Preliminary Study On The Effect And Mechanism Of B7 Family Fusion Protein On CIA Mice

Rheumatoid arthritis(RA)is a kind of chronic autoimmune disease with complex pathogenesis and unclear cause.RA is mainly with synovitis of small joints,accompanied by systemic organ damage,and even lead to disability,which affects the quality of life and life span of patients.The disease affects 1%of the global population and the incidence increases with age.The clinical drug for RA treatment has many adverse reactions and poor targeting.It is an important work to develop new biological drugs for RA treatment.In recent years,biological agents targeting T cells,B cells,cytokines,and adhesion factors have shown high efficiency and safety in the treatment of RA,so more and more attention has been paid in the biological drugs.The pathogenesis of RA is very complex.It has been found that the over activation of self-reactive T lymphocytes plays an important role in the pathogenesis of RA.At the early stage of RA,autoantigens are carried by antigen-presenting cells to activate T cells in lymphoid organs,which leads to the inflammatory response of lymphoid organs.After activation,T cells with abnormal amplification selectively enter the joint and are activated by joint antigens,which leads to pathological damage of the joint.The inflammatory cytokines TNF-α and IL-17A secreted by T cells promote the secretion of chemokines and cytokines by fibroblast like synoviocytes,promote the formation of pannus,stimulate bone absorption,cause articular cartilage damage and aggravate the degree of joint damage.The metabolic status of RA patient is disordered,and form the microenvironment of hypoxia and nutrient deficiency.The evidence of the abnormal levels of glycolipid metabolites has been found in the serum and synovial fluid of RA patients.In addition,in the process of RA immune status establishment and maintenance of chronic inflammation,T cells in hypoxia and low nutrition environment maintain their functions by regulating metabolism activities.When activated with autoantigens,T cells are characterized by increased glycolysis and pentose phosphate pathway,and abnormal fatty acid metabolism.Lactate,a glycolytic product,inhibits the movement of T cells and stimulates T cells secrete a large amount of IL-17A,which promotes the chronic inflammatory process of arthritis.Ribose-5-phosphate,a pentose phosphate pathway product,provides more sufficient precursors for T cell proliferation,and NADPH,an other product,provides more reducing power for T cell biosynthesis.Moreover,there is a large amount of fatty acid accumulation in T cells.This abnormal lipid acid metabolism is involved in the formation of an inflammatory response.B7 family immunosuppressive molecules down-regulate T cell immune response,prevent over activation of the immune response,maintain peripheral self-tolerance,and participate in the development of RA.PD-L1 and PD-L2 are both ligands of PD-1.They regulate T cell activation,tolerance,and immune-mediated tissue damage by transmitting inhibitory signals,and participate in the immune and disease development process of RA.B7H4 was induced expressing on antigen-presenting cells.By binding with T cell surface receptors,it inhibited T cell proliferation and inflammatory factors production,and its gene knockout mice has the aggravation of arthritis.VISTA can play an inhibitory role as the ligand and receptor of T cells,and it is more sensitive to the inhibition of the activity of initial T cells than memory T cells.Its potential therapeutic effect in autoimmune diseases has attracted more and more attention.The clinical treatment of RA needs more efficient and safe biological agents.As the important role of over-activated self-reactive T cells in the process of RA disease,the B7 family immune checkpoint molecules regulate the T cell immune response and participate in the disease development process of RA.In this study,we established collagen-induced arthritis(CIA)mice model to study the efficacy of B7 family fusion protein on treating CIA mice,the effect on immune response and metabolism of CIA mice,and the regulatory effect on glycolysis,pentose phosphate and fatty acid metabolism of T cells of CIA mice.The purpose of this study is to provide an experimental and theoretical basis for the development of candidate immunomodulatory drugs for RA.Methods:1.The fusion protein of the B 7 family was obtained by genetic engineering technology,and the CIA mouse model was established.2.The CIA mice were treated with B7 family fusion proteins.The therapeutic effect of B7 family fusion proteins on CIA mice was estimated by using clinical scoring,pathological analysis,and the detection of inflammatory factors(TNF-α and IL-17A)and product of glycolysis(LA)in peripheral blood with ELISA and micromethod.3.After the T cells of CIA mice were stimulated by CII,the antigen-specific T cells were treated with B7 family fusion proteins VISTA-Ig and PD-L2-Ig.The content of TNF-α and IL-17A secreted by T cells was detected by ELISA.4.Antigen-specific T cells of CIA mice were treated with fusion proteins VISTA-Ig and PD-L2-Ig,and the content of key enzymes and products in glycometabolism of T cells,such as HK、LDH、G6PDH、LA and NADPH,were determined with micromethod,and the mRNA levels of HIF-la in T cells were detected by qRT-PCR.5.Antigen-specific T cells of CIA mice were treated with fusion proteins VISTA-Ig and PD-L2-Ig,the content of the metabolite in fatty acid degradation of T cells(NEFA)were determined with micromethod,and the mRNA levels of TFEB in T cells were detected by qRT-PCR.Results:1.The B7 family fusion proteins effectively relieved the disease severity and reduced the incidence and clinical scores of the CIA mice,and reduced cartilage damage and pannus formation in joints in joint tissue of CIA mice.2.The B7 family fusion proteins reduced the content of TNF-α,IL-17A and LA in peripheral blood of CIA mice.3.The fusion proteins VISTA-Ig and PD-L2-Ig decreased the secretion of IL-17A in antigen-specific T cells of CIA mice,and fusion proteins VISTA-Ig decreased the secretion of TNF-α in antigen-specific T cell of CIA mice.4.The fusion proteins VISTA-Ig and PD-L2-Ig reduced the content of glycolysis key enzymes and product,such as HK,LDH,and LA in antigen-specific T cells,and reduced the content of pentose phosphate pathway key enzyme G6PDH in antigen-specific T cells of CIA mice,tended to reduce the content of NADPH,and depressed the relative expression of HIF-la,a transcription factor related to glucose metabolism.5.The fusion proteins VISTA-Ig and PD-L2-Ig influenced the content of the metabolite of NEFA and the relative expression of TFEB,which were related to lipid acid metabolism.Conclusion:In this study,the B7 family recombinant fusion proteins were found to reduce the disease severity of CIA mice and regulate the abnormal immune and metabolic state of CIA mice.B7 family fusion proteins may affect the immune function of T cells and play a therapeutic role in CIA mice by regulating glycolysis,pentose phosphate,and fatty acid metabolism in antigen-specific T cells.In this study,we evaluated the therapeutic effect of B7 family fusion proteins on CIA mice,and preliminarily studied their mechanism of action on CIA.It provided the experimental and theoretical basis for the development of candidate immunomodulatory drugs for RA.