SummaryBACKGROUNDTwelve months of oral cyclophosphamide (CYC) has been shown to alter the progression of scleroderma-related interstitial lung disease (SSc-ILD) when compared to placebo. However, toxicity was a concern and without continued treatment the efficacy disappeared by 24 months. We hypothesized that a two-year course of mycophenolate mofetil (MMF) would be safer, better tolerated and produce longer lasting improvements than CYC.METHODSPatients with SSc-ILD meeting defined dyspnea, pulmonary function and high-resolution computed tomography (HRCT) criteria were randomized in a double-blind, two-arm trial at 14 medical centers. MMF (target dose 1500 mg twice daily) was administered for 24 months in one arm and oral CYC (target dose 2·0 mg/kg/day) administered for 12 months followed by placebo for 12 months in the other arm. The primary endpoint, change in forced vital capacity as a percent of the predicted normal value (FVC %) over the course of 24 months, was assessed in a modified intention-to-treat analysis using an inferential joint model combining a mixed effects model for longitudinal outcomes and a survival model to handle non-ignorable missing data. The study was registered with ClinicalTrials.gov, number NCT00883129, and is closed.RESULTSBetween November, 2009, and January, 2013, 142 patients were randomized. 126 patients (63 MMF; 63 CYC) with acceptable baseline HRCT studies and at least one outcome measure were included in the analysis. The adjusted FVC % (primary endpoint) improved from baseline to 24 months by 2.17 in the MMF arm (95% CI, 0.53–3.84) and 2·86 in the CYC arm (95% confidence interval 1·19–4·58) with no significant between-treatment difference (p=0·24), indicating that the trial was negative for the primary endpoint. However, in a post-hoc analysis of the primary endpoint, within-treatment improvements from baseline to 24 months were noted in both the CYC and MMF arms. A greater number of patients on CYC than on MMF prematurely withdrew from study drug (32 vs 20) and failed treatment (2 vs 0), and the time to stopping treatment was significantly shorter in the CYC arm (p=0·019). Sixteen deaths occurred (11 CYC; 5 MMF) with most due to progressive ILD. Leukopenia (30 vs 4 patients) and thrombocytopenia (4 vs 0 patients) occurred more often in patients treated with CYC. In post-hoc analyses, within- (but not between-) treatment improvements were also noted in defined secondary outcomes including skin score, dyspnea and whole-lung HRCT scores.INTERPRETATIONTreatment of SSc-ILD with MMF for two years or CYC for one year both resulted in significant improvements in pre-specified measures of lung function, dyspnea, lung imaging, and skin disease over the 2-year course of the study. While MMF was better tolerated and associated with less toxicity, the hypothesis that it would have greater efficacy at 24 months than CYC was not confirmed. These findings support the potential clinical impact of both CYC and MMF for progressive SSc-ILD, as well as the current preference for MMF due to its b...

Background Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. Methods We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. Results Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 downregulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. Conclusions Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.)

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs that leads to profound disability and premature death. To identify novel SSc susceptibility loci we conducted the first genome wide association study (GWAS) in a population of Caucasian ancestry including a total of 2296 SSc patients and 5171 controls. Analysis of 279,621 autosomal single nucleotide polymorphisms (SNPs) followed by replication testing in an independent case-control set of European ancestry (2,753 SSc patients / 4,569 controls) identified a new susceptibility locus for systemic sclerosis at CD247 (1q22-23; rs2056626, P = 2.09 × 10−7 in the discovery samples, P = 3.39 × 10−9 in the combined analysis). Additionally, we confirm and firmly establish the role of MHC (2.31 × 10−18), IRF5 (P =1.86 × 10−13) and STAT4 (P =3.37 × 10−9) gene regions as SSc genetic risk factors.

In this study, 1,833 systemic sclerosis (SSc) cases and 3,466 controls were genotyped with the Immunochip array. Classical alleles, amino acid residues, and SNPs across the human leukocyte antigen (HLA) region were imputed and tested. These analyses resulted in a model composed of six polymorphic amino acid positions and seven SNPs that explained the observed significant associations in the region. In addition, a replication step comprising 4,017 SSc cases and 5,935 controls was carried out for several selected non-HLA variants, reaching a total of 5,850 cases and 9,401 controls of European ancestry. Following this strategy, we identified and validated three SSc risk loci, including DNASE1L3 at 3p14, the SCHIP1-IL12A locus at 3q25, and ATG5 at 6q21, as well as a suggested association of the TREH-DDX6 locus at 11q23. The associations of several previously reported SSc risk loci were validated and further refined, and the observed peak of association in PXK was related to DNASE1L3. Our study has increased the number of known genetic associations with SSc, provided further insight into the pleiotropic effects of shared autoimmune risk factors, and highlighted the power of dense mapping for detecting previously overlooked susceptibility loci.

Objective To examine the heterogeneity of global transcriptome patterns in systemic sclerosis (SSc) skin in a large sample of patients with SSc and control subjects. Methods Skin biopsy specimens obtained from 61 patients enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort and 36 unaffected control subjects with a similar demographic background were examined by Illumina HumanHT-12 bead arrays. Followup experiments using quantitative polymerase chain reaction and immunohistochemical analysis were also performed. Results We identified 2,754 differentially expressed transcripts in SSc patients compared with controls. Clustering analysis revealed 2 prominent transcriptomes in SSc patients: the keratin and fibroinflammatory signatures. Higher keratin transcript scores were associated with shorter disease duration and interstitial lung disease, while higher fibroinflammatory scores were associated with diffuse cutaneous involvement, a higher skin score at the biopsy site, and a higher modified Rodnan skin thickness score. A subgroup of patients with significantly longer disease duration had a normal-like transcript pattern. Analysis of cell type–specific signature scores revealed remarkable heterogeneity across patients. Significantly higher scores were calculated for fibroblasts (72% of patients), microvascular cells (61%), macrophages (54%), and dendritic cells (DCs) (49%). The majority of samples with significantly higher fibroblast scores (35 of 44 [80%]) had significantly increased macrophage and/or DC scores. Further analysis and immunohistochemical staining indicated that the keratin signature was not a general marker of keratinocyte activation but was in fact associated with an activation pattern in hair and adnexal structures. Conclusion Prominent fibroinflammatory and keratin signatures are present in SSc skin. Expression profiles of SSc skin show significant heterogeneity, and this finding might be useful for stratifying patients for targeted therapies or predicting the response to immunosuppression.

Objective. To investigate peripheral blood (PB) cell transcript profiles of systemic sclerosis (SSc) and its subtypes in direct comparison with systemic lupus erythematosus (SLE).Methods. We investigated PB cell samples from 74 SSc patients, 21 healthy controls, and 17 SLE patients using Illumina Human Ref-8 BeadChips and quantitative polymerase chain reaction confirmation. None of the study participants were receiving immunosuppressive agents other than low-dose steroids and hydroxychloroquine. In addition to conventional statistical and modular analysis, a composite score for the interferon (IFN)-inducible genes was calculated. Within the group of patients with SSc, the correlation of the IFN score with the serologic and clinical subtypes was investigated, as were single-nucleotide polymorphisms in a selected number of IFN pathway genes.Results. Many of the most prominently overexpressed genes in SSc and SLE were IFN-inducible genes. Forty-three of 47 overexpressed IFN-inducible genes in SSc (91%) were similarly altered in SLE. The IFN score was highest in the SLE patients, followed by the SSc patients, and then the controls. The difference in IFN score among all 3 groups was statistically significant (P < 0.001 for all 3 comparisons). SSc and SLE PB cell samples showed striking parallels to our previously reported SSc skin transcripts in regard to the IFN-inducible gene expression pattern. In SSc, the presence of antitopoisomerase and anti-U1 RNP antibodies and lymphopenia correlated with the higher IFN scores (P ‫؍‬ 0.005, P ‫؍‬ 0.001, and P ‫؍‬ 0.004, respectively); a missense mutation in IFNAR2 was significantly associated with the IFN score.Conclusion. SLE and SSc fit within the same spectrum of IFN-mediated diseases. A subset of SSc patients shows a "lupus-like" high IFN-inducible gene expression pattern that correlates with the presence of antitopoisomerase and anti-U1 RNP antibodies.

Systemic sclerosis (SSc) is a systemic autoimmune disease affecting multiple organ systems, including the lungs. Interstitial lung disease (ILD) is the leading cause of death in SSc.There are no valid biomarkers to predict the occurrence of SSc-ILD, although auto-antibodies against anti-topoisomerase I and several inflammatory markers are candidate biomarkers that need further evaluation. Chest auscultation, presence of shortness of breath and pulmonary function testing are important diagnostic tools, but lack sensitivity to detect early ILD. Baseline screening with high-resolution computed tomography (HRCT) is therefore necessary to confirm an SSc-ILD diagnosis. Once diagnosed with SSc-ILD, patients' clinical courses are variable and difficult to predict, although certain patient characteristics and biomarkers are associated with disease progression. It is important to monitor patients with SSc-ILD for signs of disease progression, although there is no consensus about which diagnostic tools to use or how often monitoring should occur. In this article, we review methods used to define and predict disease progression in SSc-ILD.There is no valid definition of SSc-ILD disease progression, but we suggest that either a decline in forced vital capacity (FVC) from baseline of ≥10%, or a decline in FVC of 5–9% in association with a decline in diffusing capacity of the lung for carbon monoxide of ≥15% represents progression. An increase in the radiographic extent of ILD on HRCT imaging would also signify progression. A time period of 1–2 years is generally used for this definition, but a decline over a longer time period may also reflect clinically relevant disease progression.

The aim of this study was to determine, through a genome-wide association study (GWAS), the genetic components contributing to different clinical sub-phenotypes of systemic sclerosis (SSc). We considered limited (lcSSc) and diffuse (dcSSc) cutaneous involvement, and the relationships with presence of the SSc-specific auto-antibodies, anti-centromere (ACA), and anti-topoisomerase I (ATA). Four GWAS cohorts, comprising 2,296 SSc patients and 5,171 healthy controls, were meta-analyzed looking for associations in the selected subgroups. Eighteen polymorphisms were further tested in nine independent cohorts comprising an additional 3,175 SSc patients and 4,971 controls. Conditional analysis for associated SNPs in the HLA region was performed to explore their independent association in antibody subgroups. Overall analysis showed that non-HLA polymorphism rs11642873 in IRF8 gene to be associated at GWAS level with lcSSc (P = 2.32×10−12, OR = 0.75). Also, rs12540874 in GRB10 gene (P = 1.27 × 10−6, OR = 1.15) and rs11047102 in SOX5 gene (P = 1.39×10−7, OR = 1.36) showed a suggestive association with lcSSc and ACA subgroups respectively. In the HLA region, we observed highly associated allelic combinations in the HLA-DQB1 locus with ACA (P = 1.79×10−61, OR = 2.48), in the HLA-DPA1/B1 loci with ATA (P = 4.57×10−76, OR = 8.84), and in NOTCH4 with ACA P = 8.84×10−21, OR = 0.55) and ATA (P = 1.14×10−8, OR = 0.54). We have identified three new non-HLA genes (IRF8, GRB10, and SOX5) associated with SSc clinical and auto-antibody subgroups. Within the HLA region, HLA-DQB1, HLA-DPA1/B1, and NOTCH4 associations with SSc are likely confined to specific auto-antibodies. These data emphasize the differential genetic components of subphenotypes of SSc.