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.