The Pathogenesis of Kawasaki Disease

描述（由申请人提供）：我的实验室的主要儿科研究重点和长期目标是确定川崎病（KD）的病因和发病机制，川崎病是发达国家儿童获得性心脏病的主要原因。KD可导致冠状动脉动脉瘤（CAA）形成，并导致心肌梗死和/或猝死。临床和流行病学数据与KD的感染原因一致，但传统方法尚未得出病因。我们发现，在急性KD中，寡克隆IgA浆细胞、CD8 T淋巴细胞和巨噬细胞浸润CAA和其他组织，表明对细胞内病原体（如病毒）的抗原驱动反应，具有粘膜入口。我们发现CAA中的巨噬细胞分泌基质金属蛋白酶和其他可能导致组织损伤的分泌产物。在之前的资助期内，我们在体外制备了寡克隆KD抗体，并将其作为鉴定其靶抗原的工具。免疫组织化学显示，KD合成抗体在急性KD中检测到抗原，但在急性KD炎症组织中不能控制支气管上皮和巨噬细胞亚群。在急性KD纤毛支气管上皮中，抗原定位于独特的核周胞浆内球体。光镜下H&E、三色及核酸染色显示纤毛支气管上皮内球形细胞为细胞质包涵体（CIB），透射电镜显示4/4急性KD患者纤毛支气管上皮内呈均匀的电子致密包涵体，与蛋白及相关核酸聚集最一致，可能来源于KD病原。因此，从浸润急性KD动脉壁的浆细胞中普遍存在的IgA序列衍生的合成抗体与急性KD纤毛支气管上皮中的CIB结合，证明了CIB在KD发病机制中的可能重要性。这些数据支持了KD药物通过呼吸道进入，感染支气管上皮，并通过巨噬细胞传播到包括冠状动脉在内的目标组织，抗原特异性IgA浆细胞和其他炎症细胞进入组织对抗病原体并导致组织损伤的假设。在急性KD肺中，与微生物、可能的病毒、蛋白质和核酸聚集一致的CIB的存在提供了结构和分子足迹，可能导致KD病原体的鉴定。本提案的具体目的是：1)鉴定急性KD中CIB形成的细胞和组织类型，并通过TEM搜索这些细胞/组织中的微生物元素；2)鉴定KD CIB的核酸/蛋白含量；3)基于我们之前对急性KD中巨噬细胞分泌因子在CAA形成中发挥作用的研究，确定肿瘤坏死因子(TNF)-和TNF受体超家族成员的失调是否有助于KD血管病变。这些研究的目的是确定CIB是否源于KD病原，并确定急性KD治疗的新靶点。KD是发达国家儿童获得性心脏病的主要原因，可导致心脏病发作和猝死。临床和流行病学特征表明是一种常见的感染因子，但病因尚不清楚。公共卫生相关性：这些研究的目的是确定KD患者组织中的细胞质包涵体是否来自KD病原，并确定KD治疗的新靶点。

DESCRIPTION (provided by applicant): A major pediatric research priority and the long-term goal of my laboratory is the identification of the etiology and pathogenesis of Kawasaki Disease (KD), the leading cause of acquired heart disease in children in developed nations. KD can result in coronary artery aneurysm (CAA) formation with resultant myocardial infarction and/or sudden death. Clinical and epidemiologic data are consistent with an infectious cause of KD, but conventional methods have not yielded the etiologic agent. We discovered that oligoclonal IgA plasma cells, CD8 T lymphocytes, and macrophages infiltrate CAA and other tissues in acute KD, indicating an antigen-driven response to an intracellular pathogen, such as a virus, with a mucosal portal of entry. We found that macrophages in CAA secrete matrix metalloproteinases and other secreted products that may result in tissue damage. In the prior funding period, we made oligoclonal KD antibodies in vitro and used them as tools to identify their target antigens. Immunohistochemistry revealed that KD synthetic antibodies detected antigen in acute KD but not control bronchial epithelium and in a subset of macrophages in acute inflamed KD tissues. In acute KD ciliated bronchial epithelium, antigen localized to distinctive perinuclear intracytoplasmic spheroids. Light microscopy using H&E, trichrome and nucleic acid stains revealed that the spheroids in ciliated bronchial epithelium were cytoplasmic inclusion bodies (CIB), and transmission electron microscopy (TEM) of these cells in 4/4 acute KD patients showed homogeneous electron-dense CIB. The CIB were most consistent with aggregates of protein and associated nucleic acid and likely derive from the KD etiologic agent. Thus, synthetic antibodies derived from IgA sequences prevalent in plasma cells infiltrating acute KD arterial wall bind to CIB in acute KD ciliated bronchial epithelium, attesting to the likely importance of the CIB in KD pathogenesis. These data support the hypothesis that the KD agent enters via the respiratory tract, infects bronchial epithelium, and travels in macrophages to targeted tissues including coronary arteries, with antigen- specific IgA plasma cells and other inflammatory cells entering the tissues to combat the pathogen and resulting in tissue damage. The presence of CIB consistent with aggregates of microbial, likely viral, proteins and nucleic acids in acute KD lung provides a structural and molecular footprint that could lead to identification of the KD etiologic agent. The specific aims of this proposal are to 1) Identify the cell and tissue types in which CIB form in acute KD, and search these cells/tissues for microbial elements by TEM; 2) identify the nucleic acid/protein contents of KD CIB; and 3), building upon our prior studies of macrophage-secreted factors that play a role in CAA formation in acute KD, determine whether dysregulation of members of the tumor necrosis factor (TNF)- and TNF-receptor superfamily contribute to KD vasculopathy. The goals of these studies are to determine whether CIB derive from the KD etiologic agent and to identify new targets for therapy of acute KD.KD is the leading cause of acquired heart disease in children in developed nations and can result in heart attacks and sudden death. Clinical and epidemiologic features point to a common infectious agent, but the cause remains unknown. PUBLIC HEALTH RELEVANCE: The goal of these studies is to determine whether cytoplasmic inclusion bodies in KD patient tissues derive from the KD etiologic agent, and to identify new targets for therapy of KD.