Association Study Of Serum And Polymorphisms In TIM-1 And TIM-3 With Systemic Lupus Erythematosus

Background Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune diseases,which affects multiple organ systems such as kidney. It involves a diverse array of autoantibody production, complement activation and immune complex deposition, causing tissue and organ damage. At present, the pathogenesis of SLE remains unclear. Na?ve T-helper cells chiefly differentiate into Th1 or Th2 cells and defects in the appropriate regulation of Th1 and Th2 cell function have been implicated in the pathophysiology of SLE, in which a predominance of Th1 can induce autoimmunity, whereas a Th2 phenotype can be associated with lupus nephritis (LN).Recent studies have indicated that inappropriate regulation of Th17 cells also participates in the pathogenesis of SLE.The T-cell immunoglobulin mucin (TIM) proteins represent a group of molecules that in concert with T-cell receptor and costimulatory signal to regulate expansion and effector functions of Th1 and Th2 cells.TIM gene family located in chromosome 5q33.2 consists of three different genes in human beings, TIM-1, TIM-3 and TIM-4, without any other intervening genes (The TIM-2 gene is found only in mice), which encode cell surface glycoproteins with common structural motifs, including a signal peptide, Ig domain, mucin domain, transmembrance region and intercellular tail with phosphorylation sites. TIM protein was initially identified through a screen for Th1-specific and Th2-specific markers. TIM-1 is preferentially expressed in Th2 cells but not Th1 cells and is involved in the development and regulation of Th2-biased immune responses, in contrast with TIM-3, which is expressed on terminally differentiated Th1 cells but not Th2 cells and functions to inhibit aggressive Th1-mediated immune responses. TIM-3 may also be expressed in Th17 cells but at lower levels than Th1 cells.TIM gene family may influence the susceptibility to Th1-and Th2-mediated conditions and several studies have suggested that polymorphisms in these genes might be associated with allergic and autoimmune disease in some populations, including rheumatoid arthritis (RA), a Th1/Th17 disease as SLE. Although TIM gene family products have been shown to play a role in immune responses, little is known about TIM regulation and the development of SLE. Thus, on the basis of SLE genetic resources, our study adopted case-control design to assess that TIM correlated with the development of SLE through serum expression and genetic susceptibility, and analyze the relationship among TIM serum expression, single-nucleotide polymorphisms (SNPs) of the TIM gene family, and SLE disease activity and organ damages.PartⅠStudy on the Corrlation between Serum TIM-1 and TIM-3 and Systemic Lupus ErythematosusObjective To compare the expression of TIM-1 and TIM-3 in serum of SLE patients and healthy controls, SLE with and without nephritis, active and inactive SLE. Combined with clinical and laboratory data, the relationship among their serum levels and SLE disease activity and organ damages were further analyzed.Methods A total of 62 patients with SLE (57 female and 5 male) were recruited from Anhui Provincial Hospital and the First Affiliated Hospital of Anhui Medical University. The diagnosis of SLE was established by the presence of four of more American College of Rheumatology (ACR) diagnostic criteria. We excluded patients who had a history of atopy-related disease, such as asthma or allergic rhinitis, because both TIM-1 and TIM-3 have been repeatedly suggested to be linked to asthma and other allergic diseases. Demographic data and clinical data were collected from hospital records or by questionnaire and reviewed by experienced physicians. Individual disease activity was quantified using the systemic lupus erythematosus disease activity index (SLEDAI) score. A total of 21 healthy blood donors were included as controls, all of whom were without any rheumatologic conditions or any allergic status. All subjects gave their written consent to participate before study. Sera were obtained from 3ml of whole blood and stored at -80℃until tested. Serum TIM-1 and TIM-3 concentrations were determined by Specific enzyme linked immunosorbent assay (ELISA) kits according to the manufacturer's recommendation (R&D Systems, Inc. Minneapolis, MN), the results were expressed as pgml. All statistical analyses were done by SPSS 10.0. For comparing the median between different groups, the nonparametric Mann-Whitney 2 sample test was used. For the correlation analysis between serun TIM-1, TIM-3 and SLEDAI, Spearman's rank correlation coefficient was used. Probability level <0.05 in two-tailed test was used as a criterion of significance.Results There was no significant difference regarding serum TIM-1 level between patients with SLE and normal controls (293.66 pg/mL (239.11-408.67) pg/mL vs 289.50 pg/mL (254. 39-443.78) pg/mL, P=0.410),SLE patients with nephritis and those without nephritis (334.87 pg/mL (264.18-417.63) pg/mL vs 267.05 pg/mL (234.09-376.43) pg/mL, P=0.134), active SLE patients and inactive ones (272.54 pg/mL (235.64-383.59) pg/mL vs 316.72 pg/mL (253.20-443.06) pg/mL, P=0.305). As for clinical and laboratory features, SLE patients with lower complement showed decreased expression level of TIM-1 (P=0.023).Compared with controls, TIM-3 level was significantly increased in the serum of SLE patients (186.41 pg/mL (144.72-305.06) pg/mL vs 138.82 pg/mL (113.90-233.36) pg/mL, P=0.032). Moreover, significant difference was found between SLE patients with nephritis and those without nephritis (209.72 pg/mL (158.94-562.36) pg/mL vs 175.87 pg/mL (130.51-286.38) pg/mL, P=0.036). However, there was no significant difference regarding serum TIM-3 level between active SLE patients and inactive patients (184.17 pg/mL(146.80-289.89)pg/mL vs 191.83 pg/mL(134.98-319.59)pg/mL, P=0.983). As for clinical and laboratory features, SLE patients with butterfly erythema showed increased expression level of TIM-3 (P=0.016).We did not find association of serum TIM-1 or TIM-3 with SLEDAI in SLE patients (P=0.916, 0.188 respectively).However, expression of TIM-3 in sera showed significant positive correlation with SLEDAI(rs=0.375,P=0.022).Conclusion In summary, our study showed a increased in serum TIM-3 levels in patients with SLE and SLE patients with nephritis, and a positive correlation between serum TIM-3 and SLEDAI in active SLE patients, which suggests that this molecule may be implicated in the pathomechanisms of SLE. Therefore, an in-depth, systemic study of the role of TIM-3 in SLE might provide more effective diagnostic and prognosis and target clinical management tools for this disease.PartⅡAssociation Study of TIM-1 and TIM-3 Polymorphisms with Systemic Lupus ErythematosusObjective To determine both the TIM-1 gene (SNP rs12522248 and SNP rs1501909) and the TIM-3 gene (SNP rs9313439 and SNP rs10515746), estimate the Hardy-Weinberg equilibrium (HWE) for those four SNPs in both SLE patients and healthy controls, and investigate whether or not genotype and allele frequencies of the TIM gene family confer susceptibility to SLE in a Chinese population. Combined with clinical and laboratory data, the relationship among the SNPs selected and SLE disease activity and kidney damage were further analyzed.Methods A total of 202 patients with SLE (172 female and 30 male) were recruited from Anhui Provincial Hospital and the First Affiliated Hospital of Anhui Medical University. The diagnosis of SLE was established by the presence of four of more American College of Rheumatology (ACR) diagnostic criteria. We excluded patients who had a history of atopy-related disease, such as asthma or allergic rhinitis, because both TIM-1 and TIM-3 have been repeatedly suggested to be linked to asthma and other allergic diseases. Demographic data and clinical data were collected from hospital records or by questionnaire and reviewed by experienced physicians. Individual disease activity was quantified using the systemic lupus erythematosus disease activity index (SLEDAI) score. A total of 217 sex-matched healthy blood donors were included as controls, all of whom were without any rheumatologic conditions or any allergic status. All subjects gave their written consent to participate before study. EDTA anti-coagulated venous blood samples were collected from all participants. Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using Flexi Gene DNA kits (Qiagen). Genotyping was performed by TaqMan SNP assay using Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA, USA). All statistical analyses were done by SPSS 10.0. The chi-square test or Fisher's exact test was used to HWE test in both SLE and healthy controls as well as to compare the genotype and allele frequencies between the two groups. Adjusted odds ratios (ORs) and 95% confidence interval (CI) for genotype frequency was performed by logistic regression analyses. Haplotype analysis was assessed using SHEsis software. Probability level <0.05 in two-tailed test was used as a criterion of significance. Results(1) HWE testNo deviations from HWE were observed in either the patients with SLE or the controls in each polymorphism.(For SNP rs1501909,χ~2=0.046,P=0.977;χ~2=0.003,P=0.999, respectively; For SNP rs12522248,χ~2=0.102,P=0.950;χ~2=1.845,P=0.398, respectively; For SNP rs9313439, Fisher P=0.258;Fisher P=0.258, respectively; For SNP rs10515746,χ~2=0,P=1.000;χ~2=0,P=1.000, respectively)(2) Association analysis of TIM-1 gene and SLETo SNP rs1501909, genotype frequencies for CC,AC and AA were 16.7%, 48.1% and 35.2% in the control group and 14.7%, 46.7% and 38.6% in the case group, respectively. Multivariate logistic regression analysis adjusted for sex and age faied to show an association between any of the genotypes and susceptibility to SLE (P>0.05). And no significant differences were either exhibited in the allelic distributions betweeen them (P>0.05).To SNP rs12522248, genotype frequencies for AA,AG and GG were 7.2%, 29.8% and 63.0% in the control group and 3.5%, 33.3% and 63.1% in the case group, respectively. Multivariate logistic regression analysis adjusted for sex and age faied to show an association between any of the genotypes and susceptibility to SLE (P>0.05). And no significant differences were either exhibited in the allelic distributions betweeen them (P>0.05).(3) Association analysis of TIM-3 gene and SLETo SNP rs9313439, genotype frequencies for CC,CG and GG were 96.3%, 2.8% and 0.9% in the control group and 96.0%, 3.0% and 1.0% in the case group, respectively. Multivariate logistic regression analysis adjusted for sex and age faied to show an association between any of the genotypes and susceptibility to SLE (P>0.05). And no significant differences were either exhibited in the allelic distributions betweeen them (P>0.05). To SNP rs10515746, genotype frequencies for GG,GT and TT were 97.2%, 2.8% and 0 in the control group and 97.5%, 2.5% and 0 in the case group, respectively. Multivariate logistic regression analysis adjusted for sex and age faied to show an association between any of the genotypes and susceptibility to SLE (P>0.05). And no significant differences were either exhibited in the allelic distributions betweeen them (P>0.05).(4) Association analysis of TIM-1 and TIM-3 gene and LNTwo hundred and two patients with SLE were included in the present study, 83 of whom were patients with LN (41.1%). To SNP rs1501909, genotype frequencies for CC,AC and AA were 12.5%, 51.3% and 36.3% in SLE patients with LN and 16.2%, 43.6% and 40.2% in those without nephritis, respectively; To SNP rs12522248, genotype frequencies for AA,AG and GG were 5.0%, 35.0% and 60.0% in SLE patients with LN and 2.5%, 32.2% and 65.3% in those without nephritis, respectively; To SNP rs9313439, genotype frequencies for CC,CG and GG were 94.0%, 4.8% and 1.2% in SLE patients with LN and 97.5%, 1.7% and 0.8% in those without nephritis, respectively; To SNP rs10515746, genotype frequencies for GG,GT and TT were 97.6%, 2.4% and 0 in SLE patients with LN and 97.5%, 2.5% and 0 in those without nephritis, respectively. No statistical differences were detected in genotype and allele frequency for each polymorphism between the patients with LN and those without nephritis (P>0.05).(5) Association analysis of TIM-1 and TIM-3 gene and SLE disease activityPatients were subdivided into active group (n=94) and inactive group (n=105) according to clinical disease activity. To SNP rs1501909, genotype frequencies for CC,AC and AA were 15.1%, 42.0% and 43.0% in active SLE patients and 14.9%, 52.5% and 32.7% in inactive patients, respectively; To SNP rs12522248, genotype frequencies for AA,AG and GG were 4.4%, 28.6% and 67.0% in active SLE patients and 2.9%, 38.5% and 58.7% in inactive patients, respectively; To SNP rs9313439, genotype frequencies for CC,CG and GG were 93.8%, 3.2% and 0 in active SLE patients and 95.2%, 2.9% and 1.9% in inactive patients, respectively; To SNP rs10515746, genotype frequencies for GG,GT and TT were 97.9%, 2.1% and 0 in active SLE patients and 97.1%, 2.9% and 0 in inactive patients, respectively. There was no significant differences in genotype and allele frequency for each polymorphism between active SLE patients and inactive patients (P>0.05).(6) TIM-1-TIM-3 haplotype analysisWe analysed the haplotypes of TIM-1-TIM-3 and determined three main haplotype: AGCG, CACG and CGCG, and found that the frequencies in cases were not significantly different from those in controls, with respect to the three haplotypes (P>0.05).Conclusion No deviations from HWE were observed in either the patients with SLE or the controls in polymorphism for SNP rs1501909, SNP rs12522248, SNP rs9313439 and SNP rs10515746. No significant differences existed between the patients with SLE and the controls as well as SLE patients with nephritis and those without nephritis, in all four SNPs, which suggests that the polymorphisms of TIM gene familly might not contribute to SLE susceptibility in the Chinese population.