Preferential infection of mature dendritic cells by mouse hepatitis virus strain JHM

Abstract

Mouse hepatitis virus strain JHM (MHV-JHM) causes acute encephalitis and acute and chronic demyelinating diseases in mice. Dendritic cells (DCs) are key cells in the initiation of innate and adaptive immune responses, and infection of these cells could potentially contribute to a dysregulated immune response; consistent with this, recent results suggest that DCs are readily infected by another strain of mouse hepatitis virus, the A59 strain (MHV-A59). Herein, we show that the JHM strain also productively infected DCs. Moreover, mature DCs were at least 10 times more susceptible than immature DCs to infection with MHV-JHM. DC function was impaired after MHV-JHM infection, resulting in decreased stimulation of CD8 T cells in vitro. Preferential infection of mature DCs was not due to differential expression of the MHV-JHM receptor CEACAM-1a on mature or immature cells or to differences in apoptosis. Although we could not detect infected DCs in vivo, both CD8(+) and CD11b(+) splenic DCs were susceptible to infection with MHV-JHM directly ex vivo. This preferential infection of mature DCs may inhibit the development of an efficient immune response to the virus.

FIG. 1.

MHV-JHM and rJHM.GFP productively replicate in BM-derived DCs. DCs were harvested from the bone marrow of naïve B6 mice and prepared as described in Materials and Methods. (A) After 6 to 7 days in culture, cells were infected with MHV-JHM at an MOI of 1 and fixed at 7 to 9 h p.i. Cells were stained for CD11c (red) and N protein (green). Nuclei were labeled with TO-PRO-3 (blue). Original magnifications: left, ×40; right, ×100. (B) DC cultures were enriched for CD11c⁺ cells by using magnetic beads prior to infection with MHV-JHM. Cells were stained as described for panel A. Initial magnification, ×38. (C) A recombinant MHV-JHM expressing GFP was engineered as described in Materials and Methods, with GFP inserted into gene 4 (rJHM.GFP). HE, hemagglutinin-esterase; E, small membrane protein; M, transmembrane protein. (D) BM-derived DCs were infected with MHV-JHM or rJHM.GFP at an MOI of 1, and samples were harvested in triplicate for titers at the indicated times. Virus titers were determined by plaque assay on HeLa-MHVR cells. A representative example of three independent experiments is shown in the figure. (E) Mice were inoculated with 6 × 10⁴ PFU rJHM.GFP intranasally. Brains were harvested and analyzed for MHV-JHM N antigen (red) and GFP expression (green). Original magnification, ×40. Scale bar, 20 μM.

FIG. 2.

rJHM.GFP-infected DCs are MHC class I/II^hi and CD86^hi. DCs were infected with rJHM.GFP at an MOI of 10. After 9 h, cells were harvested and the expression of CD11c, MHC class I, MHC class II, and CD86 molecules, as well as GFP, was assessed by FACS analysis. Shown are data for samples after gating on CD11c⁺ cells. The percentage of infected cells was determined by GFP expression. The data for the experiment shown are representative of 10 independent experiments.

FIG. 3.

rJHM.GFP and rA59.GFP preferentially infect mature DCs. (A) CD86^hi or CD86^lo DCs and CD11c⁻ DC precursors in the culture were separated using a flow cytometer prior to infection with rJHM.GFP or rA59.GFP (MOI of 10). Cells were harvested at 9 h p.i., and the percentage of GFP⁺ cells in each population was assessed by FACS analysis. (B) CD86^hi or CD86^lo DCs were infected with MHV-JHM at an MOI of 10. Virus titers were measured at the indicated times by plaque assay. The experiments shown in each panel are representative samples from three independent experiments.

FIG. 4.

Infection of DCs with MHV-JHM impairs their ability to induce T-cell proliferation. (A) BM-derived DCs were infected with MHV-JHM for 1, 3, 6, or 9 h at an MOI of 100 or mock infected. Following pulsing with gp33 peptide for 1 h, mock- or MHV-JHM-infected DCs were added to CFSE-labeled P14-Tg CD8⁺ T cells and incubated for 3 days. Ratios of 1:100 (panels a and c) and 1:500 (panels b and d) DCs to T cells are shown. (a and b) T-cell proliferation was analyzed by FACS analysis. (c and d) The percentage of the total number of cells that have divided (% Divided), as well as the average number of divisions the cell population has undergone (Division index), was calculated. Compared to mock-infected DCs and DCs harvested at 1 h p.i., the percentage of divided cells and division index in DCs harvested at 3, 6, or 9 h are significantly different for both DC/T-cell ratios shown in the figure (P < 0.05). The data are representative of three independent experiments. (B) To determine whether infected DCs induced apoptosis of T cells, DCs were infected with MHV-JHM for 6 h, pulsed with gp33 peptide, and added to P14-Tg CD8⁺ T cells at a ratio of 1:1 or 1:100. After incubation for 24 h, cells were harvested and stained with annexin V and PI.

FIG. 5.

CEACAM-1a expression levels on mature and immature DCs are equivalent. DCs were stained sequentially with biotinylated anti-CEACAM-1a MAb and SA-PerCP and analyzed by FACS analysis. Shaded, immunoglobulin control; solid line, CD86^hi DCs; dashed line, CD86^lo DCs.

FIG. 6.

Infection with MHV-JHM does not induce apoptosis in DCs. (A) BM-derived DCs were infected with rJHM.GFP (MOI = 10). Cells were harvested and stained with annexin V and PI at 9 h p.i. PI-negative cells are shown in the figure. Only a low percentage of infected (GFP⁺) or uninfected (GFP⁻) cells underwent apoptosis (annexin V⁺) within the infected culture (right hand panel). (B) CD86^hi and CD86^lo DCs were sorted using a flow cytometer and infected with MHV-JHM (MOI = 10). MHV-JHM infection did not induce apoptosis in either CD86^hi or CD86^lo DC cultures compared to mock-infected cultures at 9 h p.i.

FIG. 7.

MHV-JHM replication in DCs is not dependent on cathepsin L. DCs were treated with increasing doses of the cathepsin L inhibitor, FYdmk, 4 h prior to infection with MHV-JHM. After incubation for 20 h, viruses were harvested and titers were determined on HeLa-MHVR cells.

FIG. 8.

Infection of splenic DCs and B cells directly ex vivo. (A) Spleen cells were harvested from naïve B6 mice and directly infected with rJHM.GFP (MOI of 1). A small percentage of B220⁺ B cells were infected as showing by GFP expression. (B) CEACAM-1a expression levels were equivalent on CD8⁺ CD11b⁻ and CD8⁻ CD11b⁺ splenic DCs harvested from naïve mice. Shaded, immunoglobulin control; solid line, CD8⁻ CD11b⁺ DCs; dashed line, CD8⁺ CD11b⁻ DCs. (C) CD11c⁺ cells were enriched using magnetic beads prior to infection with rJHM.GFP at an MOI of 10. Both CD8⁺ and CD11b⁺ DCs were susceptible to infection with the virus.

FIG. 9.

MHV-JHM infection of DCs is modestly sensitive to IFN-β treatment. BM-derived DCs were treated with different concentrations of IFN-β prior to and after or only after infection with MHV-JHM (MOI = 1). Cells were harvested at 20 h p.i., and virus titers were determined by plaque assay. The dotted line shows the limit of detection of virus. One of three independent experiments is shown in the figure.