Tatiana Spadoni

A Reactive Oxygen Species–Mediated Loop Maintains Increased Expression of NADPH Oxidases 2 and 4 in Skin Fibroblasts From Patients With Systemic Sclerosis

Reactive oxygen species (ROS) contribute to the pathogenesis of fibrosis in systemic sclerosis (SSc; scleroderma), and NADPH oxidase (NOX) is an important source of ROS. Since the role of single NOX isoforms has not been previously investigated in SSc, this study was undertaken to assess the expression of NOX in SSc fibroblasts compared to normal healthy cells and to analyze their role in cell activation.

Oxidative DNA damage induces the ATM-mediated transcriptional suppression of the Wnt inhibitor WIF-1 in systemic sclerosis and fibrosis

DNA damage not only promotes a mitotic checkpoint response but also activates a pathway that can produce fibrotic disease. Connecting DNA Damage to Fibrotic Skin Thickening Systemic sclerosis (SSc) is an autoimmune disease characterized by the accumulation of collagen in the connective tissue, a process known as fibrosis. Fibrosis affects skin and internal organs and is frequently associated with activation of the Wnt signaling pathway. Svegliati et al. found loss of the Wnt inhibitor WIF-1 in skin biopsies from SSc patients compared with healthy skin. Antibodies in serum from SSc patients or oxidative DNA-damaging agents triggered a transcriptional suppression pathway that suppressed WIF-1 expression and promoted collagen production in normal fibroblasts. In fibroblasts cultured from SSc patients, inhibiting this pathway restored WIF-1 expression and reduced collagen production; in a mouse model of fibrosis, inhibiting this pathway prevented fibrotic skin thickening. Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector β-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, β-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis.

Induction of Scleroderma Fibrosis in Skin‐Humanized Mice by Administration of Anti−Platelet‐Derived Growth Factor Receptor Agonistic Autoantibodies

To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors.

Induction of scleroderma fibrosis in skin‐humanized mice by anti‐Platelet‐Derived Growth Factor receptor agonistic autoantibodies

To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors.

Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

IntroductionAbnormal oxidative stress has been described in systemic sclerosis (SSc) and previous works from our laboratory demonstrated an increased generation of reactive oxygen species (ROS) by SSc fibroblasts and monocytes. This study investigated the ability of SSc T lymphocytes to produce ROS, the molecular pathway involved, and the biological effects of ROS on SSc phenotype.MethodsPeripheral blood T lymphocytes were isolated from serum of healthy controls or SSc patients by negative selection with magnetic beads and activated either with PMA or with magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Intracellular ROS generation was measured using a DCFH-DA assay in a plate reader fluorimeter or by FACS analysis. CD69 expression and cytokine production were analyzed by FACS analysis. Protein expression was studied using immunoblotting techniques and mRNA levels were quantified by real-time PCR. Cell proliferation was carried out using a BrdU incorporation assay.ResultsPeripheral blood T lymphocytes from SSc patients showed an increased ROS production compared to T cells from healthy subjects. Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox. Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.ConclusionsSSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production. These data confirm lymphocytes as key cellular players in the pathogenesis of systemic sclerosis and suggest a crucial link between ROS and T cell activation.

Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro

One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia.

NADPH oxidase, oxidative stress and fibrosis in systemic sclerosis

ABSTRACT Systemic sclerosis (SSc) is an autoimmune disease characterized by damage of small vessels, immune abnormalities and exaggerated production of extracellular matrix. The etiology of the disease is unknown and the pathogenesis ill defined. However, there is consistent evidence that oxidative stress contributes to the establishment and progression of the disease. This review examines the most relevant research regarding the involvement of free radicals and of nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases; NOX) in the pathogenesis of systemic sclerosis. Graphical abstract Figure. No caption available. HighlightsOxidative stress may play a role in the molecular pathogenesis of fibrosis and in the development of systemic sclerosis.NADPH oxidases are involved in ROS generation in systemic sclerosis and other fibrotic disorders.Further work is needed to develop new specific NOX inhibitors for the treatment of several fibrotic disorders.

Characterization of binding and quantification of human autoantibodies to PDGFRα using a biosensor-based approach

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20-4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.

Sclerostin and Antisclerostin Antibody Serum Levels Predict the Presence of Axial Spondyloarthritis in Patients with Inflammatory Bowel Disease

Objective. The early diagnosis of inflammatory bowel disease (IBD)-associated spondyloarthritis (SpA/IBD) in patients affected by IBD represents a major topic in clinical practice; in particular, to date there are no available serum biomarkers revealing the presence of joint inflammation in these patients. Sclerostin (SOST), an antagonist of the Wnt/β-catenin pathway, and antisclerostin-immunoglobulin G (anti-SOST–IgG) have been recently studied in patients with ankylosing spondylitis (AS) as a putative marker of disease activity. Methods. SOST and anti-SOST-IgG serum levels were assayed in 125 patients with IBD, 85 with axial or peripheral SpA, and in control groups (patients with AS and rheumatoid arthritis, and healthy individuals). The diagnostic performance in discriminating the presence of SpA/IBD was assessed for both candidate biomarkers. Results. Patients affected by SpA/IBD with axial involvement displayed significantly lower levels of SOST and higher levels of anti-SOST-IgG compared to patients with only peripheral arthritis, IBD, and controls. Moreover, SOST and anti-SOST-IgG serum levels were inversely correlated and were associated with the duration of articular symptoms. Both biomarkers showed good accuracy in predicting the presence of axial SpA in patients with IBD. Conclusion. We demonstrated that in patients with IBD, SOST and anti-SOST-IgG might represent novel biomarkers to assess the presence of axial joint involvement. Moreover, the development of anti-SOST-IgG and the subsequent decrease of SOST serum levels could play a role in the pathogenesis of SpA/IBD.

FRI0242 Role of Mitochondria- and Nadph Oxidase-Derived ROS in Fibroblasts Isolated from Patients Affected by Systemic Sclerosis

Background Oxidative stress plays an important role in the development of fibrosis under various pathological conditions. Fibroblasts isolated from patients with Systemic Sclerosis (SSc) overproduce reactive oxygen species (ROS), overexpress type I collagen and α-smooth muscle actin (α-SMA) and show DNA damage and activation of checkpoint kinase ATM (1, 2). NADPH oxidase (NOX) is an important source of ROS, and its overactivity or overexpression are often associated with chronic diseases, characterized by tissue damage and fibrosis. Recently, we have demonstrated that NOX2 and NOX4 are critical components of NADPH oxidase complex in SSc fibroblasts and ROS generated by NOX play a primary role in the pathological activation of dermal fibroblasts (3). Since a recent study reported that NOX4 localizes to membranes and mitochondria and contributes to the generation of mitochondrial ROS (4), we wondered whether mitochondria also play a role in ROS production in SSc fibroblasts. Objectives The aim of this study was to investigate the contribution of mitochondrial ROS to oxidative stress in SSc. Methods ROS production was analysed by confocal microscopy using dihydroethidium (DHE, to assess all intracellular superoxide), or MitoSOX™ Red (to detect mitochondria superoxide), or using a microplate reader following Amplex® Red kit protocol (to measure H2O2). Mitochondria were purified using a mitochondria isolation kit. For protein expression, mitochondrial and cytosolic fractions were subjected to western blot with specific antibodies. Results SSc fibroblasts incubated with the mitochondria-targeted antioxidant MitoQ showed a reduced MitoSOX™ Red staining, and a partially decreased DHE fluorescence, suggesting that mitochondria contribute to the redox state in SSc fibroblasts. Mitochondria purified from SSc fibroblasts generated significantly higher levels of ROS compared to controls. Incubation of normal cells with PDGF, a profibrotic cytokine able to activate a SSc-like phenotype, led to a significant increase of total and mitochondrial ROS levels compared to unstimulated cells. Mitochondrial and cytosolic fractions from SSc and activated normal fibroblasts were also analyzed to evaluate different protein expression patterns. Conclusions In this study we demonstrated that mitochondria contribute to the abnormal redox state of SSc fibroblasts and activated normal cells. Further studies may clarify whether mitochondrial ROS are generated by a mitochondrial NOX isoform or are the result of the interplay between mitochondria and NOX enzymes located outside the organelles. References Baroni S et al, N Engl J Med 2006; 354:2667–76; Svegliati S et al, Science Signaling 2014; 7: ra84; Spadoni T et al, Arthritis Rheum 2015; 67:1611–1622; Lee DY et al, J Biol Chem 2013;288:28668–28686. Disclosure of Interest None declared