The Expressions And Significances Of The Immune Negative Regulatory Genes TIPE2 And A20 In PBMCs From Patients With SLE

ObjectiveTIPE2 (tumor necrosis factor-a induced protein 8-like 2) is a new gene found in 2002 and a single locus was identified for human TIPE2 on chromosome I(1q21.1-1q21.3). TIPE2 is thought to contain 184 amino acids, a DED-like domain and six putative conserved a helices. TIPE2, TNFAIPL1(TIPE1) and TNFAIPL3(TIPE3) belong to TNFAIP8 family. TIPE2 is mainly expressed on immune organs and lymphoid tissues, such as thymus, spleen, lymph node and intestine. Further study found that TIPE2 mice became chronically ill, characterized by body weight loss, multiorgan inflammation and splenomegaly. Serological testing showed high levels of inflammatory cytokines such as IL-1, IL-6, IL-12, and TNF-a in the blood of TIPE2"/" mice. Inflammatory reaction will become more serious in TIPE2 mice and TIPE2 will inhibit nuclear factor-KB activation, which indicate that TIPE2 is a new negative regulator of innate and adaptive immunity. However, there are only a few papers about TIPE2 and the investigation of TIPE2 mainly through mouse model. The expression of TIPE2 and its significance in autoimmune disease such as (systemic lupus erythematosus, SLE) are still unknown.SLE is a classical autoimmune disease, which is characterized by a variety of autoantibodies, leading to multiple systems and organs chronic, inflammatory autoimmune disease. There are many reports about SLE, however,the factors contributing to the onset and progression of SLE are not well understood. SLE is a serious systemic autoimmune disorder, in which the levels of a few proinflammatory cytokines such as IL-4, IL-6, IL-12, IFN-y(interferon-y) are higher. Recent evidence indicates an important role for type I interferon(IFN-I) in the pathogenesis of SLE. As IFN-I family includes 14 IFN-a subtypes and IFN-β, and all the subtypes bind to a single receptor, the serum levels of IFN-I can be represented by mRNA expression levels of MX1, one of the IFN-I inducible genes. TIPE2, as a negative regulator of innate and adaptive immunity, whether it involves in the development of SLE and what's it function in this process? The aim of this research is to detect the expression of TIPE2 in the PBMCs from SLE patients and the correlation between TIPE2 and MX1, TIPE2 and clinical indexs. To further study the function of human TIPE2 in the pathogenesis of SLE, we carry out some experiments with THP1 cell line, which is a mononuclear cell, to provide a new insight to study the mechanism of SLE and and to study whether TIPE2 plays roles during the development of SLE and its mechanism.Materials and Methods1 Detection of TIPE2 mRNA in peripheral blood mononuclear cells from SLE patients1.1 Collection of the samplesFrom October 2008 to March 2009,39 SLE patients were enrolled in QILU hospital. SLE patients fulfilled the 1982 updated revised criteria of the American Rheumatism Association (ARA). The average SLEDAI(systemic lupus erythematosus disease activity index) is 16.33±1.409. Thirty five age and sex matched healthy controls were enrolled.1.2 Extract total RNA of peripheral blood mononuclear cells and the preparation of cDNATotal RNA was extracted from peripheral blood mononuclear cells using Trizol. The purity and concentration of RNA was detected by ultraviolet spectrophotometer, if the absorbance(A) on wavelength 260/280 nm is between 1.8～2.0, it is available. cDNA was prepared with M-MLV reverse transcriptase (RNA 2.0μg).1.3 Detect TIPE2 mRNA expression by Relatively quantitative RT-PCR and Real-time quantitative RT-PCR(qRT-PCR)According to the sequence of human TIPE2 cDNA in PubMed (GI:141802951), the primers were designed by primier primer 5.0 software. The mRNA expression of TIPE2 in PBMCs from SLE patients and healthy controls were assayed by relatively quantitative RT-PCR and real-time quantitative RT-PCR.2 Correlations between TIPE2 expression in peripheral blood mononuclear cells and clinical parameters of SLE patients and MX1Correlation analysis was performed between TIPE2 expression in peripheral blood mononuclear cells and SLEDAI and MX1 with Prism Graphpad software.3 Analysis the relationship between TIPE2 and type I interferon3.1 A small interfering RNA targeting against human TIPE2 expression in THPl cellsTo knockdown TIPE2 expression, a small interfering RNA targeting against human TIPE2 was synthesized and transfected into THP1 cells with lipofectamine TM2000. The expressions of TIPE2, MX1 and TNF-a were detected by real-time quantitative RT-PCR.3.2 THPl cells cultured with serum stimulationTHPl cells were cultured with 5% and 25% SLE serum for 24h, while with the same concentration serum from healthy donors as control. TIPE2 and MX1 expression were measured by real-time quantitative RT-PCR.3.3 THPl cells stimulated with IFN-a2a and IFN-a2bTHP1 cells were cultured at a density of 1×106/ml in 24-well flat-bottomed plates. Cells were stimulated with IFN-a2a and IFN-a2b at the concentration of 1000U/ml for 8h and 12h. The control groups were stimulated with PBS. Then we measure the expression of TIPE2 and MX1 by real-time quantitative RT-PCR.4 The effection of dexamethason on the expression of TIPE2THP1 cells and PBMCs from healthy controls were cultured at a density of 1×106/ml in 24-well flat-bottomed plates. Cells were stimulated with dexamethason at the concentration of 1×10-6mol/L for 2h,12h and 24h. The control groups were stimulated with PBS. Then we measure the expression of TIPE2 by real-time quantitative RT-PCR.Results1 The expression of TIPE2 mRNA and MX1 mRNA in PBMCs from SLE patients and the correlation between the expression of TIPE2 and MX1 and SLEDAIThe expression of TIPE2 and MX1 in PBMCs from SLE patients were assayed by real-time quantitative RT-PCR,2-△△ct is used to figure the expression value of TIPE2 and MX1. Results show that the expression value of TIPE2 in PBMCs from SLE patients and healthy controls are respectively 4.207±0.5772 and 8.620±1.128. The expression of TIPE2 in PBMCs from SLE patients is relative lower than healthy controls (p=0.0004). The expression value of MX1 in PBMCs from SLE patients and healthy controls are respectively 44.480±5.400和23.081±4.866. The expression of MX1 in PBMCs from SLE patients is obvious higher than healthy controls (p=0.0.0031). The level of the expression of MX1 is in concordance with papers published before.It showed that there was a significant negative correlation of TIPE2 mRNA expression and SLEDAI(r=-0.5016, p=0.0011) in SLE patients; while there was a significant positive correlation of MX1 mRNA expression and SLEDAI(r=0.4814, p=0.0019). There was a negative correlation between TIPE2 and MX1 mRNA expression in SLE patients (r=-0.8083, P＜0.0001).2 The expressions of MX1 and TNF-a mRNA in THP1 cells which with siRNA-TIPE2RNA interference could efficiently suppress the TIPE2 expression in THP1 cells. At 24h after transfection, the TIPE2 mRNA was significantly inhibited by Relatively quantitative RT-PCR and real-time quantitative RT-PCR analysis. SiRNA inhibiting TIPE2 expression decreased the expression of MX1 and increased the expression of TNF-a.3 The expressions of TIPE2 and MX1 in THP1 cells cultured with serumThe expression of TIPE2 in THP1 cells cultured with 5% and 25% SLE serum for 24h was not obviously changed than controls, while the expression of MX1 significantly increased by real-time quantitative RT-PCR analysis.4 Stimulated with dexamethason and IFN-IThe expression of TIPE2 increased in THP1 cells and PBMCs stimulated with dexamethason for 2h,12h,24h and at 24h, it significantly increased. The expression of MX1 significantly increased in THP1 cells stimulated with IFN-α2a and IFN-α2b, while the expression of TIPE2 didn't obviously change.Conclusions1 The expression of TIPE2 in PBMCs from SLE patients is significantly lower than that of healthy controls, and negatively correlated with SLEDAI. MX1, one of the genes induced by IFN-I, plays a vital role in the pathogenesis of SLE, and negatively correlate with TIPE2.2 The expression of TIPE2 may affect the expressions of MX1 and TNF-a. When SiRNA suppressed the expression of TIPE2 in THP1 cells, the expression of MX1 decreased and the expression of TNF-a increased. As TIPE2 is a gene induced by TNF-a, its down expression may negatively feedback the expression of TNF-a.3 As dexamethason can down regulate the expression of proinflammatory factors, it plays as a kind of immunosuppressive drug in the treatment of SLE. The expression of TIPE2, a negative regulator of immune system, will increase in THP1 cells stimulated with dexamethason, involving in controlling inflammatory reaction.All above indicates that the down expression of TIPE2 in the PBMCs from SLE patients, may cannot efficient negative regulate immune reaction, leading to the high expressions of a variety of inflammatory cytokines. The down expression of TIPE2 in PBMCs from SLE patients may play an important role in the pathogenesis of SLE.Innovation and significancesTIPE2 is a newly identified gene and it's unknown its role in many human diseases. We have first detected the down expression of TIPE2 in PBMCs from SLE patients, and there is a significant negative correlation with SLEDAI, which is a clinical index of SLE. The pathogenesis of SLE hasn't been well known, and there are many limits in the treatment of SLE. As a new negative regulator of innate and adaptive immunity, it may provide a new target for the treatment of SLE. Objective:Zinc finger protein A20, a kind of new protein molecule, has been found in recent years'researches of cellular endogenous protective mechanism. It is confirmed that zinc finger protein A20 is not only an important negative feedback factor to regulate signaling pathway NF-κB but also an essential feedback immune regulator in suppressing inflammatory. responses in vivo. Systemic lupus erythematosus (SLE) is characterized by B-cell hyperactivity, auto-antibody production, and immune complex deposition in vital organs. There is a wide range of inflammatory response in SLE and high level expressions of a variety of inflammatory cytokines. Previous researches have shown that the signaling pathway NF-κB is activated in the process of SLE. In this study, we investigate the expression of zinc finger protein A20 in peripheral blood mononuclear cells(PBMCs) from SLE patients and the correlations between A20 and clinical indexs of SLE, and primarily discuss the role of zinc finger protein A20 in the pathogenesis of SLE.Methods:The levels of mRNA of zinc finger protein A20 in PBMCs from 35 SLE patients were examined by real-time quantitative reverse transcription polymerse chain reaction (real-time RT-PCR). Thirty-two cases of health people were regarded as control group.Results:The A20 mRNA expression was obviously down-regulated in SLE patients compared with healthy controls(p=0.0133) and was lower expressed in active SLE patients(SLEDAI>5) than its expression in inactive SLE patients(p=0.0006). The expression of A20 was also lower in SLE patients with nephritis than those without nephritis (p=0.0188). Furthermore, the mRNA expression levels negatively correlate with the SLE disease activity index (SLEDAI) and Erythrocyte Sedimentation Rate (r=-0.4661, p=0.0036; r=-0.5222, p=0.0009).Conclusions:Zinc finger protein A20 may play a critical role in self-protection of inflammation injury. It may be through inhibiting the activition of nuclear transcription factor NF-κB and decreasing the secretion of proinflammatory cytokines. The down expression of A20 in PBMCs from SLE patients cannot efficiently inhibit inflammatory response, and the high level expressions of inflammatory cytokines play a vital role in the pathogenesis of SLE. The study of the expression of A20 in PBMCs from SLE patients will provide a new insight in the pathogenesis of SLE.