Early events during infection with anthrax

描述（由申请人提供）：吸入后，炭疽孢子被巨噬细胞吞噬，从巨噬细胞萌发成植物形态，复制、传播并产生导致该疾病临床表现的强毒力因子。对炭疽先天免疫反应的第一步知之甚少：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。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TLRs的初次接触。我们假设TLR和/或FcgammaR激活是杀死摄入的孢子所必需的，但在缺乏免疫抗体的情况下，外孢子保护了孢子结构，特别是肽聚糖（PG）免受TLR识别和细胞激活。我将添加发芽缺陷的BA孢子（以避免营养形式污染）到转染了特定人类TLR构建物的人胚胎肾细胞中。在加入完整的BA孢子、剥去外孢子、纯化的PG和纯化的外孢子后，测量IL-8和NFkappaB活化的诱导。特异性TLRs的参与将在原发性非肺泡和肺泡人类和小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。特异性目的II将评估TLRs在fcrγ介导的Sterne菌株（毒素+，胶囊-）BA孢子杀伤中的潜在作用。在有和没有外孢子的孢子中培养的抗血清将用于调节Sterne BA孢子，并在人肺泡和非肺泡巨噬细胞以及野生型和TLR2-， TLR4-和MyD88 KO小鼠中测定这些抗血清对BA杀伤和细胞因子诱导的影响。这些后一项研究将探讨是否杀死摄入的BA孢子需要tlr和FcRgammas之间的协同作用。这些研究使用特定的人类TLR结构、内源性TLR蛋白在原代人巨噬细胞中的表达/功能抑制siRNA和细胞可渗透TLR肽抑制剂、TLR敲除小鼠和一种独特的炭疽孢子菌株，将确定巨噬细胞与BA孢子的初始相互作用，并可能提供有利于宿主的细胞因子环境的智能操纵的数据。

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.