Translational Immunology research: a support for clinical immunological research

转化免疫学研究：临床免疫学研究的支持

• 批准号: 8344974
• 负责人: Massimo Gadina
• 金额: $ 233.8万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344974
• 关键词: Adult; Affect; Autoimmune Diseases; Autoimmune Process; B-Cell Development; B-Lymphocytes; Basic Science; Behcet Syndrome; Binding; Binding Sites; Biological Assay; Biological Markers; Blood donor; CD4 Positive T Lymphocytes; Cardiac Myocytes; Cell Differentiation process; Cell Therapy; Cells; Chromatin Structure; Chromosomal translocation; Clinical; Clinical Investigator; Clinical Trials; Collaborations; Cooperative Research and Development Agreement; DNA; DNA Damage; DNA Double Strand Break; DNA Methylation; DNA repair protein; Disease; Enzyme-Linked Immunosorbent Assay; Epigenetic Process; Event; Fibroblasts; Flow Cytometry; Functional RNA; Gene Expression Profile; Genes; Genetic Determinism; Genetic Variation; Genome; Goals; Graft Rejection; HLA Antigens; Helper-Inducer T-Lymphocyte; Heterotopic Ossification; Histones; Human; IL10 gene; IgE; Immune response; Immunoassay; Immunology; Immunology procedure; In Vitro; Individual; Inflammatory; Injury; Instruction; Interleukin-10; Interleukin-17; Interleukin-4; Interleukin-6; Journals; Laboratories; Lead; Life; Magnetism; Maps; Measures; Mesenchymal Stem Cells; Messenger RNA; Methods; Modification; Molecular; Mus; Musculoskeletal Diseases; Myogenic Regulatory Factors; National Human Genome Research Institute; National Institute of Arthritis and Musculoskeletal and Skin Diseases; Nucleic Acids; Other Genetics; Pathogenesis; Pathology; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pluripotent Stem Cells; Polycomb; Population; Predisposition; Process; Production; Proteins; Publishing; RNA; RNA Polymerase II; RNA Splicing; Real-Time Systems; Regulation; Relative (related person); Research; Research Personnel; Resources; Role; STAT1 gene; STAT3 gene; STAT4 gene; STAT5A gene; Services; Signal Transduction; Single Nucleotide Polymorphism; Skeletal Muscle; Small RNA; Source; Stress; Structure of retinal pigment epithelium; System; Systemic Lupus Erythematosus; T-Lymphocyte; Techniques; Testing; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Translational Research; Trauma; Treatment Efficacy; Tyrosine Kinase Inhibitor; Variant; Western Blotting; Whole Blood; Zebularine; base; clinical application; cytokine; genome-wide; histone modification; human disease; hydroxyurea; improved; indexing; instrumentation; interleukin-22; interleukin-23; mRNA Expression; melting; member; monocyte; next generation; novel; novel therapeutic intervention; pluripotency; prevent; programs; promoter; receptor; response; tool; trait; transcription factor

The Translational immunology Section (TIS)is equipped with instrumentation to measure cellular responses at multiple levels and it supports primarily (but not solely) the research NIAMS clinical investigators. Besides having the capability to detect and quantify cell-secreted proteins and analyzing signalling events using classical techniques such as ELISA and Western blotting the facility is capable to measure the same events with powerful high-sensitivity, high-throughput instrumentation such as: Multi-mode analyzers (Bio-Plex 200; Sinergy4), magnetic cell sorter (Auto MACS), nucleic acid analyzers (one Illumina Gene Analyzer and one Illumina HiSeq2000 for high-throughput sequencing; Agilent 2100; Bio-Rad CFX96 Real Time System/C1000 Thermal Cycler). A major project in which involves the TIS is the definition of molecular biomarkers for autoimmune and autoinflammatory diseases. In collaboration NHGRI investigators we have shown that susceptibility to Behcet's disease is associated with genes which regulate the immune response. In particular, it was shown that single nucleotide polymorphisms (SNPs), in the region of the with the human leukocyte antigen (HLA)-B51 region of the MHC were highly associated with the disease. Other genetic factors were associated with the disease. Associations were found with a variant of the IL10 gene and with a variant located between the genes for the IL-23 receptor (IL23R). We showed that cells from blood donors who had two copies of the IL10 gene variant produced one-third of the IL-10 protein compared to people with one or two normal IL10 genes. We are currently investigating if the variants discovered in the IL-23R locus influence the expression levels of this receptor on T cells and monocytes of healthy individuals. We are measuring the mRNA expression levels for IL-23 in PBMCs from normal donors isolated from whole blood. Furthermore, using a flow cytometry-based assay we are measuring the expression levels of IL-23R in specific cellular population. These assays will allow us to better understand the mechanisms of inflammatory processes underlying this disease and may lead to potential novel therapeutic approaches. As mentioned above, the discovery of protein patterns as biomarkers of disease or of therapeutic efficacy is a major objective of the TIS. The laboratory of Dr. John OShea has been investigating novel therapeutic approaches for the treatment of autoimmune diseases. The TIS is currently involved in a collaboration with the O'Shea group and Pfizer (a CRADA is in place) aimed at defining the efficacy of the tyrosine kinase inhibitor Tofacitinib on the differentiation and activation programs of human T cells. We examined the mode of action of Tofacitinib (CP-690,550) on the JAK/STAT pathway involved in adaptive and innate immune responses. We analyzed cytokine stimulation of mouse and human T cells in vitro. We investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. Tofacitinib inhibited IL-4-dependent Th2 cell differentiation and interfered with Th17 cell differentiation. Expression of IL-23R and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-beta. Overall, Tofacitinib may improve autoimmune diseases and prevent transplant rejection. The results of this study were recently published in The Journal of Immunology. The TIS has also started a project aimed at generating inducible pluripotent stem cells (iPS). iPS are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somaticc cell, by inducing a "forced" expression of specific transcription factors. Starting from a commercial source of human fibroblast we have generated iPS cells and demonstrated their pluripotency by assessing specific markers and embryoid body formation capacity. Furthermore, iPSCs were differentiated into cardiomyocytes that spontaneously began beating. Our research will now be aimed at showing that such cells are a powerful new tool for studying the pathogenesis of human diseases for pharmacological and toxicological testing as well as cell-based therapy. The TIS, is also providing support to several projects carried out at NIAMS aimed at better understanding the genetic determinants of autoimmune, autoinflammatory and musculoskeletal diseases using the Illumina Genome Analyzer (GA) for ultra high-throughput sequencing. Below is the description of ongoing projects. Dr. Kambiz Mousavi in Dr. Sartorellis laboratory aims at defining the epigenome and the transcriptional landscape during skeletal muscle formation. The laboratory focused on two objectives: 1. To delineate the function of novel member of Polycomb group (PcG) protein, Ezh1, within myogenic system; and 2. To characterize genome-wide occupancy of myogenic regulatory factors (i.e. MyoD/MyoG) relative to epigenome and transcriptome. Throughout these endeavors, we demonstrated the enhancement RNA Polymerase II elongation by Ezh1; and regulation of numerous short-lived intergenic non-coding RNA by MyoD during skeletal muscle formation. With Dr. Yuka Kanno we have continued to accumulate profiles of STAT bindings in T cells, and profiled action of STAT3 and STAT5 in Th17 cells to reveal global competition between the two STATs which share the same binding sites. Histone epigenetic mapping of Tfh cells and other lineages revealed consistently open chromatin structure for Tfh master regulator Bcl-6 across all lineages, suggesting considerable plasticity of Tfh lineage. Similarly we have mapped T-bet binding events in Th1 cells and compared the role of T-bet with that of STAT4 and STAT1 in Th1 specification. We have also performed the first set of RNA-Seq analysis to characterize the T helper cells transcriptome. With Dr. Casellas we comprehensively analyzed sets of genome-wide epigenetic modifications (histone modification/DNA methylation status) and RNA (mRNA/splicing variants/small RNA) throughout B-cell development. We are using next-generation sequencing technique to elucidate how these DNA editing can produce byproducts in its process, namely double strand DNA break and chromosomal translocation, which could lead to B-cells pathologies. We also developed new sequencing applications to look at the translocation and DNA breaks. We did 4C analysis and CTCF ChIP-seq using the sequence facility. We also developed the sequencing assay, which capture genome-wide promoter melting in the cells and applying it to explore a long-standing immunological question. Dr. Chao Jiang in Dr. Riveras lab is studying a subset of SLE patients that have elevated levels of circulating auto-IgE and this trait was highly associated with the disease activity as defined by the SLEDAI Index. To understand the contribution of auto-IgE to the pathogenesis of SLE, we investigated the gene expression profile associated with high levels of auto-IgE production in humans using RNA-Seq. With Dr. Leon Nesti and Dr. Youngmi Ji we have performed RNA-Seq studies with the aim of elucidating how activation via injury affects the mesenchymal progenitor cells (MPCs) and to determine whether trauma may contribute to pathological differentiation of the MPCs and formation of heterotopic ossification. With Dr. Lai Wei and Dr. Nussenblatt from NEI we investigated how epigenetic drugs, especially Zebularine, change genome-wide H3K4me3 and H3K27me3 patterns in the retinal pigment epithelium cells under inflammatory cytokine stimulation. With Dr. Nussenzweig group from NCI we are mapping replication-induced DNA damage stress in response to the drug hydroxyurea and the recruitment of DNA repair proteins to AID-dependent and in B cells.

转化免疫学部分 (TIS) 配备了测量多个水平细胞反应的仪器，它主要（但不仅仅是）支持 NIAMS 临床研究人员的研究。除了能够检测和定量细胞分泌的蛋白质以及使用 ELISA 和蛋白质印迹等经典技术分析信号事件外，该设施还能够使用强大的高灵敏度、高通量仪器测量相同的事件，例如：多模式分析仪（Bio-Plex 200；Sinergy4）、磁性细胞分选仪（Auto MACS）、核酸分析仪（一台 Illumina Gene） 分析仪和一台Illumina HiSeq2000用于高通量测序；安捷伦2100； Bio-Rad CFX96 实时系统/C1000 热循环仪）。 TIS 涉及的一个主要项目是定义自身免疫和自身炎症疾病的分子生物标志物。 通过与 NHGRI 研究人员的合作，我们发现白塞氏病的易感性与调节免疫反应的基因有关。特别是，研究表明，MHC 人类白细胞抗原 (HLA)-B51 区域的单核苷酸多态性 (SNP) 与该疾病高度相关。其他遗传因素与该疾病有关。发现与 IL10 基因的变体以及位于 IL-23 受体 (IL23R) 基因之间的变体存在关联。我们发现，与具有一两个正常 IL10 基因的人相比，具有两个 IL10 基因变异拷贝的献血者的细胞产生了三分之一的 IL-10 蛋白。我们目前正在研究 IL-23R 位点中发现的变异是否会影响健康个体 T 细胞和单核细胞上该受体的表达水平。我们正在测量从全血中分离出的正常供体的 PBMC 中 IL-23 的 mRNA 表达水平。此外，我们使用基于流式细胞术的测定法测量特定细胞群中 IL-23R 的表达水平。这些测定将使我们能够更好地了解这种疾病的炎症过程机制，并可能带来潜在的新治疗方法。 如上所述，发现蛋白质模式作为疾病或治疗功效的生物标志物是 TIS 的主要目标。 John OShea 博士的实验室一直在研究治疗自身免疫性疾病的新方法。 TIS 目前正在与 O'Shea 集团和辉瑞（CRADA 已到位）合作，旨在确定酪氨酸激酶抑制剂托法替尼（Tofacitinib）对人类 T 细胞分化和激活程序的功效。我们检查了 Tofacitinib (CP-690,550) 对涉及适应性和先天免疫反应的 JAK/STAT 通路的作用模式。我们在体外分析了小鼠和人类 T 细胞的细胞因子刺激。我们研究了 CP-690,550 处理对幼鼠 CD4(+) T 细胞的 Th 细胞分化的影响。 Tofacitinib 抑制 IL-4 依赖性 Th2 细胞分化并干扰 Th17 细胞分化。当用 IL-6 和 IL-23 刺激初始 Th 细胞时，IL-23R 和 Th17 细胞因子 IL-17A、IL-17F 和 IL-22 的表达被阻断。相反，当 Th17 细胞在 TGF-β 存在下分化时，IL-17A 的产生增强。总体而言，托法替布可以改善自身免疫性疾病并预防移植排斥。这项研究的结果最近发表在《免疫学杂志》上。 TIS 还启动了一个旨在生成诱导性多能干细胞 (iPS) 的项目。 iPS 是一种多能干细胞，通过诱导特定转录因子的“强制”表达，人工衍生自非多能细胞（通常是成体体细胞）。从人类成纤维细胞的商业来源开始，我们生成了 iPS 细胞，并通过评估特定标记和胚状体形成能力来证明其多能性。此外，iPSC 分化为自发开始跳动的心肌细胞。我们现在的研究旨在表明，此类细胞是研究人类疾病发病机制、进行药理学和毒理学测试以及基于细胞的治疗的强大新工具。 TIS 还为 NIAMS 开展的多个项目提供支持，旨在使用 Illumina 基因组分析仪 (GA) 进行超高通量测序，更好地了解自身免疫、自身炎症和肌肉骨骼疾病的遗传决定因素。 以下是正在进行的项目的描述。 Sartorellis 博士实验室的 Kambiz Mousavi 博士旨在定义骨骼肌形成过程中的表观基因组和转录景观。该实验室主要致力于两个目标： 1. 描述多梳族 (PcG) 蛋白新成员 Ezh1 在生肌系统中的功能； 2. 表征肌源性调节因子（即 MyoD/MyoG）相对于表观基因组和转录组的全基因组占据情况。 在这些努力中，我们证明了 Ezh1 可以增强 RNA 聚合酶 II 的延伸；骨骼肌形成过程中 MyoD 对大量短寿命基因间非编码 RNA 的调节。 我们与 Yuka Kanno 博士一起继续积累 T 细胞中 STAT 结合的概况，以及 Th17 细胞中 STAT3 和 STAT5 的作用概况，以揭示共享相同结合位点的两种 STAT 之间的全局竞争。 Tfh 细胞和其他谱系的组蛋白表观遗传图谱揭示了所有谱系中 Tfh 主调节因子 Bcl-6 一致的开放染色质结构，表明 Tfh 谱系具有相当大的可塑性。同样，我们绘制了 Th1 细胞中的 T-bet 结合事件图，并将 T-bet 的作用与 STAT4 和 STAT1 在 Th1 规范中的作用进行了比较。我们还进行了第一组 RNA-Seq 分析来表征 T 辅助细胞转录组。 我们与 Casellas 博士一起全面分析了 B 细胞发育过程中的一系列全基因组表观遗传修饰（组蛋白修饰/DNA 甲基化状态）和 RNA（mRNA/剪接变体/小 RNA）。我们正在使用下一代测序技术来阐明这些 DNA 编辑如何在其过程中产生副产物，即双链 DNA 断裂和染色体易位，这可能导致 B 细胞病理。我们还开发了新的测序应用程序来观察易位和 DNA 断裂。我们使用测序工具进行了 4C 分析和 CTCF ChIP-seq。 我们还开发了测序分析，捕获细胞中全基因组启动子的熔化，并将其应用于探索长期存在的免疫学问题。 Riveras 博士实验室的 Chao Jiang 博士正在研究一组 SLE 患者，这些患者的循环自身 IgE 水平升高，这一特征与 SLEDAI 指数定义的疾病活动度高度相关。 为了了解自身 IgE 对 SLE 发病机制的贡献，我们使用 RNA-Seq 研究了与人类高水平自身 IgE 产生相关的基因表达谱。 我们与 Leon Nesti 博士和 Youngmi Ji 博士一起进行了 RNA 测序研究，目的是阐明损伤激活如何影响间充质祖细胞 (MPC)，并确定创伤是否可能导致 MPC 的病理分化和异位骨化的形成。 我们与 NEI 的 Lai Wei 博士和 Nussenblatt 博士一起研究了表观遗传药物（尤其是 Zebularine）如何在炎症细胞因子刺激下改变视网膜色素上皮细胞的全基因组 H3K4me3 和 H3K27me3 模式。 我们与 NCI 的 Nussenzweig 博士小组合作，绘制了羟基脲药物引起的复制诱导的 DNA 损伤应激，以及 AID 依赖性细胞和 B 细胞中 DNA 修复蛋白的募集。