Xiaoxia Zuo

MicroRNA Expression Abnormalities in Limited Cutaneous Scleroderma and Diffuse Cutaneous Scleroderma

Scleroderma (systemic sclerosis, SSc) is a complex autoimmune disease caused by progressive fibrotic replacement of normal tissue architecture, a progressive and ultimately fatal process that currently has no cure. Although dysregulation of microRNAs (miRNAs) is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in SSc is unclear. In comparison with the normal skin tissues, miRNAs were aberrantly expressed in limited cutaneous scleroderma and diffuse cutaneous scleroderma skin tissues. We also identified miRNAs whose expressions were correlated with SSc fibrosis: miR-21, miR-31, miR-146, miR-503, miR-145, and miR-29b were predicted to be involved. This study further confirmed that miR-21 was increased whereas miR-145 and miR-29b were decreased both in the skin tissues and fibroblasts. As predicted target genes, SMAD7, SAMD3, and COL1A1 were regulated by these miRNAs. After stimulation with transforming growth factor β, the expression of miR-21 was increased and that of SMAD7 mRNA was decreased. MiR-145 was upregulated whereas the mRNA level of SMAD3 was downregulated. The downregulation of miR-29b was correlated with the upregulation of COL1A1 mRNA. MiRNAs might play an important role in the pathogenesis of SSc and suggest a potential therapy.

MicroRNA-21 in Scleroderma Fibrosis and its Function in TGF-β- Regulated Fibrosis-Related Genes Expression

Uncontrolled fibrosis in multiple organs is the main cause of death in systemic sclerosis (SSc), and transforming growth factor-β (TGF-β) activation plays a fundamental role in the process. Our previous study demonstrated that miR-21 was significantly up-regulated in SSc fibroblasts. Here, we found that TGF-β regulated the expression of miR-21 and fibrosis-related genes, and decreased Smad7 expression. Over-expression of miR-21 in fibroblasts decreased the levels of Smad7, whereas knockdown of miR-21 increased its expression. Further study using a reporter gene assay demonstrated Smad7 was a direct target of miR-21. Similar to human SSc, the expression of miR-21 increased in the bleomycin induced skin fibrosis. Inhibition of fibrosis by treatment with anti-fibrosis drug bortezomib restored the levels of miR-21 and Smad7. MiR-21 may function in an amplifying circuit to enhance TGF-β signaling events in SSc fibrosis, and suggesting that miR-21 may act as a potential therapeutic target.Uncontrolled fibrosis in multiple organs is the main cause of death in systemic sclerosis (SSc), and transforming growth factor-β (TGF-β) activation plays a fundamental role in the process. Our previous study demonstrated that miR-21 was significantly up-regulated in SSc fibroblasts. Here, we found that TGF-β regulated the expression of miR-21 and fibrosis-related genes, and decreased Smad7 expression. Over-expression of miR-21 in fibroblasts decreased the levels of Smad7, whereas knockdown of miR-21 increased its expression. Further study using a reporter gene assay demonstrated Smad7 was a direct target of miR-21. Similar to human SSc, the expression of miR-21 increased in the bleomycin induced skin fibrosis. Inhibition of fibrosis by treatment with anti-fibrosis drug bortezomib restored the levels of miR-21 and Smad7. MiR-21 may function in an amplifying circuit to enhance TGF-β signaling events in SSc fibrosis, and suggesting that miR-21 may act as a potential therapeutic target.

Extracellular heat shock protein 70 inhibits tumour necrosis factor-α induced proinflammatory mediator production in fibroblast-like synoviocytes

IntroductionIt was recently suggested that heat shock protein (HSP)70, an intracellular protein, is a potential mediator of inflammatory disease when it is released into the extracellular compartment. Although elevated HSP70 levels have been identified in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid compared with patients with osteoarthritis and healthy individuals, it remains unclear what role extracellular HSP70 plays in the pathogenesis of RA. This study was conducted to investigate the effects of extracellular HSP70 on the production of RA-associated cytokines in fibroblast-like synoviocytes from patients with RA and to elucidate the mechanisms involved.MethodsIL-6, IL-8 and monocyte chemoattractant protein (MCP)-1 levels in culture supernatants were measured using enzyme-linked immunosorbent assays. Activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated protein kinases (ERKs), c-Jun amino-terminal kinase (JNK) and p38 MAPK, was detected using Western blotting. Nuclear translocation of nuclear factor-κB (NF-κB) and degradation of the inhibitory protein IκBα were examined using immunohistochemistry and Western blotting.ResultsHuman HSP70 downregulated IL-6, IL-8 and MCP-1 production in RA fibroblast-like synoviocytes induced by tumour necrosis factor (TNF)-α in a concentration dependent manner. HSP70 inhibited the activation of ERK, JNK and p38 MAPK in fibroblast-like synoviocytes stimulated by TNF-α. Furthermore, HSP70 also significantly inhibited nuclear translocation of nuclear factor-κB and degradation of IκBα induced by TNF-α.ConclusionExtracellular HSP70 has an anti-inflammatory effect on RA by downregulating production of IL-6, IL-8 and MCP-1 in fibroblast-like synoviocytes, which is mediated through inhibited activation of the MAPKs and NF-κB signal pathways.

Autoantigen Microarray for High-throughput Autoantibody Profiling in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of autoantibodies to a broad range of self-antigens. Profiling the autoantibody repertoire using array-based technology has emerged as a powerful tool for the identification of biomarkers in SLE and other autoimmune diseases. Proteomic microarray has the capacity to hold large number of self-antigens on a solid surface and serve as a high-throughput screening method for the determination of autoantibody specificities. The autoantigen arrays carrying a wide variety of self-antigens, such as cell nuclear components (nucleic acids and associated proteins), cytoplasmic proteins, phospholipid proteins, cell matrix proteins, mucosal/secreted proteins, glomeruli, and other tissue-specific proteins, have been used for screening of autoantibody specificities associated with different manifestations of SLE. Arrays containing synthetic peptides and molecular modified proteins are also being utilized for identification of autoantibodies targeting to special antigenic epitopes. Different isotypes of autoantibodies, including IgG, IgM, IgA, and IgE, as well as other Ig subtypes, can be detected simultaneously with multi-color labeled secondary antibodies. Serum and plasma are the most common biologic materials for autoantibody detection, but other body fluids such as cerebrospinal fluid, synovial fluid, and saliva can also be a source of autoantibody detection. Proteomic microarray as a multiplexed high-throughput screening platform is playing an increasingly-important role in autoantibody diagnostics. In this article, we highlight the use of autoantigen microarrays for autoantibody exploration in SLE.

MicroRNAs: their involvement in fibrosis pathogenesis and use as diagnostic biomarkers in scleroderma.

miRNAs are important post-transcriptional regulators. The aberrant expression of miRNAs is strongly associated with the initiation and progression of pathophysiologic processes in a wide range of human diseases. Scleroderma (systemic sclerosis; SSc) is a highly heterogeneous autoimmune disease that includes the progressive fibrotic replacement of normal tissue architecture in multiple organs. Our previous studies have suggested that SSc skin tissues display a different miRNA expression signature than that found in normal controls. miRNAs with pro- or antifibrotic properties are found to be dysregulated in SSc skin fibrosis. Serum miRNA levels are associated with SSc activity and severity. miRNAs have the potential to be therapeutic targets and serve as biomarkers for SSc diagnosis and assessment of disease state and severity. This review summarizes the SSc miRNA expression signature and the roles of dysregulation of miRNAs in SSc tissues and serum and examines the future therapeutic potential of targeting miRNAs in the management of SSc patients.

MicroRNA-130b regulates scleroderma fibrosis by targeting peroxisome proliferator-activated receptor γ.

Abstract Objectives. To investigate the role of microRNA-130b (miR-130b) in systemic sclerosis (SSc) skin fibrosis and its regulatory effect on peroxisome proliferator-activated receptor γ (PPARγ). Methods. miR-130b was identified from microarray analyses in our previous studies. The expression of miR-130b, PPARγ, and fibrosis-related genes were determined by real-time PCR analysis. PPARγ protein levels were detected by immunohistochemistry and Western blot. Cells were transfected with microRNA mimics/inhibitor/scramble of miR-130b using Lipofectamine. Luciferase reporter gene assays were used to identify the direct target of miR-130b. Transforming growth factor β (TGF-β) was used for stimulation. Results. The expression of miR-130b was significantly upregulated and level of PPARγ was decreased in the dermis of the SSc skin biopsy samples and fibroblasts. Similar to human SSc, the same expression patterns of miR-130b, PPARγ, and fibrosis-related genes were observed in the bleomycin-induced skin fibrosis model; TGF-β induced the expression of miR-130b and fibrosis-related genes expression, but downregulated the expression of PPARγ. Overexpression of miR-130b in normal or SSc skin fibroblasts significantly decreased, and accordingly, knockdown of miR-130b increased the levels of PPARγ and fibrosis-related genes. In the reporter gene assay, cotransfection with miR-130b mimics significantly decreased the relative luciferase activity, which suggested a direct regulation of PPARγ by miR-130b. Conclusions. These studies demonstrated that miR-130b played important profibrotic roles in SSc fibrosis, and enhanced TGF-β signaling through negative regulation of PPARγ expression. MiR-130b may be a potential therapeutic target in SSc fibrosis.

Attenuation of collagen induced arthritis via suppression on Th17 response by activating cholinergic anti-inflammatory pathway with nicotine

The cholinergic anti-inflammatory pathway can inhibit the inflammation of collagen induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). However, the immunologic mechanisms that provide a therapeutic effect against the auto-inflammatory disease are not yet elucidated. The present study explores the effect of cholinergic anti-inflammatory pathway on CD4+ T cell responses in CIA. Forty DBA/1 mice were divided into 4 groups: a control group, a CIA group, a vagotomy group, and a nicotine group. The degree of arthritis was measured by arthritis score and hematoxylin and eosin. ELISA was used to detect the serum concentration of IFN-γ, IL-4 and IL-17A. Flow cytometry was used to detect the cytokines and transcription factors (TFs) (the TFs of Th1, Th2, and Th17 cells are T-bet, RORγτ and GATA3 respectively) in the spleen. Immunohistochemistry was used to analyze RORγτ expression in the joint synovium. Arthritis in the nicotine group was significantly lightened compared with that in the CIA group and in the vagotomy group. Nicotine attenuated Th17 lineage by reducing IL-17A production and RORγτ expression. The expressions of IL-4 and GATA3 were increased in the same setting. However, the expressions of IFN-γ and T-bet had no difference between the nicotine and the CIA group. Nicotine may induce a shift to the Th2 lineage and improve the Th1/Th2 imbalance. Activating the cholinergic anti-inflammatory pathway with nicotine can inhibit Th17 cell responses and may improve the Th1/Th2 imbalance in CIA, providing a new justification for its application in the treatment of rheumatoid arthritis.

Whole-genome transcription and DNA methylation analysis of peripheral blood mononuclear cells identified aberrant gene regulation pathways in systemic lupus erythematosus

BackgroundRecent achievement in genetics and epigenetics has led to the exploration of the pathogenesis of systemic lupus erythematosus (SLE). Identification of differentially expressed genes and their regulatory mechanism(s) at whole-genome level will provide a comprehensive understanding of the development of SLE and its devastating complications, lupus nephritis (LN).MethodsWe performed whole-genome transcription and DNA methylation analysis in PBMC of 30 SLE patients, including 15 with LN (SLE LN+) and 15 without LN (SLE LN−), and 25 normal controls (NC) using HumanHT-12 Beadchips and Illumina Human Methy450 chips. The serum proinflammatory cytokines were quantified using Bio-plex Human Cytokine 27-plex assay. Differentially expressed genes and differentially methylated CpG were analyzed with GenomeStudio, R, and SAM software. The association between DNA methylation and gene expression were tested. Gene interaction pathways of the differentially expressed genes were analyzed by IPA software.ResultsWe identified 552 upregulated genes and 550 downregulated genes in PBMC of SLE. Integration of DNA methylation and gene expression profiling showed that 334 upregulated genes were hypomethylated, and 479 downregulated genes were hypermethylated. Pathway analysis on the differential genes in SLE revealed significant enrichment in interferon (IFN) signaling and toll-like receptor (TLR) signaling pathways. Nine IFN- and seven TLR-related genes were identified and displayed step-wise increase in SLE LN− and SLE LN+. Hypomethylated CpG sites were detected on these genes. The gene expressions for MX1, GPR84, and E2F2 were increased in SLE LN+ as compared to SLE LN− patients. The serum levels of inflammatory cytokines, including IL17A, IP-10, bFGF, TNF-α, IL-6, IL-15, GM-CSF, IL-1RA, IL-5, and IL-12p70, were significantly elevated in SLE compared with NC. The levels of IL-15 and IL1RA correlated with their mRNA expression. The upregulation of IL-15 may be regulated by hypomethylated CpG sites in the promotor region of the gene.ConclusionsOur study has demonstrated that significant number of differential genes in SLE were involved in IFN, TLR signaling pathways, and inflammatory cytokines. The enrichment of differential genes has been associated with aberrant DNA methylation, which may be relevant to the pathogenesis of SLE. Our observations have laid the groundwork for further diagnostic and mechanistic studies of SLE and LN.

Treatment with Recombinant Hsp72 Suppresses Collagen-Induced Arthritis in Mice

Although the level of heat shock protein (Hsp72) has been shown to be enhanced in rheumatoid arthritis (RA) synovial tissues and RA synovial fluid, it remains unclear what role extracellular Hsp72 plays in the pathogenesis of RA. This study was conducted to investigate the effects of recombinant human Hsp72 on collagen-induced arthritis (CIA) when administered therapeutically and elucidate its underlying mechanism. We demonstrated that recombinant Hsp72 significantly reduced disease severity. Hsp72-treated animals displayed significantly less cartilage and bone destruction than that in the controls. Hsp72 treatment also reduced the expression of tumor necrosis factor alpha and interleukin 6 in the sera. Furthermore, Hsp72 treatment significantly inhibited activation of nuclear factor kappa B (NF-κB) in synovial tissues of CIA mice. These findings suggest that recombinant Hsp72 effectively suppressed synovial inflammation and the development and progress of CIA, which is mediated through the reduction of production of proinflammatory cytokines and the suppression of activation of NF-κB pathway.

MicroRNAs Regulating Signaling Pathways: Potential Biomarkers in Systemic Sclerosis.

Systemic sclerosis (SSc) is a multisystem fibrotic and autoimmune disease. Both genetic and epigenetic elements mediate SSc pathophysiology. This review summarizes the role of one epigenetic element, known as microRNAs (miRNAs), involved in different signaling pathways of SSc pathogenesis. The expression of key components in transforming growth factor-β (TGF-β) signaling pathway has been found to be regulated by miRNAs both upstream and downstream of TGF-β. We are specifically interested in the pathway components upstream of TGF-β, while miRNAs in other signaling pathways have not been extensively studied. The emerging role of miRNAs in vasculopathy of SSc suggests a promising new direction for future investigation. Elucidation of the regulatory role of miRNAs in the expression of signaling factors may facilitate the discovery of novel biomarkers in SSc and improve the understanding and treatment of this disease.