Emma Derrett-Smith

Clinical and pathological significance of interleukin 6 overexpression in systemic sclerosis

Objective To determine the potential clinical and pathological significance of altered expression of interleukin 6 (IL-6) in systemic sclerosis (SSc). Methods Serum IL-6 and soluble IL-6 receptor levels were measured in patients with SSc (n=68) and healthy controls (n=15). Associations between serum IL-6 level and C reactive protein, platelet count and key clinical outcomes in SSc were explored. Expression of IL-6 in skin biopsies was also examined and western blot and reverse transcription PCRanalysis were performed using cultured dermal fibroblasts. The effect of IL-6 trans-signalling on production of extracellular matrix proteins was assessed and downstream signalling pathways were examined using pharmacological inhibitors. Results Serum IL-6 level was frequently elevated in patients with SSc, particularly in those with diffuse cutaneous SSc (dcSSc) with thrombocytosis and elevated acute phase markers. Prominent expression in the skin was observed in dermal fibroblasts, mononuclear cells and endothelial cells in patients with early dcSSc. In vitro experiments supported a potent profibrotic effect of IL-6 trans-signalling via the JAK2/STAT3 and ERK pathways. High IL-6 expression early in dcSSc appears to be associated with more severe skin involvement at 3 years and worse long-term survival than in those without elevated IL-6 levels. Conclusion Our results confirm the overexpression of IL-6 in dcSSc and support the potential of IL-6 as a surrogate marker for clinical outcome in this disease. The data also provide rationale for clinical studies targeting IL-6 trans-signalling as a potential antifibrotic therapy for SSc.

An Essential Role for Resident Fibroblasts in Experimental Lung Fibrosis Is Defined by Lineage-Specific Deletion of High-Affinity Type II Transforming Growth Factor β Receptor

RATIONALE Fibrotic response to lung injury depends on development of a fibrogenic population of myofibroblasts. The importance of resident interstitial fibroblasts and role of transforming growth factor β (TGFβ) in this process is unclear. OBJECTIVES To define the importance of TGFβ signaling in resident lung fibroblasts in the development of experimental pulmonary fibrosis. METHODS A compound genetic strategy in which mice homozygous for a floxed high-affinity type II TGFβ receptor (TβRII) allele were crossed with a transgenic strain harboring a fibroblast-specific transgene encoding ligand-dependent Cre-recombinase was used. TβRII was deleted by postnatal administration of tamoxifen over 5 days to compound mutant mice with appropriate littermate controls. Illumina microarray gene profiling and quantitative reverse transcriptase-polymerase chain reaction were used to confirm anergy to TGFβ in explanted lung fibroblasts. Bleomycin lung injury was used to induce lung fibrosis, which was analyzed by histology and biochemical methods. Immunofluorescence was used to define cell populations after lung injury. MEASUREMENTS AND MAIN RESULTS There was significant attenuation of fibrosis in mice after deletion of TβRII in resident fibroblasts. At 7 days after injury the number of fibrocytes and myofibroblasts was substantially reduced. Potential regulators of fibrosis were suggested by gene expression profiles that identified key candidate profibrotic genes, including connective tissue growth factor and endothelin-1 expressed by wild-type but not mutant lung fibroblasts. CONCLUSIONS Intact TGFβ signaling in resident pulmonary fibroblasts is essential for pulmonary fibrosis to develop. Our data support a key regulatory role of these cells in determining fibrocyte recruitment and myofibroblast differentiation.

Endothelial Injury in a Transforming Growth Factor β–Dependent Mouse Model of Scleroderma Induces Pulmonary Arterial Hypertension

OBJECTIVE To delineate the constitutive pulmonary vascular phenotype of the TβRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH). METHODS The TβRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor β (TGFβ) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFβ signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFβ and VEGF signaling axes in tissue sections and explanted smooth muscle cells. RESULTS In the TβRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFβ and VEGF ligand and receptor was consistent with a scleroderma phenotype. CONCLUSION In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFβ and VEGF, which provides insight into the pathogenesis of this disease.

Systemic vasculopathy with altered vasoreactivity in a transgenic mouse model of scleroderma

IntroductionVasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-β) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-β signaling in fibroblasts.MethodsThe transgenic mouse strain Tβ RIIΔk-fib is characterized by balanced ligand-dependent upregulation of TGF-β signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-β1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-β and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed.ResultsAortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-β1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-β- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-β1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wild-type, but were again resistant to exogenous TGF-β1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis.ConclusionsThe histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-β and endothelin activity in this process.

Gut fibrosis with altered colonic contractility in a mouse model of scleroderma

OBJECTIVE Gastrointestinal involvement occurs in up to 90% of patients with SSc. Animal models of SSc mimic some of the pathophysiological disease processes of SSc. The transgenic (TG) mouse strain TβRIIΔk-fib is characterized by ligand-dependent up-regulation of TGF-β signalling and has been shown to develop skin fibrosis, lung fibrosis and diminished aortic ring contractility and adventitial fibrosis. We investigated if similar changes are observed in the gut tissue in this mouse model. METHODS Colonic tissue was examined using histology and immunohistochemistry analyses. Tissue architecture was examined by haematoxylin and eosin (H&E), picrosirius red and immunohistochemical markers for α-smooth muscle actin (α-SMA), phospho-Smad 2/3 (pSmad2/3), Ki-67, protein gene product 9.5 and S-100. Fibrosis was quantified using the NIS Elements BR 2.30 system and by Sircol assay. Colonic strip contractile responses to potassium chloride (KCl) and carbachol were assessed in isolated organ baths. Confirmatory gut fibroblast and intestinal tissue biochemical assays, including cellular signalling mechanisms, were performed. RESULTS H&E staining and staining for α-SMA, Ki-67, pSmad2/3 or neural tissue staining showed no differences between TG and wild-type (WT) mice gut tissue. There was increased collagen deposition in the gut of TG mice. Quantitative PCR results of TG gut fibroblasts showed evidence of up-regulated collagen and CTGF transcription, and non-canonical TGF-β signalling pathways were also up-regulated. The organ bath studies showed diminished colonic strip contractility in TG mice compared with WT control mice to KCl and carbachol. CONCLUSION We have shown that this TG mouse model, previously shown to develop skin and lung, develops colonic fibrosis with associated effects on colonic tissue contractility. This may offer further insight in pathological processes leading to the development of gut fibrosis.

Animal models of scleroderma: lessons from transgenic and knockout mice

Purpose of reviewThe underlying pathogenesis of systemic sclerosis (SSc; scleroderma) involves a complex interplay of inflammation, fibrosis and vasculopathy that is incompletely understood. In this article, we highlight the important contributions that recent preclinical research has made to the knowledge base of pathogenesis and therapeutics in SSc, describe some of the newly developed models available for further investigation and discuss future research opportunities in this fascinating area. Recent findingsSeveral well characterized SSc models are available for the study of fibrosis. However, recent study on transgenic and knockout models has advanced knowledge both in fibrosis research and in vascular disease in SSc. In the present review, we focus on models in which altered signalling, particularly transforming growth factor-β (TGF-β), is limited to fibroblasts. We discuss contemporary models of SSc vascular disease, transgenesis in fibrocyte research, the contribution to neurological signalling research and provide examples of how preclinical models have contributed to novel therapeutics development in SSc. We also look at how research from related disciplines impacts on the SSc knowledge base. SummaryThese new models represent exciting advances. However, none completely recapitulates the vasculopathic and inflammatory components of this disease. These advances help to delineate the relative contributions of specific ligands, receptors, their signalling pathways and feedback mechanisms, in fibrotic and inflammatory processes and this will provide new targets for potential therapies in SSc.

Impaired Bone Morphogenetic Protein Receptor II Signaling in a Transforming Growth Factor-β–Dependent Mouse Model of Pulmonary Hypertension and in Systemic Sclerosis

RATIONALE Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH. OBJECTIVES To explore BMPRII expression and function in a mouse model of SSc (TβRIIΔk-fib) that is susceptible to developing pulmonary hypertension and in SSc lung. METHODS BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the TβRIIΔk-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts. MEASUREMENTS AND MAIN RESULTS Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-β-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-β activity. CONCLUSIONS We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-β-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.

Juvenile-onset systemic sclerosis: children are not small adults

The majority of childhood-onset rheumatic diseases differ markedly in presentation and management from their equivalent adult conditions, and juvenile-onset systemic sclerosis (JSSc) is no exception. Chronic ill health from any cause in childhood impacts heavily, not only on physical growth and development, but also in terms of social, educational and psychological development. Even localized scleroderma, the most common spectrum in children, which is not associated with the internal organ complications that account for the majority of mortality and morbidity in both juvenile and adult systemic disease, has profound effects on limb growth and development that cause major morbidity compared with the adult disease. SSc is the most serious form of scleroderma and is much rarer in children than adults. In the past, lessons from adult SSc have been extrapolated to JSSc but this is potentially misleading. In the present issue, Martini et al. [1] present the most recent of a series of reports examining clinical features and outcomes of JSSc in retrospective cohorts, and indirectly help to identify the characteristics of juvenile-onset disease that differentiate it from the adult condition [1–3]. The clinical heterogeneity of SSc remains one of the most challenging aspects of adult disease, but both the limited and diffuse subsets do tend to follow recognizable disease courses, in which genetic, environmental and autoantibody profiles appear to play a pathophysiological role. This study, along with those performed previously, reveal a disease that differs in terms of epidemiology, clinical characteristics, immunology and outcomes, and does not clearly follow any of the defined pathways seen in adult disease [4]. Even in late-stage disease, within an adult SSc cohort, patients with juvenile-onset disease continue to remain distinct in terms of clinical features and outcomes [5]. Incidence and prevalence of JSSc is somewhat difficult to determine, with incidence quoted between 3 and 10% of adult disease. This difficulty arises from a lack of clear definition in juvenile-onset CTDs. These present as overlap syndromes far more commonly than adult-onset disease, and so Martini et al. [1], by applying ACR preliminary classification criteria for SSc in adults, may exclude many overlap syndromes with features of SSc and a proportion of those with limited cutaneous disease. Additionally, the scleroderma research community depends heavily on the modified Rodnan skin score as a primary or secondary outcome measure in clinical trials but this measure has not been validated in JSSc. A preliminary system for diagnosis and classification of JSSc has now been agreed between PRES, EULAR and ACR to endeavour to standardize clinical research, therapeutic trials and other studies [6]. Notwithstanding these caveats, the present study again confirms the clinical suspicion that diffuse disease is more common in JSSc; in this study, 122/134 patients had diffuse disease. In adult cohorts, the majority of cases are the limited subset; for instance in our centre, only one-third of the patients under follow-up have the diffuse subset [7]. Perhaps unsurprisingly, the hallmark autoantibody of limited disease, ACA, is extremely rare in childhood disease, carried by just one patient in this cohort. Autoantibodies carried in cases of JSSc are less specific than adults in any case. Interestingly, the higher incidence in females, even prior to puberty, again challenges the dogma that oestrogens are solely responsible for the increased autoimmune propensity in females, with X-inactivation chimerism cited as an alternative mechanism. While the overall mortality of juvenile-onset disease is lower, those with a poor outcome tend to progress more rapidly than their adult counterparts. Cardiac disease in adults is rarely the primary cause of death, whereas this study supports findings from previous studies that most juvenile deaths occur due to cardiac disease, excluding right heart failure for pulmonary arterial hypertension—25% in this study and over 50% in previous studies. Remembering the increased incidence of overlap syndromes in children, this may be accounted for by an increased vasculitic or inflammatory serosal component to this disease compared with adults. An alternative explanation arises from both animal and clinical research in adult SSc, where a secondary insult obtained over time in a susceptible individual may result in a particular pattern of disease, whether that be epithelial injury as a cause for lung fibrosis or hypertension or steroid use triggering renal crisis. With the relative absence of long-term comorbidities or exposures to epithelial injury, and less specific auto-antibody profiles in children, these adjuvant factors may be less prominent resulting in the apparent overrepresentation of cardiac disease in this cohort. In adults, primary cardiac disease in SSc can involve any of the cardiac structures, and hence can present diversely as pericardial effusions, inflammatory myocarditis, diastolic dysfunction, conduction defects and restrictive or dilated cardiomyopathy. The most commonly cited cause of death in juveniles was heart failure, which often occurred in the context of multiorgan involvement. Pericardial effusion is a common finding in SSc, usually without haemodynamic significance. The stronger association of mortality and pericardial effusion in juvenile-onset disease suggests a less benign mechanism for this particular clinical finding. Interestingly, clinical or radiological evidence of pulmonary fibrosis at presentation was associated with increased mortality, though this is not reflected in the causes of death, with 2 of 16 patients dying as a result of respiratory failure. Indeed, raised creatinine levels are also strong predictors, though unlike the adult context, hypertensive renal crisis is less likely to be the cause, as vasculitic processes and hypoperfusion due to primary cardiac disease are more prevalent in the juvenile cohort. Treatment of JSSc is especially challenging. Treatment of organ-specific disease is similar to adults, with the use of endothelin receptor antagonists or phosphodiesterase inhibitors followed by intravenous prostanoids for pulmonary arterial hypertension, cyclophosphamide and steroid for pulmonary fibrosis, ACE-inhibitors and prostacyclin for hypertensive crises and vasodilators and prostacyclin (with a putative role for endothelin receptor antagonists or phosphodiesterase inhibitors) in digital vasculopathy. Again, perhaps as a reflection of the increased incidence of overlap syndromes and diffuse disease in the young, immunosuppression with MTX, cyclophosphamide or mycophenolate mofetil is central in the overall management of JSSc. Special consideration of the long-term impact of such therapies on fertility and risks of malignancy is vital in this cohort. Rheumatology 2009;48:96–97 doi:10.1093/rheumatology/ken418 Advance Access publication 21 November 2008

Resolution of paraneoplastic PM/Scl-positive systemic sclerosis after curative resection of a pancreatic tumour

SIR, Compelling evidence has recently been presented that SSc may occur as a paraneoplastic disease, especially for cases expressing hallmark anti-RNA polymerase III autoantibodies [1]. Elegant studies have confirmed expression of variant protein by tumours [2] and larger cohort analyses confirm that the association with malignancy is often contemporaneous with autoantibody development [3]. It would be predicted from this model that tumour removal at an early stage could interrupt the autoimmune process and be associated with resolution of the associated SSc. Such clinical improvement would confirm the role of antigen-driven adaptive autoimmunity driving the disease and strongly support current immunosuppressive treatment strategies. Here we describe the case of a 43-year-old woman with malignancy and clinical features of SSc and polymyositis [creatine kinase (CK)>2000 IU/L] associated with PM/Scl antibodies. The presence of tendon contractures, palmar skin thickening and diffuse distribution of skin disease [peak modified Rodnan skin score (mRSS) 11/51] with generalized hyperpigmentation prompted further radiological assessment for underlying malignancy and a cystic pancreatic lesion with a solid component was identified. A distal pancreatectomy and splenectomy successfully excised all tumour tissue and a diagnosis of solid pseudopapillary pancreatic neoplasm was confirmed on histological and immunohistochemical analysis. Following surgical recovery, the patient was rapidly weaned off all immunosuppression and has remained in clinical remission with resolution of both skin sclerosis (mRSS 4/51) and inflammatory muscle disease [normal CK and Medical Research Council (MRC) grade]. Immunostaining with anti-EXOSC10 antibody (anti-PM/Scl-100) demonstrated increased staining in normal exocrine pancreatic cytoplasm (negative in endocrine pancreas) when compared with tumour from this patient. However, nuclear staining was present universally in tumour tissue and found in very few nuclei in the normal pancreas (Fig. 1, arrows). Clinical remission following full resection and increased nuclear staining for PM/Scl-100 in tumour tissue lends support to a potential pathogenic link in this case, and this has not been previously demonstrated in SSc associated with this antibody subset. To confirm and extend the potential association of this hallmark scleroderma ANA pattern with malignancy we interrogated our SSc research database of 2200 patients and identified 80 patients who tested positive for PM/Scl by Hep-2 immunofluorescence and confirmatory counterimmunoelectrophoresis. Data were available for 70 of these patients, 80% of whom were female, with a mean age of 58.4 years (S.D. 14.0) and a mean age at SSc onset of 44.1 years ( S.D. 14.5). Forty-seven patients (67.1%) had limited cutaneous involvement and the remainder had diffuse disease, except three patients for whom these data were missing. More than one-third of the population showed the presence of calcinosis (38.6%) and inflammatory arthropathy (38.6%), while more than half of the patients in the study population were affected by lung involvement, gastrointestinal involvement and inflammatory myopathy (57.1, 62.9 and 61.4%, respectively). These demographic and clinical data are broadly representative of those published previously for this antibody subset [4]. However, a history of malignancy was found in 14/70 patients (20.0%), with cancer onset within 36 months from SSc diagnosis in 5 patients. This is far FIG. 1 Increased nuclear expression of EXOSC10 (PM/ Scl-100) in tumour tissue of a patient with PM/Scl-positive paraneoplastic scleroderma

Limited cutaneous systemic sclerosis skin demonstrates distinct molecular subsets separated by a cardiovascular development gene expression signature

BackgroundSystemic sclerosis (SSc; scleroderma) is an uncommon autoimmune rheumatic disease characterised by autoimmunity, vasculopathy and fibrosis. Gene expression profiling distinguishes scleroderma from normal skin, and can detect different subsets of disease, with potential to identify prognostic biomarkers of organ involvement or response to therapy. We have performed gene expression profiling in skin samples from patients with limited cutaneous SSc (lcSSc).MethodsTotal RNA was extracted from clinically uninvolved skin biopsies of 15 patients with lcSSc and 8 healthy controls (HC). Gene expression profiling was performed on a DNA oligonucleotide microarray chip. Differentially expressed genes (DEG) were identified using significance analysis of microarrays (SAM). Functional enrichment analysis of gene signatures was done via g:Profiler.ResultsThere were 218 DEG between lcSSc and HC samples (false discovery rate <10%): 181/218 DEG were upregulated in lcSSc samples. Hierarchical clustering of DEG suggested the presence of two separate groups of lcSSc samples: “limited 1” and “limited 2”. The limited-1 group (13 samples, 10 unique patients) showed upregulation of genes involved in cell adhesion, cardiovascular system (CVS) development, extracellular matrix and immune and inflammatory response. The CVS development signature was of particular interest as its genes showed very strong enrichment in response to wounding, response to transforming growth factor (TGF)-β and kinase cascade. Neither limited-2 samples (six samples, five unique patients) nor HC samples showed functional enrichment. There were no significant differences in demographic or clinical parameters between these two groups. These results were confirmed using a second independent cohort.ConclusionsOur study suggests the presence of molecular subsets in lcSSc based on gene expression profiling of biopsies from uninvolved skin. This may reflect important differences in pathogenesis within these patient groups. We identify differential expression of a subset of genes that relate to CVS and are enriched in fibrotic signalling. This may shed light on mechanisms of vascular disease in SSc. The enrichment in profibrotic profile suggests that dysregulated gene expression may contribute to vasculopathy and fibrosis in different disease subsets.