Shared and unique molecular signatures across different autoantibody groups in systemic sclerosis: a multiomics analysis.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

Microarray analyses of peripheral blood leukocytes have shown that patients with systemic lupus erythematosus express increased levels of type I interferon (IFN)-regulated genes. In this study we examined gene expression by peripheral blood mononuclear cells (PBMCs) from patients with systemic sclerosis (SSc) to better understand the dysregulation of the immune system in this disease. PBMC gene expression was analyzed by microarray and confirmed by real-time polymerase chain reaction (PCR). Surface protein expression of Siglec-1 was analyzed by flow cytometry in PBMCs from healthy control subjects and patients with SSc, and in control PBMCs that were cultured in vitro with Toll-like receptor (TLR) agonists. SSc patients showed increased expression of a cluster of IFN-regulated genes, including Siglec-1 (CD169, sialoadhesin). This result was verified and extended by real-time PCR, showing that a subset of the SSc patients expressed strikingly increased levels of Siglec-1 messenger RNA (mRNA). Flow cytometry of PBMCs from SSc patients and healthy controls showed increased Siglec-1 surface protein expression, which was restricted to CD14+ monocytes. In vitro studies showed that type I IFN and certain TLR agonists, including TLR-7 and TLR-9, induced Siglec-1 mRNA and protein expression. Moreover, TLR induction of surface Siglec-1 was shown to be type I IFN-dependent. Increased numbers of Siglec-1+ cells were observed by immunohistochemistry in the skin of SSc patients compared with healthy controls. Increased expression of Siglec-1 in circulating SSc monocytes and tissue macrophages suggests that type I IFN-mediated activation of monocytes occurs in SSc, possibly through TLR activation of IFN secretion. These observations indicate a potential role for type I IFN-activated monocyte/macrophages in the pathogenesis of SSc.

Activation of the type I interferon (IFN) pathway and autoreactive B cells are key immunopathogenic features of systemic lupus erythematosus (SLE), primary Sjögren's disease (pSjD) and systemic sclerosis (SSc). TANK-binding kinase 1 (TBK1) is a mediator of type I IFN and essential during B cell development in mice. We investigated the properties of the TBK1 inhibitor amlexanox in systemic autoimmune diseases. The effects of amlexanox on peripheral blood mononuclear cells (PBMCs) stimulated with Imiquimod, CpG-A, Poly:IC, G3-YsD and 3p-hpRNA were assessed. B cells from healthy controls and patients with SLE, pSjD and SSc were cultured with CD40L, IL-21, IFN, B cell activating factor (BAFF) and amlexanox. Differentiation into CD38highCD27highCD138+/- cells, proliferation, and IgM and IgG production were measured. Amlexanox inhibited production of type I IFN induced through endosomal and cytosolic routes in PBMCs. Likewise, supernatants from amlexanox-treated cells did not induce expression of BAFF and MX1. Amlexanox inhibited spontaneous MX1 expression in PBMCs from SLE, pSjD and SSc patients. Immunohistochemical staining confirmed expression of the TBK1 protein in pSjD salivary glands. Using a B cell differentiation assay, addition of amlexanox decreased B cell proliferation and differentiation into CD27highCD38highCD138+/- plasmablasts and plasma cells. Correspondingly, production of IgM and IgG was suppressed. The observations were corroborated in B cells from patients with SLE, pSjD and SSc. Our findings demonstrate inhibitory effects of amlexanox on type I IFN production and B cell differentiation in primary human cells. Inhibition of TBK1 could potentially be a therapeutic option for the treatment of type I IFN-driven systemic inflammatory diseases.

The -2518 Promotor Polymorphism in the MCP-1 Gene Is Associated with Systemic Sclerosis

ObjectiveTo characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to...

Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by widespread fibrosis affecting the skin, internal organs, and vasculature. However, there are currently no systemic, disease-modifying therapies available for the treatment of the overall condition, and the outcomes remain poor. Studies into disease pathogenesis have identified several pathways that are dysregulated in SSc, and novel targeted therapies are currently being developed. In this special issue, we invited authors to submit original research articles, review articles, or case reports/case series describing preclinical, translational, or clinical studies related to new therapies for SSc. A set of papers in this special issue focuses on identification of new therapeutic targets in preclinical and translational studies. V. J. Moulin's paper is a review article describing the role of apoptosis in the initiation and maintenance of disease in SSc, through effects on the immune system, vascular damage, and fibroblast proliferation. This report discusses potential therapies targeting apoptosis and the Fas/FasL pathway that could be investigated in patients with SSc. Other paper describes vascular changes in the bleomycin-induced mouse model of SSc. The authors discuss the use of this mouse model to investigate targeting fibrosis, apoptosis, and cellular adhesion molecules for the treatment of vascular disease in SSc. The paper by T. Radstake et al. reviews the evidence that hypoxia contributes to the pathogenesis of SSc, with a focus on the role of hypoxia inducible factor (HIF)-1 alpha in the vasculopathy, immune dysregulation, and fibrosis in SSc. This paper summarizes potential therapeutic interventions to bypass the dysfunctional hypoxic pathway in SSc. The paper by R. De Vries et al. describes an original research study evaluating the accumulation of advanced glycation end products (AGE) in the skin of patients with SSc compared with controls using a technique called skin autofluorescence. Although this study did not find a significant difference in AGE accumulation in SSc skin compared with control samples, use of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in the SSc patients may have confounded the results. The paper by E. G. Kroon et al. is an original research article evaluating the expression of Types I and III interferons (IFNs) and interferon-stimulated genes (ISG) in peripheral blood mononuclear cells from SSc patients compared with controls. This study confirmed the increased basal expression of Type I IFNs and the ISG 2′5′OAS in SSc, but found no induction of Type III IFNs. This paper provides further evidence that targeting the IFN pathway may be useful in the treatment of SSc. The paper by S. M. Violette et al. reviews the preclinical data supporting the role of the integrin αvβ6 in the activation of fibrosis via the transforming growth factor (TGF)-β pathway. This paper summarizes in vivo evidence of the utility of blocking αvβ6 for the treatment of lung fibrosis and provides rationale for pursuing this therapeutic approach in patients with SSc-associated interstitial lung disease. Another set of articles includes reviews of novel therapies that are currently being evaluated for the treatment of SSc. The paper by M. Anderson et al. reviews the role of interleukin-6 (IL-6) in SSc, summarizing evidence of effects on B cells, inflammation, fibrogenesis, and endothelial cell activation. The paper reviews the rationale for ongoing clinical trials of agents blocking IL-6 transsignaling for the treatment of SSc. The paper by S.-N. Liossis et al. reviews the published literature supporting the role of B cells in SSc, summarizing data from animal models and human studies. The authors then review the results of four clinical trials assessing the effects of B cell depletion with rituximab therapy on skin disease and lung function in patients with SSc. The paper by R. F. Spiera and J. Gordon reviews the preclinical and clinical studies of tyrosine kinase inhibitors, with a focus on experience with imatinib, in the treatment of SSc and related fibrotic conditions. The authors conclude that interpretation of the results from the completed proof-of-concept studies is difficult due to the small size and heterogeneity of the populations studied and the open-label designs. The review article by K. Phillips et al. describes published studies investigating the utility of phosphodiesterase-5 inhibitors (PDE-5-I) in the treatment of Raynaud's phenomenon (RP) and/or digital ulcers (DU), detailing results of studies using sildenafil, vardenafil, and tadalafil. The authors also list the ongoing clinical studies of PDE-5-I for RP and DU. The paper by D. F. Fiorentino et al. reviews the role of endothelin-1 in the pathogenesis of SSc-associated vascular disease and summarizes the published reports evaluating the use of endothelin receptor antagonists in the treatment of RP and DU. One of the sets in this special issue includes two articles describing rare clinical manifestations of SSc, gastric antral vascular ectasias and primary biliary cirrhosis, and management strategies for these entities. The paper B. Markewitz et al. is a case series describing the tolerability and efficacy of naltrexone for the treatment of pruritus and gastrointestinal symptoms in three patients with SSc. The paper by K. Nikolov and M. Baleva reviews the rationale for using intravenous immunoglobulins (IVIG) in the treatment of SSc. The authors also summarize the published case reports and series supporting the potential efficacy of IVIG in the treatment of skin sclerosis in SSc. In summary, this special issue provides an interesting compilation of articles addressing potential emerging therapies for the treatment of SSc, including information gleaned from preclinical, translational, and clinical studies. We, the guest editors, hope readers find that the manuscripts included herein offer a comprehensive summary of the current status of drug development and promising therapies for SSc. Lorinda Chung Oliver Distler Laura Hummers Eswar Krishnan Virginia Steen

Objective: To study serum type I interferon (IFN) activity in patients with early systemic sclerosis (SSc).Method: Serum type I IFN activity was measured in 33 consecutive patients with SSc and a disease duration of < 2 years and in 13 healthy individuals by calculating a type I IFN score according to the induction of six IFN-α regulated genes in a reporter cell line.Results: Twenty-seven per cent of the SSc patients had an increased type I IFN score compared to none of the healthy individuals (p < 0.05). The clinical SSc phenotype associated with high serum type I IFN activity did not differ from patients with low serum type I IFN activity regarding the presence of skin or lung fibrosis, pulmonary hypertension, or digital complications. Patients with high serum type I IFN activity were younger (p < 0.01) and had a lower frequency of cardiac involvement (p = 0.053), lower leucocyte count (p < 0.001), higher immunoglobulin (Ig)G levels (p < 0.05), and a higher amount of antibodies against extractable nuclear antigens (p < 0.01) than patients with low serum type I IFN activity. The presence of antibodies against topoisomerase I, Sjögren’s syndrome antigen, and nuclear ribonucleoprotein antigens was associated with higher type I IFN activity (p < 0.05 for all comparisons).Conclusions: Our study indicates that increased serum type I IFN activity in early SSc patients is associated with an antibody and laboratory profile that may reflect a subclinical overlap of SSc with other type I IFN-driven connective tissue diseases (CTDs).

Cytokine production and serum levels in systemic sclerosis

Activation of type I interferon (IFN) response has been shown to correlate with disease activity in systemic sclerosis (SSc). It is currently unknown whether the tissue-specific type I IFN activation is a consequence of the response observed in blood or rather its source. Exosomes from SSc fibroblasts were recently shown to activate macrophages in vitro. Here, we aimed to determine the source of type I IFN signature in SSc skin biopsies and the potential role of exosomes from SSc dermal fibroblasts in the process. Skin biopsies were obtained from the forearms of healthy patients and of those with SSc and processed for dermal fibroblasts and keratinocytes. Exosomes were isolated from healthy and SSc dermal fibroblast supernatants by ultracentrifugation and added to human skin keratinocytes. Keratinocyte transcriptome was analyzed by RNA sequencing (RNA-seq) analysis. TANK-binding kinase (TBK) and JAK were inhibited using a small molecule inhibitor (GSK8612) and tofacitinib, respectively. SSc skin biopsies showed the highest levels of type I IFN response in the epidermal layer. RNA-seq analysis of keratinocytes transcriptome following exposure to dermal fibroblast exosomes showed strong up-regulation of IFN signature genes induced by SSc exosomes compared to healthy control. Inhibition of TBK or JAK activity suppressed the up-regulation of the IFN signature induced by SSc exosomes. IFN activation of SSc keratinocytes is dependent on their crosstalk with dermal fibroblasts and inducible by extracellular exosomes. Our data indicate that SSc fibroblast exosomes contribute to the type I IFN activation in SSc skin through activation of pattern recognition receptors upstream of TBK.

This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD), and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways. PBMCs were collected from 19 patients with SSc (SSc group) and 18 healthy persons (control group). Among SSc patients, there were 10 patients with ILD (SSc with ILD subgroup) and 9 patients without ILD (SSc without ILD subgroup). The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip. The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated. Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup. Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup. In PBMCs of the SSc group, 35 genes in Wnt/β-catenin signaling pathway were hypomethylated, while frizzled-1 (FZD1), mitogen-activated protein kinase 9 (MAPK9), mothers against DPP homolog 2 (SMAD2), transcription factor 7-like 2 (TCF7L2), and wingless-type MMTV integration site family, member 5B (WNT5B) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of dickkopf homolog 2 (DKK2), FZD1, MAPK9 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). In PBMCs of the SSc group, 38 genes in chemokine signaling pathway were hypomethylated, while β-arrestin 1 (ARRB1), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 16 (CXCL16), FGR, and neutrophil cytosolic factor 1C (NCF1C) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of ARRB1, CXCL10, CXCL16 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD. The expression levels of multiple genes in Wnt/β- catenin and chemokine signaling pathways are upregulated, which might be associatea with the pathogenesis of SSc.

BackgroundDysregulated immune responses are major pathogenic players in systemic sclerosis (SSc). The biophysical properties (such as cell deformation, Young’s modulus (a measure of cell stiffness) and area) of circulating immune...

BackgroundChronic graft-versus-host disease (cGVHD) is a major factor of morbidity and mortality for allogeneic stem cell transplantation (aSCT). The skin and internal organ involvement is the most common systemic complication of cGVHD and closely resembles systemic sclerosis (SSc). Circulating lymphocytes characterize the autoimmune nature of both conditions. Therefore we hypothesized that the common clinical manifestation (systemic organ and skin injury) and the common underlying players (lymphocytes) justify the combined meta-analysis of these diseases.ResultsThe aSCT and SSc datasets were uploaded from Gene Expression Omnibus (GEO), a public functional genomics data repository. The available microarray studies of peripheral blood mononuclear cells (PBMCs) and isolated lymphocytes were limited to well established microarray platforms (Affymetrix, Agilent, Canvac, and Illumina) and experimental settings with ≥10 patients per group. The resulting pools of data were merged by unique gene identifier and analyzed by the expression genome-wide association studies (eGWAS) coupled with the subtraction of the cGVHD+ and cGVHD− molecular signatures. The eGWAS was applied to 47 and 50 lymphocyte profiles from aSCT and SSc patients, respectively. The identified 35 candidates were represented by 8 known cGVHD genes (including CXCR4, LTBR and PML) and 28 new candidate genes (including SEPX1 and DNJGB1). The further mutual subtraction of cGVHD+ and cGVHD− candidates and pathway analysis identified a list of 25 genes. Seven of these genes belong to the fibroblast development and function pathway, consisting of the well known cGVHD genes CCND1, JUN, and FOS, and the new molecular targets MMP2, FOSB, TNFAIP8, and DUSP1. These genes become primary candidates for a potential link of systemic effects of cGVHD and SSc.ConclusionsWe designed a new approach for meta-analysis by combining data from different diseases using common clinical manifestation as a linker. This allowed us to power up the insufficient standalone meta-analysis of aSCT microarray studies, by adding SSc samples to the data pool. This new method has successfully identified novel molecular targets for systemic effects of both aSCT and SSc. We believe that this approach is generalizable and can be applied to an array of diseases with common clinical manifestations.

Systemic sclerosis (SSc) is a chronic fibrosing disease characterized by vasculopathy, autoimmunity, and an accumulation of collagen in tissues. Numerous studies have shown that compared to healthy or diseased controls, the peripheral blood mononuclear cells (PBMC) from patients with SSc produce a variety of cytokines or proliferate when cultured with solubilized type I collagen (CI) or constituent α1(II) and α2(I) polypeptide chains. The purpose of this study was to determine whether PBMC isolated from patients with SSc and cultured in vitro with soluble CI elaborated soluble mediators that inhibit the production of collagenase (i.e., matrix metalloproteinase, MMP-1) by fibroblasts. Supernatants of CI-stimulated PBMC from juvenile and adult diffuse cutaneous (dc)SSc patients significantly reduced MMP-1 production by SSc dermal fibroblasts, while supernatants of CI-stimulated PBMC from patients with localized scleroderma (LS) did not. CI-stimulated PBMC culture supernatants from patients with dcSSc in contrast to patients with LS exhibited increased levels of platelet-derived growth factor (PDGF)-AA, PDGF-BB, TNF-α, IL-13, and EGF. Prolonged culture of SSc dermal fibroblasts with recombinant PDGF-BB or IL-13 inhibited the induction of MMP-1 in response to subsequent TNF-α stimulation. These data suggest that therapies aimed at reducing these cytokines may decrease collagen accumulation in SSc, preventing the development of chronic fibrosis.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation, microangiopathy, and progressive fibrosis in the skin and internal organs. To evaluate the pathophysiologic mechanisms and efficacies of potential therapeutics for SSc, a preclinical model recapitulating the disease phenotypes is needed. Here, we introduce a novel animal model for SSc using immunodeficient mice injected with peripheral blood mononuclear cells (PBMCs) from SSc patients. Human PBMCs acquired from SSc patients and healthy controls were transferred into NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice with concurrent bleomycin injection. Blood, skin, and lung tissues were acquired and analyzed after PBMC engraftment. In addition, we investigated whether the humanized murine model could be used to assess the efficacy of potential therapeutics for SSc. Human PBMCs from SSc patients and healthy controls were engrafted into the blood, skin, and lung tissues of NSG mice. Histological analysis of affected tissues from mice treated with SSc PBMCs (SSc hu-mice) demonstrated substantial inflammation, fibrosis and vasculopathy with human immune cell infiltration and increased expression of IL-17, TGF-β, CCL2, CCL3, and CXCL9. The proportions of circulating and tissue-infiltrating T helper 17 (Th17) cells were elevated in SSc hu-mice. These cells showed increased expression of CXCR3 and phosphorylated STAT3. SSc hu-mice treated with rebamipide and other potential Th17-cell-modulating drugs presented significantly reduced tissue fibrosis. Mice injected with patient-derived PBMCs show promise as an animal model of SSc.

BackgroundThe role of IL17 cells in the pathogenesis of Systemic Sclerosis (SSc) has recently emerged, as it has been reported that IL-17 is overproduced by T cells from the peripheral...