Phagosomal Signals Shape Inflammatory Responses to B. Burgdorferi

描述（由申请人提供）：莱姆病（LD）是一种由细胞外螺旋体伯氏疏螺旋体（Bb）引起的蜱传多系统感染性疾病。由于Bb缺乏外毒素或已知的细菌分泌系统，人们普遍认为该疾病的炎症表现是由宿主对细菌的先天和共同进化的适应性免疫反应引起的。单核细胞和巨噬细胞被认为是对螺旋体的先天免疫反应的关键细胞成分。十多年来，bb介导的细胞活化被认为主要是由于螺旋体丰富的外膜相关脂蛋白与这些细胞表面的CD14和toll样受体（TLR） 1/2相互作用的结果。我们现在有大量证据表明，人单核细胞和小鼠巨噬细胞吞噬活螺旋体产生的炎症反应比脂蛋白介导的细胞表面TLR1/2激活更强烈，范围更广。完整Bb的吞噬也会诱导干扰素-2 （IFN-2）和I型干扰素刺激基因（ISGs）的转录，而不依赖于TLR2。最近来自研究者实验室的证据提供了大量证据，证明Bb在人单核细胞中引发的tlr2非依赖性信号事件是myd88依赖性的，并通过TLR8发生。基于我们的共同发现，我们现在提出了bb诱导单核细胞活化的新模型，该模型强调吞噬作用的重要性以及TLR2和TLR8信号的协同作用。在该模型中，Bb通过尚未表征的吞噬受体与单核细胞/巨噬细胞表面结合，随后是一系列广泛的免疫信号事件，这些事件在机制上只能在细菌内化和吞噬酶体形成后整合。吞噬体信号模型的验证是我们研究策略的核心，将显著增强我们对细菌如何触发炎症过程的理解，这些炎症过程在实际疾病条件下导致组织损伤和/或促进细菌清除。为了实现我们的目标，并检查所建议的模型的机制方面，我们制定了以下具体目标。在目的1中，我们将使用离体刺激技术来详细描述Bb吞噬引发人类单核细胞激活和TLR1/2和TLR8依赖炎症信号的机制。在Aim 2中，我们将研究bb感染的人和小鼠巨噬细胞吞噬体信号模型的关键要素。大量基因敲除小鼠的使用将使我们能够从机制上定义Bb是如何被感知并在这些细胞中触发免疫反应的。在Aim 3中，我们将在体外和体内使用从已知MyD88信号通路成分先天缺陷（IRAK-4和MyD88缺陷）的儿童和成人中获得的人类单核细胞和巨噬细胞，来表征MyD88对伯氏疏螺旋体的依赖和独立反应。

DESCRIPTION (provided by applicant): Lyme disease (LD) is a tick-borne, multi-system, infectious disorder caused by the extracellular spirochetal bacterium Borrelia burgdorferi (Bb). Since Bb lacks exotoxins or known bacterial secretory system, it is widely believed that the inflammatory manifestations of the disease result from the host's innate and co-evolving adaptive immune responses to the bacterium. Monocytes and macrophages are considered to be critical cellular elements of the innate immune response to the spirochete. For more than a decade, Bb-mediated cell activation was thought to occur chiefly as a result of the interactions of the spirochete's abundant outer membrane-associated lipoproteins with CD14 and Toll-like receptors (TLR) 1/2 on the surface of these cells. We now have extensive evidence that phagocytosis of live spirochetes by human monocytes and murine macrophages generates a more intense and far broader inflammatory response than can be attributed to lipoprotein-mediated, cell surface TLR1/2 activation. Phagocytosis of intact Bb also induced transcription of interferon-2 (IFN-2) and type I interferon-stimulated genes (ISGs), independently of TLR2. Recent evidence from the investigator's laboratory provides substantial evidence the TLR2-independent signaling events elicited by Bb in human monocytes are MyD88-dependent and occur via TLR8. On the basis of our collective findings we now propose a new model of Bb-induced monocyte activation, which emphasizes the importance of phagocytosis and the cooperative role of TLR2 and TLR8 signaling. In this model, binding of Bb to the monocyte/macrophage cell surface, through a yet to be characterized phagocytic receptor, is followed by a broad sequence of immune signaling events which mechanistically can only be integrated following internalization of the bacterium and formation of the phagolysosome. The validation of the phagosomal signaling model, the centerpiece of our research strategy, will significantly enhance our understanding for how the bacterium triggers the inflammatory processes that under actual disease conditions cause tissue damage and/or that promote bacterial clearance. To accomplish our goals and examine mechanistic aspects of the proposed model, we have formulated the following Specific aims. In Aim 1 we will use a ex vivo stimulation technique to characterize in detail the mechanisms by which phagocytosis of Bb elicits activation and TLR1/2 and TLR8 dependent inflammatory signals in human monocytes. In Aim 2, we will examine key elements of the phagosomal signaling model in Bb-infected human and murine macrophages. The use a large repertoire of available knockout mice will allow us to define mechanistically how Bb is sensed and triggers immune responses in these cells. In Aim 3 we will characterize MyD88 dependent and independent responses to Borrelia burgdorferi both in vitro and in vivo using human monocytes and macrophages obtained from children and adults with known congenital deficiencies in components of the MyD88 signaling pathway (IRAK-4 and MyD88 deficient).