Vascular tropism models of blood-borne microbial dissemination

Abstract

SUMMARYSimilar to circulating tumour and immune cells, many blood-borne microbes preferentially “home” to specific vascular sites and tissues during hematogenous dissemination 1–5. For many pathogens, the “postal codes” and mechanisms responsible for tissue-specific vascular tropism are unknown and have been challenging to unravel. Members of the Lyme disease Borreliella burgdorferi species complex infect a broad range of mammalian tissues and exhibit complex strain-, species- and host-specific tissue tropism patterns. Intravenous perfusion experiments and intravital microscopy studies suggest that heterogeneous tissue tropism properties may depend on tissue-specific differences in host and microbial molecules supporting vascular interaction and extravasation. However, interpreting these studies can be complicated because of the immune-protective moonlighting (multitasking) properties of many B. burgdorferi adhesins. Here, we investigated whether B. burgdorferi vascular interaction properties measured by live cell imaging and particle tracking in aorta, bladder, brain, joint and skin microvascular flow chamber models predict strain- and tissue-specific dissemination patterns in vivo These studies identified strain- and endothelial cell type-specific interaction properties that accurately predicted in vivo dissemination of B. burgdorferi to bladder, brain, joint and skin but not aorta, and indicated that dissemination mechanisms in all of these tissues are distinct. Thus, the ability to interact with vascular surfaces under physiological shear stress is a key determinant of tissue-specific tropism for Lyme disease bacteria. The methods and model systems reported here will be invaluable for identifying and characterizing the diverse, largely undefined molecules and mechanisms supporting dissemination of Lyme disease bacteria. These methods and models may be useful for studying tissue tropism and vascular dissemination mechanisms of other blood-borne microbes.

A Live Cell Imaging Microfluidic Model for Studying Extravasation of Bloodborne Bacterial Pathogens

“…Investigation of the potential contributions of endothelial activation and junction rearrangement to transmigration mechanisms using a microfluidic transmembrane model will require the ability to cocultivate primary endothelial cells and B. burgdorferi for periods longer than 4 hours, which we have unfortunately not yet been able to technically achieve. In addition, leukocyte molecular transmigration mechanisms differ in distinct vascular beds, and genetically diverse Borrelia strains also exhibit distinct interaction affinities for endothelia from the vascular beds of different tissues under physiological shear stress [44]. Thus, factors specific to endothelia in different tissues likely also influence B. burgdorferi transendothelial migration kinetics and mechanisms.…”

“…The HUVEC flow chamber model recapitulates all major molecular and biophysical properties of B. burgdorferi interactions with the walls of postcapillary venules in the skin of mice [25][26][27]44]. Interaction of genetically diverse Borrelia strains with distinct tissue dissemination patterns with postconfluent HUVEC cultured under static conditions under postcapillary shear stress conditions also robustly predicts strain-specific dissemination to skin in intravenous (IV) perfusion infection models [44].…”

“…HUVECs were used in these proof-of-principle experiments to enable direct comparison to previous studies investigating B. burgdorferi-endothelial transmigration in Transwell devices under static conditions [42,43], as well as B. burgdorferi-endothelial interaction mechanisms under postcapillary venule shear stress [25][26][27][28]. The HUVEC flow chamber model recapitulates all major molecular and biophysical properties of B. burgdorferi interactions with the walls of postcapillary venules in the skin of mice [25][26][27]44]. Interaction of genetically diverse Borrelia strains with distinct tissue dissemination patterns with postconfluent HUVEC cultured under static conditions under postcapillary shear stress conditions also robustly predicts strain-specific dissemination to skin in intravenous (IV) perfusion infection models [44].…”

Porous Membrane Electrical Cell–Substrate Impedance Spectroscopy for Versatile Assessment of Biological Barriers In Vitro

“…Cells were cultured at 37 °C and 5% CO 2 in cell-specific media: primary human umbilical vascular endothelial cells (HUVECs, Lonza; up to passage 6) in EGM-2 (Lonza) culture medium kit; primary human brain microvascular endothelial cells (HBMVECs, Cell Systems - ACBRI 376) in complete classic medium (4Z0–500, Cell Systems) supplemented with CultureBoost (Cell Systems); immortalized HBMVECs in EGM-2MV microvascular endothelial cell growth medium (Lonza) supplemented with hygromycin-B (20 μg/mL); hCMEC/D3 BBB endothelial line (CELLutions Biosystems, Inc. CLU512) cultured according to supplier’s directions in supplemented EBM-2 endothelial basal medium (Lonza); and normal human astrocytes (NHAs, Lonza) in Dulbecco’s minimum essential medium (DMEM), supplemented with N2 supplement (1%) and fetal bovine serum (10%). Media was changed every 48 h in cell culture flasks and every 24–48 h in PM-ECIS devices.…”

A microfluidic platform with integrated porous membrane cell-substrate impedance spectroscopy (PM-ECIS) for biological barrier assessment

“…Immortalized human brain microvascular endothelial cells (HBMEC) (provided by Tara Moriarty, University of Toronto) 35 , were cultured using the EGM™-2 MV Microvascular Endothelial Cell Growth Medium BulletKit™ (Lonza, CC-3202), which contains hydrocortisone, and was supplemented with 20 µg/mL Hygromycin B Biobasic Canada (Cat #BS725) to select for transfected cells. Primary human astrocytes (Thermo Fisher, N7805100) were used for the co-culture conditions and grown in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and N-2 supplement (17502-048).…”