DNA Binding of Human and Viral Transcription Factors is Associated with Rheumatoid Arthritis Risk Loci

人类和病毒转录因子的 DNA 结合与类风湿关节炎风险位点相关

• 批准号: 10045951
• 负责人: KENNETH M KAUFMAN
• 金额: --
• 依托单位: CINCINNATI VA MEDICAL CENTER RESEARCH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045951
• 关键词: Alleles; Arthritis; Autoantibodies; Award; B-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line; Cells; ChIP-seq; Chronic; Complex; DNA; DNA Binding; Data; Degenerative polyarthritis; Dependence; Development; Diagnosis; Elements; Environment; Epstein-Barr Virus Infections; Epstein-Barr Virus latency; Etiology; Fibroblasts; Foundations; Frequencies; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Risk; Human; Human Herpesvirus 4; Immune; Immune response; Individual; Infection; Inflammatory; Inflammatory Response; Investigation; Joints; Learning; Lytic Virus; Modeling; Nature; Nuclear Antigens; Pathogenesis; Pathology; Patients; Peptides; Population; Prevention strategy; Probability; Process; Property; PubMed; Publications; Quantitative Trait Loci; Regulator Genes; Relative Risks; Rheumatoid Arthritis; Risk; Site; Specificity; Synovial Membrane; Systemic disease; T cell response; Testing; Therapeutic; Tissues; Variant; Viral; Viral Antigens; Viral Proteins; Virus; Virus Diseases; Work; base; cyclic citrullinated peptide; design; differential expression; disorder control; experience; experimental study; gene product; genomic locus; infected B cell; insight; joint destruction; medical attention; microbiome; morphogens; novel therapeutic intervention; prevent; programs; response; risk variant; single-cell RNA sequencing; small molecule libraries; transcription factor; transcriptome sequencing; viral DNA; virtual

This Merit Review application is based on our hypotheses that Fibroblast-Like Synoviocytes (FLS) from Rheumatoid Arthritis (RA) patients infected with Epstein - Barr virus (EBV) will have unique gene expression properties that alter the host immune response to EBV, and that these difference influences the risk for developing RA. These hypotheses is based on the following observations: All 12 of the FLS cell lines that we have evaluated, six from Rheumatoid Arthritis (RA) patients and six from Osteoarthritis (OA) disease controls, contained some cells infected with EBV. We discovered that FLS can be superinfected with EBV. We observed that RA (but not OA) genetic risk loci are enriched for DNA variants that are immunoprecipitated with Epstein-Barr Nuclear Antigen-2 (EBNA2) (Relative Risk (RR)=4.5, Bonferroni corrected probability (Pc)=2.4x10-14). Preliminary data showed differences between RA and OA in the expression of the genes controlled by elements in RA risk loci. These observations and the 792 publications in Pubmed, often contradictory, present many intriguing relationships between EBV and RA. In our view these observations are consistent with EBV being involved in the pathogenesis of RA. We plan three initiatives with this DVA Merit Award project. First, we will characterize the virus infection in FLS (Aim 1) by: assessing the frequency of EBV FLS infection, sequencing the EBV DNA in FLS, and comparing EBV gene expression in RA, OA, & normal (NL) FLS. We will test the hypothesis that FLS infection is common among the EBV infected human population. Second, we will explore the FLS response to EBV infection (Aim 2) by determining host gene expression with RNA-seq to assess EBV infected and secondarily influenced FLS from RA, OA, & NL subjects and by exploring allele specificity at RA loci in RA, OA, and NL FLS. Once experimental conditions are determined, single cell RNA-seq will characterize the cell population infected with EBV and the cells responding to EBV infection. These experiments will evaluate the hypotheses that RA FLS will have a different gene expression profile than the FLS from OA or NL subjects and that the expression quantitative trait loci (eQTL) will distinguish RA from OA and NL FLS. Third, we will assess the immune response to EBV infected FLS (Aim 3). Our hypothesis is that the host response to EBV infection of the FLS will differentiate RA from OA and NL subjects. In specific, our detailed working hypothesis and current model is: 1. That usually the OA & NL FLS are abortively infected with EBV, 2. That OA & NL EBV infected FLS exist at a minimal level, perhaps only expressing a few EBV latent genes, as seen in latently infected B cells, and 3. That EBV infected OA & NL FLS are usually quiescent to immune recognition. In contrast, in RA there is evidence that the early steps of the EBV lytic program are initiated with the expression of Early- Immediate EBV antigens. These changes in combination with other factors (e.g., anti-cyclic citrullinated peptide autoantibodies, possible microbiome changes, etc.) awaken the quiescent immune response and result in an overwhelming inflammatory response directed against the EBV infected RA FLS in a way that does not happen in the OA or NL synovium. As has been our experience in the previous submissions, the results obtained may contradict any of these ideas leading this work into a new conceptual and strategic direction. On the other hand, this work has the potential to provide mechanistic details of the currently unknown aspects of RA pathogenesis, which would become the basis for new therapeutic approaches and preventive strategies. Whether or not EBV is a component of RA pathogenesis, the studies proposed will provide insight into a previously unknown tissue specific site of and possible reservoir for a common chronic viral infection that infects the vast majority of human beings.

此优点审查申请基于我们的假设，即成纤维细胞样滑膜细胞 (FLS) 来自 感染爱泼斯坦-巴尔病毒（EBV）的类风湿性关节炎（RA）患者将拥有独特的基因 表达特性会改变宿主对 EBV 的免疫反应，并且这些差异会影响风险 用于开发 RA。这些假设基于以下观察： 所有 12 种 FLS 细胞系 我们评估了 6 名来自类风湿性关节炎 (RA) 患者和 6 名来自骨关节炎 (OA) 疾病的患者 对照，包含一些感染 EBV 的细胞。我们发现FLS可以被EBV重复感染。我们 观察到 RA（而非 OA）遗传风险位点富含经过免疫沉淀的 DNA 变体 Epstein-Barr 核抗原 2 (EBNA2)（相对风险 (RR)=4.5，Bonferroni 校正概率 (Pc)=2.4x10-14)。初步数据显示 RA 和 OA 在基因表达方面存在差异 由 RA 风险基因座中的元件控制。这些观察结果和 Pubmed 中的 792 篇出版物，经常 EBV 和 RA 之间存在着许多相互矛盾的关系。我们认为这些观察结果是 与 EBV 参与 RA 发病机制一致。 我们针对 DVA 优异奖项目计划了三项举措。首先我们先来了解一下病毒感染的特征 FLS（目标 1）：评估 EBV FLS 感染的频率，对 FLS 中的 EBV DNA 进行测序，以及 比较 RA、OA 和正常 (NL) FLS 中的 EBV 基因表达。我们将检验 FLS 感染的假设 在 EBV 感染人群中很常见。其次，我们将探讨 FLS 对 EBV 的反应 通过 RNA-seq 确定宿主基因表达来评估 EBV 感染和继发性感染（目标 2） 通过探索 RA、OA 和 NL 中 RA 位点的等位基因特异性，影响了 RA、OA 和 NL 受试者的 FLS FLS。一旦实验条件确定，单细胞 RNA-seq 将表征细胞群 感染了 EBV 以及对 EBV 感染做出反应的细胞。这些实验将评估假设 RA FLS 将具有与 OA 或 NL 受试者的 FLS 不同的基因表达谱，并且 表达数量性状位点 (eQTL) 将 RA 与 OA 和 NL FLS 区分开来。第三，我们将评估 对 EBV 感染的 FLS 的免疫反应（目标 3）。我们的假设是宿主对 EBV 感染的反应 FLS 将 RA 与 OA 和 NL 受试者区分开来。具体来说，我们详细的工作假设和当前 模型是： 1. 通常 OA 和 NL FLS 会流产感染 EBV，2. OA 和 NL EBV 感染 FLS 存在于最低水平，可能仅表达一些 EBV 潜伏基因，如潜伏感染 B 中所见 细胞，以及 3. EBV 感染的 OA 和 NL FLS 通常对免疫识别处于静止状态。相反，在 RA 有证据表明 EBV 裂解程序的早期步骤是随着 Early- 直接 EBV 抗原。这些变化与其他因素（例如抗环瓜氨酸 肽自身抗体、可能的微生物组变化等）唤醒静止的免疫反应并 导致针对 EBV 感染的 RA FLS 的压倒性炎症反应 不会发生在 OA 或 NL 滑膜中。根据我们之前提交的经验，结果 所获得的可能与这些想法相矛盾，从而导致这项工作进入新的概念和战略方向。在 另一方面，这项工作有可能提供目前未知方面的机制细节 RA 发病机制，这将成为新的治疗方法和预防策略的基础。 无论 EBV 是否是 RA 发病机制的一个组成部分，所提出的研究都将提供对 RA 发病机制的深入了解。 以前未知的常见慢性病毒感染的组织特定部位和可能的储存库 感染绝大多数人类。