The Role Of Defective EZH2 Expression In The Pathogenesis Of Rheumatoid Arthritis. The Role Of VSTM1 And Its Ligands In The Pathogenesis Of Systemic Lupus Erythematosus

Part Ⅰ.EZH2 deficiency contributes to the pathogenesis of rheumatoid arthritisRheumatoid arthritis(RA)is an autoimmune disease characterized by chronic,aggressive and symmetrical polyarthritis,which can cause serious damage and deformity of the joint.The course of disease is prolonged repeatedly,and the rate of disability within one year is as high as 20%,which is one of the main causes of human labor loss and disability.RA patients are characterized by T lymphocytes and other inflammatory cells infiltration and inflammatory cytokines secretion,such as TNF-α,IL-6,IL17,IFN-γ and so on.The proportion of Treg cells in peripheral blood has a downward trend,which suggests that the imbalance of CD4+T subsets may play an important role in the pathogenesis of RA.The activation and differentiation of T cells are regulated by many factors,among which epigenetic regulation plays an important role,including DNA methylation and histone modification.DNA methylation,demethylation and histone modification play an important role in the development,activation and differentiation of T cells.Recently,more and more studies show that epigenetic regulation is involved in the pathogenesis of RA.Although epigenetic regulation is involved in T cell differentiation and functional regulation in many models,it is not clear whether abnormal epigenetic regulation regulates CD4+T cell differentiation in RA patients.In this work,we screened the epigenetic regulatory genes of immune cell subsets in peripheral blood of RA patients.After the preliminary screening results were obtained,the expression of EZH2 in peripheral blood mononuclear cells,CD4+T cells,CD 19+B cells and CD 14+mononuclear cells of the newly treated RA patients and healthy control group(HC)was verified by expanding the samples.In order to further study the potential mechanism of EZH2 in the pathogenesis of rheumatoid arthritis.We used the EZH2 inhibitor GSK126 to inhibit the expression of EZH2,and measured the differentiation,proliferation and apoptosis of CD4+T cells.We found that EZH2 inhibited the differentiation and FOXP3 transcription of regulatory T cells(Treg)in CD4+T cells.The expression of FOXP3 was also inhibited by knockdown of EZH2 expression in T cell lines.In order to clarify the effect of EZH2 on Foxp3 related regulatory pathways,we further detected the TGFβ-SMAD and RUNX1 signaling pathway.It was found that EZH2 inhibitor could down regulate RUNX1 and upregulate SMAD7 expression.Finally,we studied the regulation mechanism of RA synovial fluid on EZH2 through co-culture system,and found that RA synovial fluid and fibroblast like synovial cells inhibited the expression of EZH2 on CD4+T cells.After neutralizing IL-17,a key proinflammatory cytokine in RA synovial fluid,EZH2 expression of CD4+T cells was partially restored.In conclusion,EZH2 expression in CD4+T cells of patients with rheumatoid arthritis is defective.When EZH2 is inhibited,it can inhibit FOXP3 transcription by down regulating RUNX1 and upregulating SMAD7 in CD4+T cells,and finally inhibit Treg differentiation.IL-17 in the synovial fluid of rheumatoid arthritis may be the cause of the low expression of EZH2 in CD4+T cells.The deficiency of EZH2 expression in CD4+T cells can lead to the inhibition of Treg differentiation in RA patients.Part Ⅱ.The role of VSTM1 and its ligands in the pathogenesis of systemic lupus erythematosusSystemic lupus erythematosus(SLE)is an autoimmune disease with multiple organs and systems involvement.SLE mainly occurs in women of childbearing age,mainly in the 15-45 age group.The main clinical feature of SLE is the presence of a variety of autoantibodies in serum.The most common lesion of SLE is the lesions of articular skin and mucous membrane.However,SLE is also very prone to the involvement of important organs such as kidney and nervous system,which seriously affects the prognosis of patients,resulting in a high rate of disability and death.Abnormal immune regulation and lack of autoimmune tolerance are the main immunopathological mechanisms of SLE.Both innate and adaptive immune response disorders are involved in the occurrence and development of the disease.This systemic autoimmune disease is characterized by a loss of tolerance to nuclear antigens,resulting in the deposition of immune complexes in tissues and multiple organ involvement.More and more researched found that neutrophils also play an important role in the pathogenesis of SLE.In the pathogenesis of SLE,autoantibodies can activate neutrophils,resulting that neutrophils release nets containing DNA and antimicrobial peptide complexes,also known as neutrophil extracellular traps(NETs).Histone and DNA in the structure of NETs are the main sources of autoantigens in different rheumatic diseases.As immune stimuli,it can also aggravate inflammation and activate the adaptive immune system.The mechanism of producing NETs is not completely clear,but the production of ROS and the citrullination of histone are two necessary conditions.There are two ways to regulate ROS negatively in cells:one is to use enzyme activity or reducing molecules to eliminate ROS,the other is to inhibit ROS production by immunostaining receptor.In this work,we verified the increase of ROS expression level of neutrophils in lupus patients by flow cytometry.After cross culture of the serum of lupus patients and healthy controls with the neutrophils of lupus patients and healthy controls,ROS was detected.It was found that the serum of lupus patients could significantly promote the formation of ROS and NETs of neutrophils.As an immunosuppressive receptor,VSTM1 is mainly expressed in myeloid cells,such as neutrophils and monocytes.The expression of VSTM1 in neutrophils from both mRNA and protein levels was significantly lower than that of healthy controls.Moreover,lupus serum can downregulate the expression of VSTM1 on the surface of neutrophils.Activation of VSTM1 can significantly down regulate the formation of ROS and NETs in neutrophils.Through mass spectrometry screening,we found that Galectin 1,complement C1q and complement C4 are potential physiological ligands of vstml.The interaction between Galectin 1 and VSTM1 can be further confirmed by surface plasmon resonance,Co-IP and immunofluorescence confocal.The three candidate ligands can also inhibit the production of ROS by neutrophils in function.What’s more,with the increasing concentration,Galectin 1,complement C1q and complement C4 can enhance the ability of inhibiting ROS.Through the overexpression of VSTM1 in THP1 cell line,it was confirmed that the candidate ligands could inhibit ROS production through VSTM1.In terms of ROS related regulatory pathways,serum from lupus patients can significantly promote the expression of phos-ERK and phos-p38 in neutrophils.VSTM1 can inhibit the expression of phos-p38 in neutrophils.The effect of candidate ligands on phos-p38 was consistent with the trend of VSTM1 activating antibody.To sum up,our study found that there was abnormal high expression of ROS in neutrophils of lupus patients.Lupus serum participated in the regulation of this abnormal phenomenon and promoted the formation of NETs.VSTM1 expression was defective in neutrophils of lupus patients at mRNA and protein levels.VSTM1 activation can reverse the abnormal expression of ROS and NETs in neutrophils induced by lupus serum.Galectin 1,complement Clq and complement C4,potential physiological ligands of VSTM1,can inhibit ROS expression of neutrophils through VSTM1,which may be related to the inhibition of phos-p38 protein expression in neutrophils.