Jennifer L. Sargent

Molecular Subsets in the Gene Expression Signatures of Scleroderma Skin

Background Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production. Methodology and Findings We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of ‘intrinsic’ genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynauds phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc. Conclusions and Significance Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

Wnt/β-catenin signaling is hyperactivated in systemic sclerosis and induces Smad-dependent fibrotic responses in mesenchymal cells

OBJECTIVE Fibrosis in human diseases and animal models is associated with aberrant Wnt/β-catenin pathway activation. The aim of this study was to characterize the regulation, activity, mechanism of action, and significance of Wnt/β-catenin signaling in the context of systemic sclerosis (SSc). METHODS The expression of Wnt signaling pathway components in SSc skin biopsy specimens was analyzed. The regulation of profibrotic responses by canonical Wnt/β-catenin was examined in explanted human mesenchymal cells. Fibrotic responses were studied using proliferation, migration, and gel contraction assays. The cell fate specification of subcutaneous preadipocytes by canonical Wnt signaling was evaluated. RESULTS Analysis of published genome-wide expression data revealed elevated expression of the Wnt receptor FZD2 and the Wnt target LEF1 and decreased expression of Wnt antagonists DKK2 and WIF1 in skin biopsy specimens from subsets of patients with diffuse cutaneous SSc compared to the other distinct subsets. Immunohistochemical analysis showed increased nuclear β-catenin expression in these biopsy specimens. In vitro, Wnt-3a induced β-catenin activation, stimulated fibroblast proliferation and migration, collagen gel contraction, and myofibroblast differentiation, and enhanced profibrotic gene expression. Genetic and pharmacologic approaches were used to demonstrate that these profibrotic responses involved autocrine transforming growth factor β signaling via Smads. In contrast, in explanted subcutaneous preadipocytes, Wnt-3a repressed adipogenesis and promoted myofibroblast differentiation. CONCLUSION Canonical Wnt signaling was hyperactivated in SSc skin biopsy specimens. In explanted mesenchymal cells, Wnt-3a stimulated fibrogenic responses while suppressing adipogenesis. Taken together, these results indicate that Wnts have potent profibrotic effects, and that canonical Wnt signaling plays an important role in the pathogenesis of fibrosis and lipoatrophy in SSc.

PPARγ Downregulation by TGFß in Fibroblast and Impaired Expression and Function in Systemic Sclerosis: A Novel Mechanism for Progressive Fibrogenesis

The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)-dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a “TGF-ß responsive gene signature” in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis.

Interspecies Comparison of Human and Murine Scleroderma Reveals IL-13 and CCL2 as Disease Subset-Specific Targets

Development of personalized treatment regimens is hampered by lack of insight into how individual animal models reflect subsets of human disease, and autoimmune and inflammatory conditions have proven resistant to such efforts. Scleroderma is a lethal autoimmune disease characterized by fibrosis, with no effective therapy. Comparative gene expression profiling showed that murine sclerodermatous graft-versus-host disease (sclGVHD) approximates an inflammatory subset of scleroderma estimated at 17% to 36% of patients analyzed with diffuse, 28% with limited, and 100% with localized scleroderma. Both sclGVHD and the inflammatory subset demonstrated IL-13 cytokine pathway activation. Host dermal myeloid cells and graft T cells were identified as sources of IL-13 in the model, and genetic deficiency of either IL-13 or IL-4Rα, an IL-13 signal transducer, protected the host from disease. To identify therapeutic targets, we explored the intersection of genes coordinately up-regulated in sclGVHD, the human inflammatory subset, and IL-13-treated fibroblasts; we identified chemokine CCL2 as a potential target. Treatment with anti-CCL2 antibodies prevented sclGVHD. Last, we showed that IL-13 pathway activation in scleroderma patients correlated with clinical skin scores, a marker of disease severity. Thus, an inflammatory subset of scleroderma is driven by IL-13 and may benefit from IL-13 or CCL2 blockade. This approach serves as a model for personalized translational medicine, in which well-characterized animal models are matched to molecularly stratified patient subsets.

Experimentally-Derived Fibroblast Gene Signatures Identify Molecular Pathways Associated with Distinct Subsets of Systemic Sclerosis Patients in Three Independent Cohorts

Genome-wide expression profiling in systemic sclerosis (SSc) has identified four ‘intrinsic’ subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFNα signaling showed a strong association with early disease, while TGFβ signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-κB. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGFβ and TNFα. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFNα is involved in early disease pathology, and disease severity is associated with active TGFβ signaling.

Capturing the heterogeneity in systemic sclerosis with genome-wide expression profiling

Heterogeneity in the clinical presentation and basic science findings of systemic sclerosis (SSc) has hindered the understanding of pathogenesis and development of effective treatments. Genome-wide profiling of SSc has measured this heterogeneity. Gene expression studies of diffuse SSc skin have shown reproducible, disease-specific gene expression signatures when compared with healthy controls and, surprisingly, disease-specific gene expression was found in both lesional and non-lesional skin. SSc-specific gene expression in peripheral blood cells and the lungs has also been demonstrated. Hypothesis-driven approaches that assess the contribution of individual pathways provide insight into the etiology of gene expression subsets.

Egr-1 induces a profibrotic injury/repair gene program associated with systemic sclerosis.

Transforming growth factor-ß (TGF-ß) signaling is implicated in the pathogenesis of fibrosis in scleroderma or systemic sclerosis (SSc), but the precise mechanisms are poorly understood. The immediate-early gene Egr-1 is an inducible transcription factor with key roles in mediating fibrotic TGF-ß responses. To elucidate Egr-1 function in SSc-associated fibrosis, we examined change in gene expression induced by Egr-1 in human fibroblasts at the genome-wide level. Using microarray expression analysis, we derived a fibroblast “Egr-1-responsive gene signature” comprising over 600 genes involved in cell proliferation, TGF-ß signaling, wound healing, extracellular matrix synthesis and vascular development. The experimentally derived “Egr-1-responsive gene signature” was then evaluated in an expression microarray dataset comprising skin biopsies from 27 patients with localized and systemic forms of scleroderma and six healthy controls. We found that the “Egr-1 responsive gene signature” was substantially enriched in the “diffuse-proliferation” subset comprising exclusively of patients with diffuse cutaneous SSc (dcSSc) of skin biopsies. A number of Egr-1-regulated genes was also associated with the “inflammatory” intrinsic subset. Only a minority of Egr-1-regulated genes was concordantly regulated by TGF-ß. These results indicate that Egr-1 induces a distinct profibrotic/wound healing gene expression program in fibroblasts that is associated with skin biopsies from SSc patients with diffuse cutaneous disease. These observations suggest that targeting Egr-1 expression or activity might be a novel therapeutic strategy to control fibrosis in specific SSc subsets.

Identification of Optimal Mouse Models of Systemic Sclerosis by Interspecies Comparative Genomics

Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets.

Interspecies comparative genomics identifies optimal mouse models of systemic sclerosis

Understanding the pathogenesis of systemic sclerosis (SSc) is confounded by considerable disease heterogeneity. Animal models of SSc that recapitulate distinct subsets of disease at the molecular level have not been delineated. We applied interspecies comparative analysis of genomic data from multiple mouse models of SSc and patients with SSc to determine which animal models best reflect the SSc intrinsic molecular subsets.

The role of Ifng in alterations in liver gene expression in a mouse model of fulminant autoimmune hepatitis

Background/Aims: BALB/c mice with a homozygous deficiency in the Tgfb1 gene are a model of fulminant autoimmune hepatitis (AIH), spontaneously and rapidly developing Th1‐mediated IFN‐γ‐dependent necroinflammatory liver disease. We sought to understand the molecular basis for fulminant Th1 liver disease and the specific role of the Ifng gene.