Extravasation of the Bacteria that causes Lyme Disease Using a 3D Tissue-Engineered Microvessel Model

Introduction: Lyme disease is the most widespread tick-borne disease in North America and is caused by the bacteria Borrelia burgdorferi (Bb). Roughly 10-15% of infections result in Bb dissemination to the brain which causes neuroborreliosis, common symptoms of which include headaches, facial palsy, and long term cognitive impairment. Though there are many in vivo and in vitro studies of Bb dissemination, they largely focus on quantifying transmigrated Bb at certain time points rather than assessing the active process of extravasation, which is an inherently complex and dynamic interaction. As a result, the specific biological and microenvironmental factors that govern Bb dissemination remain poorly understood. Here, we use a 3D tissue-engineered microvessel model and perfuse fluorescently labeled Bb through brain specific microvessels to model neuroborreliosis, and non-tissue-specific microvessels as a control for comparison. Using high resolution confocal microscopy, we acquire time-lapses in real-time to record the effect of wall shear stress, chronic inflammation, and glycocalyx degradation on Bb adhesion and extravasation.

Materials and

Methods: Microvessels were formed with two isogenic stem cell derived endothelial cell lines: induced brain microvascular endothelial cells (iBMECs) to model the blood-brain barrier (BBB), and induced endothelial cells (iECs) as a non-tissue-specific control. GFP-labeled Bb were perfused at a density of 5 x 106 cells mL-1, and spinning disk confocal microscopy was used to acquire time-lapse images of Bb-microvessel interactions at the equatorial plane of the microvessel over three hours. A syringe pump was used to perfuse Bb at a constant wall shear stress up to 10 dyne cm-2. Microvessels were treated with 5 ng mL-1 TNF-α for 12 hours to model chronic inflammation, or 50 mU mL-1 of heparinase III and 10 mU mL-1 of sialidase for 48 hours to degrade the glycocalyx. Bb-microvessel interactions were quantitatively assessed by measuring Bb adhesion rate, extravasation rate, frequency of successful extravasation events, and duration of adhesion and extravasation.

Results, Conclusions, and Discussions: Using a 3D tissue-engineered microvessel model and confocal microscopy, we recorded time lapse videos of Bb adhering to iECs and extravasating into the surrounding matrix (Fig. 1A), demonstrating that our system is sufficient for extravasation to occur, and that chemoattractants or additional components are not needed in the ECM to facilitate endothelial transmigration. However, Bb rarely adhere to iBMECs, suggesting that Bb are unable to cross the BBB. Bb adhesion in iEC microvessels decreased with increasing wall shear stress (Fig. 1B). This confirms that flow conditions play an important role in dissemination, and that extravasation occurs at post-capillary venules where the wall shear stress is lower compared to arterioles or capillaries. Treating iEC microvessels with TNF-α to model chronic inflammation increased Bb adhesion rate (Fig. 1C), while it had no influence on Bb-EC interactions with iBMECs. Removal of the glycocalyx significantly reduces Bb adhesion in iEC microvessels (Fig. 1D), but had no effect in iBMEC microvessels. These findings provide a further understanding of the dynamics and mechanics of Bb adhesion and extravasation during dissemination.

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