Immune Dysregulation in Pediatric SLE Pathogenesis

项目总结 系统性红斑狼疮(SLE)是一种以免疫为特征的多器官风湿病 调节失调和异质性的病程，儿科患者的疾病严重程度更高。 常规的临床和实验室参数对于检测正在进行的疾病不够敏感或特异 疾病活动或对特定免疫调节剂的反应，强调有必要更好地描述 潜在的免疫变化，导致不可预测的疾病活动爆发。知识的匮乏 关于导致SLE疾病的确切免疫细胞和分子事件构成了一个重大障碍 努力开发准确预测疾病预后的生物标记物和选择性治疗剂。我们的长- 学期目标是确定免疫细胞和分子介体，为治疗提供靶点。 干预。本研究的目的是阐明血清循环因子的作用机制。 包括外切体在儿童系统性红斑狼疮发病机制中调节免疫细胞和细胞因子的紊乱。这个 这项拟议研究的基本原理是，了解这些机制可以识别新的疾病 生物标志物，允许准确的预后和提供选择性的治疗靶点。 为了实现这一目标，谢博士将使用高维质量细胞仪平台，该平台提供单细胞 分析40多个参数，用稀土金属同位素代替荧光团作为结合的标记 抗体。在一项初步研究中，谢博士建立了一种定量的、可重复的质量细胞分析方法， 允许精确监测表型(表面标志物)和功能性(细胞因子/趋化因子)免疫 儿童系统性红斑狼疮患者外周血中可检测到的紊乱。这项研究的数据构成了 谢博士的中心假设，即儿童SLE患者共享独特的多参数单核细胞 细胞因子标志，驱动疾病活动，并由血清因子(包括外切体)传播 通过激活JAK/STAT信号通路。在强劲的初步数据的指导下，这一假说将 通过追求三个具体目标进行测试：1)确定细胞因子签名(和其他 免疫变化)与SLE疾病活动性，通过结合质量细胞仪免疫图谱数据和临床 前瞻性纵向儿童系统性红斑狼疮患者队列参数：2)阐明免疫调节作用 通过评估疾病血清外切体诱导SLE血清分离外切体的能力来研究其特性 细胞因子特征，并鉴定这种活性所必需的外体蛋白质组分；以及3) 通过以下方法确定ruxolitinib(JAK1/2抑制剂)抑制细胞因子信号表达的能力 鲁索利替尼体外免疫抑制作用(包括靶外效应)的评价 在儿童系统性红斑狼疮发病机制中观察到的紊乱。 谢博士是科罗拉多大学丹佛分校/科罗拉多州儿童医院的助理教授 免疫学和微生物学，以及儿科。作为一名临床研究员，她接受了加里博士的培训 诺兰的实验室(斯坦福大学)，在将质量细胞术应用于单细胞方面被公认为世界级 在那里，她获得了这项技术的专业知识。谢博士组建了一个指导团队 由一个在基础科学免疫学方面拥有研究专长的多学科科学家小组组成， 尤其是自身免疫中免疫信号的改变；临床研究设计和实施；外显子 生物学；和生物统计学。她的主要导师约翰·坎比尔博士和共同导师V·迈克尔·霍勒斯博士- 与知名科学家合作，专注于自身免疫性疾病和免疫系统。他们的 她的科学咨询委员会的专业知识将补充她与受训者的广泛经验， 共同导师、国际知名的外显体生物学专家迈克尔·格兰纳博士， 顾问/合作者Jennifer Soep博士，儿科风湿病专家，专注于SLE的临床研究，以及 Debashim Ghosh博士，生物统计学主席，在高通量数据分析方面拥有专业知识。总而言之， 该团队将提供出色的培训环境，填补她工具箱中的关键空白，并 增强她研究儿童系统性红斑狼疮患者免疫紊乱的能力的知识库。谢医生已经 创建了一个职业发展计划，为她成功过渡到独立做好准备， 结合教学和辅导培训：1)基础科学免疫学；2)临床研究设计和 实现；3)外显体生物学；以及4)生物统计学和计算生物学。 申请人认为，拟议的方法是创新的，因为它通过以下方式改变了现状 阐明系统性红斑狼疮炎症紊乱的具体机制(例如，血清外切体介导的 免疫调节)以及在多重免疫的背景下描述这些机制的特征 单细胞水平的细胞表型、细胞内细胞因子网络和缓解/复发疾病活动性 决议。这项拟议的研究具有重要意义，因为它可能会确定候选疾病生物标记物 系统性红斑狼疮的预后和治疗目的，并可能描绘出源于 血清外切体及其蛋白质组分。所获得的知识将具有广泛的可翻译性 通过应用该系统免疫学方法对其他系统性风湿病的重要性 精神错乱。

PROJECT SUMMARY Systemic lupus erythematosus (SLE) is a multi-organ rheumatologic disease characterized by immune dysregulation and a heterogeneous disease course, with heightened disease severity in pediatric patients. Conventional clinical and laboratory parameters are not sufficiently sensitive or specific for detecting ongoing disease activity or response to specific immunomodulators, underscoring a need to better delineate the underlying immune changes that drive unpredictable flare-ups of disease activity. The lack of knowledge regarding the precise immune cellular and molecular events leading to SLE disease poses a significant hurdle in the effort to develop accurate prognostic disease biomarkers and selective therapeutic agents. Our long- term goal is to identify immune cellular and molecular mediators that could provide targets for therapeutic intervention. The objective of this proposal is to elucidate the mechanisms by which serum-circulating factors including exosomes modulate immune cellular and cytokine derangements in pediatric SLE pathogenesis. The rationale for the proposed research is that understanding these mechanisms could identify novel disease biomarkers that permit accurate prognosis and provide selective therapeutic targets. To achieve this goal, Dr. Hsieh will use a high-dimensional mass cytometry platform, which offers single-cell analysis of over 40 parameters, utilizing rare earth metal isotopes instead of fluorophores as tags bound to antibodies. In a pilot study, Dr. Hsieh established a quantitative and reproducible mass cytometry assay that allows precise monitoring of phenotypic (surface markers) and functional (cytokines/chemokines) immune derangements detectable in peripheral blood of pediatric SLE patients. The data from this study form the basis of Dr. Hsieh's central hypothesis that pediatric SLE patients share a unique multi-parametric monocyte cytokine signature, which drives disease activity and is propagated by serum factors (including exosomes) through activation of the JAK/STAT signaling pathway. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the association of the cytokine signature (and other immune changes) with SLE disease activity, by integrating mass cytometry immune profile data and clinical parameters from a prospective longitudinal pediatric SLE patient cohort; 2) Elucidate immunomodulatory properties of SLE serum-isolated exosomes, by evaluating the ability of diseased serum exosomes to induce the cytokine signature, and identifying exosome proteomic components essential for such activity; and 3) Determine the capacity of ruxolitinib (JAK1/2 inhibitor) to abrogate expression of the cytokine signature, by evaluating ex vivo immunosuppressive effects (including off-target effects) of ruxolitinib on immune derangements observed in pediatric SLE pathogenesis. Dr. Hsieh is an Assistant Professor at the University of Colorado Denver/Children's Hospital Colorado in the Departments of Immunology and Microbiology, and Pediatrics. As a clinical fellow, she trained in Dr. Garry Nolan's laboratory (Stanford), acknowledged as world-class in the application of mass cytometry to single-cell analysis, where she acquired expertise in this technology. Dr. Hsieh has assembled a mentoring team composed of a multidisciplinary group of scientists with research expertise in basic science immunology, particularly immune signaling alterations in autoimmunity; clinical study design and implementation; exosome biology; and biostatistics. Her primary mentor Dr. John Cambier and co-mentor Dr. V. Michael Holers are well- established scientists with collaborations focused on autoimmune disorders and the immune system. Their extensive experience with trainees will be complemented by the expertise of her scientific advisory committee, co-mentor Dr. Michael Graner, internationally known expert in the field of exosome biology, advisors/collaborators Dr. Jennifer Soep, pediatric rheumatologist with a clinical research focus on SLE, and Dr. Debashis Ghosh, Chairman of Biostatistics with expertise on high-throughput data analysis. Collectively, this team will provide an outstanding training environment that will fill critical gaps in her toolbox and knowledge base to enhance her ability to study immune derangements in pediatric SLE patients. Dr. Hsieh has created a career development plan that will prepare her for successful transition to independence, incorporating didactic and mentored training in: 1) basic science immunology; 2) clinical study design and implementation; 3) exosome biology; and 4) biostatistics and computational biology. The proposed approach is innovative, in the applicant's opinion, because it departs from the status quo by elucidating specific mechanisms underlying inflammatory perturbations in SLE (e.g., serum exosome-mediated immune modulation) and by characterizing these mechanisms as they occur in the context of multiple immune cell phenotypes, intracellular cytokine networks, and remitting/relapsing disease activity, with single-cell level resolution. The proposed research is significant because it will likely identify candidate disease biomarkers for prognostic and therapeutic purposes in SLE, and will likely delineate novel therapeutic targets derived from serum exosomes and their proteomic components. The knowledge gained will have broad translational importance through application of this systems immunology approach to other systemic rheumatologic disorders.