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

细胞表观遗传学

• 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
• 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

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.

系统性硬化症(SSC)的皮肤纤维化会导致严重的发病率，其原因是毁容、疼痛和瘙痒。 皮肤和关节痉挛。我们最近通过单细胞RNA测序(scRNA-seq)表明，SSC 真皮成纤维细胞(表达增加的THBS1、PRSS23)和真皮肌成纤维细胞(也表达 SFRP4、ADAM12、TNFSF18和CTGF)增加源于健康人群中发现的表达SFRP2的祖细胞 控制皮肤。这些研究为理解这些细胞状态的促纤维化驱动因素提供了一个框架。 转录因子在调节基因表达方面起着关键作用，并为其提供了一个强大的里程碑。 细胞状态。使用Scenic，开发了一种用于检测TF相关调控网络的计算方法 (调节子)在单细胞数据集中，我们发现了可能驱动肌成纤维细胞分化的转录因子：Foxp1，HIF1a， IRF7、STAT1和FOSL2。此外，在初步结果中，我们采用了转座酶可及性检测。 染色质测序(scATAC-seq)数据支持这些转录因子在SSC肌成纤维细胞中的重要性 差异化。在我们的第一个目标中，我们将评估这些转录因子在进一步的多组研究中的重要性，并确认 通过检测Tf对成纤维细胞转录组的影响来研究它们在肌成纤维细胞分化中的作用 和表观基因组。巨噬细胞活化的标志物与皮肤的主要临床指标密切相关 疾病严重程度，改良的Rodnan皮肤评分(MRSS)，表明巨噬细胞传递促纤维化 驱动肌成纤维细胞分化的信号。最近对SSc-ILD的研究证实IL-6在SSC-ILD的发病机制中起作用。 肺部疾病，我们看到它的下游靶点CCL18也在皮肤巨噬细胞中上调。在我们的第二个 目的：我们将使用一种新的系统生物学方法论CAUSER来分析潜在的调节因素 巨噬细胞-成纤维细胞相互作用并产生tocilizumab前后的SnRNA-seq数据以更好地 了解IL-6在SSC皮肤中激活促纤维化巨噬细胞中的作用。我们预计这将告知 类似的过程发生在SSc-间质性肺疾病中。局限性硬皮病(LS)继续导致 儿童和成人的毁容和功能残疾。我们的初步结果表明IFNG为 激活LS皮肤中的巨噬细胞和成纤维细胞。在目标3中，我们将使用类似的方法来研究SSC 比较LS和SSC皮肤中的免疫和非免疫细胞群。首先，我们将合并我们现有的LS (n=14)、SSC(n=27)和健康对照(n=14)scRNA-seq数据集，并检查成纤维细胞和 髓系细胞转录组-表型和差异表达基因。然后像在目标2中一样，我们将使用 原因是找出调节这些细胞之间相互作用的潜在因素。然后我们将确定TF 使用SCEARE和MULTOME调节髓系和成纤维细胞表型。我们期望对LS的这些研究能够 为激活肌成纤维细胞的细胞因子和细胞内通路提供新的见解，从而导致皮肤 这些患者的纤维化。拟议的研究将得到临床和生物样本收集的大力支持。 通过核心B以及核心C中的辛格博士和达斯博士的系统生物学专业知识。