A. actinomycetemcomitans Cdt induces pro-inflammatory innate immune responses

说明(申请人提供)：总的来说，免疫系统在减少和/或预防感染，特别是牙周病方面发挥着主要作用。我们认为，先天免疫反应和获得性免疫反应的扰动都有助于牙周病的发病和易感性。我们研究的基本假设是，牙周病原体产生免疫调节蛋白，改变天然免疫或获得性免疫，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，伴生放线杆菌产生一种免疫毒素，即细胞致死性膨胀毒素(CDT)，它通过诱导细胞周期停滞和凋亡来损害人类T细胞。在这项建议中，我们关注CDT对组成先天免疫系统的两个关键细胞的一种新的、非致命性的影响。具体地说，我们认为CDT通过其作为PIP3磷酸酶的能力来诱导促炎反应，耗尽这种关键信号脂质的细胞，进而阻断PI-3K信号导致PACT降低到GSK3β的激活。初步研究表明，CDT可促进人巨噬细胞系THP1和人巨噬细胞合成和释放促炎细胞因子(TNF和IL-1)，并增强TLR激动剂诱导的细胞因子释放。CDT还可促进肥大细胞激活后细胞因子(IL-6和IL-13)的从头合成。这些对巨噬细胞和肥大细胞的影响与PIP3的耗竭、PI-3K信号通路的阻断以及该通路作为促炎反应的负调节因子的作用是一致的。我们现在计划扩展这些初步研究，以确定CDT改变人巨噬细胞和肥大细胞合成和释放炎症介质的程度和机制。本研究分为三个具体目的：(1)明确CDT的促炎作用 目的：(1)研究CDT对巨噬细胞和肥大细胞的先天免疫反应；(2)确定CDT对先天免疫系统的促炎作用是否依赖于PIP3磷酸酶活性和PI-3K/PIP3/Akt信号的改变；(3)确定细胞甘蛋白和GSK3在CDT介导的葡萄糖摄取和利用改变中的作用及其与促炎反应的关系。预计这些研究将有助于更详细地了解CDT的作用模式，并有助于更好地了解由CDT产生细菌引起的疾病的发病机制。这些研究不仅促进了我们对CDT作用模式的理解，还将促进我们对PI-3K信号通路调节TLR介导的细胞因子产生的机制(S)的理解。因此，这些研究将为未来的体内研究确定新的靶点，涉及对产生CDT的细菌引起的疾病进行药理学和免疫学干预。

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.