Single-Cell Transcriptome Analysis Identifies Subclusters with Inflammatory Fibroblast Responses in Localized Scleroderma.

Localized scleroderma (LS) is an autoimmune disease with both inflammatory and fibrotic components causing an abnormal deposition of collagen in the skin and underlying tissue, often leading to disfigurement and disability. Much of its pathophysiology is extrapolated from systemic sclerosis (SSc) since the histopathology findings in the skin are nearly identical. However, LS is critically understudied. Single-cell RNA sequencing (scRNA seq) technology provides a novel way to obtain detailed information at the individual cellular level, overcoming this barrier. Here, we analyzed the affected skin of 14 patients with LS (pediatric and adult) and 14 healthy controls. Fibroblast populations were the focus, since they are the main drivers of fibrosis in SSc. We identified 12 fibroblast subclusters in LS, which overall had an inflammatory gene expression (IFN and HLA-associated genes). A myofibroblast-like cluster (SFRP4/PRSS23) was more prevalent in LS subjects and shared many upregulated genes expressed in SSc-associated myofibroblasts, though it also had strong expression of CXCL9/10/11, known CXCR3 ligands. A CXCL2/IRF1 cluster identified was unique to LS, with a robust inflammatory gene signature, including IL-6, and according to cell communication analysis are influenced by macrophages. In summary, potential disease-propagating fibroblasts and associated gene signatures were identified in LS skin via scRNA seq.

Genetic Signatures From RNA Sequencing of Pediatric Localized Scleroderma Skin.

The purpose of this study was to explore the skin transcriptional profile in pediatric localized scleroderma (LS) to provide a better understanding of the altered immune and fibrotic pathways promoting disease. LS is a progressive disease of the skin and underlying tissue that causes significant functional disability and disfigurement, especially in developing children. RNA sequencing (RNAseq) technology allows for improved understanding of relevant cellular expression through transcriptome analysis of phases during LS disease progression (more active/inflammatory vs. inactive/fibrotic) and also permits the use of RNA extracted from existing paraffin-embedded skin tissue, which is important in pediatrics. A strong correlation was observed between the comparison of genes expressed between fresh (RNAlater) and paraffinized skin in healthy and LS subjects, supporting the use of paraffinized tissue. LS gene signatures compared to healthy controls showed a distinct expression of an inflammatory response gene signature (IRGS) composed of IFNγ-, IFNα-, and TNFα-associated genes. GSEA© enrichment analysis showed that the IRGS, including interferon-inducible chemokines such as CXCL9, CXCL10, CXCL11, and IFNγ itself, was more highly expressed in LS patients with more inflammatory lesions. The use of paraffinized skin for sequencing was proven to be an effective substitute for fresh skin by comparing gene expression profiles. The prevalence of the IFNγ signature in the lesion biopsies of active LS patients indicates that these genes reflect clinical activity parameters and may be the promoters of early, inflammatory disease.

DNA methylation patterns in juvenile systemic sclerosis and localized scleroderma.

Scleroderma refers to a group of chronic fibrotic immune-mediated diseases of unknown etiology. Characterizing epigenetic changes in childhood-onset scleroderma, systemic sclerosis or localized scleroderma, has not been previously performed. The aim of this study was to assess DNA methylation differences and similarities between juvenile systemic sclerosis (jSSc) and juvenile localized scleroderma (jLS) compared to matched healthy controls. Genome-wide DNA methylation changes in peripheral blood mononuclear cell samples were assessed using the MethylationEPIC array followed by bioinformatic analysis and limited functional assessment. We identified a total of 105 and 144 differentially methylated sites compared to healthy controls in jSSc and jLS, respectively. The majority of differentially methylated sites and genes represented were unique to either jSSc or jLS suggesting a different underlying epigenetic pattern in both diseases. Among shared differentially methylated genes, methylation levels in a CpG site in FGFR2 can distinguish between LS and healthy PBMCs with a high accuracy. Canonical pathway analysis revealed that inflammatory pathways were enriched in genes differentially methylated in jSSc, including STAT3, NF-κB, and IL-15 pathways. In contrast, the HIPPO signaling pathway was enriched in jLS. Our data also suggest a potential role for NOTCH3 in both jSSc and jLS, and revealed a number of transcription factors unique to each of the two diseases. In summary, our data revealed important insights into jSSc and jLS and suggest a potentially novel epigenetic diagnostic biomarker for LS.

Transcriptomic Evaluation of Juvenile Localized Scleroderma Skin With Histologic and Clinical Correlation.

OBJECTIVE: Juvenile localized scleroderma (LS) is an autoimmune disease of the skin whose pathogenesis is not well understood due to the rarity of the disease. This study was undertaken to determine the skin transcriptome in skin biopsy tissue from children with juvenile LS compared to pediatric healthy controls, with identification of significant molecular targets using RNA sequencing (RNA-Seq). In this study, differentially expressed genes (DEGs) were assessed for correlations with histopathologic and clinical features in children with juvenile LS, and were used to group the children into distinct genetic clusters based on immunophenotype. METHODS: RNA-Seq was performed on sections of paraffin-embedded skin tissue obtained from 28 children with juvenile LS and 10 pediatric healthy controls. RNA-Seq was carried out using an Illumina HTS TruSeq RNA Access library prep kit, with data aligned using STAR and data analysis using a DESeq2 platform. A standardized histologic scoring system was used to score skin sections for the severity of inflammation and levels of collagen deposition. Histologic scoring was completed by 2 pathologists who were blinded with regard to the status of each sample. Spearman's rank correlation coefficients were used to assess significant correlations between DEG expression profiles and skin histologic findings in patients with juvenile LS. RESULTS: We identified 589 significant DEGs in children with juvenile LS as compared to healthy controls. Hierarchical clustering was used to demonstrate 3 distinct juvenile LS immunophenotype clusters. The histologic scores of skin inflammation (based on numbers and categories of inflammatory cell infiltrates) were significantly correlated with the expression levels of HLA-DPB1, HLA-DQA2, HLA-DRA, and STAT1 genes (rs > 0.5, P < 0.01). Collagen thickness correlated with the expression levels of collagen organization genes as well as with genes found to be correlated with the severity of inflammation, including genes for major histocompatibility complex (MHC) class I, MHC class II, and interferon-γ signaling. CONCLUSION: Among children with juvenile LS, 3 distinct genetic signatures, or clusters, were identified. In one cluster, inflammation-related pathways were up-regulated, corresponding to the histologic skin inflammation score. In the second cluster, fibrosis-related pathways were up-regulated. In the third cluster, gene expression in the skin corresponded to the patterns seen in healthy controls. Up-regulation of HLA class II genes was observed within the first cluster (characterized by predominant inflammation), a feature that has also been observed in the peripheral blood of patients with morphea and in the skin of patients with systemic sclerosis.

Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma.

BACKGROUND: The purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to RPMI to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders. METHODS: We performed single-cell RNA sequencing on paired skin biopsy specimens from 3 patients with LS. Each patient with one sample cryopreserved in CryoStor® CS10 and one fresh in RPMI media using 10× Genomics sequencing. RESULTS: Levels of cell viability and yield were comparable between CryoStor® CS10 (frozen) and RPMI (fresh) preserved cells. Furthermore, gene expression between preservation methods was collectively significantly correlated and conserved across all 18 identified cell cluster populations. CONCLUSION: Comparable cell population and transcript expression yields between CryoStor® CS10 and RPMI preserved cells support the utilization of cryopreserved skin tissue in single-cell analysis. This suggests that employing standardized cryopreservation protocols for the skin tissue will help facilitate multi-site collaborations looking to identify mechanisms of disease in disorders characterized by cutaneous pathology.

Autoantibodies in Morphea: An Update.

Skin autoimmune conditions belong to a larger group of connective tissue diseases and primarily affect the skin, but might also involve underlying tissues, such as fat tissue, muscle, and bone. Autoimmune antibodies (autoantibodies) play a role in autoimmune skin diseases, such as localized scleroderma also termed morphea, and systemic scleroderma, also called systemic sclerosis (SSc). The detailed studies on the biological role of autoantibodies in autoimmune skin diseases are limited. This results in a few available tools for effective diagnosis and management of autoimmune skin diseases. This review aims to provide an update on the detection and most recent research on autoantibodies in morphea. Several recent studies have indicated the association of autoantibody profiles with disease subtypes, damage extent, and relapse potential, opening up exciting new possibilities for personalized disease management. We discuss the role of existing autoantibody tests in morphea management and the most recent studies on morphea pathogenesis. We also provide an update on novel autoantibody biomarkers for the diagnosis and study of morphea.