Increased Macroautophagy in Interferon-Gamma-Producing T Cells from Patients with Newly Diagnosed Systemic Lupus Erythematosus.

BACKGROUND: Imbalance of interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-17 producing by T cells is confirmed to contribute to the pathogenesis of systemic lupus erythematosus (SLE). Autophagy is now emerging as a core player in the development and the function of the immune system. Therefore, we investigated the autophagic behavior in IFN-γ-, IL-4-, and IL-17-producing T cells from patients with SLE. METHODS: Thirty patients with SLE and 25 healthy controls matched for gender and age were recruited between September 2016 and May 2017. The autophagic levels in IFN-γ+ T cells, IL-4+ T cells, and IL-17+ T cells from patients with newly diagnosed SLE and healthy controls were measured using flow cytometry. The plasma levels of IFN-γ were determined by enzyme-linked immunosorbent assay in SLE patients and healthy controls. Unpaired t-tests and the nonparametric Mann-Whitney U-test were used to compare data from patients with SLE and controls. Spearman's rank correlation coefficient was applied for calculation of the correlation between parallel variables in single samples. RESULTS: Our results showed increased percentage of autophagy in IFN-γ+ T cells from patients with SLE and healthy controls ([8.07 ± 2.72]% vs. [3.76 ± 1.67]%, t = 5.184, P < 0.001), but not in IL-4+ T cells or IL-17+ T cells (P > 0.05) as compared to healthy donors. Moreover, the plasma levels of IFN-γ in SLE patients were significantly higher than those in healthy controls ([68.9 ± 29.1] pg/ml vs. [24.7 ± 17.6] pg/ml, t = 5.430, P < 0.001). Moreover, in SLE patients, the percentage of autophagy in IFN-γ+ T cells was positively correlated with the plasma levels of IFN-γ (r = 0.344, P = 0.046), as well as the disease activity of patients with SLE (r = 0.379, P = 0.039). CONCLUSION: The results indicate that autophagy in IFN-γ+ T cells from SLE patients is activated, which might contribute to the persistence of T cells producing IFN-γ, such as Th1 cells, and consequently result in the high plasma levels of IFN-γ, and then enhance the disease activity of SLE.

WITHDRAWN Monocyte chemoattractant protein-1 promoter-2518 G allele increases renal damage in systemic lupus erythematosus patients: result from a meta-analysis.

Ahead of Print article withdrawn by publisher. Not clarified suspected conflict of interest.

Salvia miltiorrhiza injection restores apoptosis of fibroblast-like synoviocytes cultured with serum from patients with rheumatoid arthritis.

Salvia miltiorrhiza injection (SMI) is a water­soluble agent, derived from Salvia miltiorrhiza (SM), that is traditionally used to treat cardiovascular and cerebrovascular diseases. Furthermore it has been demonstrated to possess the ability to induce apoptosis of tumor cells. However, it remains unclear whether SMI can induce apoptosis of rheumatoid arthritis (RA) fibroblast­like synoviocytes (FLS), which are hyperplastic in RA due to defective apoptosis. There is also evidence that allogenic serum may be associated with the induction of apoptosis. The aim of the present study was to investigate the involvement of serum during SMI­induced apoptosis in RA FLS. The results demonstrated that SMI could induce apoptosis of RA FLS, cultured with fetal bovine serum (FBS), in a dose­dependent manner. In addition, SMI decreased the expression of nuclear factor­κB in RA FLS nuclear extracts and inhibited the secretion of tumor necrosis factor­α. Fas ligand expression was not detected in RA FLS, in either the presence or absence of SMI. The pro­apoptotic genes B­cell lymphoma 2 (Bcl­2) associated X protein (Bax) and Fas, were shown to be upregulated following SMI stimulation, whereas the expression levels of the anti­apoptotic gene Bcl­2, were downregulated. Upon replacement of FBS with normal human serum, the apoptotic rate and Bax mRNA expression levels following SMI stimulation, were unchanged. However, culturing RA FLS with patient' serum (RPS), restored the apoptotic rate and Bax mRNA expression levels following SMI stimulation. There may be numerous mechanisms by which SMI inhibits RA FLS proliferation. The present study demonstrated that SMI can restore apoptosis of RA FLS cultured with RPS. These results indicate that SMI may have a potential role in the treatment of synovial hyperplasia of RA.

Hydroxychloroquine facilitates autophagosome formation but not degradation to suppress the proliferation of cervical cancer SiHa cells.

Hydroxychloroquine (HCQ), the hydroxylated analog of chloroquine, is an antimalarial lysomotropic agent that inhibits autophagy due to lysosomal acidification, and subsequently blocks the fusion of autophagosomes with lysosomes which leads to the accumulation of autophagosomes that may accelerate tumor cell death. Given these hypothesis the aim of this study was to investigate the effects of HCQ in the inhibition of autophagy and the induction of apoptosis in cervical cancer SiHa cells. Cervical cancer SiHa cells were cultured with Hank's balanced salt solution (HBSS) as positive control of autophagy or treated with HCQ as part of the experimental groups. LC3 and P62/SQSTM1 were detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively in order to evaluate initially autophagosome formation and their degradation. Specific green fluorescent protein (GFP)-LC3 was subsequently detected by fluorescence microscopy in order to confirm the formation of autophagosomes. MTT and flow cytometry were adopted respectively to assess the proliferation and apoptosis of the SiHa cells. miRNA-9* was also investigated. The results demonstrated that HCQ increased the expressions of LC3 mRNA and LC3II protein and GFP-LC3 signalling but reduced the expression of p62/STSQM1 in cervical cancer SiHa cells. These results indicated HCQ has the ability to inhibit autophagy as incapable of degrading the autophagosome. However, HCQ may promote SiHa cell apoptosis as the MTT, apoptotic assay and miRNA-9* results revealed. HCQ has the ability to inhibit autophagy by blocking the degradation of autophagosomes and subsequently facilitates the apoptosis of cervical cancer SiHa cells.

[The association of vitamin D receptor gene ApaI and BsmI polymorphism with systemic lupus erythematosus].

OBJECTIVE: To determine the distribution of vitamin D receptor (VDR) gene ApaI and BsmI polymorphism in systemic lupus erythematosus (SLE) and the association with SLE in Chinese Han patients. METHODS: Genomic DNA from 244 Chinese SLE patients and 162 sex and ethnically matched controls were typed for VDR ApaI and BsmI polymorphism combination by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Clinical characteristics were analyzed between different ApaI and BsmI genotypes. RESULTS: There was no significant difference between the distribution frequencies of allelic gene A and a in SLE patients and the controls, but the distribution frequency of genotypes heterozygote Aa in SLE patients was higher than that in the controls (38.9% vs 22.2%, χ(2) = 12.442, P = 0.000). There was no significant difference between the distribution frequency of allelic gene and genotypes of BsmI in SLE patients and the controls (P > 0.05). However, there was significant difference between the distribution frequencies of ApaI and BsmI genotypes combination in SLE patients and the controls (χ(2) = 18.226, P = 0.006). The distribution frequency of genotypes Aa-bb in SLE patients was higher than that in the controls (32.4% vs 17.9%, χ(2) = 10.449 P = 0.001), while the distribution frequency of genotypes Aa-bb in SLE patients was lower than that in the controls (30.3% vs 42.0%, χ(2) = 5.808, P = 0.016). Furthermore, analyzing the effect of VDR ApaI and BsmI polymorphism combination to the symptoms of SLE, significant difference was observed in SLE patients carrying Aa-bb genotypes involved in serositis (P = 0.003), hematological system disorder (P = 0.021), and anti-Sm antibodies (P = 0.01) compared with other genotypes. CONCLUSION: There is significant association between ApaI and BsmI gene polymorphism Aa-bb genotypes and the incidence of SLE in the Han population of China, and genotype Aa-bb is more involved in serositis, hematological system disorder and has a positive effect on production of antibodies.

[Relationship of vitamin D receptor gene Fok I polymorphism with systemic lupus erythematosus].

AIM: To investigate the relationship of vitamin D receptor (VDR) gene Fok I polymorphism with systemic lupus erythematosus (SLE) and to observe VDR mRNA levels in Chinese Han SLE patients. METHODS: Genomic DNAs from 271 Chinese SLE patients and 130 healthy controls were determined for Fok I polymorphism by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), and VDR mRNA levels from 48 Chinese SLE patients and 38 healthy controls were detected by real-time polymerase chain reaction (RT-PCR). RESULTS: Gene frequencies of allelic F and f were significantly different between the SLE patients and the controls (P=0.001).The relative risk of SLE in the presence of allelic gene F was 1.630 (95%CI=1.210-2.196, P=0.001). The frequency of homozygote FF in the SLE patients was higher than that in the controls (42.8% vs 25.4%, x(2);=11.417, P=0.001). Serositis, anti-dsDNA antibody, anti-Sm antibody and anti-histone antibody in the SLE patients carrying homozygote FF and heterozygote Ff were higher than those in the SLE patients carrying homozygote ff (P=0.001, P=0.001, P=0.047, P=0.001, respectively). The VDR mRNA was decreased in the SLE patients, with a delta;Ct value of 9.26 ± 2.37 (P=0.026), as compared with a delta;Ct value of 7.82 ± 3.05 in the controls (the bigger of the delta;Ct value, the lower of VDR mRNA expression). The delta;Ct value of VDR mRNA in the SLE patients carrying FF and Ff was bigger than that in the SLE patients carrying ff (10.54 ± 1.88 vs 7.15 ± 3.78, P=0.019). CONCLUSION: VDR gene Fok I polymorphism is associated with SLE in the Han population of southern China. The SLE patients carrying F allel ± are more likely to have serositis and produce anti-dsDNA antibody, anti-Sm antibody and anti-Histone antibody, presumably as a result of down-regulation of VDR mRNA.

[Detecting anti-megakaryocyte antibodies in serum of systemic lupus erythematosus patients by indirect immunofluorescence].

This study was purposed to investigate the mechanism of thrombocytopenia in patients with systemic lupus erythematosus (SLE) through detecting anti-megakaryocyte antibodies in SLE patients. The serum anti-megakaryocyte antibodies in 36 SLE cases with thrombocytopenia were detected by using indirect immunofluorescence, the detected results were compared with detected results of 30 SLE cases without thrombocytopenia and 30 healthy persons. The results showed that the positive incidences of anti-megakaryocyte antibody in serum of 36 SLE cases with thrombocytopenia, 30 SLE cases without thrombocytopenia and 30 healthy persons were 19.4% (7/36), 6.7% (2/30) and 3.3% (1/30) respectively. As compared with SLE patients without thrombocytopenia and healthy persons, SLE patients with thrombocytopenia had higher incidence of anti-megakaryocyte antibodies, moreover there was significant difference between SLE patients with thrombocytopenia and healthy persons (p < 0.05), while there was no significant difference between SLE patients with or without thrombocytopenia (p > 0.05). It is concluded that autoantibodies against megakaryocytes exist in SLE patients and may partially contribute to the incidence of thrombocytopenia in SLE patients. The detection of anti-megakaryocyte antibodies with a enough case number is needed to make a final conclusion on thrombocytopenia pathogenesis in SLE.

Expression of FLICE-inhibitory protein in synovial tissue and its association with synovial inflammation in juvenile idiopathic arthritis.

OBJECTIVE: To examine the expression of FLICE-inhibitory protein (FLIP) in juvenile idiopathic arthritis (JIA) and analyze its correlation with synovial inflammation. METHODS: The expression of FLIP was assessed in 11 JIA and 3 normal synovial tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The cell types expressing FLIP were further characterized, and the correlation of FLIP expression with the degree of synovial inflammation, as well as the activity of caspase 8 was then analyzed. RESULTS: RT-PCR revealed the expression of FLIP mRNA in all 11 JIA samples, but not in 3 normal synovial tissues. In JIA, FLIP expression could be found in both the lining and sublining layers, mainly in the macrophage-like cells. Moreover, the expression of FLIP in JIA synovial tissues was positively correlated with the degree of synovial inflammation (r = 0.563, P < 0.05). CONCLUSION: The expression of antiapoptotic FLIP in JIA synovial tissue and its correlation to accumulation of inflammatory cells in synovial tissue suggests that FLIP potentially extends the lifespan of synovial cells and thus contributes to the progression of joint destruction.