Giuseppina Stifano

Proteome-wide Analysis and CXCL4 as a Biomarker in Systemic Sclerosis

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.).

Association of Interferon‐ and Transforming Growth Factor β–Regulated Genes and Macrophage Activation With Systemic Sclerosis–Related Progressive Lung Fibrosis

Systemic sclerosis (SSc)–related interstitial lung disease (ILD) is one of the leading causes of mortality. We undertook this study to analyze the gene expression of lung tissue in a prospective cohort of patients with SSc‐related ILD and to compare it with that in control lungs and with 2 prospective clinical parameters in order to understand the molecular pathways implicated in progressive lung disease.

A longitudinal biomarker for the extent of skin disease in patients with diffuse cutaneous systemic sclerosis.

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.

Chronic Toll-like receptor 4 stimulation in skin induces inflammation, macrophage activation, transforming growth factor beta signature gene expression, and fibrosis.

IntroductionThe 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.MethodsThe 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.ResultsWe 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.ConclusionsWe 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.

Impact of 111In-DTPA-octreotide SPECT/CT fusion images in the management of neuroendocrine tumours

PurposeSomatostatin receptor scintigraphy with [111In]-diethylene triamine pentaacetate acid (DTPA)-octreotide is an accurate method for detecting neuroendocrine tumours (NETs) but often does not provide clear anatomical localisation of lesions. The aim of this study was to assess the clinical usefulness of anatomical-functional image fusion.Materials and methodsFifty-four patients with known or suspected NET were included in the study. Planar and single-photon-emission computed tomography (SPECT) imaging was performed using a dual-head gamma camera equipped with an integrated X-ray transmission system, and the images were first interpreted alone by two nuclear medicine physicians and then compared with SPECT/CT fusion images together with a radiologist. The improvement provided by SPECT/CT in the interpretation of SPECT data alone and any modification in patientmanagement were recorded.ResultsFusion images improved SPECT interpretation in 23 cases, providing precise anatomical localisation of increased tracer uptake in 20 cases and disease exclusion in sites of physiological uptake in 5. In 10 patients, SPECT/CT allowed definition of the functional significance of lesions detected by diagnostic CT. SPECT/CT data modified clinical management in 14 cases by changing the diagnostic approach in 8 and the therapeutic modality in 6.ConclusionsOur study demonstrates that image fusion is clearly superior to SPECT alone, allowing precise localisation of lesions and reducing false-positive results.RiassuntoObiettivoLa scintigrafia con 111In-DTPA-octreotide rappresenta un metodo accurato per la diagnosi dei tumori neuroendocrini, ma spesso non fornisce una corretta localizzazione anatomica delle lesioni. Scopo di questo studio è valutare l’utilità clinica della fusione di immagini SPECT-TC.Materiali e metodiSono stati inclusi 54 pazienti con tumore neuroendocrino sospetto o noto. Le immagini planari e SPECT, ottenute utilizzando una gamma-camera a doppia testata con integrato sistema trasmissivo TC, sono state interpretate prima separatamente da due medici nucleari e poi confrontate con immagini di fusione insieme ad un radiologo, per valutare i miglioramenti apportati dalla fusione nell’interpretazione delle immagini SPECT e nella gestione del paziente.RisultatiLe immagini di fusione hanno migliorato l’interpretazione SPECT in 23 casi, permettendo una precisa localizzazione anatomica dei siti patologici in 20 casi e l’esclusione di malattia in siti di accumulo fisiologico in 5. In 10 pazienti SPECT/TC ha permesso la definizione funzionale di lesioni evidenziate alla TC diagnostica. SPECT/TC ha modificato il management clinico in 14 pazienti, modificando l’approccio diagnostico in 8 e quello terapeutico in 6.ConclusioniIl nostro studio dimostra che la fusione di immagini è chiaramente superiore alla sola SPECT, permettendo una precisa localizzazione delle lesioni individuate e riducendo i risultati falsi positivi.

Thymic Stromal Lymphopoietin Is Up-Regulated in the Skin of Patients With Systemic Sclerosis and Induces Profibrotic Genes and Intracellular Signaling That Overlap With Those Induced by Interleukin-13 and Transforming Growth Factor β

OBJECTIVE To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin-13 (IL-13) and transforming growth factor β (TGFβ). METHODS Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild-type, IL-4Rα1-, and TSLP-deficient mice were treated with TGFβ, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFβ, IL-13, poly(I-C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho-Smad2 and macrophage marker CD163 were tested. RESULTS TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP-treated mice showed up-regulated clusters of gene expression that overlapped strongly with those in IL-13- and TGFβ-treated mice. TSLP up-regulated specific genes, including CXCL9, proteasome, and interferon (IFN)-regulated genes. TSLP treatment in IL-4Rα1-deficient mice promoted similar cutaneous inflammation as in wild-type mice, though TSLP-induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up-regulated tumor necrosis factor α, Mx-1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP-deficient mice treated with TGFβ showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I-C)-treated mice showed high levels of cutaneous TSLP. CONCLUSION TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFβ and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.

Global chemokine expression in systemic sclerosis (SSc): CCL19 expression correlates with vascular inflammation in SSc skin

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.

miR-155 in the progression of lung fibrosis in systemic sclerosis

BackgroundMicroRNA (miRNA) control key elements of mRNA stability and likely contribute to the dysregulated lung gene expression observed in systemic sclerosis associated interstitial lung disease (SSc-ILD). We analyzed the miRNA gene expression of tissue and cells from patients with SSc-ILD. A chronic lung fibrotic murine model was used.MethodsRNA was isolated from lung tissue of 12 patients with SSc-ILD and 5 controls. High-resolution computed tomography (HRCT) was performed at baseline and 2–3 years after treatment. Lung fibroblasts and peripheral blood mononuclear cells (PBMC) were isolated from healthy controls and patients with SSc-ILD. miRNA and mRNA were analyzed by microarray, quantitative polymerase chain reaction, and/or Nanostring; pathway analysis was performed by DNA Intelligent Analysis (DIANA)-miRPath v2.0 software. Wild-type and miR-155 deficient (miR-155ko) mice were exposed to bleomycin.ResultsLung miRNA microarray data distinguished patients with SSc-ILD from healthy controls with 185 miRNA differentially expressed (q < 0.25). DIANA-miRPath revealed 57 Kyoto Encyclopedia of Genes and Genomes pathways related to the most dysregulated miRNA. miR-155 and miR-143 were strongly correlated with progression of the HRCT score. Lung fibroblasts only mildly expressed miR-155/miR-21 after several stimuli. miR-155 PBMC expression strongly correlated with lung function tests in SSc-ILD. miR-155ko mice developed milder lung fibrosis, survived longer, and weaker lung induction of several genes after bleomycin exposure compared to wild-type mice.ConclusionsmiRNA are dysregulated in the lungs and PBMC of patients with SSc-ILD. Based on mRNA-miRNA interaction analysis and pathway tools, miRNA may play a role in the progression of the disease. Our findings suggest that targeting miR-155 might provide a novel therapeutic strategy for SSc-ILD.

Macrophage Involvement in Systemic Sclerosis: Do We Need More Evidence?

The pathogenesis of systemic sclerosis is still unknown, although immune cells, mainly macrophages/monocytes, may have an important role in initiating and/or perpetuating the disease. Macrophages and monocytes are often classified as pro-inflammatory M1 phenotype or classic activation and pro-fibrotic/anti-inflammatory M2 phenotype or alternative activation. In this review, we highlighted the most relevant research regarding the involvement of macrophages/monocytes in the pathogenesis of this complex disease.

A Proteome-Derived Longitudinal Pharmacodynamic Biomarker for Diffuse Systemic Sclerosis Skin

In this study we systematically investigated alterations in the serum proteome of patients with diffuse cutaneous systemic sclerosis and identified differentially expressed proteins that correlated with disease severity. Our goal was to identify a combination of serum proteins that would provide a biological measure for the extent of skin disease and that could be combined into a longitudinal pharmacodynamic biomarker. We found that 16% of the sera proteins analyzed by SOMAscan aptamer technology, from two cohorts of patients with diffuse cutaneous systemic sclerosis, were identified as differentially regulated between diffuse cutaneous systemic sclerosis and controls and correlated with modified Rodnan skin score. This dataset showed tumor necrosis factor-α, IFN-γ, transforming growth factor-β, and IL-13 as potential upstream regulators of the serum protein patterns in the sera of patients with diffuse cutaneous systemic sclerosis. By ELISA, two analytes (ST2 and Spondin-1) best described longitudinal change in modified Rodnan skin score, using linear mixed models. This model was then validated in three independent cohorts. In this study we discovered a large array of proteins not previously associated with systemic sclerosis that provide insight into pathogenesis and potential targets for therapeutic intervention. Furthermore, we show that two of these proteins can be combined to form a robust longitudinal biomarker that might be used in clinical trials to assess changes in diffuse cutaneous systemic sclerosis skin disease over time.