Intravital assessment of Borrelia burgdorferi-immune cell interactions in skin

描述(申请人提供)：伯氏疏螺旋体(BB)通过扁虱叮咬传播会导致莱姆病的发展，莱姆病是北美主要的媒介传播疾病。如果没有用抗生素进行适当的诊断和治疗，BB可能会在无数组织中产生持久和衰弱的疾病，导致巨大的个人和公共卫生成本。在传播到宿主后，细菌在皮肤组织中局部停留至少12-48小时 然后通过皮肤传播，最终在整个宿主的多个组织中建立持续感染。BB逃避免疫清除的机制在很大程度上尚不清楚。使用不同免疫细胞群的体外分析显示，BB感染的皮肤组织对BB有有效的摄取/杀死作用，然而这种螺旋体在体内具有很高的传染性，很容易逃避免疫清除，这表明传统的体外分析无法准确评估这些专性寄生虫与皮肤中免疫细胞的关键相互作用。我们的团队最近开发了新的显微镜技术，允许直接在活着的小鼠完整的皮肤内直接和实时地评估不同免疫细胞群的荧光BB，首次为我们提供了在自然宿主环境中准确评估这些关键相互作用的工具。初步数据表明，这些技术可以准确地描绘皮肤组织的深度和距离，识别不同的细胞外基质分子及其与BB的共定位，并区分/测量两者不同的不同运动和相互作用特征 BB和关键的先天免疫细胞类型，证实我们的技术能够执行这些分析。总体目标是使用这些活体内技术，结合已建立的感染模型，更准确地评估BB与宿主皮肤组织中重要的免疫细胞群和介质的相互作用，从而更准确地识别对逃避免疫清除和导致莱姆病至关重要的毒力机制。在这些研究中，荧光BB将被用来描述它们在皮肤中的传播特征，并使用荧光敲入鼠系来区分几种关键的先天性免疫细胞类型如何对皮肤中的BB感染做出反应。这项申请的具体目标是：目标1.描述 皮肤组织中BB运动性和趋化性在逃避免疫细胞清除中的重要性。目的2.阐明BB诱导的IL-10是否与皮肤组织对BB感染的天然免疫反应短促而低效有关。目的3.确定BB特异性抗体在感染早期和晚期对皮肤BB清除的影响。这些研究应该首次准确地描述在皮肤组织中自然感染和传播期间发生的BB免疫细胞相互作用，并可能确定潜在的莱姆疗法的靶点。

DESCRIPTION (provided by applicant): Transmission of Borrelia burgdorferi (Bb) via tick-bite leads to the development of Lyme disease, which is the leading vector-borne disease in North America. If not diagnosed and treated appropriately with antibiotics, Bb can produce lasting and debilitating disease in myriad tissues, resulting in significant personal and public health costs. After transmission into a host, the bacteria remain locally in the skin tissues for at least 12-48h before disseminating via the skin to eventually establish persistent infection in multiple tissues throughout the host. The mechanisms by which Bb evades immune clearance are largely unknown. In vitro analyses using the different immune cell populations that reside within and immigrate into Bb-infected skin tissues display efficient uptake/killing of Bb, however this spirochete is highly infectious in vivo and readily evades immune clearance, indicating that traditional in vitro analyses are unable to accurately assess critical interactions of these obligae parasites with immune cells in skin. Our group has recently developed novel microscopy techniques that allow for the direct assessment of fluorescent Bb with different immune cell populations directly within the intact skin of living mice and in real-time, providing us for the frst time with the tools to accurately assess these critical interactions in the natural host environment. Preliminary data indicates these techniques can accurately delineate depth and distance in skin tissues, identify different extracellular matrix molecules and their co-localizatin with Bb, and distinguish/measure different diverse motility and interactive characteristics of both Bb and key innate immune cell types, confirming that our techniques are able to perform these analyses. The overall goal is to use these intravital techniques, together with established infection models, to more accurately assess Bb interactions with important immune cell populations and mediators within host skin tissues, and thus more accurately identify virulence mechanisms that are critical for escaping immune clearance and causing Lyme disease. For these studies, fluorescent Bb will be used to describe their dissemination characteristics in skin and use fluorescent knock-in mouse lines to distinguish how several key innate immune cell types respond to Bb infection in skin. The specific aims for this application are: Aim 1. Delineate the importance of Bb motility and chemotaxis properties in evading clearance by immune cells in skin tissues. Aim 2. Delineate whether Bb-elicited IL-10 is responsible for the abbreviated and inefficient innate immune response to Bb infection within skin tissues. Aim 3. Determine the effects of Bb-specific Abs on mediating Bb clearance in skin during early and late stages of infection. These studies should provide the first accurate description of the Bb-immune cell interactions that occur during natural infection and dissemination in skin tissues, and may identify targets for potential Lyme therapies.