Vaccinia virus interleukin-18-binding protein promotes virulence by reducing gamma interferon production and natural killer and T-cell activity

Abstract

Interleukin-18 (IL-18) is a proinflammatory cytokine that promotes natural killer (NK) and T-cell activation. Several poxviruses, including vaccinia virus (VV), encode a soluble IL-18-binding protein (IL-18bp). The role of the VV IL-18bp (gene C12L) in vivo was studied with wild-type (vC12L), deletion mutant (vDeltaC12L), and revertant (vC12L-rev) viruses in a murine intranasal model of infection. The data show that vDeltaC12L was markedly attenuated, characterized by a mild weight loss and reduced virus titers in lungs, brain, and spleen. Three days after infection, NK cytotoxic activity was augmented in the lung, spleen, and mediastinal lymph nodes (MLNs) of vDeltaC12L-infected mice compared to controls. Seven days after infection, vDeltaC12L-infected mice displayed heightened VV-specific cytotoxic T-lymphocyte (CTL) responses in the lungs, spleen, and MLNs. Gamma interferon (IFN-gamma) levels were also dramatically elevated in lavage fluids and cells from lungs of mice infected with vDeltaC12L. Finally, we demonstrate that IL-18 is produced in vitro and in vivo after VV infection. Taken together, these data demonstrate a role for the vIL-18bp in counteracting IL-18 in both the innate and the specific immune response to VV infection and indicate that the ability of IL-18 to promote vigorous T-cell responses (cytotoxic activity and IFN-gamma production) is a critical factor in the accelerated clearance of the vDeltaC12L mutant.

FIG. 1.

Virulence of recombinant VVs in the murine intranasal model. Groups of five BALB/c mice were mock-infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). (A) Mice were weighed daily, and the results are expressed as the mean percent weight loss of each group ± standard error compared with the weight immediately prior to infection. Data are from five mice, except for the group infected with vC12L-rev, in which one mouse was sacrificed at day 9 and another at day 10. P values were determined by using Student's t test and indicate mean percent weight changes of mice infected with vΔC12L that were significantly different from those of mice infected with vC12L or vC12L-rev. (B) Animals were monitored daily for signs of illness, which was scored from 1 to 4 as described previously (2). Data from each day are expressed as the mean ± standard error from five mice, except for the group infected with C12L-rev, in which 1 mouse was sacrificed at day 9 and another at day 10. P values were determined by using Student's t test and indicate mean signs of illness of mice infected with vΔC12L that were significantly different from those of mice infected with vC12L or vC12L-rev.

FIG. 2.

Replication and spread of recombinant VVs in vivo. Mice were infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). On the indicated day p.i., five animals infected with each virus were sacrificed, and the titer of infectious virus in the lungs, brains, and spleens was determined by plaque assay on BS-C-1 cells. Virus titers are expressed as PFU per organ. The broken line indicates the minimum detection limit of the plaque assay.

FIG. 3.

NK cytotoxicity in BAL, lung, spleen, and MLN after VV infection. Groups of six mice were mock infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). At day 3, NK cytotoxicity was determined in cell suspensions prepared from the BAL, lung, spleen, and MLN of mice. Specific lysis of YAC-1 cells was assessed by ⁵¹Cr-release assay. Data are expressed as the mean ± standard error from two groups of mice (n = 3 per group). Cells were also stained for expression of the pan-NK cell marker DX5 and examined by flow cytometry with a minimum of 20,000 cells analyzed in a lymphocyte gate. Results are expressed as the percentage of DX5⁺ cells in the total viable cell population. P values were determined by Student's t test and indicate the mean percentage of DX5⁺ cells from mice infected with vΔC12L that were significantly different from those from mice infected with vC12L or vC12L-rev. *, P < 0.05.

FIG. 4.

CTL activity in BAL, lung, spleen, and MLN after VV infection. Groups of six mice were mock infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). At day 7, CTL activity was determined in cell suspensions prepared from BAL, lung, spleen, and MLN of mice. Specific lysis of WR-infected P815 cells was assessed by ⁵¹Cr-release assay. Data are expressed as the mean ± standard error from two groups of mice (n = 3 per group). Lysis of noninfected P815s by day 7 effector cell populations was always <10% at an E:T ratio of 100:1 and is not shown. Cells were also stained for expression of CD8 and examined by flow cytometry with a minimum of 20,000 cells analyzed in a lymphocyte gate. Results are expressed as the percentage of CD8⁺ cells in the total viable cell population. P values were determined by Student's t test and indicate the mean percentage of CD8⁺ cells from mice infected with vΔC12L that were significantly different from those from mice infected with vC12L or vC12L-rev. *, P < 0.05.

FIG. 5.

Production of IFN-γ in the lungs of VV-infected mice. Groups of four to six BALB/c mice were mock infected (⋄) or infected with 10⁴ PFU of vC12L (▪), vΔC12L (○), or vC12L-rev (▴). At days 3 and 7, mice were sacrificed, BALs were performed, and single-cell suspensions were prepared from lung tissue. (A and B) Levels of IFN-γ in BAL fluids of VV-infected mice. BAL samples were centrifuged, and the levels of IFN-γ present in the supernatant were determined by ELISA at days 3 and 7. Values for individual mice are shown. The broken line represents the minimum detection level of the assay. (C and D) Intracellular production of IFN-γ by lung lymphocytes from mice 7 days pi. Lung cells were stimulated with PMA and iomnomycin for 4 h, and brefeldin A was added to retain cytokines in the cytoplasm. Cells were stained with FITC-labeled anti-CD8, QR-labeled anti-CD4, and, after permeabilization with saponin, PE-labeled anti-IFN-γ before analysis by three-color flow cytometry. Shown is the percentage of CD4⁺ (C) or CD8⁺ (D) T lymphocytes producing IFN-γ. Values are averaged from two groups (n = 3 per group). P values were determined by Student's t test and indicate the mean percentage of CD8⁺ cells from mice infected with vΔC12L that were significantly different from those from mice infected with vC12L or vC12L-rev. *, P < 0.05; **, P < 0.02.

FIG. 6.

Nitrite levels in the cell-free BAL of mock-infected mice or mice infected with 10⁴ PFU of vC12L, vΔC12L, or vC12L-rev. The data for each time point represent the mean nitrite level ± standard error of four to five individual mice. Columns marked with an asterisk indicate nitrite levels in BAL fluid from vΔC12L-infected mice that were statistically different from those from both vC12L- and vC12L-rev-infected animals. **, P < 0.02.

FIG. 7.

IL-18 production in response to VV infection in vivo and in vitro. (A) IL-18 levels in BAL. Mice were infected with 10⁴ PFU of vC12L, vΔC12L, or vC12L-rev and were sacrificed 3 or 7 days later. BALs from four to five mice were pooled, and the level of total IL-18 protein was determined by ELISA. Data are expressed as the mean ± standard error from two independent experiments. (B) IL-18 release by lung macrophages. Lung macrophages were isolated from naive BALB/c mice and cultured in the presence of 10 PFU of vC12L, vΔC12L, or C12L-rev per cell. Supernatants were measured for IL-18 by ELISA. Results are expressed as the mean ± standard error of duplicate wells.