The inflammatory environment in CIA mice and TNF-α stimulation in vitro could induce UC-MSC to undergo apoptosis and autophagy,affecting UC-MSC’s therapeutic effects,and sTNFRⅡ-MSC could reduce their apoptosis and autophagy by neutralizing inflammatory cytokines,such asTNF-α.5.TNF-α inhibited the differentiation of UC-MSC into chondrocytes and affected the cartilage repair function of UC-MSC,while sTNFRⅡ-MSC could neutralize TNF-α and enhance its cartilage differentiation ability.