Pro-inflammatory S100A11 is elevated in inflammatory myopathies and reflects disease activity and extramuscular manifestations in myositis.

BACKGROUND: S100A11 (calgizzarin), a member of the S100 family, is associated with oncogenesis, inflammation and myocardial damage. Our aim was to analyse S100A11 in idiopathic inflammatory myopathies (IIMs) and its association with disease activity features and cancer development. METHODS: S100A11 in muscle was determined by immunohistochemistry in polymyositis (PM), dermatomyositis (DM), myasthenia gravis (MG) and in subjects without autoimmune inflammatory disease (HC). S100A11 in plasma was measured in 110 patients with IIMs (PM, DM, and cancer associated myositis (CAM) patients) and in 42 HC. Disease activity was assessed by myositis disease activity assessment (MYOACT), muscle enzymes and C-reactive protein (CRP) were measured by routine laboratory techniques; autoantibodies by immunoprecipitation or by immunoblot. RESULTS: We observed an accumulation of S100A11 in the cytoplasm of regenerating and necrotizing muscle fibres of PM and DM patients. S100A11 was increased in plasma of all myositis patients compared to HC (3.8 (1.5-16.8) vs 2.8 (1.7-11.2) ng/ml, p = 0.011) and in DM and CAM patients compared to HC (4.0 (2.2-14.9) and 4.5 (1.5-9.1) vs 2.8 (1.7-11.2) ng/ml, p < 0.001 and p = 0.022, respectively). In all myositis patients, S100A11 correlated with the levels of lactate dehydrogenase (r = 0.256, p = 0.011), aspartate aminotransferase (AST) (r = 0.312, p = 0.002), CRP (r = 0.254, p = 0.022) and MYOACT (r = 0.245, p = 0.022). S100A11 was associated with MYOACT (r = 0.377, p = 0.030) and pulmonary and cutaneous disease activity in DM patients (r = 0.408, p = 0.017 and r = 0.417, p = 0.01, respectively). S100A11 was related to the levels of AST (r = 0.412, p = 0.027) in PM and to the levels of creatine phosphokinase (r = 0.432, p = 0.028) in CAM patients. CONCLUSIONS: We show for a first time a potential implication of S100A11 in the local inflammatory and tissue remodelling processes in myositis and an association of circulating S100A11 with disease activity and extra muscular manifestations in DM.

Circulating S100 proteins effectively discriminate SLE patients from healthy controls: a cross-sectional study.

S100 proteins are currently being investigated as potential diagnostic and prognostic biomarkers of several cancers and inflammatory diseases. The aims of this study were to analyse the plasma levels of S100A4, S100A8/9 and S100A12 in patients with incomplete systemic lupus erythematosus (iSLE), in patients with established SLE and in healthy controls (HCs) and to investigate the potential utility of the S100 proteins as diagnostic or activity-specific biomarkers in SLE. Plasma levels were measured by ELISA in a cross-sectional cohort study of 44 patients with SLE, 8 patients with iSLE and 43 HCs. Disease activity was assessed using the SLEDAI-2K. The mean levels of all S100 proteins were significantly higher in SLE patients compared to HCs. In iSLE patients, the levels of S100A4 and S100A12 but not S100A8/9 were also significantly higher compared to HCs. There were no significant differences in S100 levels between the iSLE and SLE patients. Plasma S100 proteins levels effectively discriminated between SLE patients and HCs. The area under the curve (AUC) for S100A4, S100A8/9 and S100A12 plasma levels was 0.989 (95% CI 0.976-1.000), 0.678 (95% CI 0.563-0.792) and 0.807 (95% CI 0.715-0.899), respectively. S100 levels did not differentiate between patients with high and low disease activity. Only the S100A12 levels were significantly associated with SLEDAI-2K and with cSLEDAI-2K. S100 proteins were significantly higher in SLE patients compared HCs and particularly S100A4 could be proposed as a potential diagnostic biomarker for SLE.

Composition of TWIST1 dimers regulates fibroblast activation and tissue fibrosis.

OBJECTIVES: TWIST1 is a member of the class B of basic helix-loop-helix transcription factors that regulates cell lineage determination and differentiation and has been implicated in epithelial-to-mesenchymal transition. Here, we aimed to investigate the role of TWIST1 for the activation of resident fibroblasts in systemic sclerosis (SSc). METHODS: The expression of Twist1 in fibroblasts was modulated by forced overexpression or siRNA-mediated knockdown. Interaction of Twist1, E12 and inhibitor Of differentiation (Id) was analysed by co-immunoprecipitation. The role of Twist1 in vivo was evaluated using inducible, conditional knockout mice with either ubiquitous or fibroblast-specific depletion of Twist1. Mice were either challenged with bleomycin or overexpressing a constitutively active transforming growth factor (TGF)ß receptor I. RESULT: The expression of TWIST1 was increased in fibroblasts in fibrotic human and murine skin in a TGFß/SMAD3-dependent manner. TWIST1 in turn enhanced TGFß-induced fibroblast activation in a p38-dependent manner. The stimulatory effects of TWIST1 on resident fibroblasts were mediated by TWIST1 homodimers. TGFß promotes the formation of TWIST1 homodimers by upregulation of TWIST1 and by induction of inhibitor of DNA-binding proteins, which have high affinity for E12/E47 and compete against TWIST1 for E12/E47 binding. Mice with selective depletion of Twist1 in fibroblasts are protected from experimental skin fibrosis in different murine models to a comparable degree as mice with ubiquitous depletion of Twist1. CONCLUSIONS: Our data identify TWIST1 as a central pro-fibrotic factor in SSc, which facilitates fibroblast activation by amplifying TGFß signalling. Targeting of TWIST1 may thus be a novel approach to normalise aberrant TGFß signalling in SSc.

The transcription factor GLI2 as a downstream mediator of transforming growth factor-ß-induced fibroblast activation in SSc.

OBJECTIVES: Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways. METHODS: The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-ß (TGF-ß) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-ß receptor I. RESULTS: TGF-ß upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBRact-induced fibrosis. Combined targeting of canonical and non-canonical hedgehog signalling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signalling with SMO inhibitors in experimental dermal and pulmonary fibrosis. CONCLUSIONS: Our data demonstrate that hedgehog pathways and TGF-ß signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

Interleukin-20 is triggered by TLR ligands and associates with disease activity in patients with rheumatoid arthritis.

BACKGROUND: Interleukin (IL)-20 is a pro-inflammatory cytokine that may be implicated in the pathogenesis of rheumatoid arthritis (RA). This study aimed to determine the association between IL-20 and disease activity in patients with RA. METHODS: The levels of serum and synovial fluid IL-20 were measured in patients with RA and OA. The disease activity was assessed based on the Disease Activity Score of 28 joints (DAS28). The expression of IL-20 in synovial tissue samples from patients with RA and OA were determined by immunohistochemistry. Immunofluorescence staining was used to co-localize IL-20 with selected cells. The secretion of IL-20 was analysed in human peripheral blood mononuclear cells (PBMCs) of patients with RA. RESULTS: Synovial fluid and synovial tissue IL-20 were significantly increased in patients with RA compared with patients with OA. The expression of IL-20 in RA synovial tissue was particularly associated with macrophages and neutrophil granulocytes, but also with synovial fibroblasts and lymphocytes. The IL-20 levels in synovial fluid correlated with DAS28 (r=0.434; p=0.015) and were significantly elevated in anti-CCP positive RA compared with anti-CCP negative RA (122.3±104.1pg/ml and 45.9±35.8pg/ml; p=0.008). IL-20 production from PBMCs was induced by Poly I:C and LPS but not with pro-inflammatory cytokines, such as TNF-α or IL-1. CONCLUSION: Our data showed that IL-20 is independently associated with RA disease activity and may be triggered by TLR ligands at local sites of inflammation.

Sirt1 regulates canonical TGF-ß signalling to control fibroblast activation and tissue fibrosis.

BACKGROUND: Sirt1 is a member of the sirtuin family of proteins. Sirt1 is a class III histone deacetylase with important regulatory roles in transcription, cellular differentiation, proliferation and metabolism. As aberrant epigenetic modifications have been linked to the pathogenesis of systemic sclerosis (SSc), we aimed to investigate the role of Sirt1 in fibroblast activation. METHODS: Sirt1 expression was analysed by real-time PCR, western blot and immunohistochemistry. Sirt1 signalling was modulated with the Sirt1 agonist resveratrol and by fibroblast-specific knockout. The role of Sirt1 was evaluated in bleomycin-induced skin fibrosis and in mice overexpressing a constitutively active transforming growth fac-tor-ß (TGF-ß) receptor I (TBRIact). RESULTS: The expression of Sirt1 was decreased in patients with SSc and in experimental fibrosis in a TGF-ß-dependent manner. Activation of Sirt1 potentiated the profibrotic effects of TGF-ß with increased Smad reporter activity, elevated transcription of TGF-ß target genes and enhanced release of collagen. In contrast, knockdown of Sirt1 inhibited TGF-ß/SMAD signalling and reduced release of collagen in fibroblasts. Consistently, mice with fibroblast-specific knockdown of Sirt1 were less susceptible to bleomycin- or TBRIact-induced fibrosis. CONCLUSIONS: We identified Sirt1 as a crucial regulator of TGF-ß/Smad signalling in SSc. Although Sirt1 is downregulated, this decrease is not sufficient to counterbalance the excessive activation of TGF-ß signalling in SSc. However, augmentation of this endogenous regulatory mechanism, for example, by knockdown of Sirt1, can effectively inhibit TGF-ß signalling and exerts potent antifibrotic effects. Sirt1 may thus be a key regulator of fibroblast activation in SSc.

Tribbles homologue 3 stimulates canonical TGF-ß signalling to regulate fibroblast activation and tissue fibrosis.

OBJECTIVES: Tribbles homologue 3 (TRB3) is a pseudokinase that modifies the activation of various intracellular signalling pathways to control fundamental processes extending from mitosis and cell activation to apoptosis and modulation of gene expression. Here, we aimed to analyse the role of TRB3 in fibroblast activation in systemic sclerosis (SSc). METHODS: The expression of TRB3 was quantified by quantitative PCR, western blot and immunohistochemistry. The role of TRB3 was analysed in cultured fibroblasts and in experimental fibrosis using small interfering RNA (siRNA)-mediated knockdown and overexpression of TRB3. RESULTS: TRB3 expression was increased in fibroblasts of patients with SSc and in murine models of SSc in a transforming growth factor-ß (TGF-ß)/Smad-dependent manner. Overexpression of TRB3 stimulated canonical TGF-ß signalling and induced an activated phenotype in resting fibroblasts. In contrast, knockdown of TRB3 reduced the profibrotic effects of TGF-ß and decreased the collagen synthesis. Moreover, siRNA-mediated knockdown of TRB3 exerted potent antifibrotic effects and ameliorated bleomycin as well as constitutively active TGF-ß receptor I-induced fibrosis with reduced dermal thickening, decreased hydroxyproline content and impaired myofibroblast differentiation. CONCLUSIONS: The present study characterises TRB3 as a novel profibrotic mediator in SSc. TGF-ß induces TRB3, which in turn activates canonical TGF-ß/Smad signalling and stimulates the release of collagen, thereby inducing a positive feedback loop that may contribute to aberrant TGF-ß signalling in SSc.

S100A4 amplifies TGF-ß-induced fibroblast activation in systemic sclerosis.

OBJECTIVES: S100A4 is a calcium binding protein with regulatory functions in cell homeostasis, proliferation and differentiation that has been shown to promote cancer progression and metastasis. In the present study, we evaluated the role of S100A4 in fibroblast activation in systemic sclerosis (SSc). METHODS: The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4(-/-)) mice. Transforming growth factor ß (TGF-ß) signalling was assessed by reporter assays, staining for phosphorylated Smad2/3 and analyses of target genes. RESULTS: The expression of S100A4 was increased in SSc skin and in experimental fibrosis in a TGF-ß/Smad-dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-ß and decreased the release of collagen. S100A4(-/-) mice were protected from bleomycin-induced skin fibrosis with reduced dermal thickening, decreased hydroxyproline content and lower myofibroblast counts. Deficiency of S100A4 also ameliorated fibrosis in the tight-skin-1 (Tsk-1) mouse model. CONCLUSIONS: We characterised S100A4 as a downstream mediator of the stimulatory effects of TGF-ß on fibroblasts in SSc. TGF-ß induces the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-ß and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies.

High levels of metastasis-inducing S100A4 protein and treatment outcome in early rheumatoid arthritis: data from the PERAC cohort.

Abstract The aim of this study was to evaluate the role of S100A4 as a biomarker in patients with early rheumatoid arthritis (RA). S100A4 levels were measured in 59 patients with early RA and in 41 healthy controls. The association between the S100A4 levels and the treatment outcome after 12 months was determined using multivariate regression analysis. Serum S100A4 levels were significantly higher in the patients with early RA than in the healthy subjects and significantly decreased after 3 months of treatment. Diseases activity at 12 months was significantly higher in female patients who had initially high levels of S100A4. Persistently high S100A4 levels predicted poor treatment outcome and S100A4 may thus represent promising biomarker for assessing treatment response in patients with RA.

S100A4 is elevated in axial spondyloarthritis: a potential link to disease severity.

BACKGROUND: S100A4 is a member of calcium binding S100 protein family well known for its role in cancer progression and metastasis. Nevertheless, S100A4 also serves as a negative regulator of bone formation. Dickkopf-1 (DKK-1), marker of bone remodelling, is also implicated in the process of syndesmophyte formation in ankylosing spondylitis. The aim of our study was to evaluate plasma levels of S100A4 in patients with axial spondyloarthritis and to determine the potential association of S100A4 with disease severity, clinical manifestations and with bone changes in a cross-sectional study. METHODS: Fifty-eight patients with axial spondyloarthritis and 40 healthy controls were studied. Biological samples were analysed for S100A4 and Dickkopf-1. Disease activity was assessed according to the Bath Ankylosing Spondylitis Disease Activity Index. C-reactive protein (CRP) was used as a marker of inflammation. Radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). RESULTS: The plasma levels of S100A4 were significantly higher in patients with axial spondyloarthritis compared to heathy controls (p < 0.0001). The levels of S100A4 were higher in early stages of the disease and lower in patients with the presence of syndesmophytes (p = 0.009). Furthermore, we found weak but significant inverse correlation of plasma S100A4 with the mSASSS (r = - 0.363, p = 0.030). Levels of S100A4 were negatively associated with disease duration (r = - 0.404, p = 0.002) and positively with Dickkopf-1 binding capacity (r = 0.312, p = 0.023). CONCLUSIONS: This is the first study showing elevated circulating levels of S100A4 in patients with axial spondyloarthritis, particularly in early stages of the disease prior to spinal involvement, and its significantly lower levels in patients with syndesmophytes. The role of S100A4 in the pathogenesis of axial spondyloarthritis can be suggested.

Calgizzarin (S100A11): a novel inflammatory mediator associated with disease activity of rheumatoid arthritis.

BACKGROUND: Calgizzarin (S100A11) is a member of the S100 protein family that acts in different tumors by regulating a number of biologic functions. Recent data suggest its association with low-grade inflammation in osteoarthritis (OA). The aim of our study is to compare S100A11 expression in the synovial tissues, synovial fluid and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to characterize the potential association between S100A11 and disease activity. METHODS: S100A11 protein expression was detected in synovial tissue from patients with RA (n = 6) and patients with OA (n = 6) by immunohistochemistry and immunofluorescence. Serum and synovial fluid S100A11 levels were measured by ELISA in patients with RA (n = 40) and patients with OA (n = 34). Disease activity scores in 28 joints based on C-reactive protein (DAS28-CRP) were used to assess disease activity. Cytokine content in peripheral blood mononuclear cells (PBMCs), synovial fibroblasts (SFs) and synovial fluid was analysed by ELISA, western blotting or cytometric bead array. RESULTS: S100A11 expression was significantly up-regulated in the synovial lining and sublining layers (p < 0.01) and vessels (p < 0.05) of patients with RA compared to patients with OA, and was associated with fibroblasts and T cells. S100A11 was significantly increased in synovial fluid (p < 0.0001) but not in serum (p = 0.158) from patients with RA compared to patients with OA when adjusted for age and sex. Synovial fluid S100A11 correlated with DAS28 (r = 0.350, p = 0.027), serum CRP (r = 0.463, p = 0.003), synovial fluid leukocyte count (r = 0.677, p < 0.001), anti-cyclic citrullinated peptide antibodies (anti-CCP) (r = 0.424, p = 0.006) and IL-6 (r = 0.578, p = 0.002) and IL-8 (r = 0.740, p < 0.001) in synovial fluid from patients with RA. PBMCs and SFs isolated from patients with RA synthesized and spontaneously secreted higher levels of S100A11 in comparison with PBMCs and SFs from patients with OA (p = 0.011 and 0.03, respectively). S100A11 stimulated the production of the pro-inflammatory cytokine IL-6 by PBMCs (p < 0.05) and SFs (p < 0.01). CONCLUSIONS: Our data provide the first evidence of S100A11 up-regulation and its association with inflammation and disease activity in patients with RA.

Activation of STAT3 integrates common profibrotic pathways to promote fibroblast activation and tissue fibrosis.

Signal transducer and activator of transcription 3 (STAT3) is phosphorylated by various kinases, several of which have been implicated in aberrant fibroblast activation in fibrotic diseases including systemic sclerosis (SSc). Here we show that profibrotic signals converge on STAT3 and that STAT3 may be an important molecular checkpoint for tissue fibrosis. STAT3 signaling is hyperactivated in SSc in a TGFß-dependent manner. Expression profiling and functional studies in vitro and in vivo demonstrate that STAT3 activation is mediated by the combined action of JAK, SRC, c-ABL, and JNK kinases. STAT3-deficient fibroblasts are less sensitive to the pro-fibrotic effects of TGFß. Fibroblast-specific knockout of STAT3, or its pharmacological inhibition, ameliorate skin fibrosis in experimental mouse models. STAT3 thus integrates several profibrotic signals and might be a core mediator of fibrosis. Considering that several STAT3 inhibitors are currently tested in clinical trials, STAT3 might be a candidate for molecular targeted therapies of SSc.

Interleukin 35 Synovial Fluid Levels Are Associated with Disease Activity of Rheumatoid Arthritis.

OBJECTIVES: To study the association of systemic and local interleukin-35 (IL-35) levels in rheumatoid arthritis. METHODS: 37 patients with treatment naïve early RA, 49 with established RA and 29 control patients with osteoarthritis (OA) were studied. Serum and paired synovial fluid samples were analysed for IL-35. Disease activity of RA patients was assessed according to the 28-Joint Count Disease Activity Score (DAS28). RESULTS: The levels of serum IL-35 were significantly higher in patients with treatment naïve early RA compared to those with established disease and control OA subjects. In addition, serum levels of IL-35 significantly decreased 12 weeks after initiation of glucocorticoids and conventional synthetic disease modifying antirheumatic drugs in patients with treatment naïve early RA. Synovial fluid IL-35 levels were significantly higher in RA compared to OA patients, were significantly elevated compared to serum counterparts and correlated with synovial fluid leukocyte count (r=0.412; p<0.01), serum CRP levels (r=0.362; p<0.05) and DAS28 (r=0.430, p<0.01). CONCLUSION: This is the first study showing elevated circulating levels of IL-35 in treatment naïve early RA, its significant decrease after treatment initiation and positive association between increased synovial fluid IL-35 and disease activity in patients with long-lasting RA.