Molecular Epidemiology Study On Association Between Interleukin-10 And Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus（SLE）is a chronic autoimmune disease characterized by the production of autoantibodies and the deposition of immune complexes in tissues.The clinical manifestations of SLE vary from mild skin lesions to severe organ damage,about 60%of patients with glomerulonephritis,and glomerulonephritis may eventually lead to end-stage renal disease.SLE occurs in young women,poor quality of life in patients,thus having a serious impact on the physical and mental health of women of childbearing age.However,the etiology and pathogenesis of SLE is not yet completely clear.It may be affected by genetic factors,environmental factors,immune factors,etc.Interleukin-10（IL-10）is an anti-inflammatory cytokine that plays a central role in infection by restricting the immune response to pathogens preventing damage to the host.Inflammatory diseases such as SLE are characterized by abnormal expression of cytokines in vivo,and the balance of pro-inflammatory and anti-inflammatory cytokines is tipped toward inflammatory reactions.IL-10 plays a key role in maintaining this balance,because it blocks the secretion of inflammatory cytokines and chemokines and affects the production of inflammatory enzymes and costimulatory molecules（including CD80,CD86 and class II major Histocompatibility complex）expression.In addition,IL-10 also promotes the production of IL-1 receptor antagonists（IL-1RA）and soluble tumor necrosis factorα（TNF-α）receptors against inflammation.However,in some cases,IL-10 plays an immunostimulating role and causes proliferation and differentiation of B cells.It is an effective cofactor for class switching and antibody production.IL-10 is thought to play an important role in the regulation of human immune system.In recent years,more and more attention has been paid to the regulation of IL-10 expression in different immune cells,and it is considered to play an important role in autoimmune diseases.Currently,there are limited studies on the association between IL-10 gene polymorphism and susceptibility to SLE in Chinese Han population,and they are confined to the IL-10 promoter region.ObjectiveTo detect the expression levels of IL-10 mRNA in peripheral blood mononuclear cells（PBMCs）and plasma from patients with SLE and healthy controls,as well as the association between single nucleotide polymorphisms（SNP）of IL-10 and susceptibility to SLE in a Chinese Han population.In addition,we analyzed the association of IL-10SNPs with IL-10 mRNA levels and plasma levels in patients with SLE.MethodsThis study used case-control design.74 cases of SLE and 57 healthy controls were included in the detection of IL-10 mRNA expression levels in PBMCs by quantitative real-time polymerase chain reaction（qRT-PCR）.Plasma IL-10 level was detected by enzyme linked immunosorbent assay（ELISA）and included 74 cases of SLE and 74healthy controls.Genotyping of 9 SNPs was performed using the Taq Man SNP genotyping assays by a Fluidigm EP1 platform.The clinical data of the study subjects were collected,and the disease activity of SLE patients was scored according to the systemic lupus erythematosus disease activity index 2000（SLEDAI-2K）scale.848 SLE cases and 461 healthy controls were included in this stage.α=0.0056（0.05/9）when Bonferroni correction was performed in genetic association analysis,α=0.05 in the other statistical analysis.Results（1）Comparison of IL-10 mRNA and plasma IL-10 expression in SLE patients and healthy controls:The expression levels of IL-10 in PBMCs and plasma were significantly higher in SLE patients than healthy controls（Z₁=-2.066,P₁=0.039;Z₂=-2.174,P₂=0.030）.Compared with the control group,the levels of IL-10 mRNA in PBMCs in active SLE patients and LN patients were higher（Z₁=-2.989,P₁=0.003;Z₂=-2.709,P₂=0.007）.There was a significant difference in plasma IL-10 level between active SLE patients and control group（Z=-2.153,P=0.031）.（2）Comparison of IL-10 expression in patients with SLE in different clinical groups:The expression levels of IL-10 mRNA in PBMCs were significantly higher in positive proteinuria than negative proteinuria（Z=-2.134,P=0.033）;The expression levels of IL-10 mRNA in PBMCs were lower in SLE patients taking HCQ than those in non-HCQ-bearing SLE patients（Z=-2.504,P=0.012）.The dosage of HCQ and the expression levels of IL-10 mRNA was negatively correlated（r_s=-0.235,P=0.047）.The plasma IL-10 levels was significantly higher in patients complicated by visual impairment than that in patients without visual impairment（Z=-2.172,P=0.030）.（3）Correlation of IL-10 mRNA and plasma IL-10 expression with disease activity in SLE patients:There were no significant correlation between SLEDAI-2K and the levels of IL-10 mRNA and plasma IL-10（all P<0.05）.There was no significant correlation between the expression level of IL-10 mRNA in PBMCs and the level of IL-10 in plasma（r_s=-0.193,P=0.100）.（4）Association between SNP of IL-10 and susceptibility to SLE:Significant association was observed between the allele frequencies（C vs.T）of rs3024498 and susceptibility to SLE（χ²=9.566,P=0.002,OR=5.118,95%CI:1.819-14.405）.However,there was no significant association with the genotype frequency and allele frequency of other 8SNPs between SLE cases and healthy controls（all P>0.0056）.The genotype effects of recessive and dominant models were also analyzed,but no significant evidence for association was detected（all P>0.0056）.（5）Correlation analysis of IL-10 SNPs and clinical manifestations and laboratory parameters:Association analysis of IL-10 gene polymorphism with clinical manifestations and laboratory indicators found that the distribution of genotypes at rs1518110 and rs1518111 were significant association with SLE patients with headache（all P<0.05）.The genotype frequency and allele frequency of the rs1800890 were associated with SLE patients who combined with thrombocytopenia（all P<0.05）.The genotype frequency and allele frequency of the rs1800893 were the significant difference in patients with tubular urine and proteinuria（all P<0.05）.The allele frequency of the rs1800893 were the significant difference in patients with thrombocytopenia（χ²=5.957,P=0.015）.There were significant association between SLE patients with or without arthritis in genotype frequency and allele frequency of rs3024498（all P<0.05）.The genotype frequency of rs3024498 was the significant difference in patients with malar rash（Fisher,P=0.040）.The allele frequency of rs3024498 was the significant difference in patients with anti-ds-DNA（χ²=4.624,P=0.032）.There were significant association between SLE patients with or without thrombocytopenia in genotype frequency and allele frequency of rs6667202（all P<0.05）.（6）Haplotype analysis of IL-10 SNPs and its correlation with IL-10 mRNA and plasma IL-10 expression levels:Haplotype analysis found that the two haplotypes were not associated with the risk of SLE（all P>0.05）.The level of IL-10 in PBMCs and plasma of SLE patients had no significant association with the 9 SNPs in SLE patients（all P>0.05）.ConclusionsThe expression levels of IL-10 in PBMCs and plasma were higher in SLE patients than healthy controls.Further analysis showed that the high expression level of SLE patients is easy to produce positive proteinuria.Meanwhile,HCQ dose also affects the expression level of IL-10 mRNA.The expression level of IL-10 mRNA was negatively correlated with the dosage of HCQ.In addition,high levels of plasma IL-10 expression can make visual impairment in SLE patients.SNP study found that rs3024498 is associated with susceptibility to SLE.And some SNPs are associated with some clinical phenotypes and laboratory indicators of SLE.The results showed that IL-10 is associated with SLE susceptibility.