Molecular Epidemiology Study On The Role Of Tristetraprolin In The Pathogenesis Of Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus(SLE)is a chronic autoimmune disease,characterized by a multitude of autoantibody production,complement activation and immune complexes deposition,resulting in multiple systems and organs damage.To date,the etiology and pathogenesis of SLE has not been fully elucidated.The current researchers generally believe that the pathogenic factors of SLE include genetic susceptibility,environmental factors,endocrine status and etc.These factors interact with each other,then leading to the disorder of immune function in patients with SLE.Messenger RNA decay is a critical mechanism to control the expression of many inflammation-associated genes.A conserved mRNA sequence element found in a majority of these inflammation-associated genes is the adenylate and uridylate-rich element(ARE)within the 3' untranslated region(3'UTR)of the mRNA.RNA-binding proteins affect the mRNA stability through binding to the AU-rich element(ARE).One of the best-characterized RNA-binding proteins is tristetraprolin(TTP),also named ZFP36,TIS11,NUP475,GOS24.TTP has been shown to regulate the ARE acting element on 3' untranslated region(3'-UTR)of mRNA,then affects the mRNA stability and the protein expression,such as IL-6?IL-10?TNF-? and so on.It has been reported that attenuation of NF-kappaB activity by TTP expression was in part due to TTP's ability to interfere with the nuclear translocation of the p65 subunit of NF-kappaB.In addition,TTP could also physically interact with histone deacetylases(HDACs),and function as a co-repressor for NF-kappaB-dependent transcription.Together,these findings have identified novel cellular interactions of TTP that not only synergize with its mRNA-decay function to contribute to efficient regulation of pro-inflammatory gene expression,but also implicate a potential role in regulating inflammatory signal transduction.T helper 17(Th17)cells has been recently discoveried as a novel subset of Th cells,which selectively secrete several pro-inflammatory cytokines,mainly IL-17,as well as IL-21?IL-22 and etc.Our previous studies have suggested that Th17 cells and related cytokines participate in the pathogenesis of SLE.LPS-stimulated bone marrow-derived dendritic cell(BMDC)from TTP-/-mice produced increased IL-17?IL-23p19 and IL-23.In skin lesions and Lymph nodes,high IL-17?IL-22?IL-23p19 were detected,and increased frequencies of CD4+IL-17+T cells and CD4+IL-22+T cells were also detected.Additionally,TTP-/-mice spontaneously develop an inflammatory syndrome characterized by dermatitis and erosive arthritis.These results showed that TTP has a strong impact on IL-23 production and IL-23p19 mRNA stability and the clinical and immunological parameters associated with TTP deficiency were completely dependent on the IL-23/IL-17A axis.Over expression of TTP in human T cell lines decreased the expression of IL-17.Conversely,TTP inhibition by siRNA increased IL-17 production.In addition,TTP bound directly to the AREs of IL-17 and IL-22,then enhanced decay of IL-17 and IL-22 transcripts.The phenotype of the mice is a complex syndrome consisting of patchy alopecia,erosive arthritis,dermatitis,conjunctivitis autoimmunity,glomerular mesangial thickening,focal segmental thickening of peripheral capillary loops,with high titers of antinuclear and anti-DNA antibodies in the serum.A study on the mesangial cells of NZB/W mice given that modulating the expression of TTP may have potential implications on the production of IL-6 and SLE mesangial cell hyperplasia.Previous studies have found that TTP play an important role in the development of cancer?infection and metabolic syndrome.However,there are only a few studies on the role of TTP and autoimmune diseases.Evidence has showed that TTP is involved in the onset and severity of RA.To date,the relationship between TTP and SLE has not been reported.Thus,Firstly,we carried out a case-control association study to examine the single nucleotide polymorphisms in TTP by using TaqMan genotyping assay to investigate whether TTP gene confer susceptibility to SLE in the Chinese population.Secondly,we use real time quantitative polymerase chain reaction(RT-PCR)to analyze TTP mRNA level from peripheral blood mononuclear cells(PBMC)between patients with SLE and normal controls,active and inactive group and lupus nephritis(LN)group and non-LN group,explore its association with disease activity,clinical symptoms and laboratory parameters.Next,we used ELISA to detect TNF-a,IL-6,IL-10,IL-17,IL-21,IL-22 and IL-23 levels in SLE plasma and analyzed the relationship between the mRNA expression level of TTP and plasma TNF-?,IL-6,IL-10,IL-17,IL-21,IL-22 and IL-23 levels in SLE patients.Thirdly,by using the method of Western blot,we analyzed TTP protein level from CD4+T lymphocytes in SLE patients and normal controls,lupus nephritis(LN)group and non-LN group,active and inactive group,and explore its association with disease activity,clinical symptoms and laboratory parameters.The study has three parts.Part 1 The association between the polymorphisms of TTP gene and SLE susceptibility in a Chinese populationObjective To examine the association of two TTP single nucleotide polymorphisms(rs251864 and rs3746083)in a Chinese Han population by comparing the genotype and allele frequencies between SLE patients and normal controls,LN patients and non-LN patients,and explore the relationship between rs251864 and rs3746083 polymorphisms and clinical manifestations.Methods A total of 1167 patients with SLE were recruited from the First Affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital.All patients fulfilled the 1997 revised criteria of the American College of Rheumatology for the classification of SLE.1194 normal controls were recruited from health examination center of the First Affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital and students of Anhui Medical University.After getting their informed consent,blood samples and clinical information were collected.After extracting DNA,TTP gene rs251864 and rs3746083 polymorphisms were specified by TaqMan genotyping assay.The data were analyzed by SPSS 19.0 software.P<0.05 was considered statistically significant.Results(1)HWE testThere were no deviations from HWE observed in both SLE patients and normal controls in two polymorphisms(P>0.05).(2)Association analysis of TTP gene rs251864 and SLEFor SNP rs251864,we found no significant difference the allelic distributions between SLE patients and health controls(G versus A:?2=1.091,P=0.296).We also failed to showed an association between the genotypes and SLE(GG vs.AA:?2=1.077,P=0.299,OR=1.188,95%CI:0.858-1.646;AG vs.AA:?2=0.007,P=0.931,OR=0.992,95%CI:0.833-1.182).There was no significant association between rs251864 polymorphism and SLE in either dominant or recessive model(GG+GA vs.AA:?2=0.124,P=0.725,OR=1.030,95%CI:0.873-1.216;GG vs.GA+AA:?2=1.175,P=0.278,OR=1.192,95%CI:0.868-1.638).In addition,no significant difference was detected in the allelic and genotype distribution between LN patients and non-LN patients(P>0.05).However,we found a significant difference in the distribution of allelic frequencies between patients with oral ulcers and patients without this feature(?2=4.367,P=0.037).(3)Association analysis of TTP gene rs3746083 and SLEWith regard to SNP rs3746083,it is worth mentioning that,the allele T at rs3746083 revealed a trend but not significant difference towards less frequent in SLE than controls(T vs.C:?2=3.310,P=0.069,OR=0.749,95%CI:0.548-1.024).We also detected no significant difference in the genotype distributions between SLE patients and controls(TT vs.CC:?2=0.013,P =0.908,OR=1.093,95%CI:0.241-4.963;CT vs.CC:?2=2.761,P=0.097,OR=1.335,95%CI:0.949-1.878),as well as in the dominant model and recessive model(TT+TC vs.CC:?2=2.940,P=0.086,OR=1.337,95%CI:0.959-1.865;TT vs.TC+CC:?2=0.008,P=0.929,OR=1.071,95%CI:0.234-4.899).No significant difference was detected in the allelic and genotype distributions between LN patients and non-LN patients(P>0.05).Besides,we failed to found any positive results of rs3746083 with these main clinical symptoms of SLE(P>0.05).Conclusions Our findings suggest that the TTP gene polymorphisms(rs251864 and rs3746083)might not contribute to SLE susceptibility in the Chinese population.Rs251864 polymorphism may be associated with SLE patients who combined with oral ulcers.Part 2 Expression of TTP mRNA in PBMC from patients with systemic lupus erythematosus and their correlations with TNF-?,IL-6,IL-10,IL-17,IL-21,IL-22 and IL-23 levels in SLE plasmaObjective The objective of this study was to compare the TTP mRNA level in peripheral mononuclear blood cell(PBMC)between systemic lupus erythematosus(SLE)patients and controls,LN patients and non-LN patients,active and inactive patients,and explore the relationship between mRNA level and disease activity,clinical and laboratory data.Next,we analyzed the relationship between the expression of TTP mRNA level and plasma TNF-a,IL-6,IL-10,IL-17,IL-21,L-22 and IL-23 levels in SLE patients.Methods A total of 42 SLE patients were randomly selected from samples of Part 1.All patients with SLE met 1997 revised ACR criteria for the classification of SLE.A total of 40 healthy controls were enrolled from healthy volunteers of Hefei Blood Center and students of Anhui Medical University.After getting their informed consent,the data of demographic,clinical and laboratory features were obtained.Disease activity was evaluated by the scores of SLE disease activity index(SLEDAI).5 ml blood samples were collected.PBMC were isolated from Peripheral Blood by Ficoll Hypaque method.RNA was extracted with trizol from PBMC.The level of TTP mRNA in PBMC from 42 patients with SLE and 40 healthy controls was detected by relative quantitation PCR.The plasma levels of TNF-a,IL-6,IL-10,IL-17,IL-21,IL-22 and IL-23 from these 42 patients were detected by ELISA.The data were analyzed by SPSS 19.0 software.P<0.05 was considered statistically significant.Results(1)TTP mRNA level in PBMCThere was no significant difference regarding TTP mRNA level between SLE patients and healthy controls(Z=-1.299,P=0.194).There was a trend but no significant difference between active SLE patients and inactive ones(Z=-1.693,P=0.090).However,lower TTP mRNA level was found between SLE patients with nephritis and those without nephritis(Z=-2.695,P=0.007).(2)Association of TTP mRNA level and clinical and laboratory featuresThere is no significant difference between TTP mRNA level and these clinical features.The level of TTP mRNA were significantly associated with proteinuria(Z=-2.323,P=0.020)and elevation of anti-dsDNA antibodies(Z=-2.299,P=0.022).(3)Correlation analysis of TTP mRNA level in PBMC from SLE patients and SLEDAI Spearman rank correlation analysis results suggested a trend but no significant negative correlation between TTP mRNA level in PBMC from SLE patients and SLEDAI(r =-0.293,P?0.060).(4)Comparisons of plasma TNF-a,IL-6,IL-10,IL-17,IL-21,IL-22 and IL-23 levels between different groupsSignificant differences of plasma IL-17 level was found between SLE patients with nephritis and SLE patients without nephritis(t=2.132,P=0.039).No significant difference was found between SLE patients with more active SLE and less active SLE(P>0.05).(5)Association of plasma TNF-a,IL-6,IL-10,IL-17,IL-21,IL-22,IL-23 levels and clinical and laboratory featuresPlasma TNF-?,IL-17 levels were significantly associated with pleurisy(t=2.346,P=0.024;t=2.102,P=0.042).Plasma TNF-?,IL-17,IL-21 and IL-22 levels were significantly associated with leukopenia(t=2.314,P=0.026;t=2.232,P=0.031;t=3.140,P=0.003;t =2.229,P=0.031).No significant association was found between plasma IL-6?IL-10 and IL-23 levels and clinical data(P>0.05).(6)Correlations between plasma TNF-?,IL-6,IL-10,IL-17,IL-21,IL-22,IL-23 levels and SLEDAINo significant association was found between plasma TNF-?,IL-6,IL-10,IL-17,IL-21,IL-22 and IL-23 levels and SLEDAI(P>0.05).(7)Correlations between TTP mRNA level and plasma TNF-?,IL-6,IL-10,IL-17,IL-21,IL-22,IL-23 levelsTTP mRNA level was significantly associated with plasma TNF-a level(rs=-0.316,P=0.042)and IL-17 level(rs=-0.338,P=0.029)in SLE patients.No significant association was found between TTP mRNA level and plasma IL-6,IL-10,IL-21,IL-22 and IL-23 levels(P>0.05).(8)Association of TTP gene polymorphisms and mRNA levelWe analyzed TTP gene(SNP rs251864 and SNP rs3746083)polymorphisms with m RNA expression among 42 SLE patients.There was no significant difference between rs251864 and rs3746083 and mRNA level(P>0.05).Conclusions We found no significant difference regarding TTP mRNA level between SLE patients and healthy controls,active SLE patients and inactive ones.There was also no significant correlation between TTP mRNA level and SLEDAI in SLE patients.We found lower TTP mRNA level in LN patients when compared to non-LN patients.We also found significant association of TTP mRNA level with proteinuria and elevation of anti-dsDNA antibodies.TTP mRNA level was significantly associated with plasma TNF-a and IL-17 levels in SLE patients.No significant positive correlation was found between TTP mRNA level in PBMCs from SLE patients and SLEDAI.No association was found between the TTP gene(SNP rs251864 and SNP rs3746083)and mRNA level.Part 3 Expression of TTP in peripheral blood CD4+T cells from patients with systemic lupus erythematosusObjective To compared the protein level of TTP in peripheral blood CD4+T Cells between systemic lupus erythematosus(SLE)patients and controls,LN patients and non-LN patients,active and inactive patients,and explore the relationship between TTP protein level and clinical,laboratory data and disease activity.Methods A total of 25 SLE patients were collected from the First Affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital.All patients with SLE met 1997 revised ACR criteria for the classification of SLE.A total of 23 healthy controls were enrolled from the staff and students of Anhui Medical University.After getting their informed consent,20 ml blood samples were collected,and the data of demographic,clinical and laboratory features were also obtained.Disease activity was evaluated by the scores of SLEDAI.PBMC were isolated from Peripheral Blood by Ficoll Hypaque method and CD4+T lymphocytes were isolated from the PBMC by Magnetic Labelled Bead cell Separation(MACS)method and the purity of CD4+T were tested by flow cytometry,before and after being sorted,respectively.Then the level TTP of CD4+T lymphocytes were detected by Western blotting technique.The data were analyzed by SPSS 19.0 software.P<0.05 was considered statistically significant.Results Compared with the non-sorted T lymphocytes,the purity of CD4+T lymphocytes was up to 95%or more after being sorted by MACS.(1)TTP protein level in CD4+T lymphocytesThere was no significant difference of TTP protein level between SLE patients and healthy controls(Z=-1.249,P=0.212).We detected a trend but not significant difference regarding TTP protein level between active SLE patients and inactive ones(Z=-1.654,P=0.098).We also detected a trend but not significant difference between LN patients and non-LN patients(Z=-1.689,P=0.091).(2)Association of TTP protein level in CD4+T lymphocytes and clinical,laboratory features of SLE patientsSLE patients with fever showed decreased level of TTP protein(Z=-2.272,P=0.023).There is no significant difference between TTP protein level and other clinical and laboratory features.(3)Correlation analysis of TTP protein level in CD41+T lymphocytes from SLE patients and SLEDAISpearman rank correlation analysis results suggested that no significant positive correlation was found between TTP protein level in CD4+T lymphocytes from SLE patients and SLED AI(rs=-0.132,P=0.528).Conclusions There was no significant difference of TTP protein level between SLE patients and healthy controls,active SLE patients and inactive ones,as well as LN and non-LN patients.Significant association of TTP protein level with fever in SLE patients was detected.No significant positive correlation was found between TTP protein level in CD4+T lymphocytes from SLE patients and SLEDAI.