Hepatitis C virus infects the endothelial cells of the blood-brain barrier

Abstract

Background & aims: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS.

Methods: We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication.

Results: Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis.

Conclusions: Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Figure 1

HCV receptor expression in human brain tissue. Formalin-fixed, paraffin-embedded brain sequential sections were costained with antibodies specific for von Willebrand factor (vWF), a marker for endothelial cells, and HCV receptors SR-BI, CD81, claudin-1, occludin, and LDL-R. Brain endothelium expressed all the factors required for HCV entry. Original magnification 100×.

Figure 2

HCV receptor expression in microvascular endothelial cells. CD81, claudin-1, occludin, ZO-1, and LDL-R expression in (A) hCMEC/D3 or (B) HBMEC. Flow cytometric analysis of HCV entry factor expression in hCMEC/D3, HBMEC, liver sinusoidal endothelial cells (LSEC), and human umbilical vein endothelial cells (HUVEC), together with the permissive Huh-7 hepatoma and nonper-missive U87 cells. (C and D) The percent receptor-positive cells and mean fluorescent intensity (MFI) are shown. Anti-receptor antibodies showed negligible binding to receptor-negative Chinese hamster ovary cells with MFI values between 4 and 8. Isotype control antibodies gave MFI values between 5 and 11 for all cells tested. Data are representative of 2 independent experiments.

Figure 3

Microvascular brain endothelial cells support HCV entry. HCVpp-H77 infection of hCMEC/D3, HBMEC, human umbilical vein endothelial cells (HUVEC), liver sinusoidal endothelial cells (LSEC), primary human hepatocytes (PHH), and Huh-7 cells. (A) HCVpp and VSV-Gpp entry is expressed as relative light units (RLU). (B) Normalized HCVpp entry relative to VSV-Gpp. (C) Infectivity of HCVpp bearing primary envelopes cloned from 4 acutely infected patients (Pt 11, Pt 28, Pt 110, and Pt 18) for hCMEC/D3. HCVpp infection of hCMEC/D3 and HBMEC was neutralized by antibodies targeting CD81, SR-BI, or claudin-1 and viral glycoproteins (anti-HCV E2 3/11, 9/27, 11/20, and pooled anti-HCV IgG). (D) Anti-HIV 10/76B and irrelevant globulin had no effect. Data are representative of 3 independent experiments.

Figure 4

Microvascular brain endothelial cells support HCV replication. hCMEC/D3, HBMEC, and Huh-7 cells were infected with HCVcc J6/JFH or SA13/ JFH, and infection was expressed as focus forming units per milliliter (FFU/mL). (A) Interferon alfa (IFN-α; 100 IU/mL) inhibited infection of all cell lines. hCMEC/D3 and Huh-7 cells were infected with HCVcc J6/ JFH for 8 hours and treated with increasing concentrations of antiviral drugs targeting HCV protease and polymerase. (B) The concentration of inhibitor that reduced infection by 90% was determined (IC₉₀). (C) Focus of HCV NS5A-expressing hCMEC/D3 cells. (D) Antibodies specific for HCV entry factors CD81, SR-BI, claudin-1, LDL-R, and ApoE, anti-HCV Ig, or irrelevant Ig were incubated with hC-MEC/D3 and Huh-7 cells for 1 hour before (white bars) or 8 hours after (black bars) infection. Infected cells were incubated for 72 hours and NS5A-positive cells enumerated. Statistically significant neutralization relative to the irrelevant IgG is indicated (*P < .0001). Data are representative of 3 independent experiments.

Figure 5

HCV RNA and antigen expression in brain endothelial and hepatoma cells. hCMEC/D3 and Huh-7 cells were infected with HCVcc SA13/JFH for12hours, and unbound virus was removed by washing. (A) HCV RNA copies and (B) the frequency of NS5A-positive cells were determined at the indicated times. Infectivity is presented as focus forming units per milliliter (FFU/mL) and HCV RNA copies relative to GAPDH.

Figure 6

HCV increases brain endothelial permeability and apoptosis. hCMEC/D3 cells were cultured on permeable filters and infected with HCVcc SA13/JFH for 72 hours or treated with recombinant human tumor necrosis factor α and interferon gamma at 10 ng/mLfor24 hours. Paracellular permeability to fluorescein isothiocyanate/dextran 70 kilodaltons was measured. (A) HCV induced a significant increase in permeability (***P < .001), which was inhibited by anti-HCV Ig (**P < .05). Significant increases in permeability were also observed in response to tumor necrosis factor α/interferon gamma (IFN-γ). (B) hCMEC/D3 and Huh-7 cells were infected with HCVcc SA13/JFH for 72 hours at comparable multiplicities of infection and costained for NS5A (red) and DNA strand breaks by using TUNEL (green). Although some apoptosis was associated with HCV-infected Huh-7 cells, apoptosis was pronounced in infected hCMEC/D3 cells. Data are representative of 3 independent experiments.