Figure 1.
WNV infection of HMBVE cells, but not of leukocytes, mediates the disruption of the in vitro BBB model.
The effect of leukocyte migration on the permeability of the in vitro BBB model was determined by TEER measurements. (A) WNV infected monocytes (2.5 x 105) at 24 hrs after infection were added in the upper compartment of the uninfected BBB model and permeability was measured at 2 and 4 hrs after incubation. (B) At 72 hrs after WNV infection of BBB model, transmigration of uninfected monocytes significantly decreased the TEER as compared to control. (C and D) A chemotactic gradient was established by the addition of 100 ng/mL recombinant human CCL2 to the lower compartment and uninfected monocytes and lymphocytes were allowed to migrate across infected BBB. Results are the mean of data from two independent experiments in duplicate. *p<0.05 compared to mock. C, inserts with control HBMVE cells without leukocytes.
Figure 2.
WNV induces expression of specific CAMs in the HBMVE cells and mouse brain.
(A) The changes in the mRNA of multiple CAMs was measured in HBMVE cells at 72 hours after infection. (B) qRT-PCR was conducted on the RNA extracted from WNV-infected human monocytes to determine fold-change in the VLA-4, MAC-1 and L-selectin gene expression. (C) WNV titers and fold-change of ICAM-1, VCAM-1 and E-selectin in WNV-infected mouse brain as determined by qRT-PCR. The data are normalized to the values of GAPDH and are expressed as relative fold increase compared to uninfected controls. (D) Validation of the increase in the protein expression of ICAM-1 and E-selectin in HBMVE cells at 72 hrs after infection by immunostaining. The data are expressed as means ± SD from two independent experiments in duplicate. *p<0.05 compared to mock.
Figure 3.
WNV-Eg101 efficiently infects HBMVE cells and induces CAMs.
Primary human HBMVE cells were infected with WNV-NY99 or Eg101 (MOI5). (A) Culture supernatants recovered at 48 and 72 hrs after infection were used to determine titers using plaque assay on Vero cells. Data represent the mean ± SD PFU per mL of supernatant from two independent experiments. (B) qRT-PCR analysis of CAMs in Eg101 infected HBMVE cells at 48 and 72 hrs after infection. The data are normalized to the values of GAPDH and are expressed as relative fold increase compared to uninfected controls.
Figure 4.
Blocking of cell adhesion molecules limits leukocyte adhesion to the HBMVE cells.
HBMVE cell monolayers on the coverslips were infected with WNV for 725 monocytes or lymphocytes were added to the cells in the presence or absence of the cocktail of neutralizing antibodies against CAMs (ICAM-1, VCAM-1 and E-slectin). After two hrs, the non-adhered leukocytes were vigorously washed off and adherent cells were stained with CD45 (white arrows). (A) Immunofluorescence micrograph of CD45-stained monocytes and lymphocytes. (B) Quantitative representation of CD45+ cells from six different areas per coverslip from three independent experiments. *p<0.05 compared to mock.
Figure 5.
Neutralizing antibodies against cell adhesion molecules partially reverse the disruption of the in vitro BBB model during leukocyte transmigration.
The integrity of the in vitro BBB model was determined by measuring the TEER before and after four hrs of incubation with monocytes and lymphocytes in the presence or absence of the cocktail of neutralizing antibodies against CAMs. The decrease in the TEER values was represented as percentage change as compared to mock-infected controls. The data is mean ± SD of two independent experiments. *p<0.05 compared to mock.
Figure 6.
Blocking CAMs limits the transmigration of monocytes across the in vitro BBB model.
Human monocytes stained with leukotracker dye were added to the upper compartment of the in vitro BBB model infected with NY99 strain of WNV in the presence or absence of neutralizing antibodies against ICAM-1, VCAM-1 and E-slectin. Treatment with TNF-α (0.125 ng/mL) was used as a positive control. After four hrs of incubation, the media of the lower compartment was collected and the flourescence was measured. (A) Flourescence absorbance (RFU) of the transmigrated monocytes in the lower compartment. (B) Absolute number of transmigrated monocytes extrapolated based on the standard curve generated using RFU of known number of leukotracker stained monocytes. The data is representative of two independent experiments. *p<0.05 compared to mock and **p<0.05 compared to WNV-infected.
Table 1.
Primer sequences used for qRT-PCR.