Intravital assessment of Borrelia burgdorferi immune clearance in skin

通过扁虱叮咬传播伯氏疏螺旋体会导致莱姆病的发展，这是一种 在北美领先的媒介传播疾病。如果没有得到适当的诊断和抗生素治疗， 可以在无数的组织中产生持久的和衰弱的疾病，导致重大的个人和公众 医疗费用。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，最终在全身多个组织中建立持续感染 主持人。BB逃避免疫清除的机制在很大程度上尚不清楚。体外分析表明 表明它们缺乏准确识别寄主对这种独特的专性寄生虫的反应的能力， 强调了更好的活体模型的必要性。我们小组最近开发了一种新型活体显微镜 允许直接评估具有不同免疫细胞群的荧光BB的技术 在活体小鼠的完整皮肤内并实时地，第一次为我们提供了准确地 评估自然宿主环境中的这些关键相互作用。初步数据表明，这些技术 能准确描绘皮肤组织的深度和距离，识别不同的细胞外基质分子和 它们与BB共同定位，并区分/测量不同的不同运动性和交互特征 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，以及已建立的感染模型，以实现更多 准确评估BB与宿主皮肤内重要免疫细胞群和介质的相互作用 组织，从而更准确地识别对逃避免疫至关重要的毒力机制 清除并导致莱姆病。我们的初步研究描绘了Bb的种群动力学。 皮肤覆盖&>2年，以及确认BB-诱导的IL-10对整体免疫的中介抑制作用 功能和BB清除。基于这些发现，荧光BB将被用于提供实时 确定不同免疫机制在控制BB数中的相对重要性的反馈 在皮肤内，不同的荧光标记的BB特异性抗体池将被用来描述抗体如何相互作用 在LD急性期使用BB，以及利用不同的IL-10缺陷小鼠模型来确定 IL-10抑制小鼠组织对BB的有效清除。这项建议的具体目标是： 目的1.阐明BB诱导的IL-10抑制先天免疫应答的机制。 皮肤组织内的BB感染。 目的2.阐明抑制抗体介导的Bb清除的机制。 这些研究应该提供第一个准确的描述BB免疫细胞相互作用发生在 自然感染和在皮肤组织中的传播，并可能确定潜在莱姆疗法的靶点。

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 have been shown to be lacking in their ability to accurately identify host responses to this unique obligate parasite, highlighting the need for better in vivo models. Our group has recently developed novel intravital 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 first 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-localization 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. Our preliminary studies have delineated the population kinetics of Bb in skin over >2 years, as well as identified Bb-elicited IL-10 as mediating suppressive effects on overall immune functions and Bb clearance. Based on these findings, fluorescent Bb will be used to provide real-time feedback to determine the relative importance of different immune mechanisms in controlling Bb numbers within skin, different pools of fluorescently-labeled Bb-specific Ab will be used to delineate how Abs interact with Bb during the acute phases of LD, and different IL-10-deficient mouse models utilized to determine how IL-10 suppresses efficient clearance of Bb from mouse tissues. The specific aims for this proposal are: Aim 1. Delineate the mechanisms for Bb-elicited IL-10-mediated suppression of innate immune responses to Bb infection within skin tissues. Aim 2. Delineate the mechanisms that inhibit Ab-mediated clearance of Bb. 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.