Potential involvement of Notch1 signalling in the pathogenesis of primary cutaneous CD30-positive lymphoproliferative disorders

Background  The central role of Notch signalling in T‐cell development and oncogenesis raises the question of the importance of this pathway in cutaneous T‐cell lymphomas. Objectives  To investigate the pattern of expression of Notch and its ligands, Jagged and Delta, in skin samples of primary cutaneous CD30+ lymphoproliferative disorders. Methods  Immunohistochemistry of formalin‐fixed, paraffin‐embedded skin samples from 12 patients with lymphomatoid papulosis (LyP) and 11 patients with primary cutaneous anaplastic large cell lymphoma (ALCL). Immunofluorescence studies of fresh skin samples obtained from three patients with LyP and two patients with primary cutaneous ALCL. Results  We identified single Notch1‐positive cells or small clusters of atypical cells in LyP. Similarly, strongly positive Jagged1 cells tended to be localized in clusters. Primary cutaneous ALCL had higher expression of Notch1 and Jagged1 compared with LyP. Cells expressing Notch1 and Jagged1 were colocalized and a subset of cells expressed both the receptor and the ligand. The expression of the ligand Delta1 was low to undetectable in both types of lymphoproliferations. A subpopulation of lymphoma cells was found to coexpress Notch1 and activated Akt kinase. Conclusions  These results imply a potential role for the Notch signalling pathway in the pathogenesis of primary cutaneous CD30+ lymphoproliferative disorders and provide a rationale for the exploration of the activity of Notch antagonists in the therapy of these diseases.     

Increased expression of GAB1 promotes inflammation and fibrosis in systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease mainly characterized by persistent inflammation and fibrosis. The receptor tyrosine kinase (RTK) signal pathway plays an important role in the process of SSc, and Grb2‐associated binding protein (GAB) is crucial in activating RTK signalling. A previous study found elevated levels of GAB1 in bleomycin (BLM)‐induced fibrotic lungs, but the effects of GAB1 in SSc remain unclear. Our aim was to investigate whether GAB1 was dysregulated and its potential role in SSc. Compared with healthy donors, we found GAB1 expression was 1.6‐fold higher in peripheral blood mononuclear cells (PBMC), 2.5‐fold higher in CD4 + T cells, and 2‐fold higher in skin from of SSc patients (P < .01). At the same time, the levels of type one collagen (COLI) were also significantly increased (1.8‐fold higher) in SSc skin. Additionally, BLM‐induced SSc mice showed mRNA levels of Gab1 2‐fold higher than saline‐treated controls, and Gab1 expression correlated positively with collagen content. A further in vitro study showed silencing of GAB1 suppressed inflammatory gene expression in TNF‐α induced fibroblasts. Additionally, GAB1 deficiency prominently inhibited cell proliferation and reduced COLI protein levels in TGF‐β induced fibroblasts. Taken together, these data suggest that GAB1 has a relatively high expression rate in SSc, and knockdown of GAB1 may attenuate SSc by stimulating inflammatory and fibrotic processes.     

Canonical Wnt signalling as a key regulator of fibrogenesis – implications for targeted therapies?

Canonical Wnt signalling belongs to the so‐called morphogen pathways and plays essential roles in development and tissue homeostasis. Being such a crucial regulatory pathway, Wnt signalling is tightly controlled at different levels. However, uncontrolled activation of canonical Wnt signalling has been implicated into the pathogenesis of various human disorders. In the last years, aberrant Wnt signalling has been demonstrated in fibrotic diseases including systemic sclerosis (SSc). In this review, we will discuss the current state of research on canonical Wnt signalling in SSc. Activation of canonical Wnt signalling induces fibroblast activation with subsequent myofibroblast differentiation and excessive collagen release resulting in tissue fibrosis. Genetic or pharmacological blockade of Wnt activation ameliorates experimental fibrosis in different preclinical models. These findings have direct translational implications because several small molecule inhibitors of Wnt signalling are currently evaluated in clinical trials and some already showed first promising results.     

Osteopontin in systemic sclerosis and its role in dermal fibrosis

Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Here, we determined whether OPN levels are increased in a large cohort of patients with systemic sclerosis (SSc) and whether OPN contributes to the development of dermal fibrosis. The plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared with healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin (bleo)-induced dermal fibrosis model. OPN-deficient (OPN(-/-)) mice developed less dermal fibrosis compared with wild-type (WT) mice in the bleo-induced dermal fibrosis model. Additional in vivo studies have demonstrated that lesional skin from OPN(-/-)mice had fewer Mac-3-positive cells, fewer myofibroblasts, decreased transforming growth factor (TGF)-β and genes in the TGF-β pathway, and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and extracellular signal-regulated kinase. In vitro, OPN(-/-) dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGF-β. Finally, TGF-β production by OPN-deficient macrophages was reduced compared with WT. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that it may be a new therapeutic target in SSc.     

Abnormal B Lymphocyte Activation and Function in Systemic Sclerosis

Systemic sclerosis (SSc) is characterized by tissue fibrosis and autoimmunity. Although the pathogenic relationship between autoimmunity and clinical manifestations of SSc remains unknown, SSc patients display abnormal immune responses including the production of disease-specific autoantibodies. Previous studies have demonstrated that B cells play a critical role in systemic autoimmunity and disease expression through various functions such as induction of the activation of other immune cells in addition to autoantibody production. CD19 is a crucial regulator of B cell activation. Recent studies demonstrated that B cells from SSc patients showed an up-regulated CD19 signaling pathway that induced SSc-specific autoantibody production in SSc mouse models. CD19 transgenic mice lost tolerance for autoantigen and generated autoantibodies spontaneously. B cells from SSc patients exhibited an overexpression of CD19 that induced SSc-specific autoantibody production in transgenic mice. Moreover, SSc patients displayed intrinsic B cell abnormalities characterized by chronic hyper-reactivity of memory B cells, which was possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a genetic model of SSc, showed augmented CD19 signaling. In bleomycin-induced SSc mouse models, endogenous ligands for toll-like receptor 4 induced by bleomycin stimulated B cells to produce various fibrogenic cytokines and autoantibodies. Remarkably, the loss of CD19 resulted in the inhibition of B cell hyper-reactivity and autoantibody production, which are associated with improvements in fibrosis and a parallel decrease in fibrogenic cytokine production by B cells. Taken together, the findings suggest that altered B cell function may result in tissue fibrosis as well as autoimmunity in SSc.     

Does therapeutic intervention in atopic dermatitis normalize epidermal Notch deficiency?

This viewpoint presents a unifying concept for the treatment of atopic dermatitis (AD) that is based on the improvement of deficient Notch signalling, which appears to represent the fundamental epithelial defect of AD resulting in epidermal and immunological barrier dysfunction. One study of AD patients demonstrated a marked epidermal deficiency of Notch receptors and several mouse models with genetically suppressed Notch signalling exhibit dry skin, signs of scratching, skin barrier abnormalities, increased transepidermal water loss and Th2 cell‐mediated immunological changes closely resembling human AD. Notch signalling is critically involved in the differentiation of regulatory T cells, in the feedback inhibition of activated innate immunity, in the repression of activating protein‐1 (AP‐1), the regulation of late epidermal differentiation associated with filaggrin‐ and stratum corneum barrier lipid processing, in aquaporin 3‐ and claudin‐1 expression and in keratinocyte‐mediated release of thymic stromal lymphopoietin (TSLP), which promotes Th2‐driven immune responses with TSLP‐ and IL‐31‐mediated stimulation of cutaneous sensory neurons involved in the induction of itch. Translational evidence will be provided that all major therapeutic regimens employed for the treatment of AD such as glucocorticoids, calcineurin inhibitors and UV radiation may converge in the upregulation of impaired Notch signalling, the proposed pathogenic defect of AD.     

Morphogen pathways as molecular targets for the treatment of fibrosis in systemic sclerosis

Wnt-, Hedgehog- and Notch-signaling cascades are morphogen pathways that play crucial roles in development and tissue homeostasis. While morphogen pathways are tightly regulated at multiple levels, inappropriate activation of Wnt, Hedgehog and Notch signaling has been implicated into the pathogenesis of various diseases. In particular, Wnt, Hedgehog and Notch signaling have emerged as central players in the pathogenesis of fibrotic diseases. Here, we will review the pro-fibrotic effects of Wnt, Hedgehog and Notch signaling in systemic sclerosis (SSc), prototypical systemic fibrotic disease. Wnt, Hedgehog and Notch pathways are activated in SSc. They potently stimulate fibroblasts to differentiate into myofibroblasts and to release collagen and other extracellular matrix components. Genetic or pharmacological inhibition of morphogen pathways effectively prevents experimental fibrosis in different preclinical models and induces regression of pre-established fibrosis. As several inhibitors of Wnt, Hedgehog and Notch have recently been developed with first ones being already approved for clinical trials, morphogen pathways maybe a novel approach for the treatment of fibrosis.     

Increased expression of Wnt2 and SFRP4 in Tsk mouse skin: role of Wnt signaling in altered dermal fibrillin deposition and systemic sclerosis

Systemic sclerosis (SSc) is a complex human disorder characterized by progressive skin fibrosis. To better understand the molecular basis of dermal fibrosis in SSc, we analyzed microarray gene expression in skin of the Tight-skin (Tsk) mouse, an animal model where skin fibrosis is caused by an in-frame duplication in fibrillin-1 (Fbn-1). Tsk skin showed increased mRNA levels of several genes involved in Wnt signaling, including Wnt2, Wnt9a, Wnt10b and Wnt11; Dapper homolog antagonist of beta-catenin (DACT1) and DACT2; Wnt-induced secreted protein 2; and secreted frizzled-related protein (SFRP)2 and SFRP4. RNase protection and northern blot confirmed microarray results. Furthermore, Wnt3a markedly stimulated matrix assembly of microfibrillar proteins, including Fbn-1, by cultured fibroblasts, suggesting that Wnts contribute to increased microfibrillar matrices in Tsk skin. Further analysis showed that SFRP4 expression is specifically increased in tissues expressing Tsk-Fbn-1, such as skeletal muscle and skin. The increase in SFRP4 mRNA in Tsk skin started 2 weeks after birth, following the increase in Wnt2 mRNA that occurred at birth. This suggests that SFRP4 may modulate Wnt functions in Tsk skin fibrosis. Lesional skin from SSc patients also showed large increases in SFRP4 mRNA and protein levels in the deep dermis compared to healthy skin, suggesting that the Wnt pathway might regulate skin fibrosis in SSc.     

A potential contribution of altered cathepsin L expression to the development of dermal fibrosis and vasculopathy in systemic sclerosis

Cathepsin L (CTSL) is a lysosomal proteolytic enzyme involved in inflammation and vascular and extracellular matrix remodelling, which are the three cardinal pathological events associated with systemic sclerosis (SSc). To elucidate the potential role of CTSL in the development of SSc, we here investigated CTSL expression in the lesional skin of patients with SSc and SSc animal models and the clinical correlation of serum CTSL levels. CTSL expression was elevated in dermal small vessels of SSc patients compared with those of healthy controls. Consistently, CTSL mRNA levels were increased in SSc lesional skin samples, but not in cultivated SSc dermal fibroblasts, compared with corresponding control samples from healthy individuals. Serum CTSL levels were significantly higher in SSc patients than in healthy controls and inversely correlated with skin score. Furthermore, the elevation of serum CTSL levels was linked to SSc vasculopathy. Supporting these results, Ctsl mRNA levels were decreased in the skin of bleomycin‐treated mice, an SSc animal model recapitulating its fibrotic aspect, and CTSL expression was enhanced in dermal small vessels of endothelial cell‐specific Fli1 knockout mice, reminiscent of SSc vasculopathy. Importantly, gene silencing of FLI1 induced CTSL mRNA expression and Fli1 occupied the CTSL promoter in human dermal microvascular endothelial cells. Collectively, these results suggest that endothelial CTSL up‐regulation partially due to Fli1 deficiency may contribute to the development of vasculopathy, while the decrease in dermal CTSL expression is likely associated with dermal fibrosis in SSc.     

Role of protease-activated receptors in human skin fibrosis and scleroderma

Proteases and their receptors have poorly understood roles in skin fibrosis and systemic scleroderma (SSc). We examined the role of protease-activated receptors (PAR(1) and PAR(2) ) in the pathophysiology of human SSc and skin fibrosis. Immunohistochemistry showed that PAR(1) immunoreactivity was positive in fibroblasts of SSc skin and healthy skin. PAR(2) immunoreactivity was positive in SSc skin, but negative in endothelial cells and fibroblasts of healthy skin. Double immunofluorescence using an antibody against smooth muscle actin (α-SMA) as a marker for myofibroblasts verified a certain percentage of myofibroblasts positive for PAR(1) and PAR(2) in SSc skin. In human dermal cultured fibroblasts (HDF), PAR(1) stimulation with or without bleomycin pretreatment mobilized intracellular calcium, indicating that the expressed PARs are functional and have effects on downstream signalling by calcium release. PAR(2) -induced intracellular calcium mobilization was only measurable in HDF after bleomycin pretreatment. Thus, PAR(1) - and PAR(2) -positive fibroblasts are increased in SSc, indicating a regulatory role. Intriguingly, bleomycin activated PAR(2) in HDF indicating that fibrosis-promoting factors have a direct effect on PAR(2) expression and functionality.     

CXCL13 produced by macrophages due to Fli1 deficiency may contribute to the development of tissue fibrosis,vasculopathy and immune activation in systemic sclerosis

CXCL13, a chemokine for B cells, follicular T cells, T helper 17 cells, and regulatory T cells, is reported to contribute to the development of systemic sclerosis (SSc), reflecting aberrant activation of immune system. To better understand the role of CXCL13 in SSc, we investigated the influence of Fli1 deficiency, a potential predisposing factor of this disease, on CXCL13 expression and assessed the clinical correlation of serum CXCL13 levels by multivariate regression analysis. Haploinsufficient loss of Fli1 remarkably induced CXCL13 expression in murine peritoneal macrophages, while gene silencing of FLI1 did not affect the expression of CXCL13 in human dermal fibroblasts and human dermal microvascular endothelial cells. Serum CXCL13 levels were elevated in SSc patients compared with healthy controls and correlated positively with skin score and negatively with pulmonary function test results. SSc patients with elevated serum CXCL13 levels had longer disease duration, diffuse cutaneous involvement, interstitial lung disease (ILD), heart involvement, pulmonary arterial hypertension, Raynaud's phenomenon, pitting scars, digital ulcers, telangiectasia, and high serum IgG levels more frequently than the other patients. In particular, serum CXCL13 levels were associated with ILD and digital ulcers by multivariate regression analysis. Taken together, these results indicate that CXCL13 expression is upregulated by Fli1 deficiency in macrophages, potentially contributing to the development of tissue fibrosis, vasculopathy and immune activation in SSc, especially ILD and digital ulcers.     

Bleomycin inhibits adipogenesis and accelerates fibrosis in the subcutaneous adipose layer through TGF‐β1

Systemic sclerosis [scleroderma (SSc)]‐associated skin fibrosis is characterized by increased fibrosis in the dermis and a reduction in the thickness of the subcutaneous adipose tissue layer. Although many studies have examined fibrosis in SSc, only a few studies have focused on the associated reduction in the thickness of the subcutaneous adipose tissue layer. In this study, we investigated the effects of SSc‐induced fibrosis on adipose tissue. We found that bleomycin suppresses adipogenesis in adipose‐derived stem cells (ASCs) and stimulates ASCs to express transforming growth factor β1 (TGF‐β1), which suppresses adipogenesis and promotes fibrosis. Furthermore, we found that adipocyte‐conditioned medium suppressed collagen synthesis by fibroblasts in fibrosis‐like conditions. We concluded that in the skin affected by bleomycin‐induced fibrosis, increased TGF‐β1 expression suppresses adipogenesis and promotes adipocyte fibrosis. It was also suggested that adipocytes have an inhibitory effect on the progression of fibrosis.     

Long non‐coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization

Long non‐coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real‐time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real‐time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)‐β1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease‐related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half‐lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF‐β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.     

Notch signalling in primary cutaneous CD30+ lymphoproliferative disorders: a new therapeutic approach?

Background The oncogenic potential of deregulated Notch signalling has been described in several haematopoietic malignancies. We have previously reported an increased expression of Notch1 in primary cutaneous CD30+ lymphoproliferative disorders, lymphomatoid papulosis and primary cutaneous anaplastic large‐cell lymphoma (pcALCL). Objectives To investigate the functional importance of Notch signalling in cell lines derived from pcALCL. Methods Cell lines derived from pcALCL (Mac1, Mac2a and JK) were treated with different γ‐secretase inhibitors (GSIs) (GSI I, IX, XX and XXI). The effects of GSIs on cell viability, apoptosis and cell cycle progression were measured as well as the impact of GSI I on the known prosurvival pathway Akt–mTOR–FOXO3a. Results Notch family members were expressed in all investigated pcALCL cell lines. GSI I had a marked proapoptotic effect, but GSI IX, XX and XXI were much less potent. The GSI I‐triggered apoptosis was preceded by an accumulation of cells in the G2/M, cyclin B1‐controlled phase of the cell cycle accompanied by an increase in the cyclin‐dependent kinase inhibitor, p21^WAF/Cip. GSI I induced the nuclear translocation of proapoptotic FOXO3a, probably via an Akt‐independent pathway. Conclusions Notch signalling may be a future therapeutic target for the treatment of advanced pcALCL.     

B lymphocytes: Shedding new light on the pathogenesis of systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disease characterized by autoimmunity and tissue fibrosis. In patients with SSc, there is a close association between the presence of specific autoantibodies and the development of clinical features. Although it is known that cytokines, including transforming growth factor-β, can modulate the synthesis of extracellular matrix by fibroblasts, it is not clear how autoimmunity and tissue fibrosis are interrelated. Several recent lines of evidence indicate a potential role for B cells in the development of SSc. CD19 is a critical regulator of B-cell signaling thresholds, and B cells from SSc patients exhibit increased expression of CD19, a molecule that induces SSc-specific autoantibody production in transgenic mice. Both SSc patients and tight-skin mice, a genetic model of SSc, have intrinsic B-cell abnormalities characterized by chronic B-cell activation. Remarkably, CD19 loss or B-cell depletion using antimouse CD20 antibody suppresses the development of skin hyperplasia and autoimmunity in tight-skin mice. Additionally, a recent study revealed a possible beneficial effect of antihuman CD20 antibody (rituximab) therapy for SSc patients. As B cells have a variety of functions, further investigation into the pathogenic roles of B cells, as well as trials of B-cell-targeting therapies, may shed new light on the pathogenesis of SSc.