Real Time PCR analysis of HLA gene transcription.

@<p>Data represent Mean fold change relative to uninfected.</p><p>controls ± SEM of triplicate assays.</p>*<p>P value <0.001,</p>#<p>P value <0.01.</p

JEV infection and expression of HLA antigens.

<p>As labeled, ECV, HBMEC and HFF were stained for the cell surface expression of total HLA class I (Panel A) and HLA-E (Panel B) at 30 h after JEV infection. Alterations in cell surface expression of total HLA class I and HLA-E were not observed when tested at earlier times of infection and have not been shown. Filled histograms represent cells stained with control antibody while dotted lines represent antigen-specific staining with uninfected cells and solid lines represent antigen-specific staining with JEV-infected or IFN-γ treated cells. Insets in both panels represent cells treated for 24 h with 500 IU/ml IFN-γ. Panel C represents Western blotting analysis of cell lysates from uninfected cells (Con) and cells infected for the labeled times. Numbers at the bottom indicate the fold change in banding intensities of HLA-E after normalization to β-tubulin.</p

JEV infection of human endothelial cells.

<p>Top Panel: Total RNA from ECV, HBMEC and HFF cells was subjected to semiquantitative RT-PCR analysis for JEV envelope and control 18s rRNA. Bottom Panel: Cell lysates (100 µg protein) from ECV, HBMEC and HFF were subjected to Western blotting using rabbit anti JEV-NS3 and control goat anti-β-tubulin antibodies. Both panels show uninfected cells (Con) and cells infected for 12 h, 18 h, 24 h or 30 h.</p

Real Time PCR analysis of MMP-9 gene transcription.

@<p>Data represent Mean fold change relative to untreated.</p><p>controls ± SEM of triplicate assays.</p>§<p>ECV cells that were mock infected for 18, 24 and 30 h.</p><p>did not result in significant changes in gene expression.</p>*<p>P value <0.001,</p>#<p>P value <0.01.</p>†<p>nd: not determined.</p

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

<div><p>Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-α as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-α and IFN-β as well as the dsRNA analog, poly (I:C). Both IFN-β and TNF-α further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.</p></div

Stimulation of sHLA-E shedding and HLA-E gene expression in uninfected cells.

<p>Panel A: Uninfected (Lanes 3 and 7) and JEV infected ECV cells (Lanes 4 and 8) were cultured in the presence of untreated (Lanes 1 to 4) or UV inactivated culture supernatants (Lanes 5 to 8) obtained from 24 h-infected ECV cells. The supernatants harvested from these cultures after a period of 24 h were concentrated 10× and analysed for the release of sHLA-E by Western blotting on the same gel. Uninfected ECV culture supernatants were loaded on lanes 1 and 5 while the infected ECV culture supernatants used for stimulation were loaded on lanes 2 and 6. Panel B: UV inactivated culture supernatants obtained previously from 24 h-infected ECV cells were added to uninfected ECV cells and cultured in the presence of 3 µg and 6 µg of anti-TNFα, anti-IFNAR antibodies or both for 12 h. Cells were harvested and used for RNA extraction and analysis of HLA-E gene expression by real time quantification. Controls (JEV Con) included infected cells cultured in the presence of 6 µg of the appropriate mouse isotype antibody. Data represent mean fold change over control RNA ± SEM. Panel C: Cell culture supernatants were harvested and concentrated 10× and analysed for sHLA-E by ELISA. Data represent the mean sHLA-E released per culture ± SEM (*P<0.001, ^#P<0.01, ^†P<0.05).</p

Shedding of sHLA-E into JEV-infected culture supernatants.

<p>Panel A: Western blot for sHLA-E in cell-culture supernatants from uninfected (Con) and infected HBMEC and ECV at various times p.i. as labeled. ‘Gelatinase’ represents enzyme activity in normal (Con) ECV cells that were either treated with IFN-γ or infected with JEV for the indicated times. Panel B: Cell-culture supernatants were collected from ECV or HBMEC cells that were infected for 24 h with JEV and cultured in the presence of 0 µM, 5 µM and 10 µM GM6001. Panel C: Cell-culture supernatants were collected from uninfected (Con) and 24 h JEV-infected (Top) or TNF-α treated (Bottom) ECV cells that were left untreated or treated with 10 µM GM6001, 10 µM leupeptin, 1 mM PMSF, and 100 µM chloroquine. Panel D: Total RNA was isolated from uninfected (Con) and 24 h JEV-infected ECV cells that were left untreated or treated with 10 µM GM6001, 10 µM leupeptin, 1 mM PMSF, and 100 µM chloroquine. RT-PCR was performed for the JEV envelope and 18s RNA controls. All inhibitors used in panels B, C and D were present only during the last 8 h of the 24 h-culture.</p

Shedding of HLA-E by activating agents.

<p>Panel A: Shedding of sHLA-E from ECV and HFF cells is shown for cells that were either uninfected (Con), 24 h JEV-infected or treated with TNF-α, LPS, or IFN-γ as labeled. Panel B: Top panel shows shedding of sHLA-E from normal cells (Con) and cells that were treated for 24 h with different amounts of poly I:C. Middle panel shows gelatinase activity in normal cells (Con) and cells that were treated with TNF-α or poly I:C (100 µg) for the labeled times. Bottom panel represents sHLA-E shedding from normal cells (Con) and cells that were treated with IFN-β for 24 h, in the absence or presence of GM6001 during the last 8 h of culture.</p

Enhanced release of sHLA-E from ECV by TNF-α and IFN-β.

<p>Panel A represents sHLA-E shedding from cells that were treated with either TNF-α or IFN-β and analysed by Western blotting using anti-HLA-E antibody. Treatment with 0, 50, 200, 500 and 1200 IU of cytokine for 24 h is shown on the left while results obtained with 500 IU at 0, 6, 12, 18, 24 and 24 h of treatment is on the right. Panel B represents sHLA-E shedding from cells treated for 30 h with TNF-α (25 and 100 IU) and IFN-β (200 and 500 IU), either alone or in combination. Panel C represents real time RNA quantification for the HLA-E gene. Cells were treated for 24 h with TNF-α (25 and 100 IU) and IFN-β (200 and 500 IU), either alone or in combination. Data represent mean fold change over untreated control (con) RNA ± SEM.</p