Shunle Chen

Modulatory Effects of 1,25-Dihydroxyvitamin D3 on Human B Cell Differentiation

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)2D3 levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)2D3 on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)2D3, although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1α-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)2D3 and/or activation. Importantly, 1,25(OH)2D3 up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)2D3 may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.

Evidence for linkage of a candidate chromosome 1 region to human systemic lupus erythematosus.

Genetic susceptibility confers significant risk for systemic lupus erythematosus (SLE). The MHC region and other polymorphic loci have been associated with SLE. Because more compelling evidence for an involvement of a genetic locus includes linkage, we tested a candidate region homologous to a murine SLE susceptibility region in 52 SLE-affected sibpairs from three ethnic groups. We analyzed seven microsatellite markers from the human chromosome 1q31-q42 region corresponding to the telomeric end of mouse chromosome 1, the region where specific manifestations of murine lupus, including glomerulonephritis and IgG antichromatin, have been mapped. Comparing the mean allele sharing in affected sibpairs of each of these seven markers to their expected values of 0.50, only the five markers located at 1q41-q42 showed evidence for linkage (P = 0.0005-0.08). Serum levels of IgG antichromatin also showed evidence for linkage to two of these five markers (P = 0.04), suggesting that this phenotype is conserved between mice and humans. Compared to the expected random distribution, the trend of increased sharing of haplotypes was observed in affected sibpairs from three ethnic groups (P < 0.01). We concluded that this candidate 1q41-q42 region probably contains a susceptibility gene(s) that confers risk for SLE in multiple ethnic groups.

Analysis of gene expression profiles in human systemic lupus erythematosus using oligonucleotide microarray

Epidemiologic studies suggest a strong genetic component for susceptibility to systemic lupus erythematosus (SLE). To investigate the genetic mechanism of pathogenesis of SLE, we studied the difference in gene expression of peripheral blood cells between 10 SLE patients and 18 healthy controls using oligonucleotide microarray. When gene expression for patients was compared to the mean of normal controls, among the 3002 target genes, 61 genes were identified with greater than a two-fold change difference in expression level. Of these genes, 24 were upregulated and 37 downregulated in at least half of the patients. By the Welchs ANOVA/Welchs t-test, all these 61 genes were significantly different (P<0.05) between SLE patients and normal controls. Among these genes with differential expression, IFN-ω and Ly6E (TSA-1/Sca-2) may play an important role in the mechanism of SLE pathogenesis. TSA-1 antigens may represent an important alternative pathway for T-cell activation that may be involved in IFN-mediated immunomodulation. Hierarchical clustering showed that patient samples were clearly separated from controls based on their gene expression profile. These results demonstrate that high-density oligonucleotide microarray has the potential to explore the mechanism of pathogenesis of systemic lupus erythematosus.

miR-155 and its star-form partner miR-155* cooperatively regulate type I interferon production by human plasmacytoid dendritic cells

The recent discovery of microRNAs (miRNAs) has revealed a new layer of gene expression regulation, affecting the immune system. Here, we identify their roles in regulating human plasmacytoid dendritic cell (PDC) activation. miRNA profiling showed the significantly differential expression of 19 miRNAs in PDCs after Toll-like receptor 7 (TLR7) stimulation, among which miR-155* and miR-155 were the most highly induced. Although they were processed from a single precursor and were both induced by TLR7 through the c-Jun N-terminal kinase pathway, miR-155* and miR-155 had opposite effects on the regulation of type I interferon production by PDC. Further study indicated that miR-155* augmented interferon-α/β expression by suppressing IRAKM, whereas miR-155 inhibited their expression by targeting TAB2. Kinetic analysis of miR-155* and miR-155 induction revealed that miR-155* was mainly induced in the early stage of stimulation, and that miR-155 was mainly induced in the later stage, suggesting their cooperative involvement in PDC activation. Finally, we demonstrated that miR-155* and miR-155 were inversely regulated by autocrine/paracrine type I interferon and TLR7-activated KHSRP at the posttranscriptional level, which led to their different dynamic induction by TLR7. Thus, our study identified and validated novel miRNA-protein networks involved in regulating PDC activation.

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.

Pulmonary hypertension in systemic lupus erythematosus.

Abstract A prospective echocardiographic and clinical study was performed on 84 Chinese patients with systemic lupus erythematosus (SLE) and 99 controls to investigate the prevalence and the mechanism of pulmonary hypertension (PH) in SLE. Comparison between Doppler estimation and catheterization measurement was made in 12 cases to validate the predictive method. Compared to normal subjects, lupus patients had significantly increased sys-tolic pulmonary artery pressure (SPAP) (29.59±12.52 vs 19.64±5.82, P<0.001), mean pulmonary artery pressure (MPAP) (15.11±7.36 vs 10.21±4.72, P<0.001) and total pulmonary resistance (TPR) (315.85±190.65 vs 220.37± 55.92, P<0.001). Nine of the 84 patients presented PH, defined as SPAP >30 mmHg and MPAP >20 mmHg. Pulmonary hypertensive patients had higher serum endothelin (ET) than non-pulmonary hypertensive patients, were more commonly in active stages, and presented Raynauds phenomenon and rheumatoid factors. ET level was correlated with echocardiographic pulmonary pressure. Pulmonary hypertension commonly occurs in Chinese patients with SLE (11%), and it correlates with the lupus activity and the elevation of serum endothelin.

Adult clinically amyopathic dermatomyositis with rapid progressive interstitial lung disease : a retrospective cohort study

The aim of the study was to investigate the characteristics of adult clinically amyopathic dermatomyositis (CADM) with rapid progressive interstitial lung disease (ILD). Hospitalized patients with dermatomyositis (DM) and polymyositis (PM) between 1998 and 2005 in the Shanghai Renji Hospital were retrospectively studied. One hundred and forty-five patients were classified into CADM, classic DM or PM according to the modified Sontheimer’s definition or Bohan–Peter’s classification criteria. They were further stratified based on the presence or absence of clinical ILD. The Kaplan–Meier survival analysis and COX regression were performed. The predictive factors for ILD and other clinical properties of CADM-ILD were explored. The presence of clinical ILD was a significant risk factor for the poor outcome of DM/PM (OR = 4.237, CI 95%: 1.239–14.49, p = 0.021). Other risk factors are the presence of rashes and elevated urea nitrogen. Patients with DM/PM complicated by ILD had different clinical courses. Patients with CADM-ILD showed a rapidly progressive pattern with 6-month survival rate of 40.8%. The DM-ILD manifested a progressive pattern with a 5-year survival rate of 54%, while PM-ILD was chronic with 5- and 10-year survival rate of 72.4% and 60.3%, respectively. Better preserved muscle strength, elevated erythrocyte sedimentation rate, and hypoalbuminemia may herald ILD in DM/PM. Patients with CADM-ILD who later died had lower PO2, higher lactate dehydrogenase, and prominent arthritis/arthralgia compared with those who survived. The presence of antinuclear antibody seems to be protective. Rapid progressive CADM-ILD is refractory to conventional treatment. ILD is a common complication in over 40% of our hospitalized DM/PM cohort and is also a prominent prognostic indicator. CADM is a special phenotype of DM/PM. CADM-ILD, which is usually rapidly progressive and fatal, requires further investigation.

Identification of microRNA-31 as a novel regulator contributing to impaired interleukin-2 production in T cells from patients with systemic lupus erythematosus

OBJECTIVE MicroRNAs (miRNAs) function to fine-tune the control of immune cell signaling. It is well established that there are abnormalities in the interleukin-2 (IL-2)-related signaling pathways in systemic lupus erythematosus (SLE). The miR-31 microRNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-31 in IL-2 defects in lupus T cells. METHODS Expression levels of miR-31 were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-31. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-31 targets and effects. RESULTS The expression of miR-31 was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-31 in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-31 reduced IL-2 production. RhoA expression was directly repressed by miR-31 in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-31 in lupus T cells. Manipulation of miR-31 expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels. CONCLUSION MicroRNA-31 is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-31 and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.

Prognostic factors of lupus myelopathy

Myelopathy is a rare but severe neurological manifestation of systemic lupus erythematosus (SLE) with a high morbidity. The factors that contribute to prognosis are unknown. In this study, 14 patients with lupus myelopathy (LM) from our centre were retrospectively studied. Another 23 patients with other neuropsychiatric SLE (NPSLE) features were enrolled as a comparison group. The morbidity of LM was evaluated by the ASIA Impairment Scale. The clinical and serological characteristics and prognostic factors for LM were investigated. The age, gender, duration of SLE, non-CNS disease activity and autoantibody profile in patients with LM was not different in the NPSLE cohort. A relatively low prevalence of anti–phospholipid antibodies (aPL) in LM sera compared to NPSLE (28.6% vs 52.2%, P = 0.19) was observed. Longitudinal lesion detected by magnetic resonance imaging (MRI) was identified in 33.3% of patients with LM, whereas 50% showed focal speckle-like lesions. The morbidity of LM is 50%. Muscle strength of grade 3 or higher on admission was a strong indicator for a better prognosis (P = 0.006), whereas other parameters including longitudinal lesion, sensory deficit level, disease activity and aPL did not discriminate good from poor outcome in LM. Early aggressive immunosuppressive therapy (within 2 weeks of onset of myelopathy) tend to associate with a favourable outcome (P = 0.07).

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.