Early events during infection with anthrax

炭疽感染期间的早期事件

• 批准号: 7028848
• 负责人: Alan S. Cross
• 金额: $ 18.13万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028848
• 关键词: anthrax; antibody receptor; antigen antibody reaction; bacteria infection mechanism; biological signal transduction; bioterrorism /chemical warfare; clinical research; host organism interaction; human subject; immunologic memory; laboratory mouse; laboratory rabbit; macrophage; nuclear factor kappa beta; pathologic process; polymerase chain reaction; receptor expression; small interfering RNA; spores; toll like receptor; transfection; western blottings; wild animals

DESCRIPTION (provided by applicant): Following inhalation, anthrax spores are phagocytosed by macrophages from which they germinate into a vegetative form that replicates, disseminates and produces potent virulence factors responsible for the clinical manifestations of the disease. Relatively little is known about an initial step in the innate immune response to anthrax: recognition of spores by host Toll-like receptors (TLR), and their role in the pathogenesis of anthrax infection. We will explore the initial contact of B. anthracis (BA) spores with TLRs expressed on human and murine cells. We hypothesize that TLR and/or FcgammaR activation is required for killing of the ingested spore, but in the absence of immune antibody, the exosporium shields underlying spore structures, particularly the peptidoglycan (PG) from TLR recognition and cell activation. Specific Aim I will add germination-deficient BA spore (to avoid vegetative form contamination) to human embryonal kidney cells transfected with specific human TLR constructs. Induction of IL-8 and NFkappaB activation will be measured following addition of intact BA spores, spores stripped of exosporium, purified PG and purified exosporium. Involvement of specific TLRs will be confirmed in primary non-alveolar and alveolar human and murine macrophages, including those of TLR2-, TLR4- and MyD88 knockout mice. The adaptor protein MyD88 is required for most TLR-mediated signaling. Specific Aim II will assess the potential role of TLRs in FcRgamma-mediated killing of Sterne strain (toxin+, capsule -) BA spores. Antisera raised to spores with and without exosporium will be used to opsonize Sterne BA spores, and the effect of these antisera on BA killing and cytokine induction will be determined in human alveolar and non-alveolar macrophages as well as those from wild type and TLR2-, TLR4- and MyD88 KO mice. These latter studies will address whether killing of ingested BA spores requires cooperativity between TLRs and FcRgammas. These studies using specific human TLR constructs, inhibition of expression/function of endogenous TLR proteins in primary human macrophages with siRNA and cell permeable TLR peptide inhibitors, TLR knockout mice and a unique strain of anthrax spore will define the initial interaction of macrophages with BA spores, and may provide data enabling intellingent manipulation of the cytokine milieu in favor of the host.

描述（由申请人提供）：吸入后，炭疽孢子被巨噬细胞吞噬，从中发芽成营养体形式，复制、传播并产生导致疾病临床表现的强效毒力因子。关于炭疽先天免疫反应的第一步：宿主 Toll 样受体 (TLR) 识别孢子，以及它们在炭疽感染发病机制中的作用，人们知之甚少。我们将探索炭疽芽孢杆菌 (BA) 孢子与人类和小鼠细胞上表达的 TLR 的初始接触。我们假设 TLR 和/或 FcgammaR 激活是杀死摄入孢子所必需的，但在没有免疫抗体的情况下，孢子外膜会屏蔽潜在的孢子结构，特别是肽聚糖 (PG)，使其免受 TLR 识别和细胞激活。具体目标 我将向用特定人 TLR 构建体转染的人胚胎肾细胞中添加萌发缺陷的 BA 孢子（以避免营养形式污染）。在添加完整的BA孢子、剥离外孢子的孢子、纯化的PG和纯化的外孢子后，将测量IL-8和NFκB活化的诱导。特定 TLR 的参与将在原代非肺泡和肺泡人和鼠巨噬细胞中得到证实，包括 TLR2、TLR4 和 MyD88 敲除小鼠的巨噬细胞。大多数 TLR 介导的信号转导都需要接头蛋白 MyD88。具体目标 II 将评估 TLR 在 FcRgamma 介导的杀死 Sterne 菌株（毒素+，荚膜 -）BA 孢子中的潜在作用。针对有和没有外孢子的孢子产生的抗血清将用于调理 Sterne BA 孢子，并且将在人肺泡和非肺泡巨噬细胞以及来自野生型和 TLR2-、TLR4-和 MyD88 KO 小鼠的巨噬细胞中确定这些抗血清对 BA 杀伤和细胞因子诱导的影响。后面的这些研究将解决杀死摄入的 BA 孢子是否需要 TLR 和 FcRgamma 之间的协同作用。这些研究使用特定的人 TLR 构建体、用 siRNA 和细胞渗透性 TLR 肽抑制剂抑制原代人巨噬细胞中内源性 TLR 蛋白的表达/功能、TLR 敲除小鼠和独特的炭疽孢子菌株，将定义巨噬细胞与 BA 孢子的初始相互作用，并可能提供能够智能操纵有利于宿主的细胞因子环境的数据。