Replication of recently identified systemic lupus erythematosus genetic associations: a case–control study

Introduction

We aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.

Methods

We selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel–Haenszel approach to account for heterogeneity between sample collections.

Results

A previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 × 10-⁵), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.

Conclusions

Our results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.     

Patients with systemic lupus erythematosus have abnormally elevated Epstein–Barr virus load in blood

Various genetic and environmental factors appear to be involved in systemic lupus erythematosus (SLE). Epstein–Barr virus (EBV) is among the environmental factors that are suspected of predisposing to SLE, based on the characteristics of EBV itself and on sequence homologies between autoantigens and EBV antigens. In addition, higher titers of anti-EBV antibodies and increased EBV seroconversion rates have been observed in SLE patients as compared with healthy control individuals. Serologic responses do not directly reflect EBV status within the body. Clarification of the precise status of EBV infection in SLE patients would help to improve our understanding of the role played by EBV in this disease. In the present study we determined EBV types in SLE patients (n = 66) and normal control individual (n = 63) by direct PCR analysis of mouthwash samples. We also compared EBV load in blood between SLE patients (n = 24) and healthy control individuals (n = 29) using semiquantitative PCR assay. The number of infections and EBV type distribution were similar between adult SLE patients and healthy control individuals (98.5% versus 94%). Interestingly, the EBV burden in peripheral blood mononuclear cells (PBMCs) was over 15-fold greater in SLE patients than in healthy control individuals (mean ± standard deviation: 463 ± 570 EBV genome copies/3 μg PBMC DNA versus 30 ± 29 EBV genome copies/3 μg PBMC DNA; P = 0.001), suggesting that EBV infection is abnormally regulated in SLE. The abnormally increased proportion of EBV-infected B cells in the SLE patients may contribute to enhanced autoantibody production in this disease.     

T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?

Introduction

A hallmark of systemic autoimmune diseases like systemic lupus erythematosus (SLE) is the increased expression of interferon (IFN) type I inducible genes, so-called IFN type I signature. Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6⁺ memory T-helper cells producing IL-17A, IL-17F, IL-21, and/or IL-22 are increased in SLE patients and whether this increase is related to the presence of IFN type I signature.

Methods

In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive (IFN⁺) (n = 16) and negative (IFN^-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4⁺CD45RO⁺CCR6⁺ T cells (CCR6⁺ cells) was measured with flow cytometry and compared between IFN⁺, IFN^- patients and HCs.

Results

Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ cells were observed in IFN⁺ patients compared with IFN^- patients and HCs. IL-17A and IL-17F expression within CCR6⁺ cells correlated significantly with IFIG expression. In addition, we found significant correlation between B-cell activating factor of the tumor necrosis family (BAFF)–a factor strongly correlating with IFN type I - and IL-21 producing CCR6⁺ cells.

Conclusions

We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6⁺ memory T-helper cells in IFN⁺ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.     

IFN-α confers resistance of systemic lupus erythematosus nephritis to therapy in NZB/W F1 mice

The critical role of IFN-α in the pathogenesis of human systemic lupus erythematosus has been highlighted in recent years. Exposure of young lupus-prone NZB/W F1 mice to IFN-α in vivo leads to an accelerated lupus phenotype that is dependent on T cells and is associated with elevated serum levels of BAFF, IL-6, and TNF-α, increased splenic expression of IL-6 and IL-21, formation of large germinal centers, and the generation of large numbers of short-lived plasma cells that produce IgG2a and IgG3 autoantibodies. In this study, we show that both IgG2a and IgG3 autoantibodies are pathogenic in IFN-α-accelerated lupus, and their production can be dissociated by using low-dose CTLA4-Ig. Only high-dose CTLA4-Ig attenuates both IgG2a and IgG3 autoantibody production and significantly delays death from lupus nephritis. In contrast, BAFF/APRIL blockade has no effect on germinal centers or the production of IgG anti-dsDNA Abs but, if given at the time of IFN-α challenge, delays the progression of lupus by attenuating systemic and renal inflammation. Temporary remission of nephritis induced by combination therapy with cyclophosphamide, anti-CD40L Ab, and CTLA4-Ig is associated with the abrogation of germinal centers and depletion of short-lived plasma cells, but relapse occurs more rapidly than in conventional NZB/W F1 mice. This study demonstrates that IFN-α renders NZB/W F1 relatively resistant to therapeutic intervention and suggests that the IFN signature should be considered when randomizing patients into groups and analyzing the results of human clinical trials in systemic lupus erythematosus.     

NKT cell stimulation with α-galactosylceramide results in a block of Th17 differentiation after intranasal immunization in mice

In a previous study we demonstrated that intranasal (i.n.) vaccination promotes a Th17 biased immune response. Here, we show that co-administration of a pegylated derivative of α-galactosylceramide (αGCPEG) with an antigen, even in the presence of Th17-polarizing compounds, results in a strong blocking of Th17 differentiation. Additional studies demonstrated that this phenomenon is specifically dependent on soluble factors, like IL-4 and IFNγ, which are produced by NKT cells. Even NK1.1 negative NKT cells, which by themselves produce IL-17A, are able to block Th17 differentiation. It follows that the use of αGCPEG as adjuvant would enable to tailor Th17 responses, according to the specific clinical needs. This knowledge expands our understanding of the role played by NKT cells in overall control of the cytokine microenvironment, as well as in the overall shaping of adaptive immune responses.     

Association between anti-nucleophosmin and anti-cardiolipin antibodies in (NZW × BXSB)F1 mice and human systemic lupus erythematosus

We showed previously that nucleophosmin (NPM), a nucleolar phosphoprotein, is recognized by sera from (NZW × BXSB)F₁ (WB) mice, a model of systemic lupus erythematosus (SLE) and anti-phospholipid syndrome. In the present study we analysed the prevalence and kinetics of anti-NPM autoantibodies in WB mice by a solid-phase ELISA with recombinant human (rh) NPM as the antigen and showed that most male WB mouse sera had anti-NPM antibodies that were responsible for their indirect immunofluorescence staining pattern on Hep-2 cells. Anti-NPM antibodies were significantly associated with anti-cardiolipin (aCL) antibodies. This antibody profile mirrored that observed in certain human SLE sera because anti-NPM antibodies were detected in 28% of the sera from patients with SLE and were similarly associated with aCL antibodies. The demonstration that rhNPM bound to cardiolipin (CL) in vitro and increased the CL-binding activity of a WB-derived aCL monoclonal antibody indicates that NPM can interact with CL to form SLE-related immunogenic particles that might be responsible for the concomitant production of anti-NPM and aCL antibodies.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4⁺CD45RO⁺ T cells from peripheral blood, the percentage of ICOS⁺ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [³H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.     

The association of IL1α and IL1β polymorphisms with susceptibility to systemic lupus erythematosus: A meta-analysis

Many epidemiological studies have investigated IL1α and IL1β polymorphisms with SLE risk, but no conclusions are available because of conflicting results. This meta-analysis was performed to more precisely estimate the relationships. The databases of PubMed updated to September 1st, 2012 were retrieved. Odds ratio (OR) and corresponding 95% confidence interval (95% CI) as effect size were calculated by a fixed- or random-effect model. In total, six case–control studies for IL1β − 511C/T, four studies for IL1β + 3953C/T, three studies for IL1α − 889C/T and three studies for IL1α + 4845G/T were involved in this analysis. The results indicated that for IL1α − 889C/T polymorphism T allele was associated with decreased risk of SLE (OR (95% CI)) (T vs. C: 0.802 (0.679–0.949); TT + CT vs. CC: 0.615 (0.380–0.995); TT vs. CC: 0.679 (0.466–0.989)). However, when analysis for TT vs. CT + CC was conducted, the result indicated that IL1α − 889C/T polymorphism was not associated with SLE (OR (95% CI): 0.847 (0.595–1.205)). Combined analysis indicated that IL1β − 511C/T polymorphism was not overall associated with risk of SLE (OR (95% CI)) (T vs. C: 1.113 (0.954–1.298); TT vs. CT + CC: 1.146 (0.889–1.447); TT + CT vs. CC: 1.145 (0.903–1.452); TT vs. CC: 1.255 (0.928–1.698)). When subgroup analysis for Asian ethnicity was conducted, the results indicated that IL1β − 511C/T polymorphism was associated with SLE only for TT vs. CT + CC (OR (95% CI): 1.468 (1.001–2.152)), but was not associated for T vs. C (OR (95% CI): 1.214 (0.955–1.544)), TT + CT vs. CC (OR (95% CI): 1.112 (0.765–1.615)) and TT vs.CC (OR (95% CI): 1.411 (0.896–2.222)). In addition, overall analyses indicated that IL1β + 3953C/T and IL1α + 4845G/C polymorphisms were also not associated with risk of SLE (OR (95% CI)) (for IL1β + 3953C/T T vs. C: 0.996 (0.610–1.626), TT vs. CT + CC: 0.658 (0.318–1.358), TT + CT vs. CC: 1.021 (0.618–1.687), TT vs. CC: 0.640 (0.309–1.325); for IL1α + 4845G/T T vs. G: 1.067 (0.791–1.440), TT + GT vs. GG: 0.934 (0.646–1.351)).This study inferred that IL1α − 889C/T polymorphism might be moderately associated with SLE, but no sufficient evidence was available to support any associations between IL1β + 3953C/T or IL1α + 4845G/C polymorphisms and SLE. We could not draw a definite conclusion between IL1β − 511C/T polymorphism and risk of SLE owing to the limited data. Further large sample-sized studies should be required.     

Role of HLA DRB1*15 and HLA DRB1*16alleles in the genetic susceptibility to develop systemic lupus erythematosus (SLE) after Chikungunya and Zika viruses infection in México

Systemic lupus erythematosus (SLE) is a clinically and genetically heterogeneous disease particularly prevalent in Mexico. Althoughits etiology is unknown, genetic factors strongly influence its presenceas well as triggering factors, such as viral infections, including Cytomegalovirus and Epstein-Barr virus. Here,the study presents the appearance of de novoSLE (patients who did not present SLE before de virus infection, corroborated by serological analysis and negative for antinuclear antibodies) cases in Mexicans who live near the southern border of Mexico, who presented clinical symptoms of arthritic, hematological, mucocutaneous and renal SLE, after Zika and/ or Chikungunya virus infection. Low resolution class Ⅱ HLA typing was performed, which found a significantly increased frequency of HLA DRB1*02 (15 and 16)when compared to a group of 99 healthy individuals (P =0.001, OR=4.5, IC95% 1.8~11.0). All the patients were diagnosed with SLE 1 to 3 years after being confirmed with the Zika, and/or Chikungunya infection. At the point of acute viral infection, none of the patients presented clinical signs or symptoms of autoimmunity or were negative for antinuclear antibodies. In genetically susceptible individuals, Zika and Chikungunya viral infection can trigger SLE.     

<Emphasis Type="Italic">SDF1-3</Emphasis>′ G801A polymorphisms in Polish patients with systemic lupus erythematosus

It has been reported that stromal cell-derived factor-1 (SDF1), currently also designated CXCL12, plays a significant role in the development of nephritis and death in the lupus mice model. Using restriction length fragment polymorphism (RFLP) analysis we assessed the frequencies of SDF1-3′ G801A (rs 1801157) polymorphic variants between systemic lupus erythematosus (SLE) patients (n = 150) and controls (n = 300). There were no significant differences in the prevalence of SDF1-3′ G801A polymorphic variants in SLE patients and healthy individuals. However, we observed that the SDF1-3′ A/A and G/A genotypes (recessive model) contributed to renal manifestations of SLE OR = 3.042 (95% CI = 1.527–6.058, P = 0.002), and the p value stayed statistically significant after Bonferroni correction (p _corr = 0.032) in SLE patients. We also found an association of the SDF1-3′ A/A and G/A genotypes (recessive model) with dermal manifestations of SLE OR = 2.510 (95% CI = 1.247–5.052, P = 0.0122), (p _corr = 0.1952) but this did not remain statistically significant after Bonferroni correction. Our observations suggest that the SDF1-3′ G801A genotype may be associated with some clinical manifestations in patients with SLE.     

Role of HLA DRB1*15 and HLA DRB1*16alleles in the genetic susceptibility to develop systemic lupus erythematosus (SLE) after Chikungunya and Zika viruses infection in México

Systemic lupus erythematosus (SLE) is a clinically and genetically heterogeneous disease particularly prevalent in Mexico. Althoughits etiology is unknown, genetic factors strongly influence its presenceas well as triggering factors, such as viral infections, including Cytomegalovirus and Epstein-Barr virus. Here,the study presents the appearance of de novoSLE (patients who did not present SLE before de virus infection, corroborated by serological analysis and negative for antinuclear antibodies) cases in Mexicans who live near the southern border of Mexico, who presented clinical symptoms of arthritic, hematological, mucocutaneous and renal SLE, after Zika and/ or Chikungunya virus infection. Low resolution class Ⅱ HLA typing was performed, which found a significantly increased frequency of HLA DRB1*02 (15 and 16)when compared to a group of 99 healthy individuals (P =0.001, OR=4.5, IC95% 1.8~11.0). All the patients were diagnosed with SLE 1 to 3 years after being confirmed with the Zika, and/or Chikungunya infection. At the point of acute viral infection, none of the patients presented clinical signs or symptoms of autoimmunity or were negative for antinuclear antibodies. In genetically susceptible individuals, Zika and Chikungunya viral infection can trigger SLE.     

DNA synthesis,mitotic index,drug-metabolising systems and cytogenetic analysis in regenerating rat liver: Comparison with bone marrow test after ‘in vivo’ treatment with cyclophosphamide

Rat-liver cells can be used to reveal “in vivo” clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54–66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione-S-epoxide transferase were chosen as markers.All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed.DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22–25 and 28–31 h after partial removal of the liver.The sensitivity to clastogenic damage induced by “in vivo” treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure).The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.     

Determination of 1-methyl-1,2,3,4-tetrahydroisoquinoline in mouse brain after treatment with haloperidol by gas chromatography–selected ion monitoring

The content of the endogenous amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), in mouse brain, treated with the antipsychotic agent haloperidol (HP) was determined by GC–SIM (selected ion monitoring) system. 1-MeTIQ in brain was extracted with chloroform at pH 11–12 and was detected as PFP derivative by GC–SIM. The 1-MeTIQ contents in mouse brains following intraperitoneal administration of HP or its dehydrated product, HPTP (1 and 4 mg/kg per day, for four days), were markedly reduced compared with control groups. This result agrees well with the findings in human idiopathic parkinsonianism and in MPTP-treated mouse brain. In addition, this finding suggests that the change of the endogenous amine 1-MeTIQ content in the brain plays an important role in the pathogenesis of toxin-induced parkinsonism.