Date Published: December 18, 2015
Publisher: Public Library of Science
Author(s): Devender Kumar, Laura C. Ristow, Meiqing Shi, Priyanka Mukherjee, Jennifer A. Caine, Woo-Yong Lee, Paul Kubes, Jenifer Coburn, George Chaconas, D. Scott Samuels.
Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration.
Lyme disease is a spirochetal illness caused by various members of the genus Borrelia, and the most prevalent vector-borne illness in North America and Europe [1–5]. The disease is transmitted to humans during the feeding of infected hard-shelled ticks that have acquired the spirochetes during an earlier blood meal on infected reservoir animals. Once inoculation of the skin has occurred, the highly motile Borrelia species multiply and migrate in the skin, often resulting in an erythema migrans lesion or “bulls-eye rash”. As the disease progresses (see Fig 1), spirochetes invade the vasculature, which provides a mechanism for dissemination throughout the body, followed by extravasation into a variety of tissue types. Dissemination of spirochetes can result in non-specific illness, arthralgia, carditis and neuroborreliosis. Persistent untreated infection can result in acrodermatitis and a variety of neurological problems, as well as Lyme arthritis, a common feature of the disease in North America that results from the inflammatory response to spirochete invasion into the joints .
In this work we report a new approach to study vascular transmigration. This approach utilizes assessment of transmigration in the mouse peripheral knee vasculature by intravital microscopy in Cd1d-/- mice. The intravital methodology allows direct visualization and enumeration of transmigrated spirochetes in living mice. The use of Cd1d-/- mice makes this assay possible by eliminating iNKT cells, a component of the innate immune response that disrupts dissemination of B. burgdorferi into mouse joints through a granzyme dependent pathway [17, 18]. The peripheral knee vasculature also provides a desirable target for observation since transmigrating spirochetes remain in the local environment, in contrast to flank skin where they rapidly leave the transmigration site . The general applicability to other tissues of conclusions drawn herein from intravital data obtained from the peripheral knee vasculature is currently unknown as this work is the first reported study of spirochete transmigration using intravital microscopy and the peripheral knee vasculature currently the only location where quantitative imaging of this process is currently viable.