Early Diffuse Systemic Sclerosis Patients Research Articles

Elevated interleukin-11 in systemic sclerosis and role in disease pathogenesis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease in which there is elevated inflammation, aberrant cytokine expression, and subsequent fibrosis. Interleukin-11 (IL-11) is a recently described profibrotic cytokine that can mediate fibrosis in the heart, lungs, and skin and is upregulated by transforming Growth Factor-β (TGF-β1). The objective of this study was to quantify the serum levels of IL-11 in early diffuse SSc patients. Also, if IL-11 could regulate the alarmin IL-33 in dermal fibroblasts was quantified. Early diffuse SSc patient sera was isolated and IL-11 was quantified by specific commercial ELISA compared to healthy control (n = 17). Healthy dermal fibroblasts were cultured in vitro and then serum starved and incubated with or without recombinant IL-11. At specific early and late time points the supernatant was quantified for the alarmin IL-33 by specific ELISA. In early diffuse SSc patients it was demonstrated that they have elevated IL-11 in their sera. In a subgroup of SSc patients with interstitial lung disease (ILD) this elevation was particularly pronounced compared to those devoid of fibrotic lung disease. In vitro incubation of healthy dermal fibroblasts led to a significant induction of IL-33 cytokine release into the cell media. IL-11 is a profibrotic cytokine that is elevated in early diffuse SSc and is particularly elevated in those with ILD. This suggests that IL-11 could be a possible biomarker of ILD in SSc. It was also found that IL-11 led to release of the cytokine alarmin IL-33 in fibroblasts at earlier time points but not late time points, suggesting early stimulation elicits an inflammatory response in the local microenvironment but prolonged stimulation leads to fibrosis.

Interleukin-36α is elevated in diffuse systemic sclerosis and may potentiate fibrosis.

Systemic sclerosis (SSc) is an autoimmune prototypical connective tissues disease that results in alterations in vasculature, inflammation and fibrosis of the skin. Interleukin-1 family cytokines has been implicated in the disease including IL-1. IL-36α is an IL-1 family member that is clearly implicated in psoriatic skin disease but its role in systemic sclerosis disease is not clear. The aim of this work is to evaluate the levels and role of IL-36α in systemic sclerosis. Early diffuse SSc patients sera was isolated along with healthy controls and IL-36 levels quantified by ELISA. In vitro analysis was also undertaken with primary dermal fibroblasts with recombinant IL-36α and keratinocyte cells were also incubated with IL-36α. Cytokines were measured by ELISA. Serum IL-36 was significantly elevated compared to healthy controls. Elevated neutrophil elastase was also elevated in the matched sera. IL-36 was not directly pro-fibrotic in dermal fibroblasts but did induce pro-inflammatory cytokines that were dependant on the MAPK pathway for their release. IL-36α also led to release of CCL20 and CCL2 in keratinocytes which may potentiate fibrosis. IL-36α is elevated in SSc serum and whilst not directly pro-fibrotic it may through keratinocytes, potentiate fibrosis through keratinocyte-immune fibroblast cross-talk.

Circulating Gremlin-1 is elevated in systemic sclerosis patients.

Introduction:Systemic sclerosis is an autoimmune connective tissue disease in which there is activation of the immune system, vascular disease and fibrosis. Activation of quiescent fibroblasts to myofibroblasts is key to disease pathogenesis. Gremlin-1 is a bone morphogenetic protein antagonist which is important in development and we recently reported in skin fibrosis. The aim of this study was to determine the serum circulating levels of Gremlin-1 in early diffuse systemic sclerosis.Methods:Twenty-one early diffuse systemic sclerosis patients (less than 2 years from first non-Raynaud’s symptom) were included and age and sex-matched healthy controls. Serum was isolated from blood and measured with a specific enzyme-linked immunoassay for Gremlin-1. Clinical variables were also measured.Results:Significantly elevated Gremlin-1 was found in sera of early diffuse systemic sclerosis patients (p < 0.001). In patients with interstitial lung disease, this compared to systemic sclerosis without evidence of interstitial lung disease, Gremlin-1 was significantly elevated (p < 0.0007). A correlation was found between circulating Gremlin-1 and modified Rodnan Skin Score, albeit weak.Discussion:In early diffuse systemic sclerosis patients, elevated Gremlin-1 is found in serum. This is particularly prominent in systemic sclerosis–associated interstitial lung disease. This suggests that Gremlin-1 may be a biomarker for systemic sclerosis interstitial lung disease.

Defining the optimal disease duration of early diffuse systemic sclerosis for clinical trial design.

Clinical trials in early diffuse cutaneous systemic sclerosis (SSc) using the modified Rodnan skin score (mRSS) as the primary outcome measure have most often been negative. We wanted to assess how the definition of disease onset (first SSc manifestation vs first non-Raynaud manifestation) and varying lengths of disease duration at trial entry as an inclusion criteria functioned. Our objective was to optimize trial inclusion criteria. We used the prospective, observational University of Pittsburgh Scleroderma Cohort to identify early diffuse SSc patients first evaluated between 1980 and 2015. All had <3 years from first SSc (n = 481) or first non-Raynaud manifestation (n = 514) and three or more mRSS scores. We used descriptive, survival and group-based trajectory analyses to compare the different definitions of disease onset and disease duration as inclusion criteria for clinical trials. There was no appreciable difference between using first SSc manifestation compared with first non-Raynaud manifestation as the definition of disease onset. Compared with other disease durations, <18 months of disease had >70% of patients fitting into trajectories with worsening cutaneous disease over 6 months of follow-up. Longer disease durations demonstrated the majority of patients with trajectories showing an improvement in mRSS (regression to the mean) over 6 months. Regardless of whether the first SSc or first non-Raynaud manifestation is used to define disease onset, duration of <18 months at enrolment is preferable. A longer disease duration criterion more frequently results in regression to the mean of the mRSS score, and likely contributes to negative trial outcomes.

MicroRNA27a-3p mediates reduction of the Wnt antagonist sFRP-1 in systemic sclerosis

ABSTRACT Systemic Sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin and lung fibrosis. The Wnt pathway is clearly elevated in SSc and is pro-fibrotic via activation of canonical Wnt signalling. sFRP-1 is a Wnt antagonist that acts as a negative regulator of Wnt signalling. We sought to measure the levels of serum sFRP-1 in early diffuse SSc patients compared to healthy controls and if this is regulated by microRNA27a-3p. Ten early diffuse SSc patients and healthy controls sera were taken and sFRP-1 quantified by ELISA. Skin biopsies were also taken in five SSc patients and controls. Fibroblasts were quantified for microRNA27-3p expression by Taqman qRT-PCR with an internal microRNA to normalize. 3ʹUTR luciferase assays were performed to confirm direct targets of microRNA27a-3p with microRNA overexpression. Fibroblasts were transfected with microRNA27a mimics or scramble controls and using ELISA sFRP-1 was quantified. Furthermore, Collagen, Axin-2, TIMP-1 and MMP-1 were measured. Serum sFRP-1 was significantly reduced in early diffuse SSc patients. We identified microRNA27a-3p-3p as regulating sFRP-1 in dermal fibroblasts. We found significantly elevated microRNA27a-3p in isolated dermal fibroblasts from SSc patients. We confirmed that sFRP-1 is a direct target of microRNA27a-3p through cloning of the 3ʹUTR into a luciferase vector. ECM genes were also upregulated by microRNA27a-3p-3p and the matrix-degrading enzyme MMP-1 was suppressed. Serum sFRP-1 is reduced in diffuse SSc patients and is regulated by microRNA27a-3p and this is a direct regulation. Modulation of microRNA27a-3p levels could mediate fibrosis regression.

Wnt antagonist DKK-1 levels in systemic sclerosis are lower in skin but not in blood and are regulated by microRNA33a-3p.

Systemic sclerosis is an autoimmune skin disease which is associated with inflammation and resulting skin fibrosis. Myofibroblasts are the key cell type associated with the fibrosis but how they are differentiated is not clear. DKK-1 is a Wnt antagonist that blocks Wnt-mediated fibrosis and is reduced in fibrotic conditions. Thus, DKK-1 is a clear negative regulator of fibrosis in systemic sclerosis and its regulation is unknown. The aim of this work is to determine the levels of DKK-1 in serum and tissues of SSc and its regulation. Skin biopsies were taken from early diffuse systemic sclerosis patients and healthy controls and DKK-1 measured by ELISA; serum was also isolated and DKK-1 quantified. DKK-1 was also measured by qRT-PCR. MicroRNA33a-3p was measured by TaqMan PCR. miR mimics and controls were transfected into dermal fibroblasts. Bleomycin mouse model was employed and compared to vehicle control treated mice, and gene expression was employed for DKK-1 and various extracellular matrix genes. DKK-1 is reduced in SSc skin and fibroblasts but is not reduced in the circulation in patients. MicroRNA33a-3p regulates DKK-1 levels epigenetically and is significantly reduced in SSc cells and whole tissue. DKK-1 is also reduced in the bleomycin mouse model and pro-fibrotic genes elevated. DKK-1 is reduced in SSc cells and is regulated by miR33a-3p, and restoring DKK-1 levels through epigenetic means could be a therapeutic target in systemic sclerosis.

Global skin gene expression analysis of early diffuse cutaneous systemic sclerosis shows a prominent innate and adaptive inflammatory profile

ObjectivesDetermine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease.MethodsSkin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic...

Identification of regulators of the myofibroblast phenotype of primary dermal fibroblasts from early diffuse systemic sclerosis patients

Systemic sclerosis (SSc or scleroderma) is an auto-immune disease characterized by skin fibrosis. While primary cells from patients are considered as a unique resource to better understand human disease biology, the effect of in vitro culture on these cells and their evaluation as a platform to identify disease regulators remain poorly characterized. The goal of our studies was to provide insights into the utility of SSc dermal fibroblast primary cells for therapeutic target discovery. The disease phenotypes of freshly isolated and in vitro cultured SSc dermal fibroblasts were characterized using whole transcriptome profiling, alpha smooth muscle actin (ASMA) expression and cell impedance. SSc dermal fibroblasts retained most of the molecular disease phenotype upon in vitro culture for at least four cell culture passages (approximatively 10 cell doublings). We validated an RNA interference high throughput assay that successfully identified genes affecting the myofibroblast phenotype of SSc skin fibroblasts. These genes included MKL1, RHOA and LOXL2 that were previously proposed as therapeutic anti-fibrotic target, and ITGA5, that has been less studied in fibrosis biology and may be a novel potential modifier of SSc fibroblast biology. Together our results demonstrated the value of carefully-phenotyped SSc dermal fibroblasts as a platform for SSc target and drug discovery.

CD4+CD45RA+ T-cells from early diffuse systemic sclerosis patients produce high levels of interleukin-17

Background In Systemic Sclerosis (SSc) the detrimental role of the immune system is a topic of great interest. In recent genetic association studies most genes identified to date are immunity related genes. A contribution of T-cells as important cytokine producing cells is shown and the skewing of this population can be an important player in the maintenance and progression of inflammation and fibrosis.