Xinfang Huang

MicroRNA-146a contributes to abnormal activation of the type I interferon pathway in human lupus by targeting the key signaling proteins

OBJECTIVEnMicroRNA have recently been identified as regulators that modulate target gene expression and are involved in shaping the immune response. This study was undertaken to investigate the contribution of microRNA-146a (miR-146a), which was identified in the pilot expression profiling step, to the pathogenesis of systemic lupus erythematosus (SLE).nnnMETHODSnTaqMan microRNA assays of peripheral blood leukocytes were used for comparison of expression levels of microRNA between SLE patients and controls. Transfection and stimulation of cultured cells were conducted to determine the biologic function of miR-146a. Bioinformatics prediction and validation by reporter gene assay and Western blotting were performed to identify miR-146a targets.nnnRESULTSnProfiling of 156 miRNA in SLE patients revealed the differential expression of multiple microRNA, including miR-146a, a negative regulator of innate immunity. Further analysis showed that underexpression of miR-146a negatively correlated with clinical disease activity and with interferon (IFN) scores in patients with SLE. Of note, overexpression of miR-146a reduced, while inhibition of endogenous miR-146a increased, the induction of type I IFNs in peripheral blood mononuclear cells (PBMCs). Furthermore, miR-146a directly repressed the transactivation downstream of type I IFN. At the molecular level, miR-146a could target IFN regulatory factor 5 and STAT-1. More importantly, introduction of miR-146a into the patients PBMCs alleviated the coordinate activation of the type I IFN pathway.nnnCONCLUSIONnThe microRNA miR-146a is a negative regulator of the IFN pathway. Underexpression of miR-146a contributes to alterations in the type I IFN pathway in lupus patients by targeting the key signaling proteins. The findings provide potential novel strategies for therapeutic intervention.

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.

MicroRNA-125a contributes to elevated inflammatory chemokine RANTES levels via targeting KLF13 in systemic lupus erythematosus

OBJECTIVEnMicroRNA (miRNA) have received increasing attention as posttranscriptional regulators that fine-tune the homeostasis of the inflammatory response. This study aimed to clarify whether miR-125a, which was identified in a pilot expression profiling step, is involved in the inflammatory chemokine pathway in systemic lupus erythematosus (SLE).nnnMETHODSnIndependent verification of miR-125a expression in amplified samples from SLE patients and normal controls was performed by TaqMan quantitative polymerase chain reaction (PCR) analysis. A combination of 3 bioinformatic prediction techniques and reporter gene assays was used to identify miR-125a targets. In vitro systems of overexpression by transfection and inducible expression by stimulation were performed to investigate the function of miR-125a, which was followed by real-time quantitative PCR and enzyme-linked immunosorbent assay.nnnRESULTSnIn SLE patients, the expression of miR-125a was reduced and the expression of its predicted target gene, KLF13, was increased. Bioinformatics predicted that miR-125a base-paired with sequences in the 3-untranslated region of KLF13. Overexpression of miR-125a led to a significant reduction in the expression of RANTES and KLF13. MicroRNA-125a inhibited endogenous KLF13 expression in a dose-dependent manner, as determined using gain- and loss-of-function methods. A luciferase reporter system confirmed the miR-125a binding sites. Notably, miR-125a expression was induced in T cells in a dose- and time-dependent manner. Finally, the introduction of miR-125a into T cells from SLE patients alleviated the elevated RANTES expression.nnnCONCLUSIONnMicroRNA-125a negatively regulates RANTES expression by targeting KLF13 in activated T cells. The underexpression of miR-125a contributes to the elevated expression of RANTES in SLE. Our findings extend the role of miRNA in the pathogenesis of lupus and provide potential strategies for therapeutic intervention.

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

OBJECTIVEnMicroRNAs (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.nnnMETHODSnExpression 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.nnnRESULTSnThe 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.nnnCONCLUSIONnMicroRNA-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.

Let-7/miR-98 regulate Fas and Fas-mediated apoptosis.

Fas is ubiquitously expressed on a variety of cells and triggers apoptosis, which have critical roles in the immune system. MicroRNAs (miRNAs) have been recently identified as regulators that modulate target gene expression and are involved in diverse biological processes, such as cell proliferation and apoptosis. This study was undertaken to investigate the contribution of miRNA in the regulation of Fas expression and Fas-mediated apoptosis. Bioinformatics analysis indicated that Fas was a potential target of let-7/miR-98 family. Indeed ectopic expression of let-7/miR-98 reduced, whereas knockdown of endogenous let-7/miR-98 increased the expression of Fas at both mRNA and protein levels. Let-7/miR-98 was verified to target Fas 3′ untranslated region directly by site-directed gene mutagenesis and reporter gene assay. More importantly, introduction of let-7/miR-98 could decrease the sensitivity to Fas-induced apoptosis. Furthermore, let-7/miR-98 expression was reduced in activation-induced cell death process, accompanied by increased expression of Fas. In conclusion, our study first demonstrated that let-7/miR-98 regulated Fas expression and the sensitivity of Fas-mediated apoptosis.

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.

Multidimensional Single Cell Based STAT Phosphorylation Profiling Identifies a Novel Biosignature for Evaluation of Systemic Lupus Erythematosus Activity

Introduction Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE), a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE. Methods Phospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA) patients and 13 healthy donors (HDs). A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10) were also monitored. Results We observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF), and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells. Conclusions The basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.

MicroRNA-125b/Lin28 pathway contributes to the mesendodermal fate decision of embryonic stem cells.

MicroRNAs (miRNAs) are important regulators of cell fate decisions, while the miRNAs and their targets in the regulation of stem cell differentiation are largely unidentified. Here we report novel functions of miR-125b/Lin28 axis in the regulation of mouse embryonic stem cell (mESC) lineage specification and cardiomyocyte differentiation. With a MicroRNA Array screen, we identified a number of miRNAs significantly changed during ESC differentiation, among which miR-125b showed a marked reduction during early differentiation. The abundantly expressed miR-125b in undifferentiated mESCs was dramatically downregulated to a level hardly detected during differentiation day 3 to 5, with a concomitant upregulation of Lin28. Ectopically expressing miR-125b did not alter characteristics of undifferentiated mESCs, whereas it impaired the endoderm and mesoderm development, but not the ectoderm, and inhibited cardiomyocyte formation. We further demonstrate that miR-125b targeted the 3-untranslated region of Lin28 and reduced the abundance of Lin28 at both mRNA and protein levels. Moreover, phenotypes of miR-125b overexpressing cells were mimicked by downregulation of Lin28 and rescued by reintroduction of Lin28. In addition, the impaired cardiogenesis in miR-125b-introduced cells was greatly recovered when mimicking endoderm environment by cultivation with the condition medium from a visceral endoderm-like cell line, END-2. These results reveal that the miR-125b/Lin28 axis is an important regulator of early lineage specification and cardiomyocyte differentiation of ESCs.

A genetic role for macrophage migration inhibitory factor (MIF) in adult-onset Still's disease

IntroductionAdult-onset stills disease (AOSD) is a rare systemic inflammatory disorder in which abnormalities in inflammatory cytokines production appear to play a pathophysiological role. Our previous work has reported increased expression of macrophage migration inhibitory factor (MIF) and revealed its correlation with disease severity and activity in AOSD. A -173 G/C single nucleotide polymorphism (SNP) (rs755622) and a -794 CATT5-8 repeat (rs5844572) in the MIF promoter have been reported. In this study, we sought to explore the relationship between functional MIF promoter polymorphisms and MIF expression in AOSD.Methods100 patients and 200 controls were recruited in the study. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was utilized to analyze the -173 G/C SNP (rs755622) and PCR-based size discrimination assay was applied to detect the -794 CATT5-8 repeat (rs5844572) in the MIF promoter. Plasma MIF levels were measured by ELISA. MIF mRNA levels were quantified by real-time reverse transcription (RT)-PCR. Bisulfate genomic sequencing was employed to evaluate DNA methylation status within the MIF promoter.ResultsWe identified that the frequencies of MIF -794 CATT5 (P = 0.001) allele and the expression of MIF (P <0.001) were increased in patients compared to healthy controls. Plasma levels of MIF in patients with CC genotype were higher than those of patients with GC or GG genotypes (P = 0.05). In patients with established AOSD, a higher frequency of -794 CATT7 containing MIF genotypes was observed in those with liver dysfunction (P = 0.009). Haplotype analysis revealed a higher representation of the MIF haplotype defined by -173*C/-794 CATT5 (C5) in AOSD patients (P = 0.001).ConclusionFunctional promoter polymorphisms in the MIF gene influence plasma MIF levels in AOSD and may contribute to disease susceptibility or clinical presentation of AOSD.

Aberrant activation of the WNT/β-catenin signaling pathway in lupus nephritis.

Objective The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis. Methods The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA. Results Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05). β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels. Conclusions The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis.