Early events during infection with anthrax

描述（由申请方提供）：吸入后，炭疽孢子被巨噬细胞吞噬，从中萌发成繁殖体，繁殖体复制、传播并产生导致疾病临床表现的强效毒力因子。对炭疽先天免疫反应的初始步骤知之甚少：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。我们将探讨B的最初接触。炭疽（BA）孢子与在人和鼠细胞上表达的TLR。我们假设TLR和/或Fc γ R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，孢子外壁保护下面的孢子结构，特别是肽聚糖（PG）免受TLR识别和细胞激活。特定目的I将向用特定人TLR构建体转染的人胚肾细胞中加入萌发缺陷的BA孢子（以避免营养体污染）。在添加完整的BA孢子、剥去孢子外壁的孢子、纯化的PG和纯化的孢子外壁后，测量IL-8和NF κ B活化的诱导。将在原代非肺泡和肺泡人和鼠巨噬细胞中证实特异性TLR的参与，包括TLR 2-、TLR 4-和MyD 88敲除小鼠的巨噬细胞。衔接蛋白MyD 88是大多数TLR介导的信号传导所必需的。特定目的II将评估TLR在FcR γ介导的Sterne菌株（毒素+，荚膜-）BA孢子杀伤中的潜在作用。将使用针对具有和不具有外孢壁的孢子产生的抗血清调理Sterne BA孢子，并将在人肺泡和非肺泡巨噬细胞以及来自野生型和TLR 2-、TLR 4-和MyD 88 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。这些后面的研究将解决杀死摄入的BA孢子是否需要TLR和FcR γ之间的协同性。这些研究使用特定的人TLR构建体、用siRNA和细胞可渗透的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敲除小鼠和炭疽孢子的独特菌株，将定义巨噬细胞与BA孢子的初始相互作用，并且可以提供使细胞因子环境的主动操纵有利于宿主的数据。

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.