The Mechanism Of Th17 Cells In Immunity And Inflammation Response Of The Patients With Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a typical and complex autoimmune disease which violates the skin and multiple organs, characterized by the appearance of a variety of autoantibodies. The disease is recurrent alternating with remission. Young women suffer SLE much more than men. The prevalence of SLE in China is higher than that in the western countries, which may be associated with genetic factors. The research of etiology and pathogenesis has always been of great concerned. At present the view is that the fundamental reason is the body losses the immune tolerance of autoantigen and self-regulation of reactive immune cells, resulting in producing a large number of autoantibodies and influencing humoral immunity, cellular immunity and complement system. Although the mortality of SLE has decreased over the past decade, it is still a huge threat to human health. The pathogenesis and therapeutic approach has always been a hot spot in the research field of medicine.For a long time, people thought that T help cells included only two subsets which were Th1 and Th2 cells.The autoimmune diseases mainly were the results of Th1 cells. The traditional concept was broken with the discovery of a new T cell subset-Th17 cells.In the study of experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA) mouse models, we found a new independent T helper cell subset-Th17 which was closely related to inflammatory and autoimmune diseases which is derived from CD4+T cells and be characterized by secreting IL-17. Recently, the researchers have found that human Th17 cells may involve in rheumatoid arthritis,SLE,multiple sclerosis and other autoimmune diseases, making the role of the Th17 cells in autoimmune diseases has gradually been payed attention to.Primary CD4+T cells (Thp) can be induced to differentiate into Th1,Th2,Th17 and regulatory T cells (Treg). Treg cell was first reported by Sakaguchi et al. in 1995 which is a T cell subset with the effect of immune inhibition. It plays a vital role in the immune balance and the maintaining of immune tolerance. It controls the generation,activation and proliferation of autoreactive T cells mainly through the inhibition of cell contacting dependent mechanism or mechanisms of active regulation of cytokine dependent. At present the effection of Treg in the pathogenesis of SLE has caused much attention of the scholars.Th17 and Treg cells are derived from common precursor cells—CD4+ T cells. Th17 cells are related with autoimmune inflammatory damage; Treg cells play an important role in the induction of immune tolerance and suppressing the autoimmune diseases. The regulation of differentiation depends on the direction of the key transcription factor. RORyt (retinoic acid-related orphan nuclear hormone receptor), as the specific transcription factor which promotes the expression of activation of precursor cells to differentiate into Th17 cells. RORyt coding in RORc sites is a family member of retinoic acid-an orphan nuclear nuclear hormone receptors, RORyt plays a key role on the Th17 cell differentiation and secretion. The key transcription factor of Treg cells is Foxp3. Th17 cells involve in inflammation, autoimmune diseases, but the mechanism is not clear so far. Th17 is the factor of immune injury, Treg is the immune protective factor. The scientists speculate that the incidence of SLE is the result of the two factors fighting with each other.The imbalance between the two subsets may involve in the pathogenesis of SLE.By testing the levels of IL-17 in peripheral blood mononuclear cells (PBMC) supernatant of SLE patients and healthy controls,ritio of Th17 cells and Treg cells in the proportion of CD4+T cells, and the RORγt mRNA expression in the PBMC. Further explore the mechanism of Th17 cells in immunity and inflammatory response of the patients with SLE.Objectives:1. Detect the relationship between the Th17 cells and Treg cells accounting for CD4+T cell in the peripheral blood of the SLE patients and healthy control group.Assess the relationship between Th17/Treg and the disease activity and the relationship between lupus nephritis and non-nephritis. Discuss the correlation of Th17/Treg and SLEDAI.Analysis the possible mechanism of the imbalance of Thl7/Treg which may involve in inflammatory response of SLE.2. Detect the level of IL-17 in the PBMC supernatant of SLE patients and healthy controls stimulated with PMA+Ionomycin. Compare the level of IL-17 of PBMC supernatant in patients with lupus nephritis and non-nephritis.Assess the relationship between IL-17 and the age of patients, disease activity,ESR,complement C3,C4,anti-ds-DNA and other laboratory parameters. Analysis the possible mechanisms of the IL-17 involving in the pathogenesis of SLE.3. By detecting the level of RORyt mRNA in PBMC of patients with SLE, make research on the relationship between RORyt mRNA and SLEDAI, the relationship between RORyt mRNA and Th17 cells, and further study the mechanism of Th17 cells in SLE immune and inflammatory response at the genetic level.Methods:1. There were totally 21 patients with SLE who were outpatients and inpatients of Nanfang Hospital during July to December in 2010. The diagnosis was consistent with the 1982 revised American Rheumatism. All the patients were excluded of recent infection, asthma, cancer, RA, inflammatory bowel disease and allergies.It includes 20 female,1 male, aged 14-52 years. On the basis of SLEDAI, there were 12 cases in active group (SLEDAI≥9 points),and 9 patients in non-active group (SLEDAI<9 points). With 12 healthy persons as healthy control group, of which 11 were female, 1 male, aged 15-72.The mean age was (29.58±13.95).The proportion of their gender and age group were matched with the SLE disease group. The patients with SLE were divided into non-nephritis and nephritis group.7 cases were included in nephritis group and 14 cases were included into non-nephritis group. There's no significant difference between the patients with SLE and normal control group (t=-0.519, P= 0.607).They were comparable.2. In subjects of early morning, we venous 12ml blood when the subjects were fasting and resting,2ml of which was used to detect Th17 and Treg cells,10ml of which was used to separate PBMC.Detected the ratio of CD3+CD8-IL-17+T cells (Th17 cells) and CD4+CD25+FoxP3+T cells (Treg cells) accounting for CD4+T cells in the peripheral blood of SLE patients and healthy controls by flow cytometry.①Stimulated the peripheral blood:We mixed the same amount of anticoagulant and the medium.Then added PMA (25ng/ml),Ionomycin (1μg/ml) and monensin (1.7μg /ml).Then incubated them at 37℃95% CO2 incubator for 5 hours;②Cell surface staining:We added to the Th17 test tube with CD8-FITC, CD3-PE-CY5, Treg test tube by adding CD4-FITC and CD25-PE, control tubes were added to the respective fluorescence of the same type of control. All test tubes were added to 100ul sample, which were incubated at room temperature away from light. Added 2ml hemolysin to each tube, then incubated at room temperature away from light 10min, centrifuged supernatant; Whsh them with PBS.③Fixing cells:Add 0.5ml of 1% paraformaldehyde PBS to each tube, mixed,4℃refrigerator dark 30min;③Rupture of membrane;⑤Intracellular staining:We added IL-17-PE to Th17 test tube and add FoxP3-Alexa Fluor R64 to Treg test tube, the same type of control tubes were added at room temperature away from light incubated 30min, Wash with PBS for 2 times;⑥Re-suspending.Tested them in 24h. Analysised the cells obtained by Cellquest software.3. We got the PBMC of SLE patients and normal by density gradient centrifugation. Counted the cells obtained and took to the six-well plate, then the cells and culture medium were spread out in the six-well plate,making the concentration of 1×106/ml. Added each hole with PMA (50μg/l) and Ionomycin (750ng/l) and cultured them in 37℃95% CO2 incubator for 8 hours. Took the culture supernatant and detected the level of IL-17 secretion by ELISA. Concrete steps were provided by IL-17ELISA kit as below:①Plused samples and standard, and incubated them in the dark 36℃ incubator for 90 minutes;②Washed the plates for 5 times;③Plused biotinylated antibody working solution, and incubated in the dark 36℃incubator for 60 minutes.④Plused enzyme conjugate working solution,⑤ashed the plates for 5 times;⑥Added the TMB, incubated in the dark 36℃incubator for 15 minutes;⑦dded stop solution, measured the OD450 values during 3 min.4. Used the method of RT-PCR to measure RORyt mRNA in PBMC. Collected PBMC and added Trizol. Then added appropriate amount of DEPC treatment product deionized water soluble and electrophoresis.The samples were tested in 260/280nm A value and the ratio of to determine the concentration of total RNA and observed under the UV light,photographed, recorded. The steps of RT-PCR were as below:①Got sterilized PCR tube.Then added the RNA product,Oligo Dt and dNTPs;②eat them at 65℃for 5min and on ice for 5min.③Added to the PCR tubes Rnase inhibitor (40u/μl),10 x AMV Reaction Buffer, DTT, reverse transcriptase (AMV) after mixing 2000rpmL centrifugal 20s;④Insulated at 42℃insulation for 1hr, then 70℃for 15min;⑤ook another sterile centrifuge tube for each group, followed by adding sterile distilled wate,10 x Taq Buffer, dNTPs,RORyt,or P-actin primers I (upstream 10μM),primerⅡ(downstream 10μM), cDNA (RT reaction product) template,Taq DNA Polymerase and MgCl2 on ice.⑥Mixed gently, after PCR amplification centrifugating with the speed of 2000rpm for 20s. Determined the optimal reaction conditions and the number of cycles by pre-test.The PCR amplification conditions were as below:94℃,5min; 94℃denaturation,60s; 55℃annealing,60s; 72℃extension, 1min; 35 cycles.⑦Confirmed the products of PCR reaction by agarose gel electrophoresis, recorded the results with gel imaging system. Use Quantity One (BIO-RAD) image analysis software to analyze optical density values of RORyγ/β-actin optical density ratio to represent RORyt mRNA expression.5. Statistical treatment:All datas were entried SPSS 13.0 software processing package.The comparison of two samples was used of two independcal samplesT-test. If our datas fit for test of homogeneity of variances, we dealed them with One-Way ANOVAand LSD. If our datas were not fit for it, we analysed all datas through Welch and Dunnett's T3. The relationship between two variables was used with bivariate correlation analysis. The relationship between the two variables was used with the curve estimation. If our datas meeted the bivariate normal distribution, we analysed the datas through a Pearson correlation analysis; if our datas didnot meet the bivariate normal distribution, we analysed the datas through Spearman correlation analysis.We used crosstabs x2 test to compare the rate of multiple samples. It was significant when P value less than<0.05.The results were indicated by x±S.Results:1. The proportion of Th17 cells in CD4+T cells in SLE patients was significantly higher than the healthy control group (t=2.178,P=0.037).The proportion of Treg cells in CD4+T cells in SLE patients was lower than the healthy control group without statistically significance (t=-0.503,P=0.619).There was significant difference among active,inactive SLE patients and healthy control group(F=9.457,P=0.001).The Th17/Treg of active SLE patients was significantly higher than in the in-active SLE patients (P=0.002) and healthy control group (P=0.000).The level of Th17/Treg of in-active SLE patients was higher than in healthy control group without statistically significance (P=0.744). The Th17/Treg in patients with SLE was significantly correlated with SLEDAI (P=0.001); There was no significant difference of Th17/Treg between nephritis and non-nephritis (t=1.621,P=0.149).2. There was significant difference of IL-17 among active,inactive SLE patients and healthy control group(F=11.235,P=0.000).The level of IL-17 in the PBMC supernatant of active SLE patients was significantly higher than that of in-active SLE patients (P=0.003) and healthy control group (P=0.000). The level of IL-17 of in-active SLE patients was higher than that of healthy control group without statistically significance (P=0.299). The level of IL-17 of nephritis patients was significantly higher than that of non-nephritis (t=2.346,P=0.030). The level of IL-17 of patients with SLE was significantly correlated with SLEDAI (rp=0.732,P=0.000); And it was also significantly correlated with anti-ds-DNA and C3. But it was not correlated with age,ESR and C4.3. There was significant difference of ROR Y t mRNA among active,inactive SLE patients and healthy control group(F=23.386,P=0.000).The expression of RORyt mRNA in active SLE patients was significantly higher than that of patients with inactive SLE(P=0.002) and healthy control group (P=0.000).The expression of RORyt mRNA of inactive SLE patients was also significantly higher than healthy control group (P=0.047); The expression of RORyt mRNA in PBMC of patients with SLE was significant positively associated with SLEDAI (rp=0.623, P=0.003).Conclusions:1. The quantity of Th17 cells was increased in the patients with SLE. The quantity of Treg cells was reduced. There was imbalance of Th17/Treg,the degree of which was significantly associated with the SLEDAI. It means that in the patients with SLE, under the effect of cytokines as IL-6, the CD4+T cells differentiate to the Thl7 cells much more than others. The appearance of the phenomenon of the high expression of Thl7 cells indicates that the Th17 cells participate in the immune inflammatory response of SLE. And with the continuous inclining of the balance of Th17/Treg and reducing of the inhibitory cytokines as IL-17, more autoreactive cytokines T lymphocyte in SLE would be activated and exacerbate the SLE disease.2. IL-17 is a cytokine which is secreted by Thl7 cells. We found that the level of IL-17 in the PBMC of active SLE patients was significantly increased which is significantly correlated with SLE. This is confirmed the conclusion of that the high expression of Th17 cells derived from SLE patients at the protein level.Thl7 cells may play a role in the immune and inflammatory responses of SLE by secreting IL-17.3. RORyt is the specific transcription factor of Thl7 cells. We found that the level of RORyt mRNA of SLE patients was significantly higher than the healthy controls and was associated with SLEDAI. It confirms our previous conclusion at the genetic level, indicating the phenomenon of Th17 cells poling in patients with SLE.4. If we block the transcription factor RORyt which is the upstream of Thl7 cells differentiation or block the IL-17 which is the downstream of Th17 cells differentiation with anti-IL-17 antibody, we both likely to reduce the immune inflammatory response to treatment of SLE. This will be the direction of our next research.