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Objective: To define a pharmacodynamic biomarker based on gene expression in skin that would provide a biologic measure of the extent of disease in patients with diffuse cutaneous systemic sclerosis (dcSSc) and could be used to monitor skin disease longitudinally., Methods: Skin biopsy specimens obtained from a cohort of patients with dcSSc (including longitudinal specimens) were analyzed by microarray. Expression of genes correlating with the modified Rodnan skin thickness score (MRSS) were examined for change over time using a NanoString platform, and a generalized estimating equation (GEE) was used to define and validate longitudinally measured pharmacodynamic biomarkers composed of multiple genes., Results: Microarray analysis of genes parsed to include only those correlating with the MRSS revealed prominent clusters of profibrotic/transforming growth factor β-regulated, interferon-regulated/proteasome, macrophage, and vascular marker genes. Using genes changing longitudinally with the MRSS, we defined 2 multigene pharmacodynamic biomarkers. The first was defined mathematically by applying a GEE to longitudinal samples. This modeling method selected cross-sectional THBS1 and longitudinal THBS1 and MS4A4A. The second model was based on a weighted selection of genes, including additional genes that changed statistically significantly over time: CTGF, CD163, CCL2, and WIF1. In an independent validation data set, biomarker levels calculated using both models correlated highly with the MRSS., Conclusion: Skin gene expression can be used effectively to monitor changes in SSc skin disease over time. We implemented 2 relatively simple models on a NanoString platform permitting highly reproducible assays that can be applied directly to samples from patients or collected as part of clinical trials., (© 2015, American College of Rheumatology.)

Toll-like receptor 9 (TLR9) drives innate immune responses after recognition of foreign or endogenous DNA containing unmethylated CpG motifs. DNA-mediated TLR9 activation is highly implicated in the pathogenesis of several autoimmune skin diseases, yet its contribution to the inflammation seen in these diseases remains unclear. In this study, TLR9 ligand, CpGB DNA, was administered to mice via a subcutaneous osmotic pump with treatment lasting 1 or 4 weeks. Gene expression and immunofluorescence analyses were used to determine chemokine expression and cell recruitment in the skin surrounding the pump outlet. CpGB DNA skin treatment dramatically induced a marked influx of CD11b+ F4/80+ macrophages, increasing over 4 weeks of treatment, and induction of IFNγ and TNFα expression. Chemokines, CCL2, CCL4, CCL5, CXCL9 and CXCL10, were highly induced in CpGB DNA-treated skin, although abrogation of these signalling pathways individually did not alter macrophage accumulation. Flow cytometry analysis showed that TLR9 activation in the skin increased circulating CD11b+ CD115+ Ly6C(hi) inflammatory monocytes following 1 week of CpGB DNA treatment. Additionally, skin-resident CD11b+ cells were found to initially take up subcutaneous CpGB DNA and propagate the subsequent immune response. Using diphtheria toxin-induced monocyte depletion mouse model, gene expression analysis demonstrated that CD11b+ cells are responsible for the CpGB DNA-induced cytokine and chemokine response. Overall, these data demonstrate that chronic TLR9 activation induces a specific inflammatory response, ultimately leading to a striking and selective accumulation of macrophages in the skin., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To characterise global chemokine expression in systemic sclerosis (SSc) skin in order to better understand the relationship between chemokine expression and vascular inflammation in this disease., Methods: We investigated chemokine mRNA expression in the skin through quantitative PCR analysis comparing patients with diffuse cutaneous (dcSSc) or limited cutaneous (lcSSc) disease with healthy controls. We tested correlations between the most regulated chemokines and vascular inflammation and macrophage recruitment. CCL19 expression was examined in human primary immune cells treated with innate immune activators., Results: The chemokines, CCL18, CCL19 and CXCL13, were upregulated in dcSSc skin, and CCL18 in lcSSc skin. Expression of CCL19 in dcSSc skin correlated with markers of vascular inflammation and macrophage recruitment. Immunofluorescence data showed CCL19 colocalisation with CD163 macrophages in dcSSc skin. In vitro studies on human primary cells demonstrated that CCL19 expression was induced after toll-like receptor activation of peripheral blood mononuclear cells and separated populations of CD14 monocytes., Conclusions: CCL18, CCL19 and CXCL13-chemoattractants for macrophage and T cell recruitment-were three of six chemokines with the highest expression in dcSSc skin. Increased CCL19 expression in the skin suggests a role for CCL19 in the recruitment of immune cells to the peripheral tissue. Induction of CCL19 in macrophages but not structural cells indicates a role for skin-resident or recruited immune cells in perivascular inflammation. This study demonstrates that CCL19 is a sensitive marker for the perivascular inflammation and immune cell recruitment seen in dcSSc skin disease., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis., Methods: The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry., Results: We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes., Conclusions: We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.

Background: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon., Methods: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo., Results: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis., Conclusions: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Introduction: Sterile tissue injury induces an inflammatory response involving cytokines that have crucial roles in the tissue repair that follows., Methods: MyH3 and type 1 interferon (IFN) were assessed by qPCR after cardiotoxin (CTX)-induced muscle injury., Results: CTX-induced injury increased expression of IFN-regulated genes, IFIT1 and MX-2, which was blocked in type 1 IFN receptor (IFNR)-deficient mice. However, IFNR-deficient mice showed no significant differences in muscle regeneration as assessed by MyH3 expression. MyH3 was significantly reduced in TLR3-deficient but not MyD88-deficient mice. TLR3-deficient mice also showed altered expression of proinflammatory cytokines, IL-6, IL-1β, and TNF-α., Conclusions: CTX-induced muscle injury increased markers of innate immune activation, but blocking type 1 IFN signaling had no effect on muscle regeneration. Taken together, these results suggest a role for TLR3, and perhaps other innate immune signals, in the inflammatory response to CTX-induced muscle injury and consequent muscle regeneration., (Copyright © 2011 Wiley Periodicals, Inc.)