The Pathogenesis of Kawasaki Disease

描述(由申请人提供)：我的实验室的一个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，KD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)的形成，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，但常规方法尚未得出病因。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渗入CAA和其他组织，表明对具有粘膜入口的细胞内病原体(如病毒)的抗原驱动反应。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。在之前的资助期间，我们在体外制备了寡克隆性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，而在对照的支气管上皮和急性炎症KD组织中的巨噬细胞亚群中未检测到抗原。在急性KD纤毛支气管壁上皮，抗原定位于独特的核周胞浆内球体。HE染色、三色染色和核酸染色光镜下可见纤毛支气管壁上皮内的球体为胞质包涵体(CIB)，4/4急性期KD患者纤毛上皮细胞的透射电子显微镜显示为均匀的电子致密的CIB。CIB与蛋白质和相关核酸的聚集体最一致，很可能来自KD病原体。因此，从急性KD动脉壁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。这些数据支持这样的假设，即KD毒剂通过呼吸道进入，感染支气管上皮，并通过巨噬细胞进入包括冠状动脉在内的靶组织，与抗原特异性的IgA浆细胞和其他炎症细胞进入组织以对抗病原体，导致组织损伤。在急性KD肺中，CIB的存在与微生物、可能是病毒、蛋白质和核酸的聚集体一致，提供了一个结构和分子足迹，可能导致KD病原体的鉴定。这项建议的具体目的是：1)确定急性KD形成CIB的细胞和组织类型，并通过电子显微镜寻找这些细胞/组织中的微生物元素；2)确定KD CIB的核酸/蛋白质含量；以及3)基于我们先前对在急性KD形成CAA中起作用的巨噬细胞分泌因子的研究，确定肿瘤坏死因子(TNF)及其受体超家族成员的调节失调是否有助于KD血管病变。这些研究的目的是确定CIB是否来自KD病原体，并确定治疗急性KD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源，但病因尚不清楚。公共卫生相关性：这些研究的目标是确定KD患者组织中的细胞质包涵体是否来源于KD病原体，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.