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(申请人提供)：炭疽孢子吸入后，被巨噬细胞吞噬，从巨噬细胞萌发成营养状态，复制、传播并产生导致疾病临床表现的强大毒力因子。对于炭疽先天免疫应答的第一步：宿主Toll样受体(TLR)对孢子的识别及其在炭疽感染发病机制中的作用，人们知之甚少。我们将探索炭疽杆菌(BA)孢子与人和小鼠细胞上表达的TLRs的初始接触。我们假设TLR和/或FcGammaR的激活是杀死吞噬的孢子所必需的，但在没有免疫抗体的情况下，外孢子菌保护下面的孢子结构，特别是肽聚糖(PG)，使其免受TLR识别和细胞激活。具体目的I将在人胚胎肾细胞中加入萌发缺陷的BA孢子(以避免繁殖体污染)，并将其导入特定的人TLR构建体。加入完整的BA孢子、去掉外孢子体的孢子、纯化的PG和纯化的外孢子体后，将检测对IL-8和NFkappaB的诱导激活。在原代非肺泡性和肺泡性人和小鼠巨噬细胞，包括TLR2-、TLR4-和MyD88基因敲除小鼠的巨噬细胞中，将证实特定TLRs的参与。接头蛋白MyD88是大多数TLR介导的信号转导所必需的。特殊目的II将评估TLRs在FcRGamma介导的对Sterne菌株(毒素+，被膜-)BA孢子的杀灭中的潜在作用。用含有和不含外孢子体的抗血清调理Sterne BA孢子，这些抗血清对人肺泡巨噬细胞、非肺泡巨噬细胞以及野生型和TLR2-、TLR4-和MyD88KO小鼠的BA杀伤和细胞因子诱导的影响将被测定。这些后一项研究将解决杀死摄入的BA孢子是否需要TLRs和FcRGamma之间的协同作用。这些研究使用特定的人TLR结构，用siRNA和细胞通透性TLR肽抑制剂抑制原代人巨噬细胞内源性TLR蛋白的表达/功能，TLR基因敲除小鼠和一株独特的炭疽孢子将确定巨噬细胞与BA孢子的最初相互作用，并可能提供有利于宿主的细胞因子环境的智能操纵数据。