Study On The Genetic Risk Factors For Rheumatoid Arthritis In Chinese Han Population And Th17 Cells In Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune arthritis characterized by progressive joint destruction. In China the occurrence of RA is somewhat lower (about 0.4 percent), whereas it is substantially higher in other ethnic groups. Although the etiology of RA remains a mystery, a variety of studies suggest that a blend of environmental , immunological and genetic factors is responsible.Similar to what has been postulated for the majority of common diseases, a polygenic mode of inheritance has been proposed for RA. The heritability of RA estimated from twin studies is 53-65%, and the contribution from the MHC is estimated at ~30% of the total genetic effect. Previous genetic studies have identified and validated some risk loci for autoantibody-positive RA including HLA-DR,PTPN22,TNFAIP3 , STAT4 and so on. Less than half of genetic variation can be explained by the known RA risk alleles. However, previous results of genetic variation studies have not always been consistent between Caucasians and Asians. These divergent results suggest genetic heterogeneity of RA across the major racial groups. As such, a number of candidate risk alleles of RA found in Caucasians by genome-wide association scanning of functional SNPs should be replicated in major racial groups.Th17 cell, which has been identified as a new subset of CD4+T cells, can produce IL-17, IL-22 and other effective cytokines. IL-17 is a proinflammatory cytokine that plays an important role in host defense against extracellular bacteria, protozoa, and fungi. It has also become recognized as a key mediator of chronic inflammation in animal models of immune-mediated inflammatory diseases such as RA, multiple sclerosis, inflammatory bowel disease and psoriasis. And now, there is growing evidence that Th17 cell is important in the human disease counterparts.The aim of this study is to test the hypothesis that genes associated with RA in Caucasians are also associated with RA in Han Chinese. Another purpose of this study is to study on the role of Th17 cell in peripheral blood of patients with RA and observe the effect of anti- TNF-αtherapy on the number and function of Th17 cell. Part I: Study on the genetic risk factors for rheumatoid arthritis in Chinese Han populationObjective: To test the hypothesis that genes associated with RA in Caucasians are associated with RA in Han Chinese. Specifically, we will perform case-control association testing of genes previously reported to be associated with RA in Caucasians in a large cohort of RA cases and controls, assessing the role of genes already implicated in the disease in other populations.Methods:We genotyped the initial 821 RA cases and 1000 healthy controls for 142 SNPs covering the genes previously implicated in the disease including the SNPs in the genes PADI4, PTPN22, STAT4,CDK6, IL2RA, IL2RB, TRAF1, IL2, IL21,MMEL1, CTLA-4 and so on. For SNPs genotyping, Sequenom iPLEX and MALDI-TOF MS were used. Association tests for SNPs and haplotypes in patients and control subjects and the regional linkage disequilibrium structure for analyzed SNPs were determined using Haploview software, version 4.1.Results: RA association tests: The STAT4 SNPs(rs11685878和rs7574070)were noted to be disease-associated, with a P value of 0.0459,0.0045 respectively. The CDK6 SNPs(rs42041),IL21 SNPs(rs2221903), IL2RB SNPs(rs228979) were also associated with disease. After analyzing the linkage disequilibrium structure of the SNPs, we found 4 blocks. CDK6 haplotype constructed by rs42041 and rs42042 is associated with RA. STAT4 haplotype constructed by rs11685878/rs7574070 is also associated with RA. Further more, the gene of MMEL1 is showed strong association with RA. In contrast, a negative result was obtained on analyzing SNPs in the gene of PTPN22.Conclusions: IL21 is possibly a susceptible gene in Chinese Han RA patients. CDK6, STAT4 and MMEL1 are the common risk genes in both Caucasians and Asians. PTPN22 gene shown to be significantly associated with disease in Caucasians appears not play a role in Chinese patients with RA..Part II: Study on the role of Th17 cell in peripheral blood of patients with rheumatoid arthritis1. Expression of cytokines related with Th17 cell in peripheral blood of patients with rheumatoid arthritisObjective: To find out the effectiveness of Th17 cell in the RA pathogenic mechanism.Methods: 74 RA patients are collected and dived into two groups based on their disease activity. Normal controls are 82 samples. The levels of cytokines including IL-17,IL-23,IL-1β,IL-6, and TGFβ-1 in serum were measured by ELISA. The expression levels of these cytokines mRNA were evaluated by real-time quantitative PCR.Results: Quantitative realtime PCR and ELISA analysis demonstrated elevated mRNA expression levels and protein concentrations of four cytokines including IL-17(41.33±1.40 vs 17.66±1.40pg/ml,P<0.001), IL-1β(23.65±2.89 vs 6.82±2.76 pg/ml,P<0.05),IL-6(32.43±4.92 vs 3.37±0.70 pg/ml,P<0.001)and TGFβ-1(1117.04±232.25 vs 134.92±71.38 pg/ml,P<0.05). In addition, ELISA analysis showed that the concentration of IL-17(41.33±1.40 vs 27.54±2.81 pg/ml),IL-1β(23.65±2.89 vs 20.68±5.61 pg/ml)and TGFβ-1(1117.04±232.25 vs 403.59±93.35 pg/ml)in the serum of active patients were much higher than that in stable patients(P<0.05).Conclusions: As the main effect cytokine induced by Th17 cell, the level of IL-17 is remarkably elevated in active RA patients and correlated with disease activity. As IL-1β,IL-6 and TGFβ-1 can promote na?ve CD4+T cells to differentiate into Th17 cell, the levels of these cytokines were also elevated in RA patients. The function of IL-23 is to maintain the survival and expansion of Th17 cell, which did not depend on the dose of IL-23.2. The proportion of Th17 cell in PBMC in RA patients and effects of different cytokine environments on Th17 cell differentiationObjective: To investigate the proportion of CD4+IL-17+T cells in PBMC in RA patients and effects of different cytokine environments on Th17 cell differentiation.Methods: 10 active RA patients, 8 stable RA patients and 10 normal controls were recruited for study. PBMC was isolated by Ficoll desity gradient centrifugation methods first, then the PBMC was stimulated 5 hours by phorbol ester and calcium ionophore. The cells were stained by CD4-FITC and anti IL-17-PE, and CD4+IL-17+T cell was detected by Flow Cytometry. Part of PBMC has been cultured in different cytokine environment: such as IL-1β+IL-6,IL-1β+IL23,IL-6+IL23,IL-1β+IL23+IL-6 for five days. Then the cells were stained by CD4-FITC and anti IL-17-PE and CD4+IL-17+T cell was detected by Flow Cytometry.Results: The proportion of CD4+IL-17+ lymphocyte in the peripheral blood of active RA patients is higher than in stable RA patients and normal controls(0.52±0.04% vs 0.28±0.03% vs 0.06±0.02%,P<0.001).IL-1βand IL-6 are stronger stimulator for Th17 differentiation.Conclusions: The proportion of CD4+IL-17+ lymphocyte in the peripheral blood of active RA patients is much higher. IL-1βand IL-6 are stronger stimulator for Th17 differentiation..3. The effect of anti- TNF-αtherapy on the number and function of Th17 cell Objective: To analyze the effect of of anti- TNF-α(adalimumab)therapy on the number and function of Th17 cell.Methods: seven patients with RA were recruited and administrated adalimumab 40mg once two weeks for more than six months. The proportion of Th17 cell and mRNA expression levels of cytokines including IL-17, IL-23, IL-1β, IL-6 and TGFβ-1 at the baseline and at 6 months were evaluated.Results: A significant decrease in mRNA expression levels of IL-23(0.0873±0.01589 vs 0.0225±0.00445 ) ,IL-1β( 22.9281±3.95391 vs 0.7859±0.44970 ) and IL-6 (395.9832±110.8752 vs 102.2152±22.5869)was observed at 6 months after initial treatment of adalimumab(P<0.05). The expression levels of IL17 mRNA and TGFβ-1 mRNA did not show a significant change at 6 months after the initial injection compared with pretreatment levels, respectively(0.0061±0.00262 vs0.0038±0.00212,1.0547±0.33605 vs0.6669±0.10660,P>0.05).The flow cytometry. analysis also showed that the number of CD4+IL-17+ lymphocyte in the peripheral blood of RA patients did not decrease significantly after treatment(0.5286±0.00516% vs 0.4857±0.04592%,P>0.05). Conclusions: This study demonstrated that the reduction of IL-23 production in RA patients was a newly determined function of adalimumab. IL-17 production in RA patients in vivo may not depend on the direct stimulation by TNF-α. Combination use of TNF-αinhibitor and anti-IL17 may control the disease effectively in future.