Phagosomal Signals Shape Inflammatory Responses to B. Burgdorferi

描述（由申请人提供）：莱姆病（LD）是由胞外螺旋体细菌伯氏疏螺旋体（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，因此广泛认为该疾病的炎性表现是由宿主对细菌的先天和共同进化的适应性免疫应答引起的。单核细胞和巨噬细胞被认为是对螺旋体的先天免疫应答的关键细胞成分。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上的CD 14和Toll样受体（TLR）1/2相互作用的结果。我们现在有大量的证据表明，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TLR 1/2激活更强烈和更广泛。吞噬完整的Bb也诱导转录的干扰素-2（IFN-2）和I型干扰素刺激的基因（ISG），独立于TLR 2。来自研究者实验室的最新证据提供了大量证据，证明Bb在人单核细胞中引起的TLR 2非依赖性信号传导事件是MyD 88依赖性的，并通过TLR 8发生。在我们的集体研究结果的基础上，我们现在提出了一个新的模型，BB诱导的单核细胞活化，强调吞噬作用的重要性和TLR 2和TLR 8信号的合作作用。在该模型中，Bb通过尚待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传导事件，这些事件只能在细菌内化和吞噬溶酶体形成后机械地整合。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，并检查所提出的模型的机制方面，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表征在人单核细胞中吞噬Bb受体激活和TLR 1/2和TLR 8依赖的炎症信号的机制。在目标2中，我们将研究在BB感染的人类和小鼠巨噬细胞的吞噬体信号转导模型的关键要素。使用大量可用的基因敲除小鼠将使我们能够从机械上定义Bb如何被感知并触发这些细胞中的免疫反应。在目标3中，我们将使用从MyD 88信号传导途径的组分中具有已知先天性缺陷（IRAK-4和MyD 88缺陷）的儿童和成人获得的人单核细胞和巨噬细胞在体外和体内表征MyD 88对伯氏疏螺旋体的依赖性和独立性应答。

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).