| Objective:Systemic lupus erythematosus(SLE)is a complex autoimmune disease with a strong genetic predisposition.There are lots of differences in prevalence and disease signature of SLE among variety races.The prevalence risk of SLE is 35-50% in identical twins and first-degree relatives,which is 20 times of the general population.In Dominican Republic(DR)population,the incidence and severity of the disease are significantly higher than those in the other groups.However,the genetic risk factors and disease parameters of SLE are unknown in the DR population.SLE is characterized by over-production of autoantibodies and the tissue-deposition of immune complexes(ICs).Lupus nephritis(LN)is one of the most common complication found in about 60% of SLE patients.Autoantibodies are believed to play a critical role in the etiology of SLE.Although a variety of autoantibodies have been identified in SLE patients,how autoantibodies lead to kidney manifestation of SLE remain elusive.The present study is to identify susceptible alleles for SLE in the DR population by single nucleotide polymorphism(SNP)analysis.In addition,we identified novel MC(mesangial cell)-reactive autoantibodies in SLE patients,which might contribute to LN and SLE.A large collection of recombinant antibodies derived from B cell of SLE patients was screened for their reactivity towards MCs.Using flow cytometry analysis,antibodies that reacted with MC were identified.Cognate antigens to which the respective antibodies bound were identified using immunoprecipitations and mass spectrometry analyses.ELISA detection was performed to evaluate if the identified renal-reactive autoantibodies were associated with lupus nephritis and SLE disease activity.Our study may provide scientific basis in the pathogenesis of LN.Methods:Materials1.Human serum samples: Human serum samples from SLE patients,osteoarthritis(OA)patients,(rheumatoid arthritis)RA patients and healthy donors were obtained after informed consent in accordance with Institutional Review Board-approved protocols at the University of Nebraska Medical Center(UNMC,Omaha,NE),the Pontificia Universidad Catolica Madre y Maestra(PUCMM,Santiago De Los Caballeros,Dominican Republic).All the SLE and LN patients met the international classification standard of lupus revised by American College of Rheumatology.2.Cell lines: mesangial cells(American Type Culture Collection,ATCC,Manassas,VA,US).3.Antibodies: anti-14-3-3 eta goat polyclonal antibody was obtained from Santa Cruz Biotechnology,Dallas,TX.The secondary antibodies,FITC-conjugated AffiniPure F(ab')2 Fragment Donkey anti-Human IgG(H+L)antibodies,Peroxidase-conjugated AffiniPure F(ab')2 Fragment Donkey anti-Human IgG(H+L)antibodies,and Peroxidase-conjugated AffiniPure F(ab')2 Fragment Donkey anti-Mouse IgG(H+L)antibodies were bought from Jackson ImmunoResearch(West Grove,PA,US).Methods:1.Genotype detection: Sequenom's MassARRAY IPLEX genotyping was used for SNPs' detection2.Recombinant antibody generation: Recombinant antibodies were previously obtained in our lab using a single-cell RT PCR technique3.Cell culture4.Flow cytometry screening for antibodies binding to mesangial cells5.Competition assay6.Apoptosis assay7.Immunoprecipitation detection8.Mass spectrometry9.Western blot10.ELISA(enzyme-linked immunosorbent assay)11.Statistical analysis Results:1.Genotype analysis result showed significant difference in 19 SNPs distribution from 15 of 38 SLE-related predisposing genes(ITGAM?BLK?TNFSF4?PTPN22?TNIP1 ? STAT4 ? CARD11 ? TNXB ? TNFAIP 3 and HLA-DQA1 ? HLA-DRB2 ?HLA-DRA? HLA-DQA2 ? HLA-DRB1? HLA-DQB1)between SLE and the control groups in DR population.Patients with homozygous HLA-DRA haplotype A-T displayed higher risk to develop LN and have a gene dose effect of this haplotype in LN.The minor “AA” allele in ITGAM SNP rs1143679 was significantly associated with LN.HLA-DQA2 rs2301271 and “T” allele in TNFSF4 rs2205960 are significantly associated with NPSLE and show gene-dose effects in conferring the risk to develop NPSLE.2.Three hundred and thirty-four recombinant antibodies were screened by flow cytometry,sixteen out of which were demonstrated being high reactivity to MC.3.Immunoprecipitation assay was used to pull down cognate antigens from MC membrane protein extracts,which were then detected by mass spectrometry.The results indicated that three proteins including basement membrane-specific heparan sulfate proteoglycan core protein(HSPG),aminoacyl tRNA synthetase complex multifunctional protein 1(AIMP1),and 14-3-3 eta were identified to bind to the sixteen recombinant antibodies.4.ELISA result showed that 5 out of 16 mesangial cell binding antibodies,including S3P1D11,S3P1F3,S3P1G9,S12P2D4 and S12P2H9,reacted with 14-3-3eta in a does dependent manner.5.Western blot result showed that 5 out of 16 mesangial cell binding antibodies,including S3P1D11,S3P1F3,S3P1G9,S12P2D4 and S12P2H9,reacted with 14-3-3eta.6.Antigen competition assay result showed that reactivities of 5 recombinant antibodies(S3P1D11,S3P1F3,S3P1G9,S12P2D4 and S12P2H9)to mesangial cells completely or partially shift back when the antibodies were pre-incubated with excessive amount of 14-3-3 eta protein,suggesting that 14-3-3 eta mediated orpartially mediated the binding of these antibodies to mesangial cells.7.ELISA method was used to analyze the serum titers of autoantibodies recognizing 14-3-3 eta from SLE patients and healthy donors.As results,the serum levels of autoantibodies recognizing 14-3-3 eta was significantly elevated in SLE patients compared to the control(P<0.0001 and P=0.0003,respectively).The levels of anti-14-3-3 eta antibodies in both OA and RA patients are significantly higher than those in the healthy controls(P=0.0012 and P=0.0013,respectively),but significantly lower than those in SLE patients(P =0.0027 and P =0.0057,respectively).The level of anti-14-3-3 eta antibodies was also significantly higher in SLE patients with active nephritis compared to those with inactive nephritis(P <0.0001).8.When mesangial cells were treated with commercial or SLE derived anti-14-3-3 antibodies,they exhibited significantly lower rates of apoptosis,indicating that anti-14-3-3 antibody might contribute to LN pathogenesis via inducing apoptosis.Conclusions:1.In the DR population,19 SNPs in the ITGAM?BLK?TNFSF4?PTPN22?TNIP1?STAT4?CARD11?TNXB?TNFAIP3?HLA-DQA1?HLA-DRB2?HLA-DRA?HLA-DQA2 ?HLA-DRB1 ?HLA-DQB1 genes are discovered as predisposing SNPs related to SLE.HLA-DRA haplotype and minor “AA” allele in ITGAM displayed higher risk to develop LN.HLA-DQA2 and TNFSF4 are associated with NPSLE2.14-3-3 eta,HSPG and AIMP1 are potential autoantigens in SLE.3.Anti-14-3-3 eta antibody can inhibit apoptosis of mesangial cell.4.Anti-14-3-3 eta antibody could be an novel biomarker for evaluation of LN and SLE activity. |
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