Phagosomal Signals Shape Inflammatory Responses to B. Burgdorferi

描述(申请人提供)：莱姆病(LD)是一种由壁虱传播的多系统传染病，由细胞外伯氏疏螺旋体细菌(BB)引起。由于BB缺乏外毒素或已知的细菌分泌系统，人们普遍认为该病的炎症表现是宿主对细菌的先天和共同进化的适应性免疫反应造成的。单核细胞和巨噬细胞被认为是螺旋体先天免疫反应的关键细胞成分。十多年来，BB介导的细胞激活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。我们现在有广泛的证据表明，与脂蛋白介导的细胞表面TLR1/2激活相比，人类单核细胞和小鼠巨噬细胞吞噬活的螺旋体产生了更强烈和更广泛的炎症反应。完整BB的吞噬作用也诱导了干扰素-2和I型干扰素刺激基因(ISGs)的转录，而不依赖于TLR2。来自研究人员实验室的最新证据提供了大量证据，证明BB在人单核细胞中诱导的TLR2非依赖性信号事件是MyD88依赖的，并通过TLR8发生。在我们集体发现的基础上，我们现在提出了一个新的BB诱导单核细胞激活的模型，该模型强调了吞噬作用的重要性以及TLR2和TLR8信号的协同作用。在这个模型中，BB通过一个尚未确定的吞噬细胞受体与单核/巨噬细胞表面结合，随后是一系列广泛的免疫信号事件，从机械上讲，这些事件只有在细菌内化和吞噬溶酶体形成后才能整合。吞噬体信号模型的验证是我们研究策略的核心，它将大大提高我们对细菌如何触发炎症过程的理解，这些过程在实际疾病条件下会导致组织损伤和/或促进细菌清除。为了实现我们的目标并审查拟议模型的机械方面，我们制定了以下具体目标。在目标1中，我们将使用体外刺激技术来详细描述吞噬BB引起人单核细胞活化以及TLR1/2和TLR8依赖的炎症信号的机制。在目标2中，我们将在BB感染的人和小鼠巨噬细胞中检测吞噬体信号模型的关键要素。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是如何被感知的，并在这些细胞中触发免疫反应。在目标3中，我们将使用从已知MyD88信号通路组件先天缺陷(IRAK-4和MyD88缺失)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MyD88对伯氏疏螺旋体的依赖和独立反应。 与公共卫生相关：莱姆病(LD)是由伯氏疏螺旋体细菌(BB)引起的一种由壁虱传播的传染病，该疾病在流行地区持续增加，并在地理上传播，与其主要媒介--复合硬蜱的分布和白尾鹿种群的爆炸性增长平行。为了研究对LD螺旋体的炎症反应，研究人员将使用一个强大的刺激模型，该模型允许非常完整地描述细菌如何激活人类免疫细胞，并在平行实验中利用现有的大量基因敲除小鼠来研究这些反应。使用这种翻译-实验相结合的方法，在这项应用中，研究人员将验证提出的螺旋体识别新模型的关键要素，其中吞噬小体是识别不同支链配体的中央平台，涉及TLR2和TLR8在促炎和抗炎细胞因子反应中的合作相互作用，以及TLR8在IRF-7介导的干扰素-2诱导中的合作作用。

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). PUBLIC HEALTH RELEVANCE: Lyme disease (LD) is a tick-borne infectious disorder caused by the spirochetal bacteria Borrelia burgdorferi (Bb), which has continued to increase in endemic areas and has spread geographically, paralleling the distribution of its primary vector, Ixodes ricinus complex, and the explosive growth in the white-tailed deer population. To study the inflammatory responses to the LD spirochete, the investigator will use a powerful stimulation model that allows a very complete characterization for how the bacterium activates human immune cells and in parallel experiments takes advantage of the large repertoire of available knockout mice to study these responses. Using this combined translational-experimental approach, in this application the investigator will validate key elements of a proposed new model of spirochetal recognition, where the phagosome is a central platform for recognition of diverse borrelial ligands and which involves a cooperative interaction between TLR2 and TLR8 in pro- and anti-inflammatory cytokine responses, and TLR8 in IRF-7 mediated induction of IFN-2.