Fibroblast differentiation is a key cellular process that underlies the process of fibrosis, a deadly complication of fibrotic diseases like scleroderma (SSc). This transition coincides with the overproduction of collagen type I (COL1) and other extracellular matrix proteins. High-level expression of the collagen type 1α2 subunit (COL1A2), requires the engagement of a far-upstream enhancer, whose activation is strongly dependent on the AP1 factor JunB. We now report that STAT3 also binds the COL1A2 enhancer and is essential for RNA polymerase recruitment, without affecting JunB binding. STAT3 is required for the increased COL1A2 expression observed in myofibroblasts. We also report that TGFβ partially activates STAT3 and show that inhibiting STAT3 potently blocks TGFβ signaling, matrix remodeling, and TGFβ-induced myofibroblast differentiation. Activation of STAT3 with IL6 transsignaling alone, however, only increased COL1A2 protein expression, leaving COL1A2 mRNA levels unchanged. Our results suggest that activated STAT3 is not the limiting factor for collagen enhancer activation in human lung fibroblasts. Yet, a certain threshold level of STAT3 activity is essential to support activation of the COL1A2 enhancer and TGFβ signaling in fibroblasts. We propose that STAT3 operates at the posttranscriptional as well as the transcriptional level.

Systemic sclerosis (SSc) is characterized by the presence of SSc-specific or SSc-associated antibodies (SSc-Abs): anti-topoisomerase I (ATA), anti-centromere (ACA), anti-RNA polymerase III (ARA), anti-U3RNP (U3RNP), anti-U1RNP (U1RNP), anti-PmScl (PmScl), anti-Ku (Ku) and anti-Th/To (Th/To), each being associated with specific clinical features and prognosis. The detection of more than one SSc-Abs in SSc patients is rare and only few data about these patients' clinical phenotype is available. The aim of our study was to evaluate the frequency and the disease's features associated with the presence of > 1 SSc-Abs positivity in a large cohort of SSc patients. The autoantibody profiles of 2799 SSc patients from February 2001 to June 2017 were retrospectively reviewed. Patients with > 1 SSc-Abs were identified. Clinical features were collected and compared to a large historical cohort of SSc patients with single SSc-Ab positivity. SSc patients were excluded if previously treated with rituximab, intravenous immunoglobulins or stem cell transplantation. Non-parametric tests were used for statistical analysis. Nearly 5% of SSc patients from our cohort had ≥ 2 autoantibody positivity, and 2.3% (n = 72) had ≥ 2 SSc-Abs positivity. Th e most common combination was U1RNP and ATA (35%). These patients were younger than patients with single autoantibody positivity and showed more commonly a diffuse cutaneous SSc form. They also had higher rates of overlap features compared to ATA patients. Other combinations included U1RNP and ACA (13%), ATA and ACA (7%) and U1RNP and PmScl (5%). In our study we observed that, while infrequently, SSc patients can present with a combination of two SSc-Abs and that the double positivity can influence their clinical phenotype compared to patients with single SSc-Ab positivity. The importance of re-testing SSc-Abs in patients with changing clinical phenotypes was also highlighted, as this may confer a differing risk stratification.

The TβRII∆k-fib transgenic (TG) mouse model of scleroderma replicates key fibrotic and vasculopathic complications of systemic sclerosis through fibroblast-directed upregulation of TGFβ signalling. We have examined peroxisome proliferator-activated receptor (PPAR) pathway perturbation in this model and explored the impact of the pan-PPAR agonist lanifibranor on the cardiorespiratory phenotype.

The aim of this study was to explore outcomes in a cohort of dcSSc patients fulfilling eligibility criteria for stem cell transplantation (SCT) studies but receiving standard immunosuppression.

The origin of myofibroblasts in fibrotic conditions remains unknown and in systemic sclerosis (SSc) it has been proposed that activation of local fibroblasts, trans-differentiation of perivascular or vascular cells, recruitment of fibrocyte progenitors, or epithelial to mesenchymal transition (EMT) could be contributing. Data from our laboratory indicate that the epidermis in scleroderma is activated with the keratinocytes exhibiting a phenotype normally associated with tissue repair, including phosphorylation profiles indicative of TGFβ signaling. Since TGFβ is a known inducer of EMT, we investigated if there is evidence of this process in the SSc epidermis. In order to validate antibodies and primers, EMT was modeled in HaCaT cells cultured in the presence of TGFβ1. Skin sections were stained with phosho-SMAD2/3, as well as with epithelial and mesenchymal markers. Moreover, mRNA levels of transcription factors associated with EMT were studied in epidermal blister sheets. We observed critical changes in the scleroderma epidermis; showing significantly increased nuclear translocation of phosphorylated Smad2/3, consistent with active TGFβ signaling in SSc keratinocytes. While profound EMT could be induced in keratinocytes in vitro with the appearance of SNAI1/2 and FSP-1, and an accompanying loss of E-cadherin, in the scleroderma skin active TGFβ signaling was accompanied by only partial EMT-like changes characterised by induction of SNAI1 alone and with no loss of E-cadherin. Together, our findings support a model of altered differentiation and TGFβ dependent activation of scleroderma epithelial cells leading to a partially evoked EMT like process in the fibrotic skin.

The aim was to define clinical characteristics and long-term survival of patients with dcSSc and positive ACA.

Systemic 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).

Vasculopathy, 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-beta) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-beta signaling in fibroblasts.

Several common and rare risk variants have been reported for systemic sclerosis (SSc), but the effector cell(s) mediating the function of these genetic variants remains to be elucidated. While innate immune cells have been proposed as the critical targets to interfere with the disease process underlying SSc, no studies have comprehensively established their effector role. Here we investigated the contribution of monocyte-derived macrophages (MDMs) in mediating genetic susceptibility to SSc.

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-β signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-β/SMAD3 co-operates with Wnt3a/β-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of β-catenin and GLI expression or activity abolishes TGF-β/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-β, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors.

Although several genetic associations with scleroderma (SSc) are defined, very little is known on genetic susceptibility to SSc-associated interstitial lung disease (SSc-ILD). A number of common polymorphisms have been associated with SSc-ILD, but most have not been replicated in separate populations. Four SNPs in IRF5, and one in each of STAT4, CD226 and IRAK1, selected as having been previously the most consistently associated with SSc-ILD, were genotyped in 612 SSc patients, of European descent, of whom 394 had ILD. The control population (n = 503) comprised individuals of European descent from the 1000 Genomes Project. After Bonferroni correction, two of the IRF5 SNPs, rs2004640 (OR (95% CI)1.30 (1.10-1.54), pcorr = 0.015) and rs10488631 (OR 1.48 (1.14-1.92), pcorr = 0.022), and the STAT4 SNP rs7574865 (OR 1.43 (1.18-1.73), pcorr = 0.0015) were significantly associated with SSc compared with controls. However, none of the SNPs were significantly different between patients with SSc-ILD and controls. Two SNPs in IRF5, rs10488631 (OR 1.72 (1.24-2.39), pcorr = 0.0098), and rs2004640 (OR 1.39 (1.11-1.75), pcorr = 0.03), showed a significant difference in allele frequency between controls and patients without ILD, as did STAT4 rs7574865 (OR 1.86 (1.45-2.38), pcorr = 6.6 × 10-6). A significant difference between SSc with and without ILD was only observed for STAT4 rs7574865, being less frequent in patients with ILD (OR 0.66 (0.51-0.85), pcorr = 0.0084). In conclusion, IRF5 rs2004640 and rs10488631, and STAT4 rs7574865 were significantly associated with SSc as a whole. Only STAT4 rs7574865 showed a significant difference in allele frequency in SSc-ILD, with the T allele being protective against ILD.Key points• We confirm the associations of the IRF5 SNPs rs2004640 and rs10488631, and the STAT4 SNP rs7574865, with SSc as a whole.• None of the tested SNPs were risk factors for SSc-ILD specifically.• The STAT4 rs7574865 T allele was protective against the development of lung fibrosis in SSc patients.• Further work is required to understand the genetic basis of lung fibrosis in association with scleroderma.

Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis.

The aim of these analyses was to investigate the rate of decline in forced vital capacity (FVC) in patients with SSc-associated interstitial lung disease (SSc-ILD) with and without cough or dyspnoea in the SENSCIS trial.

To assess associations between the extent of fibrotic interstitial lung disease (ILD) and forced vital capacity (FVC) at baseline and change in FVC over 52 weeks in patients with systemic sclerosis-associated ILD (SSc-ILD) in the SENSCIS trial.

The excessive deposition of extracellular matrix, including type I collagen, is a key aspect in the pathogenesis of connective tissue diseases such as systemic sclerosis (SSc; scleroderma). To further our understanding of the mechanisms governing the dysregulation of type I collagen production in SSc, we investigated the role of the activator protein 1 (AP-1) family of transcription factors in regulating COL1A2 transcription.

To investigate the course of interstitial lung disease (ILD) and the effects of nintedanib in patients with limited cutaneous systemic sclerosis (lcSSc).

To investigate the rate of decline in forced vital capacity (FVC), and the effect of nintedanib on the rate of decline in FVC, in subjects with systemic sclerosis-associated interstitial lung disease (SSc-ILD) who had risk factors for rapid decline in FVC.

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-β. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.

To evaluate the evidence concerning systemic pharmacological treatments for SSc digital ulcers (DUs) to inform the development of evidence-based treatment guidelines.