Date Published: July 18, 2014
Publisher: Public Library of Science
Author(s): Kelsey Roe, Beverly Orillo, Saguna Verma, Michal Toborek.
Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via ‘Trojan horse’ route, and improve WNV disease outcome.
Since its introduction to the United States in 1999, West Nile virus (WNV), a mosquito-borne flavivirus classified as an NIAID Category B Priority Pathogen, has emerged as a leading cause of viral encephalitis, with more than 5,000 cases including nearly 250 deaths in 2012. WNV is an enveloped positive stranded RNA virus and is closely related to other human pathogens including dengue, yellow fever, Japanese encephalitis and tick-borne encephalitis viruses. Currently there are no therapeutic drugs or vaccines for WNV approved for human use . The fatality rate is approximately 10% for hospitalized WNV cases and up to 70% of the survivors of WNV-encephalitis experience persistent neurological deficits for several months . The pathogenesis of WNV in humans is not well characterized but WNV infection in mice mimics human WNV disease, thus making it a good model to understand the mechanisms that cause WNV disease. WNV infection triggers effective innate immune responses, which collectively mediate virus clearance from the periphery and control its dissemination in the brain, however in subset of patients WNV enters the central nervous system (CNS) . Therefore, WNV neuropathogenesis is mainly dependent on the ability of the virus to enter the brain and replicate within resident cells including neurons and astrocytes . Increased leukocyte infiltration, specifically CD8+ T cells are critical for clearing virus infection from the CNS, although migrating inflammatory monocytes and T cells also contribute to neuropathology by potentiating inflammation , . Leukocytes entering the CNS must cross the blood-brain barrier (BBB) and one of the routes of WNV-CNS entry is also proposed to be via ‘Trojan horse’ mechanism by infected leukocytes .
Infection with WNV in the mouse model of WNV-encephalitis is characterized by BBB disruption and massive leukocyte infiltration into the brain. The actual basis of BBB disruption, however, has not been elucidated. We previously demonstrated that WNV infection in the HBMVE cells can be one of the routes of cell-free virus entry into the brain, however it does not compromise the integrity of the BBB model, suggesting that events other than direct virus infection are responsible for the BBB disruption. Here, we demonstrate that (i) transmigration of leukocytesacross the WNV-infected BBB model compromises its integrity, (ii) WNV-induced ICAM-1, VCAM-1 and E-selectin are critical in mediating the adherence of leukocytes to the HBMVE cells thereby facilitating their transmigration across the in vitro BBB model and (iii) blocking these CAMs significantly reduce disruption of the BBB model.