Abnormal histone modification patterns in lupus CD4+ T cells

OBJECTIVE: To investigate alterations in histone modifications in patients with systemic lupus erythematosus (SLE). METHODS: Global histone H3/H4 acetylation and H3K4/H3K9 methylation in CD4+ T cells from 20 SLE patients and 10 healthy control subjects were assayed using the EpiQuik global histone H3/H4 acetylation and H3K4/H3K9 methylation assay kits. mRNA levels of 12 members of 3 classes of chromatin modifier genes were measured by real-time quantitative polymerase chain reaction. RESULTS: Global histone H3 and H4 hypoacetylation was observed in active lupus CD4+ T cells compared with controls (p = 0.002 and p = 0.009, respectively). The degree of histone H3 acetylation correlated negatively with increased disease activity in lupus patients as measured by SLEDAI (r = -0.889, p = 0.044). We found global histone H3K9 hypomethylation in both active and inactive lupus CD4+ T cells, compared with controls (p = 0.001, p = 0.003, respectively). However, global levels of H3K4 methylation were not different between patients and controls. SIRT1 mRNA levels were significantly increased in active lupus CD4+ T cells compared with controls (p < 0.001), while mRNA levels of CREBBP, P300, HDAC2, HDAC7, SUV39H2, and EZH2 were significantly downregulated in patients with active lupus (p < 0.001, p < 0.001, p = 0.01, p < 0.001, p = 0.003, p = 0.001, respectively). CONCLUSION: Histone modifications appear abnormal in CD4+ T cells in SLE.     

Histone acetylation is associated with differential gene expression in the rapid and robust memory CD8(+) T-cell response

To understand the molecular basis for the rapid and robust memory T-cell responses, we examined gene expression and chromatin modification by histone H3 lysine 9 (H3K9) acetylation in resting and activated human naive and memory CD8(+) T cells. We found that, although overall gene expression patterns were similar, a number of genes are differentially expressed in either memory or naive cells in their resting and activated states. To further elucidate the basis for differential gene expression, we assessed the role of histone H3K9 acetylation in differential gene expression. Strikingly, higher H3K9 acetylation levels were detected in resting memory cells, prior to their activation, for those genes that were differentially expressed following activation, indicating that hyperacetylation of histone H3K9 may play a role in selective and rapid gene expression of memory CD8(+) T cells. Consistent with this model, we showed that inducing high levels of H3K9 acetylation resulted in an increased expression in naive cells of those genes that are normally expressed differentially in memory cells. Together, these findings suggest that differential gene expression mediated at least in part by histone H3K9 hyperacetylation may be responsible for the rapid and robust memory CD8(+) T-cell response.     

系统性红斑狼疮患者CD4+CD25+调节性T细胞的变化

目的检测系统性红斑狼疮(SLE)患者外周血CD4 CD25 T细胞和CD4 CD25highT细胞占CD4 T细胞的比率,探讨其在SLE发病中的意义。方法采用流式细胞术检测93例SLE患者及47 名正常对照外周血CD4 CD25 调节性T细胞和CD4 CD25highT细胞占CD4 T细胞的比率,分析其与SLE 临床及实验室指标的关系。结果活动性SLE患者CD4 CD25 调节性T细胞及CD4 CD25highT细胞比例较正常人降低;CD4 CD25highT细胞水平与系统性红斑狼疮疾病活动指数(SLEDAI)呈负相关;CD4 CD25 调节性T细胞与抗核抗原抗体(ANA)、抗可提取的核抗原(ENA)抗体、抗dsDNA抗体、免疫球蛋白、补体C3、C4无相关。结论CD4 CD25 T细胞和CD4 CD25highT细胞可能参与SLE的发病机制。     

MicroRNA‐126 regulates DNA methylation in CD4+ T cells and contributes to systemic lupus erythematosus by targeting DNA methyltransferase 1

Objective

To identify microRNA genes with abnormal expression in the CD4+ T cells of patients with systemic lupus erythematosus (SLE) and to determine the role of microRNA‐126 (miR‐126) in the etiology of SLE.

Methods

MicroRNA expression patterns in CD4+ T cells from patients with SLE and healthy control subjects were analyzed by microRNA microarray and stem loop quantitative polymerase chain reaction (qPCR). Luciferase reporter gene assays were performed to identify miR‐126 targets. Dnmt1, CD11a, and CD70 messenger RNA and protein levels were determined by real‐time qPCR, Western blotting, and flow cytometry. CD11a, CD70, and EGFL7 promoter methylation levels were detected by bisulfite sequencing. IgG levels in T cell–B cell cocultures were determined by enzyme‐linked immunosorbent assay.

Results

The expression of 11 microRNA was significantly increased or decreased in CD4+ T cells from patients with SLE relative to that in CD4+ T cells from control subjects. Among these, miR‐126 was up‐regulated, and its degree of overexpression was inversely correlated with Dnmt1 protein levels. We demonstrated that miR‐126 directly inhibits Dnmt1 translation via interaction with its 3′–untranslated region, and that overexpression of miR‐126 in CD4+ T cells can significantly reduce Dnmt1 protein levels. The overexpression of miR‐126 in CD4+ T cells from healthy donors caused the demethylation and up‐regulation of genes encoding CD11a and CD70, thereby causing T cell and B cell hyperactivity. The inhibition of miR‐126 in CD4+ T cells from patients with SLE had the opposite effects. Expression of the miR‐126 host gene EGFL7 was also up‐regulated in CD4+ T cells from patients with SLE, possibly in a hypomethylation‐dependent manner.

Conclusion

Our data suggest that miR‐126 regulates DNA methylation in CD4+ T cells and contributes to T cell autoreactivity in SLE by directly targeting Dnmt1.

     

Decreased CD161+CD8+ T cells in the peripheral blood of patients suffering from rheumatic diseases

OBJECTIVES: Although it has been reported that the numbers of both CD4(-)CD8(-) and CD4(+) natural killer T (NKT) cells are selectively decreased in the peripheral blood of patients with rheumatic diseases, there have been no reports concerning a novel subpopulation of CD8(+) NKT cells. To examine whether CD161(+)CD8(+) T cells, which are closely related to CD8(+) NKT cells, are also decreased in patients with rheumatic diseases, we have investigated the expression of CD161, together with that of CD28, CD25 and CD62L, on T cells in the peripheral blood of these patients. METHODS: The rheumatic diseases evaluated in this study were systemic lupus erythematosus (SLE) (n= 54), mixed connective-tissue disease (MCTD) (n= 15), systemic sclerosis (SSc) (n= 14), polymyositis/dermatomyositis (PM/DM) (n= 13) and rheumatoid arthritis (RA) (n= 24). Healthy donors were examined as controls (n= 18). The expression of CD161, CD28, CD25 and CD62L on T cells was analysed by flow cytometry. RESULTS: Both the frequency of CD161 expression on CD8(+) cells and the absolute number of CD161(+)CD8(+) cells were significantly decreased in patients with SLE, MCTD, SSc and PM/DM. Only the absolute number of CD161(+)CD8(+) T cells was significantly decreased in RA. CD161 expression on CD28(-)CD8(+) T cells was significantly decreased in SLE, MCTD and SSc. The absolute number of CD161(+)CD8(+)CD62L(-) T cells was significantly decreased in SLE, MCTD and SSc. CONCLUSIONS: Both the frequency and the absolute number of CD161(+)CD8(+) T cells were decreased in the peripheral blood of patients suffering from SLE, MCTD, SSc and PM/DM. This result suggests that there is also an abnormality of NKT cells in the CD8(+) population.     

Anti-nucleosome antibodies and T-cell response in systemic lupus erythematosus

Evidence accumulated in recent years suggests that nucleosomes play a pivotal role in the induction phase and pathogenesis of systemic lupus erythematosus (SLE). Apoptotic cells are an important source of nucleosomes and apoptosis defects have been described in patients with SLE as well as in lupus mice. Moreover, it has been demonstrated that the intravenous injection of apoptotic cells in normal mice generated the production of anti-nuclear antibodies and led to the development of symptoms associated with lupus disease. In this review, we briefly summarize these results and describe recent findings on the characterization of histone T-cell epitopes recognized by CD4(+) cells from different strains of lupus mice. We have tested a panel of overlapping peptides spanning the whole sequences of H4 and H3 histones for recognition by CD4(+) T cells from unprimed (NZBxNZW) F1 and MRL/lpr lupus mice. We have also immunized na?ve BALB/c mice with nucleosomes or syngeneic apoptotic and non-apoptotic spleen cells, and tested the activation of Th cells reacting ex vivo with H4 and H3 peptides. Our results suggest that nucleosomes and apoptotic cells may effectively act as initiator of autoreactive Th cell development in lupus mice. In the BW lupus model, the region 53-85 of H3, which also contains B-cell epitopes recognized by antibodies from (NZBxNZW) F1 mice and lupus patients, might be important.     

Apoptosis-induced acetylation of histones is pathogenic in systemic lupus erythematosus

OBJECTIVE: In systemic lupus erythematosus (SLE), inadequate removal of apoptotic cells may lead to challenge of the immune system with immunogenic self antigens that have been modified during apoptosis. We undertook this study to evaluate whether apoptosis-induced histone modifications are targets for the immune system in SLE. METHODS: The epitope of KM-2, a monoclonal antihistone autoantibody derived from a lupus mouse, was mapped by random peptide phage display. The reactivity of KM-2 and plasma with (acetylated) histone H4 (H4) peptides and with nonapoptotic, apoptotic, and hyperacetylated histones was determined by immunofluorescence staining, enzyme-linked immunosorbent assay, and Western blotting. RESULTS: KM-2 recognized apoptosis-induced acetylation of H4 at lysines 8, 12, and 16. The majority of plasma samples from SLE patients and lupus mice showed higher reactivity with triacetylated H4 peptide (residues 1-22) and with hyperacetylated and apoptotic histones than with nonacetylated H4 peptide and normal histones. Importantly, administration of triacetylated H4 peptide to lupus-prone mice before disease onset accelerated the disease by enhancing mortality and aggravating proteinuria, skin lesions, and glomerular IgG deposition, while the nonacetylated H4 peptide had no pathogenic effect. The delayed-type hypersensitivity response in lupus mice against the triacetylated H4 peptide was higher than that against the nonacetylated H4 peptide. Bone marrow-derived dendritic cells (DCs) cultured in the presence of hyperacetylated nucleosomes showed increased expression/production of CD40, CD86, interleukin-6 (IL-6), and tumor necrosis factor alpha compared with DCs cultured in the presence of normal nucleosomes. Finally, DCs cultured in the presence of hyperacetylated nucleosomes were able to activate syngeneic T cells, because IL-2 production increased. CONCLUSION: Apoptosis-induced acetylation of nucleosomes may represent an important driving force in the development of lupus.     

Association of polymorphisms in complement component C3 gene with susceptibility to systemic lupus erythematosus

OBJECTIVE: Identification of the genes responsible for systemic lupus erythematosus (SLE). METHODS: All the exons and putative promoter regions of 53 candidate genes (TNFRSF6/Fas, TNFSF6/FasL, Fli1, TNFSF10/TRAIL, TNFSF12/TWEAK, Bcl-2, PTEN, FADD, TRADD, CDKN1A, TNFRSF1A/TNFR1, TNFRSF4/OX40, TNFSF4/OX40L, TNFSF5/CD40L, TNFSF13B/BAFF, ICOS, CTLA4, CD28, FYN, G2A, CR2, PTPRC/CD45, CD22, CD19, Lyn, PDCD1, PTPN6, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, TGFBR3, CD3Z, DNASE1, APCS, MERTK, C3, C1QA, C1QB, C1QG, C2, MBL2, IGHM, IL-2, IL-4, IL-10, IFNG, TNFA, MAN2A1, TNFRSF11A/RANK, TNFRSF11B/OPG, TNFSF11/OPGL) were screened for single nucleotide polymorphisms (SNPs) and their association with SLE was assessed by case-control studies. A total of 509 cases and 964 controls of Japanese descent were enrolled. RESULTS: A total of 316 SNPs was identified. When analysed in the Japanese population, the allele frequencies of T at rs7951 and G at rs2230201 of the C3 gene were 0.110 and 0.626, respectively, in SLE patients; significantly higher than the frequencies of 0.081 and 0.584, respectively, in controls [odds ratio (OR) = 1.40, 95% confidence interval (CI) = 1.05-1.86, P = 0.016 and OR=1.19, 95% CI = 1.01-1.41, P = 0.038, respectively]. The mean serum C3 level of carriers of the rs7951 T allele was significantly lower than that of non-carriers of the T allele in 87 SLE patients whose medical records were available (P = 0.0018). CONCLUSION: rs7951 T allele of the C3 gene was significantly associated with SLE, and decreased serum level of C3 seems to be correlated with this allele.     

IL-7Rαlow memory CD8+ T cells are significantly elevated in patients with systemic lupus erythematosus

Objective. Human effector memory (EM) CD8(+) T cells include IL-7Rα(high) and IL-7Rα(low) cells with distinct cellular characteristics, including the expression of cytotoxic molecules. Both NK cells and the NK cell-associated molecule 2B4 that is expressed on CD8(+) T cells promote cytotoxicity. Here we analysed the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells and its contribution to cytotoxicity. We also analysed the frequency of IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells in patients with SLE or lupus and in healthy individuals given the potential role of cytotoxic CD8(+) T cells in the pathogenesis of lupus. Methods. We used flow cytometry to measure the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells as well as the frequency of these cell populations in the peripheral blood of healthy individuals and patients with SLE. Also, 2B4-mediated cytotoxicity was quantitated in IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells using target cells with CD48 antigen. Results. We found that IL-7Rα(high) EM CD8(+) T cells had higher levels of 2B4 expression compared with IL-7Rα(low) EM CD8(+) T cells. Triggering 2B4 enhanced the cytotoxic function of IL-7Rα(low) EM CD8(+) T cells against target cells. We also noticed that patients with SLE had an increased frequency of IL-7Rα(low) EM CD8(+) T cells that correlated with disease manifestation. Conclusion. Our findings show that SLE patients have increased IL-7Rα(low) EM CD8(+) T cells, possibly contributing to tissue damage through 2B4-mediated cytotoxicity.     

外周血单核细胞CD69/CD3比值在评估系统性红斑狼疮疾病活动性中的作用

目的　通过检测系统性红斑狼疮 (SLE)患者外周血中T细胞CD6 9表达 ,以探讨CD6 9/CD3比值在评估SLE疾病活动指数中的作用。方法　利用流式细胞分析法对 5 4例SLE患者和 18名健康志愿者外周血T细胞上CD6 9表达进行分析 ,同时检测患者抗dsDNA抗体、C3和C4水平。结果　SLE患者CD6 9/CD3的比值显著高于正常人群 (P <0 0 1) ,当抗dsDNA抗体、C3和C4水平与疾病活动性缺乏相关关系时 ,CD6 9/CD3仍与SLE活动指数 (SLEDAI)有明显相关性 (P <0 0 5 )。结论　T细胞活化 (CD6 9/CD3)程度与SLEDAI指数有明显的相关性 ,可用于评估SLE疾病活动性     

外周血CD+4CD+25T细胞表达在系统性红斑狼疮患者中的意义

目的了解系统性红斑狼疮(SLE)患者外周血CD4 CD2 5T细胞表达及其临床意义。方法用流式细胞仪检测53例SLE患者外周血CD4 CD2 5T细胞表达,根据CD25表达荧光强度>100者为CD4 CD25brightT细胞,并与SLE活动指数评分(SLEDAI)、血清补体C3水平、抗双链DNA抗体、抗核抗体滴度进行相关分析。结果SLE患者外周血CD4 CD2 5T细胞表达率为(7·84±1·85)%,显著低于对照组(9·18±2·01)%(P<0·05),且活动期组[(6·72±1·16)%]较稳定期组更低[(8·57±1·91)%,P<0·01]。外周血CD4 CD25brightT细胞表达率在活动期组[(0·85±0·24)%]和稳定期组[(0·91±0·25)%]之间相比差异无统计学意义(P=0·686),但均低于对照组[(1·43±1·08)%,P<0·01]。随治疗后SLEDAI评分的下降,活动期组SLE患者外周血CD4 CD25brightT细胞表达率无明显变化。进一步分析外周血CD4 CD25brightT细胞表达率与SLEDAI评分、抗核抗体、抗双链DNA抗体滴度和血清C3水平的相关性,分别为ρ=-0·188,P=0·178;ρ=-0·216,P=0·121;ρ=0·082,P=0·560;ρ=0·010,P=0·944,差异均无统计学意义(P>0·05)。结论外周血CD4 CD2 5T细胞的减少可能与SLE的发病有关。     

Phenotypic and genetic analyses of T-cell-mediated immunoregulation in patients with Type 1 diabetes.

AIMS: To investigate the contribution of regulatory T cells and co-stimulatory molecules in CD4(+) T cells to the development of Type 1 diabetes (T1D). METHODS: Twelve patients with T1D, nine patients with systemic lupus erythematosus (SLE), and 12 age-matched healthy control subjects participated. We analysed the proportions of CD25(+)CD4(+) T cells and natural killer T cells (NKT cells), and the expression levels of Foxp3, CTLA-4, CD28, ICOS, PD-1 and BTLA in peripheral blood mononuclear cells and purified CD4(+) T cells. RESULTS: There were no significant differences in the proportions of CD25(+) CD4(+) T cells or NKT cells among the three groups. PD-1 expression levels of peripheral CD4(+) T cells from T1D patients were significantly lower than those from healthy control subjects (P = 0.00066). In contrast, PD-1 expression levels were similar in SLE patients and healthy control subjects. The expression levels of Foxp3, CTLA-4, CD28, ICOS and BTLA were similar in the three groups. CONCLUSIONS: Decreased expression of the PD-1 gene in CD4(+) T cells may contribute to the development and/or maintenance of autoimmune T1D. As the population studied was small and heterogeneous, further studies are required to confirm the findings.     

Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus.

OBJECTIVE. Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought. METHODS. Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity. RESULTS. 5-azaC-treated normal T cells had increased CD11a (leukocyte function-associated antigen 1 alpha) expression relative to other membrane molecules. A T cell subset with similar CD11a expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells.