Targeting abnormal lipid metabolism of T cells for systemic lupus erythematosus treatment.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the immune system attacks its own tissues and organs. However, the causes of SLE remain unknown. Dyslipidemia is a common symptom observed in SLE patients and animal models and is closely correlated to disease activity. Lipid metabolic reprogramming has been considered as a hallmark of the dysfunction of T cells in patients with SLE, therefore, manipulating lipid metabolism provides a potential therapeutic target for treating SLE. A better understanding of the underlying mechanisms for the metabolic events of immune cells under pathological conditions is crucial for tuning immunometabolism to manage autoimmune diseases such as SLE. In this review, we aim to summarize the cross-link between lipid metabolism and the function of T cells as well as the underlying mechanisms, and provide light on the novel therapeutic strategies of active compounds from herbals for the treatment of SLE by targeting lipid metabolism in immune cells.

Clinical analysis of chinese patients with rheumatoid arthritis treated with leflunomide and methotrexate combined with different dosages of glucocorticoid.

OBJECTIVE: To analyze the safety of combined leflunomide (LEF), methotrexate (MTX), and glucocorticoid (GC) therapy, we investigated the adverse effects of such combination therapy in patients with early active rheumatoid arthritis (RA). METHODS: Two hundred sixty-six patients with RA who were receiving LEF and MTX therapy were randomly assigned to 3 groups, as follows: group 1 received no GC, group 2 received 7.5 mg prednisone, and group 3 received 15 mg prednisone. Adverse effects were analyzed using the χ(2) test at week 4 or the Fisher exact test at week 12. RESULTS: Patients in group 1 had a higher incidence of skin rash, oral ulcers, leukopenia, and liver damage than did those in groups 2 and 3 (all, P ≤ 0.05). However, the rates of osteoporosis, diabetes, hyperlipidemia, and hypertension in group 3 were statistically higher than in groups 1 and 2 (P ≤ 0.05). CONCLUSION: In the treatment of RA, the incidence of skin rash, liver dysfunction, and oral ulcers may be decreased with combination therapy using LEF, MTX, and 7.5 mg prednisone, and blood pressure, blood glucose concentration, and bone density are not increased. Most important, 7.5 mg prednisone was synergistic with LEF and MTX, and such combination therapy could be a useful option as initial treatment of early active RA.

[Evaluating type I interferon-inducible gene expression in patients with systemic lupus erythematosus.].

OBJECTIVE: To explore the expression levels of interferon-inducible genes in patients with systemic lupus erythematosus (SLE), and to validate these gene expressions as potential biomarkers for the differentiation of disease flare and infection. METHODS: Peripheral blood was obtained from 48 SLE, 16 rheumatoid arthritis (RA) patients and 26 normal controls, and total RNA was extracted and reverse transcribed into complementary DNA. Real-time PCR technique was used to determine the gene expressions of MX1, OASL, OAS1, ISG15 and LY6E at transcription level. Univariate logistic regression analysis and multivariate conditional logistic regression model were applied to analyze 5 related factors for infection or activity. RESULTS: (1) The expression levels of MX1, OASL, OAS1, ISG15, and LY6E mRNA in SLE patients were significantly increased as compared with normal controls (P all < 0.01), while the expression levels of OASL, OAS1, ISG15 and LY6E mRNA in SLE patients were also higher than those in RA patients (P all < 0.05). (2) There were no significant difference between male and female patients of the 5 gene expression in SLE patients. (3) By logistic regression analysis, ISG15 and LY6E were independent risk factors for active SLE patients (P < 0.01), OASL expression was an independent risk factor for SLE patients with infection (P = 0.003). CONCLUSION: All the 5 interferon inducible genes are highly expressed in SLE patients, in which ISG15 and LY6E are independently associated with disease flare, while OASL may be helpful for the evaluation of infection in SLE patients.

[Gene expression of OAZ pathway in the patients of systemic lupus erythematosus].

OBJECTIVE: To explore the role of Olf/EBF associated zinc finger protein (OAZ) pathway in the RNA expression level in patients with systemic lupus erythematosus (SLE). METHODS: The expression levels of OAZ, BMP6, BMP4, Id3, Smad6, EHZF genes were valuated in bone marrow progenitor cells of 5 SLE patients and 5 normal subjects and replicated in peripheral blood cells of 30 SLE patients and 20 normal individuals by using real-time quantitative PCR technique. Relationships of the expression levels of OAZ, BMP6, BMP4, Id3 mRNA with disease activity and other clinical indices were analyzed. RESULTS: The expression levels of OAZ and Id3 mRNA (deltaCt) in the bone marrow (10.6 +/- 0.5, 5.8 +/- 3.2) and peripheral blood (14.1 +/- 2.7, 7.5 +/- 1.8) of SLE patients significantly increased than those observed in normal controls (16.5 +/- 0.9, 10.4 +/- 2.6, 16.1 +/- 2.2, 9.5 +/- 1.7), which was found to negatively correlate with SLEDAI score, renal lesion index , titers of anti-dsDNA and anti-RNA antibodies, but positively correlate with serum complement C3. Expression levels of BMP4 and BMP6 were differentially expressed in peripheral blood cells but not bone marrow progenitor cells. CONCLUSIONS: OAZ pathway is involved in the pathogenesis of SLE. Further investigation is required for the understanding of the function of these abnormally expressed genes.

Abnormal surface markers expression on bone marrow CD34+ cells and correlation with disease activity in patients with systemic lupus erythematosus.

Defects of hematopoietic stem cells (HSCs) have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the phenotypic characteristics of bone marrow (BM) CD34(+) cells in patients with SLE and its relationship with SLE disease activity. Ten SLE patients and 10 healthy subjects were recruited and their BM CD34(+) cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L, and HLA-DR. The percentage of BM CD34(+) cells was significantly decreased in active SLE patients (1.48 +/- 0.41%, n = 7) compared to the healthy controls (2.31 +/- 0.75%, n = 10, p < 0.01), but no significant difference was found between the inactive patients (2.04 +/- 0.44%, n = 3) and the controls. The expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in SLE patients (48.31 +/- 10.59%, 44.9 +/- 21.5%, 30.9 +/- 19.54%, respectively, n = 10) when compared with the control subjects (24.33 +/- 11.1%, 19.5 +/- 4.4%, 10.7 +/- 5.5%, respectively, n = 10, p < 0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r = -0.700, p < 0.05, n = 10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r = 0.472, p < 0.05, n = 10) and 24 h proteinuria (r = 0.558, p < 0.05, n = 10), but negatively correlated with serum C3 level (r = -0.712, p < 0.01, n = 10). Our study found that the surface marker expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of the HSC in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study.

Lymphocyte apoptosis and macrophage function: correlation with disease activity in systemic lupus erythematosus.

Increased lymphocyte apoptosis and defects in macrophage removal of apoptotic cells have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the relationship between peripheral lymphocyte apoptosis, macrophage function as determined by the serum levels of neopterin and interferon-gamma (IFN-gamma), and SLE disease activity. Peripheral apoptotic lymphocytes (AL) were detected by annexin V-fluorescein isothiocyanate (FITC) staining and flow cytometry. Serum levels of neopterin and IFN-gamma were measured by enzyme-linked immunosorbent assay (ELISA). SLE disease activity was determined using the systemic lupus activity measure (SLAM) and the serum titer of anti-dsDNA antibodies. The percentage of AL in the peripheral blood of active SLE patients was significantly higher (13.07+/-7.39%, n=30) than that of the inactive SLE patients (4.08+/-3.55%, n=8, p<0.01) and normal controls (5.13+/-3.37%, n=11, p<0.01). Serum levels of neopterin in active SLE patients were significantly higher (1.39+/-1.10 microg/dl, n=22) than in controls (0.26+/-0.19 microg/dl, n=20, p<0.01). Serum levels of IFN-gamma in active SLE patients were elevated (58.97+/-34.52 ng/l, n=15) when compared with controls (28.06+/-2.35 ng/l, n=16, p<0.05). The percentage of AL correlated significantly with serum levels of neopterin (r=0.446, p<0.05, n=22) and SLAM score (r=0.533, p<0.001, n=38), but not with the serum levels of IFN-gamma. The SLAM score also correlated with the serum levels of neopterin (r=0.485, p<0.05, n=22), but not with those of IFN-gamma. Our study supported the hypothesis that increased lymphocyte apoptosis has a pathogenic role in SLE. The increased levels of serum neopterin may suggest an attempt of the patients' macrophage system to remove the apoptotic cell excess. Since serum levels of neopterin correlated with the overall lupus disease activity, they may be regarded as an index of SLE disease activity.

[Variations within OLF1/EBF-associated zinc finger protein gene confer susceptibility to lupus nephritis in Chinese population].

OBJECTIVE: To observe whether single nucleotide polymorphisms (SNPs) within the OLF1/EBF-associated zinc finger protein (OAZ) gene are associated with lupus nephritis (LN) susceptibility in Chinese population. METHODS: Eight SNPs located around the positive microsatellite marker D16S517 within OAZ gene with relatively high heterozygosity were chosen for genotyping on 184 systemic lupus erythromatosus (SLE) patients, including 101 non-LN patients and 83 LN patients, and 286 normal controls using TaqMan MGB allelic discrimination method. Data were collected by SDS 2.0 software. Haplotypes and their frequencies were constructed and estimated, and linkage disequilibrium analysis between pairs of SNPs was evaluated by calculating the D Prime using Helixtree program. Case-control study was performed between the SLE, LN, and non-LN groups and normal control group. RESULTS: (1) The frequency of SNP rs1344531 T allele was 47.0% in the SLE patients, significantly higher than that in the controls [38.1%,; chi(2) = 7.300, P = 0.008, OR (95% CI) = 1.441 (1.105 - 1.878)], which showed that the frequency of SNP rs1344531 T allele is associated with SLE susceptibility. The genotypic distribution of SNP rs1344531(CC/CT/TT) differed significantly between the SLE patients (25.5%/54.9%/19.6%) and normal controls (38.1%/47.6%/14.3%) (chi(2) = 8.394, P = 0.015). The CC genotype frequency of the SLE patients was 25.5%, significantly lower than that of the normal controls [38.1%; chi(2) = 7.976, P = 0.005, OR (95% CI) = 0.557 (0.370 - 0.838)] (2) The SNP rs1344531 T allele frequency of the SLE patients was 53.0%, significantly higher than that of the normal controls [38.1%; chi(2) = 11.769, P = 0.001, OR (95% CI) = 1.832 (1.293 - 2.596)], which showed an associated between SNP rs1344531 T allele frequency and LN susceptibility. The genotypic distribution of SNP rs1344531 (CC/CT/TT) differed significantly between the LN patients (22.9%/48.2%/28.9%) and the normal controls (38.1%/47.6%/14.3%) (chi(2) = 12.065, P = 0.002). The CC genotype frequency of the LN patients was 22.9%, significantly lower than that of the normal controls (38.1%) [chi(2) = 6.578, P = 0.013, OR (95% CI) = 0.481 (0.274 - 0.848)]. The TT genotype frequency of the LN patients was 28.9%, significantly higher than that of the normal controls (14.3%) [chi(2) = 9.423, P = 0.003, OR (95% CI) = 2.431 (1.363 - 4.334)]. No statistical significance was observed between the non-LN patients and normal controls in TT genotype frequency. (3) The frequencies of haplotypes containing rs1344531:rs1420676-rs1344531(C-T) [chi(2) = 11.731, P = 0.001, OR (95% CI) = 1.867 (1.302 - 2.676)], rs3803665-rs1420676-rs1344531(C-C-T) [chi(2) = 8.876, P = 0.004, OR (95% CI) = 1.772 (1.213 - 2.589)], and rs2292155-rs3803665-rs1420676-rs1344531(C-C-C-T) [chi(2) = 9.962, P = 0.002, OR (95% CI) = 1.915 (1.274 - 2.880)] were all significantly higher in the LN patients in comparison with the normal control group (41.0% versus 27.1%; 33.3% versus 21.8%; and 27.0% versus 16.2%); while the frequencies of other haplotypes: rs1344531-rs2080353(C-A) [chi(2) = 8.06, P = 0.005, OR (95% CI) = 0.603 (0.424 - 0.856)], rs1344531-rs2080353-rs933564 (C-A-G) [chi(2) = 7.929, P = 0.006, OR (95% CI) = 0.602 (0.422 - 0.859)] were significantly lower than those of the normal control group (39.5% versus 52.2%, and 36.6% versus 49.2%), which produced additional support for such association. CONCLUSION: SNP rs1344531 and some haplotypes containing SNP rs1344531 within OAZ are significantly associated with LN susceptibility. Genetic variants of the OAZ gene are involved in the pathogenesis of LN.

Single nucleotide polymorphisms of deoxyribonuclease I and their expression in Chinese systemic lupus erythematosus patients.

BACKGROUND: Previous studies have suggested that interrupted clearance of nuclear DNA-protein complexes after cell death might initiate and propagate systemic lupus erythematosus (SLE). Deoxyribonuclease I (DNaseI) may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover, thus preventing the onset of SLE. The purpose of this study was to genotype the single nucleotide polymorphisms (SNPs) of DNase1 and characterize its gene expression and alternatively spliced transcripts in Chinese patients with SLE in order to understand the pathogenic role of DNase1 in human SLE. METHODS: Four SNPs located at the 3' end of the DNase1 gene, as listed on the SNP website, were selected for analysis. Those SNPs with relatively high heterozygosity were chosen for genotyping in 312 Chinese SLE families using the Taqman minor groove binder (MGB) allelic discrimination method. Haplotypes were constructed and linkage disequilibrium tests were performed using GeneHunter. DNase1 mRNA expression was detected using real-time polymerase chain reaction (PCR), and alternatively spliced transcripts were isolated using capillary electrophoresis. Any effects the specific SNP haplotypes had on DNase1 gene expression and the alternatively spliced transcripts were also assessed. RESULTS: rs179982 and rs1053874 had high heterozygosity, about 0.5 in this Chinese cohort, while rs1059857 was also found to be heterozygous. Analysis of the haplotype combining rs179982-rs1030874 (C-G) and rs179982-rs1030874-rs1059857 (C-G-G) revealed a skewed transmission in favor of affected offspring. DNase1 gene expression was higher in SLE patients than in normal controls (P < 0.001), but this was not related to disease activity or SNP haplotype. Capillary electrophoresis revealed that the pattern of alternatively spliced transcripts in patients differed from that of normal controls. Furthermore, different SNP haplotype combinations generated different transcript patterns in SLE patients. CONCLUSIONS: The SNP haplotypes are in linkage disequilibrium in Chinese SLE patients and may induce the disease through a modification of DNase1 mRNA splicing rather than at the level of mRNA expression. There is a relatively unique transcript band in SLE patients independent of special haplotype, which suggests that other unknown factors might be involved in adjusting gene expression.

[Deoxyribonuclease I gene expression in systemic lupus erythematosus patients].

OBJECTIVE: To observe whether deoxyribonuclease I (DNASE1) gene expression and its DNASE1 mRNA expression was detected by real-time polymerase chain reaction and its alternatively spliced transcripts were performed by capillary electrophoresis. An analysis was also made to disclose whether specific single nucleotide polymorphisms (SNPs) haplotype had effects onDNASE1 gene expression and its alternatively spliced transcripts. RESULTS: DNASE1 gene expression was higher in SLE patients than in normal controls (P<0.001), and in patients it was found to be of no relationship with SLE disease activity index score. However, it was increased in female patients. Capillary electrophoresis revealed that the pattern of alternatively spliced transcripts in patients was not the same as that in normal controls. Moreover, it seemed that different SNPs haplotype combination might show different transcript pattern in SLE patients. CONCLUSION: In SLE patients, DNASE1 gene expression is abnormal and there are alternatively spliced transcripts different from those in normal controls. DNASE1 gene is a critical factor in the pathogenesis of SLE.

[Susceptibility gene of systemic lupus erythematosus in 16q12 in Chinese cohort].

OBJECTIVE: To localize susceptibility loci in Chinese systemic lupus erythematosus (SLE) cohort by linkage disequilibrium mapping the genomic interval in human chromosome 16 so as to understand whether the pathogenesis of human SLE is related to chromosome 16. METHODS: Five microsatellite markers in chromosome 16 spanning from 57.79 cM to 65.1 cM were used for fluorescence based genotyping in 157 SLE families. Evidence for linkage disequilibrium was assessed using the extended transmission disequilibrium test (ETDT) program and Genehunter software. Susceptibility gene was searched in the positive locus, and real-time PCR method was used to detect gene expression. RESULTS: With the ETDT, evidence for linkage disequilibrium of the marker D16S409 (P=0.0278) and D16S517 (P<0.0001) with SLE were found. It was observed that 271 bp allele of D16S517 was much in evidence for preferential transmission, while 277 bp allele was found to be transmitted less often than expected. Genehunter analysis is concordant with ETDT. By real-time PCR, OAZ(OLF1/ EBF-associated zinc finger protein) gene which is located in the positive locus showed higher expression in SLE patients than in normal controls. CONCLUSION: Results reveal that 16q12 (58.46 cM) is associated with Chinese SLE. OAZ is a likely susceptibility gene in this locus for SLE.

[OAZ gene polymorphism in Chinese patients with systemic lupus erythematosus].

OBJECTIVE: To observe the association between systemic lupus erythematosus (SLE) and gene polymorphisms of OLF-1/EBF associated zinc finger protein(OAZ). METHODS: Verified single nucleotide polymorphisms (SNPs) with relatively high heterozygosity were chosen for allelic discrimination in 244 Chinese SLE pedigrees. Then transmissions of single SNP, and haplotypes were calculated by Genehunter software..OAZ mRNA level was also measured for comparing gene expression in patients of different haplotypes. RESULTS: Genotyping of five SNPs within OAZ gene introns indicated there was no preferential transmission of single SNP, and haplotype T-A-G-G for rs1344531-rs2080353-rs933564-rs1345431 showed only weak linkage with the disease (P=0.04). However, haplotypes combining SNPs and the SLE-associated D16S517 allele showed significant association with SLE susceptibility (for rs933564-d16s517 G-271bp t:non-t=93:29 P<0.000001, for rs2080353-rs933564-d16s517 A-G-271bp t:non-t=88:35 P=0.000002). The haplotype A-G-271bp-G of Rs2080353-rs933564-D16s517-rs1345431 was also transmitted to patients preferentially (P=0.0084) and it showed a tendency to affect gene expression. CONCLUSION: Special polymorphism haplotype of OAZ gene is associated with Chinese SLE. OAZ may suggest a new pathway for lupus.