Qiong Fu

Sex-specific association of X-linked Toll-like receptor 7 (TLR7) with male systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multisystem, autoimmune disease that predominantly affects women. Previous findings that duplicated Toll-like receptor 7 (Tlr7) promotes lupus-like disease in male BXSB mice prompted us to evaluate TLR7 in human SLE. By using a candidate gene approach, we identified and replicated association of a TLR7 3′UTR SNP, rs3853839 (G/C), with SLE in 9,274 Eastern Asians (Pcombined = 6.5 × 10−10), with a stronger effect in male than female subjects [odds ratio, male vs. female = 2.33 (95% CI = 1.64–3.30) vs. 1.24 (95% CI = 1.14–1.34); P = 4.1 × 10−4]. G-allele carriers had increased TLR7 transcripts and more pronounced IFN signature than C-allele carriers; heterozygotes had 2.7-fold higher transcripts of G-allele than C-allele. These data established a functional polymorphism in type I IFN pathway gene TLR7 predisposing to SLE, especially in Chinese and Japanese male subjects.

Association of a functional IRF7 variant with systemic lupus erythematosus

OBJECTIVE A previous genome-wide association study conducted in a population of European ancestry identified rs4963128, a KIAA1542 single-nucleotide polymorphism (SNP) 23 kb telomeric to IRF7 (the gene for interferon regulatory factor 7 [IRF-7]), to be strongly associated with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether genetic polymorphism within IRF7 is a risk factor for the development of SLE. METHODS We genotyped one KIAA1542 SNP (rs4963128) and one IRF7 SNP (rs1131665 [Q412R]) in an Asian population (1,302 cases, 1,479 controls), to assess their association with SLE. Subsequently, rs1131665 was further genotyped in independent panels of Chinese subjects (528 cases, 527 controls), European American subjects (446 cases, 461 controls), and African American subjects (159 cases, 115 controls) by TaqMan genotyping assay, to seek confirmation of association in various ethnic groups. A luciferase reporter assay was used to assess the effect of Q412R polymorphism on the activation of IRF-7. RESULTS Consistent association of rs1131665 (Q412R) with SLE was identified in Asian, European American, and African American populations (total 2,435 cases and 2,582 controls) (P(meta) = 6.18 × 10(-6) , odds ratio 1.42 [95% confidence interval 1.22-1.65]). Expression of the IRF7 412Q risk allele resulted in a 2-fold increase in interferon-stimulated response element transcriptional activity compared with expression of IRF7 412R (P = 0.0003), suggesting that IRF7 412Q confers elevated IRF-7 activity and may therefore affect a downstream interferon pathway. CONCLUSION These findings show that the major allele of a nonsynonymous SNP, rs1131665 (412Q) in IRF7, confers elevated activation of IRF-7 and predisposes to the development of SLE in multiple ethnic groups. This result provides direct genetic evidence that IRF7 may be a risk gene for human SLE.

Association of elevated transcript levels of interferon-inducible chemokines with disease activity and organ damage in systemic lupus erythematosus patients

IntroductionSystemic lupus erythematosus (SLE) is a multi-system autoimmune disease with a heterogeneous course and varying degrees of severity and organ damage; thus, there is increasing interest in identifying biomarkers for SLE. In this study we correlated the combined expression level of multiple interferon-inducible chemokines with disease activity, degree of organ damage and clinical features in SLE, and we investigated their roles as biomarkers.MethodsPeripheral blood cells obtained from 67 patients with SLE patients, 20 patients with rheumatoid arthritis (RA) and 23 healthy donors were subjected to real-time PCR in order to measure the transcriptional levels of seven interferon-inducible chemokines (RANTES, MCP-1, CCL19, MIG, IP-10, CXCL11, and IL-8). The data were used to calculate a chemokine score for each participant, after which comparisons were performed between various groups of SLE patients and control individuals.ResultsChemokine scores were significantly elevated in SLE patients versus RA patients and healthy donors (P = 0.012 and P = 0.002, respectively). Chemokine scores were correlated positively with SLE Disease Activity Index 2000 scores (P = 0.005) and negatively with C3 levels (P < 0.001). Compared with patients without lupus nephritis and those with inactive lupus nephritis, chemokine scores were elevated in patients with active lupus nephritis, especially when their daily prednisone dosage was under 30 mg (P = 0.002 and P = 0.014, respectively). Elevated chemokine scores were also associated with the presence of cumulative organ damage (Systemic Lupus International Collaborating Clinics/American Society of Rheumatology Damage Index ≥ 1; P = 0.010) and the occurrence of anti-Sm or anti-RNP autoantibodies (both P = 0.021).ConclusionsThe combined transcription level of interferon-inducible chemokines in peripheral blood leucocytes is closely associated with disease activity, degree of organ damage, and specific autoantibody patterns in SLE. The chemokine score may serve as a new biomarker for active and severe disease in SLE.

Interferon-α priming promotes lipid uptake and macrophage-derived foam cell formation: a novel link between interferon-α and atherosclerosis in lupus.

OBJECTIVE An increased risk of premature atherosclerosis has been associated with systemic lupus erythematosus (SLE), and type I interferon (IFN) has been shown to play a pathogenic role in human SLE. The aim of this study was to determine whether IFNα is involved in the development of atherosclerosis in patients with SLE by promoting lipid uptake and macrophage-derived foam cell formation, which is a crucial step in early atherosclerosis. METHODS The effects of IFNα on lipid uptake and foam cell formation were determined by flow cytometry and oil red O staining. Messenger RNA and protein expression of scavenger receptors (SRs) was examined. Promoter activity was detected by luciferase reporter assay. Expression of macrophage SR class A (SR-A) and IFN-inducible genes (IFIGs) was measured in peripheral blood mononuclear cells obtained from 42 patients with SLE and 42 healthy donors. RESULTS IFNα priming increased the uptake of oxidized low-density lipoprotein and hence enhanced foam cell formation by up-regulating SR-A expression. IFNα increased SR-A expression via enhancing its promoter activities. Examination using signaling inhibitors revealed that a phosphatidylinositol 3-kinase/Akt signaling pathway appeared to be involved in this process. Notably, SR-A messenger RNA was significantly increased in patients with SLE compared to normal subjects and positively correlated with IFIG expression. CONCLUSION Our data suggest that IFNα priming up-regulated the expression of SR-A in human monocyte/macrophages, leading to increased lipid uptake and foam cell formation. Activation of the IFN signaling pathway may be linked to the risk of atherosclerosis by affecting plaque formation in patients with SLE. These findings provide novel insights into the mechanisms of and potential therapeutic approaches to premature atherosclerosis in patients with SLE.

T-614, a novel immunomodulator, attenuates joint inflammation and articular damage in collagen-induced arthritis.

IntroductionT-614 is a novel oral antirheumatic agent for the treatment of rheumatoid arthritis. Whether it has immunomodulatory or disease-modifying properties and its mechanism of action are largely undetermined.MethodsRats with collagen-induced arthritis (CIA) were treated with T-614 (5 and 20 mg/kg) daily. Animals receiving methotrexate (1 mg/kg every 3 days) and the nonsteroidal anti-inflammatory agent nimesulide (10 mg/kg per day) were used as controls. A combination therapy group was treated with both T-614(10 mg/kg per day) and methotrexate (1 mg/kg every 3 days). Hind paw swelling was evaluated and radiographic scores calculated. Serum cytokine levels were assessed by Bio-plex analysis. Quantitative PCR was used to evaluate expression of mRNA for interferon-γ, IL-4 and IL-17. Serum IL-17 and anti-type II collagen antibodies (total IgG, IgG1, IgG2a, IgG2b and IgM) were measured using ELISA.ResultsOral T-614 inhibited paw swelling and offered significant protection against arthritis-induced cartilage and bone erosion, comparable to the effects of methotrexate. CIA rats treated with T-614 exhibited decreases in both mRNA expression of IL-17 in peripheral blood mononuclear cells and lymph node cells, and circulating IL-17 in a dose-dependent manner. T-614 also reduced serum levels of tumor necrosis factor-α, IL-1β and IL-6. A synergistic effect was observed for the combination of methotrexate and T-614. In addition, T-614 (20 mg/kg per day) depressed production of anti-type II collagen antibodies and differentially affected levels of IgG2a subclasses in vivo, whereas IgM level was decreased without any change in the IgG1 level. Together, the findings presented here indicate that the novel agent T-614 has disease-modifying effects against experimental arthritis, as opposed to nimesulide.ConclusionsOur data suggested that T-614 is an effective disease-modifying agent that can prevent bone/cartilage destruction and inflammation in in CIA rats. Combination with methotrexate markedly enhances the therapeutic effect of T-614.

Targeting miR-155 to Treat Experimental Scleroderma

Scleroderma is a refractory autoimmune skin fibrotic disorder. Alterations of microRNAs in lesional skin could be a new approach to treating the disease. Here, we found that expression of miR-155 was up regulated in lesional skin tissue from patients with either systemic or localized scleroderma, and correlated with fibrosis area. Then we demonstrated the potential of miR-155 as a therapeutic target in pre-clinical scleroderma models. MiR-155−/− mice were resistant to bleomycin induced skin fibrosis. Moreover, topical antagomiR-155 could effectively treat mice primed with subcutaneous bleomycin. In primary skin fibroblast, miR-155 silencing could inhibit collagen synthesis function, as well as signaling intensity of two pro-fibrotic pathways, Wnt/β-catenin and Akt, simultaneously. We further showed that miR-155 could regulate the two pathways via directly targeting casein kinase 1α (CK1α) and Src homology 2-containing inositol phosphatase-1 (SHIP-1), as previous reports. Mice with miR-155 knockout or topical antagomir-155 treatment showed inhibited Wnt/β-catenin and Akt signaling in skin upon bleomycin challenge. Together, our data suggest the potential of miR-155 silencing as a promising treatment for dermal fibrosis, especially in topical applications.

Prevention of Immune Nephritis by the Small Molecular Weight Immunomodulator Iguratimod in MRL/lpr Mice

Objective This study was performed to investigate the therapeutic effects of iguratimod in a lupus mouse model. Methods Female MRL/lpr mice were treated with iguratimod, vehicle solution or cyclophosphamide. Proteinuria was monitored and kidney injury was blindly scored by a renal pathologist. Serum anti-double-stranded DNA antibodies were monitored by radioimmunoassay. Kidney IgG and CD20 were stained by immunohistochemistry. Splenic lymphocyte phenotypes were analyzed by flow cytometry. BAFF, IL-17A, IL-6, and IL-21 levels in serum and splenic lymphocytes were detected by ELISA or quantitative PCR. Results Compared with the vehicle-treated controls, MRL/lpr mice treated with iguratimod showed less protenuria, less acute pathological lesions and no chronic changes in the kidneys. There were significant differences in glomerular injury and vasculitis scores, as well as in the semi-quantitave analysis of immune complex deposition between the two groups. Disease activity markers in sera (anti-dsDNA antibodies and immunoglobulin levels) were reduced and hypocomplementemia was attenuated. Lymphocyte expression of BAFF, IL-6, IL-17A and IL-21 was decreased. The abnormal splenic B220+ T cell and plasma cell populations in MRL/lpr mice were reduced by iguratimod treatment, with recovery of the total B cell population and inhibition of B cell infiltration of the kidney tissue. The dosage of iguratimod used in this study showed no significant cytotoxic effects in vivo and no overt side-effects were observed. Conclusion Iguratimod ameliorates immune nephritis in MRL/lpr mice via a non-antiproliferative mechanism. Our data suggest a potential therapeutic role of iguratimod in lupus.

Type I interferons promote the survival and proinflammatory properties of transitional B cells in systemic lupus erythematosus patients

A hallmark of systemic lupus erythematosus (SLE) is the breaking of B-cell tolerance with the generation of high-affinity autoantibodies; however, the antibody-independent features of the B-cell compartment in SLE are less understood. In this study, we performed an extensive examination of B-cell subsets and their proinflammatory properties in a Chinese cohort of new-onset SLE patients. We observed that SLE patients exhibited an increased frequency of transitional B cells compared with healthy donors and rheumatoid arthritis patients. Plasma from SLE patients potently promoted the survival of transitional B cells in a type I IFN-dependent manner, which can be recapitulated by direct IFN-α treatment. Furthermore, the effect of IFN-α on enhanced survival of transitional B cells was associated with NF-κB pathway activation and reduced expression of the pro-apoptotic molecule Bax. Transitional B cells from SLE patients harbored a higher capacity to produce proinflammatory cytokine IL-6, which was also linked to the overactivated type I IFN pathway. In addition, the frequency of IL-6-producing transitional B cells was positively correlated with disease activity in SLE patients, and these cells were significantly reduced after short-term standard therapies. Thus, the current study provides a direct link between type I IFN pathway overactivation and the abnormally high frequency and proinflammatory properties of transitional B cells in active SLE patients, which contributes to the understanding of the roles of type I IFNs and B cells in the pathogenesis of SLE.

319 Association of functional irf7 variants with systemic lupus erythematosus

Background and aims Previous study identified rs1131665 in IRF7 associated with SLE among multiple ethnic groups. This study was undertaken to investigate whether other genetic polymorphisms within KIAA1542/IRF7 confers risk for the development of SLE. Methods Four SNPs, including rs4963128, rs702966, rs1131665 (Q412R), rs1061502 (K179E) within KIAA1542/IRF7 were genotyped in 784 Chinese SLE patients and 899 controls/IRF7 by using Taqman genotyping assay. Luciferase reporter assay, Co-IP and EMSA were used to assess the effect of K179E polymorphism on the activation of IRF7. Results Q412R and K179E were significantly associated with SLE in Chinese Han population (p=5.8X10-3, OR=2.33[1.26–4.33], p=2.9X10-3, OR=2.82[1.38–5.76], respectively. IRF7 3’UTR SNP rs702966 was associated with renal involvement (p=0.01 OR=0.46[0.25–0.85]. Compared with expression of IRF7 179E, expression of IRF7 179K risk allele resulted in a 4-fold increase in ISRE transcriptional activity and stronger ISRE binding activity in EMSA (p=0.0002), suggesting IRF7 179K confers elevated IRF7 activity. Further study found 179K (lysine) carrying IRF7 protein showed higher acetylation compared to 179 E (glutamic acid) IRF7. Conclusions We detected a novel association between rs1061502 (K179E) and SLE susceptibility. K179E could change the acetylation of IRF7 in vitro, which might contribute to the transcriptional activity of IRF7.

318 Autophagy-related protein p62 expression is associated with clinicopathologic features and prednisone plus ctx induction treatment efficacy in lupus nephritis

Background and aims Previous studies found autophagy contributes to the pathogenesis of systemic lupus erythematosus (SLE). Whether autophagy is involved in lupus nephritis (LN) is not elucidated. P62 is a specific substrate that is degraded through autophagy-lysosomal pathway. Methods Immunohistochemistry Staining was performed to evaluate expressions of p62 in the biopsy kidney tissue of LN patients (n=128) and normal control (n=6). One hundred and five patients were given prednisone+CTX pulse therapy as induction treatment and followed by 24 weeks. Clinicopathologic features and induction phase remission efficacy were recorded and correlated with renal p62 expression level. Results Compared with the controls, the expression of p62 was significantly decreased in LN biopsy tissues (p=0.0013), suggesting increased autophagy in LN kidney. Patients with low expression of p62 had less severe nephritis, showing significantly less proteinuria, fewer interstitial fibrosis score and higher estimated creatinine clearance rates (p=0.0122, p=0.0048, p=0.0231, respectively). Logistic regression analysis revealed that lower renal p62 expression was an independent factor associated with CR(p=0.025) (Table 1). Patients with low p62 were more likely and quicker to achieve CR (Person Chi-Square test, p=0.001; Kaplan-Meier test, p=0.0294). Conclusions Low renal p62 expression was associated with less severe nephritis and better short-time outcome. Because low p62 expression is the result of high level of autophagy, this data suggested that autophagy might play a protective role in LN kidney. More studies are needed to evaluate the role autophagy plays in multiple organs and cell subtypes in SLE.