Cell epigenetics & communication in systemic sclerosis and localized scleroderma skin disease

细胞表观遗传学

基本信息

批准号：
10705648
负责人：
ROBERT A. LAFYATIS
金额：
$ 30.21万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10705648
关键词：
Adult Aftercare Automobile Driving Binding Biological Assay Biopsy Blood Vessels CCL18 gene Cells Child Chromatin Chromatin Structure Cicatrix Clinical Collection Communication Computing Methodologies Contracture Cutaneous Cutaneous sclerosis DNA Data Data Set Dermal Diffuse Drug Targeting Epigenetic Process FOSL2 gene FOXP1 gene Fibroblasts Gene Expression Genes Genome HIF1A gene IL-6 inhibitor Immune In Vitro Inflammatory Interleukin-6 Interstitial Lung Diseases Joints Link Localized scleroderma Lung Lung diseases MADH3 gene Macrophage Macrophage Activation Measures Mediator Methodology Molecular Morbidity - disease rate Myelogenous Myeloid Cells Myofibroblast Pain Pathogenesis Pathway interactions Patients Phenotype Population Predictive Factor Process Profibrotic signal Regulon Rheumatism Role SFRP4 gene STAT1 gene Sampling Severity of illness Signal Transduction Skin Source Systemic Scleroderma Systemic disease Systems Biology THBS1 gene Transposase biomarker identification clinical translation connective tissue growth factor cytokine differential expression drug development epigenome functional disability insight interest knock-down mortality multiple omics novel predictive marker progenitor single nucleus RNA-sequencing single-cell RNA sequencing skin disorder skin fibrosis tocilizumab tool transcription factor transcriptome transcriptomics translational study

项目摘要

Skin fibrosis in systemic sclerosis (SSc) leads to significant morbidity resulting from disfiguring, painful and itchy skin, and joint contractures. We have recently shown by single cell RNA-sequencing (scRNA-seq) that SSc dermal fibroblasts (expressing increased THBS1, PRSS23) and dermal myofibroblasts (also expressing increased SFRP4, ADAM12, TNFSF18 and CTGF) arise from SFRP2-expressing progenitors found in healthy control skin. These studies provide a framework for understanding the profibrotic drivers of these cell states. Transcription factors (TFs) are pivotal in regulating gene expression and provide a powerful landmark for these cell states. Using SCENIC, a computational method developed for detecting TF-associated regulatory networks (regulons) in single cell datasets, we identified putative TFs driving myofibroblast differentiation: FOXP1, HIF1A, IRF7, STAT1 and FOSL2. Additionally, in preliminary results we employed Assay for Transposase Accessible Chromatin by Sequencing (scATAC-seq) data supporting the importance of these TFs in SSc myofibroblast differentiation. In our first aim, we will assess the importance of these TFs in further multiome studies, and confirm their roles in myofibroblast differentiation by measuring the effects of TF knock-down on fibroblast transcriptome and epigenome. Markers of macrophage activation correlate strongly with the main clinical measure of skin disease severity, the modified Rodnan skin score (MRSS), suggesting that macrophages deliver profibrotic signals to drive myofibroblast differentiation. Recent studies in SSc-ILD have confirmed IL-6 in pathogenesis of lung disease and we see its downstream target CCL18 also upregulated in skin macrophages. In our second aim, we will use a novel system biology methodology, CausER, to analyze latent factors regulating the macrophage-fibroblast interaction and generate snRNA-seq data before and after tocilizumab to better understand the role of IL-6 in activating profibrotic macrophages in SSc skin. We expect that this will inform the similar process occurring in SSc-interstitial lung disease. Localized scleroderma (LS) continues to cause disfiguring and functional disabilities in children as well as adults. Our preliminary results implicate IFNg as activating macrophages and fibroblasts in LS skin. In aim 3, using similar approaches to study of SSc, we will compare the immune and non-immune cell populations in LS to SSc skin. First, we will combine our existing LS (n=14), SSc (n=27) and healthy control (n=14) scRNA-seq datasets, and examine differences in fibroblast and myeloid cell transcriptome-phenotypes and differentially expressed genes. Then as in aim 2, we will employ CausER to identify latent factors regulating the interaction between these cells. We will then identify TFs regulating myeloid and fibroblast phenotypes using SCENIC and multiome. We expect these studies of LS to provide new insights into the cytokines and intracellular pathways activating myofibroblasts that lead to skin fibrosis in these patients. The proposed studies will be strongly supported by clinical and biosample collection through Core B and the systems biology expertise by Drs. Singh and Das in core C.

全身性硬化症（SSC）的皮肤纤维化导致毁容，痛苦和瘙痒导致明显的发病率皮肤和关节染色。我们最近通过单细胞RNA-sevening（SCRNA-Seq）表明了SSC 皮肤成纤维细胞（表达THBS1，PRSS23）和皮肤肌纤维细胞（也表达 SFRP4，ADAM12，TNFSF18和CTGF的增加是由在健康中发现的表达SFRP2的祖细胞引起的控制皮肤。这些研究为理解这些细胞态的纤维化驱动因素提供了一个框架。转录因子（TFS）在调节基因表达方面是关键的，并为这些提供了有力的地标细胞状态。使用Scenic，开发了用于检测与TF相关的调节网络的计算方法（调节）在单细胞数据集中，我们确定了推定的TFS驱动肌纤维细胞分化：FOXP1，HIF1A， IRF7，STAT1和FOSL2。此外，在初步结果中，我们采用了用于转座酶的测定通过测序（SCATAC-SEQ）数据支持这些TF在SSC肌纤维细胞中的重要性分化。在我们的第一个目标中，我们将评估这些TF在进一步的多群体研究中的重要性，并确认通过测量TF敲低对成纤维细胞转录组的影响，它们在肌纤维细胞分化中的作用和表观基因组。巨噬细胞激活的标志物与皮肤的主要临床指标密切相关疾病的严重程度，改良的Rodnan皮肤评分（MRS），表明巨噬细胞提供纤维化信号驱动肌纤维细胞分化。在SSC-ILD的最新研究已经证实了IL-6的发病机理肺部疾病，我们看到其下游靶标CCL18在皮肤巨噬细胞中也上调。在我们的第二个目的，我们将使用一种新型的系统生物学方法论Causer来分析调节的潜在因素巨噬细胞 - 纤维细胞相互作用并在Tocilizumab之前和之后生成SnRNA-Seq数据以更好了解IL-6在激活SSC皮肤中纤维化巨噬细胞中的作用。我们希望这将告知在SSC间隙肺部疾病中发生类似的过程。局部硬皮病（LS）继续引起儿童和成人的毁容和功能障碍。我们的初步结果暗示IFNG 激活LS皮肤中的巨噬细胞和成纤维细胞。在AIM 3中，使用类似的SSC研究方法，我们将比较LS中的免疫和非免疫细胞群与SSC皮肤。首先，我们将结合现有的LS （n = 14），SSC（n = 27）和健康对照（n = 14）SCRNA-SEQ数据集，并检查成纤维细胞的差异和髓样细胞转录组 - 表型和差异表达的基因。然后像AIM 2一样，我们将雇用为了确定调节这些细胞相互作用的潜在因素。然后，我们将确定TFS 使用风景和多重组来调节髓样和成纤维细胞表型。我们期望这些对LS的研究提供有关细胞因子和细胞内途径的新见解，激活导致皮肤的肌纤维细胞这些患者的纤维化。拟议的研究将得到临床和生物样品收集的强烈支持通过核心B和Drs的系统生物学专业知识。 Singh和Das在CoreC中。