A. actinomycetemcomitans Cdt induces pro-inflammatory innate immune responses

描述（由申请人提供）：总的来说，免疫系统在减少和/或预防感染方面起着主要作用，特别是牙周病。我们提出先天性和适应性免疫反应的扰动有助于牙周病的发病机制和易感性。我们研究的基本假设是牙周病原体产生免疫调节蛋白，改变先天或适应性免疫，从而增强自身和/或其他机会微生物的毒力。我们的研究表明，放线菌聚合杆菌产生一种免疫毒素，即细胞致死膨胀毒素（Cdt），通过诱导细胞周期阻滞和细胞凋亡来损害人类t细胞。在这个建议中，我们专注于Cdt对构成先天免疫系统的两个关键细胞的一种新的、非致命性的作用。具体来说，我们提出Cdt通过其作为PIP3磷酸酶的能力诱导促炎反应，耗尽细胞中这种关键的信号脂质，进而阻断PI-3K信号传导，导致pAkt减少，从而激活GSK3。初步研究表明，用Cdt处理人巨噬细胞系、THP1和人巨噬细胞可导致促炎细胞因子（TNF¿和IL-1¿）的合成和释放，并增强TLR激动剂诱导的细胞因子释放。Cdt还增加肥大细胞活化后细胞因子（IL-6和IL-13）的新生合成。这些对巨噬细胞和肥大细胞的影响与PIP3耗竭、PI-3K信号通路阻断以及该通路作为促炎反应负调节因子的作用一致。我们现在计划扩展这些初步研究，以确定Cdt改变人类巨噬细胞和肥大细胞炎症介质合成和释放的程度和机制。本研究分为三个具体目的：(1)明确Cdt对细胞的促炎作用

DESCRIPTION (provided by applicant): The immune system, by and large, plays a primary role to minimize and/or prevent infection, in general, and periodontal disease, in particular. We propose that perturbation of both innate and adaptive immune responses contribute to the pathogenesis of and susceptibility to periodontal disease. The fundamental hypothesis of our studies is that periodontal pathogens produce immunomodulatory proteins that alter either innate or adaptive immunity, thereby enhancing their own virulence and/or that of other opportunistic microorganisms. Our investigations have demonstrated that Aggregatibacter actinomycetemcomitans produces an immunotoxin, the cytolethal distending toxin (Cdt), that impairs human T-cells by inducing cell cycle arrest and apoptosis. In this proposal we focus on a novel, nonlethal, effect of Cdt on two critical cells that comprise the innate immune system. Specifically, we propose the Cdt induces a pro-inflammatory response by virtue of its ability to act as a PIP3 phosphatase, deplete cells of this critical signaling lipid and in turn block PI-3K signaling leading to decreased pAkt to activation of GSK3¿. Preliminary studies indicate that treatment of the human macrophage cell line, THP1, and human macrophages with Cdt results in the synthesis and release of pro-inflammatory cytokines (TNF¿ and IL-1¿) as well as enhancement of TLR agonist-induced cytokine release. Cdt also augments de novo synthesis of cytokines (IL-6 and IL-13) following mast cell activation. These effects on both macrophages and mast cells are consistent with PIP3 depletion, blockade of the PI-3K signaling pathway and the role of this pathway as a negative regulator of the pro-inflammatory response. We now plan to extend these preliminary studies to define the extent and mechanism by which Cdt alters the synthesis and release of inflammatory mediators by human macrophages and mast cells. The study is divided into three specific aims: (1) To define the pro-inflammatory effects of Cdt on the innate immune response of macrophages and mast cells; ( 2) To determine if the Cdt-mediated pro-inflammatory effects on the innate immune system ais dependent upon PIP3 phosphatase activity and altered PI-3K/PIP3/Akt signaling; and (3) To determine the role of cellugyrin and, possibly GSK3¿, in Cdt-mediated altered glucose uptake and utilization and its relationship to the pro-inflammatory response. It is anticipated that these studies will lead to a more detailed understanding of Cdt mode of action and contribute to a greater understanding of the pathogenesis of disease caused by Cdt-producing bacteria. In addition to advancing our understanding of Cdt mode of action, these studies will advance our understanding of the mechanism(s) by which the PI-3K signaling pathway regulates TLR-mediated cytokine production. Thus these studies will identify new targets for future in vivo studies involving pharmacologic and immunologic intervention in disease caused by bacteria that produce Cdt.