Steroid hormone synthesis by vaccinia virus suppresses the inflammatory response to infection

Abstract

The 3beta-hydroxysteroid dehydrogenase (3beta-HSD) isoenzymes play a key role in cellular steroid hormone synthesis. Vaccinia virus (VV) also synthesizes steroid hormones with a 3beta-HSD enzyme (v3beta-HSD) encoded by gene A44L. Here we examined the effects of v3beta-HSD in VV disease using wild-type (vA44L), deletion (vDeltaA44L), and revertant (vA44L-rev) viruses in a murine intranasal model. Loss of A44L was associated with an attenuated phenotype. Early (days 1-3) after infection with vDeltaA44L or control viruses the only difference observed between groups was the reduced corticosterone level in lungs and plasma of vDeltaA44L-infected animals. Other parameters examined (body weight, signs of illness, temperature, virus titres, the pulmonary inflammatory infiltrate, and interferon [IFN]-gamma levels) were indistinguishable between groups. Subsequently, vDeltaA44L-infected animals had reduced weight loss and signs of illness, and displayed a vigorous pulmonary inflammatory response. This was characterized by rapid recruitment of CD4+ and CD8+ lymphocytes, enhanced IFN-gamma production and augmented cytotoxic T lymphocyte activity. These data suggest that steroid production by v3beta-HSD contributes to virus virulence by inhibiting an effective inflammatory response to infection.

Figure 1.

3β-HSD activity in VV-infected cells. CV-1 cells were mock-infected or infected at 10 PFU/cell with (A) vA44L, vΔA44L or A44L-rev, or (B) different VV strains, and assayed for 3β-HSD activity at 8–10 h p.i. All infections were performed in triplicate and results are expressed as mean ± SEM. The background values from nonenzymatic conversion to progesterone (measured in ethanol-fixed monolayers) were subtracted from each value.

Figure 2.

Deletion of A44L attenuates VV WR infection in a murine intranasal model. Groups of five BALB/c mice were mock-infected (⋄) or infected with 10⁴ PFU of vA44L (▪), vΔA44L (○), or A44L-rev (▴). (A) Mice were weighed daily and results are expressed as the mean percentage weight change of each group ± SEM compared with the weight immediately before infection. (B) Animals were monitored daily for signs of illness, scored from 1 to 4. Data are expressed as the mean ± SEM from five mice. P values were determined using the Student's t test and indicate the mean % weight changes or signs of illness of mice infected with vΔA44L that were significantly different from both those of mice infected with vA44L or A44L-rev.

Figure 3.

Titers of vA44L, vΔA44L, and A44L-rev in the lungs (A), brains (B), spleens (C), and livers (D). Groups of five mice were infected intranasally with 10⁴ PFU of VV and the lungs, brains, spleens, and livers were harvested on the days indicated. Organs were homogenized and stored at –70°C and the titer of infectious virus was determined by plaque assay on BS-C-1 cells. Virus titers are expressed as mean log₁₀ PFU per organ, with SEM. The broken line indicates the minimum detection limit of the plaque assay. Columns marked with an asterisk represent virus titers from vΔA44L-infected mice that were significantly different to those from both vA44L- and vA44L-infected animals. *, P < 0.05, **, P < 0.02.

Figure 4.

Characterization of BAL cell suspensions from mice infected with 10⁴ PFU of vA44L, vΔA44L, or vA44L-rev. BAL cells were recovered, counted, and stained to determine the numbers of (A) total cells, (B) macrophages, and (C) lymphocytes from mock-infected and VV-infected mice. Columns represent the mean cell yield per mouse ± SEM from groups of 4–5 mice. Columns marked with an asterisk represent mean cell numbers recovered from vΔA44L-infected mice that were significantly different (*, P < 0.05) to those of both vA44L and vA44L-rev-infected mice. (D and E) At 7 d pi, BAL cells were recovered, stained for expression of CD3, CD4, and CD8 and analyzed by flow cytometry. Lymphocytes were identified by their characteristic FSC/SSC profile and by expression of CD3. Data shown are the mean percentage of BAL cells ± SEM from 4–5 individual mice, and are representative of two independent experiments.

Figure 5.

Production of IFN-γ in the lungs of VV-infected mice. Groups of 4–6 BALB/c mice were mock-infected or infected with 10⁴ PFU of vA44L, vΔA44L, or vA44L -rev. At days 3, 7, and 10 mice were killed, BALs were performed and single cell suspensions were prepared from lung tissue. (A) Levels of IFN-γ in BAL fluids of VV-infected mice. BAL fluid was centrifuged and the level of IFN-γ present in the supernatant was determined by ELISA. Values represent the mean, with SEM, from two groups (n = 3/group). The dashed line represents the detection limit of the IFN-γ ELISA (50 pg/ml). (B) IFN-γ production from lung cells. Lung cell suspensions were stimulated with PMA and ionomycin for 5 h as described in Materials and Methods. Cells were pelleted and level of IFN-γ present in the supernatant was determined by ELISA. Values represent the mean ± SEM, from two groups (n = 3/group). The dashed line represents the detection limit of the IFN-γ ELISA (50 pg/ml). (C and D) Intracellular production of IFN-γ by lung lymphocytes from mice 7 d after intranasal VV. Lung cells were stimulated with PMA and ionomycin for 4 h; brefeldin A was added to retain cytokines in the cytoplasm. Cells were stained with FITC-labeled anti-CD8, APC-labeled anti-CD4, and after permeabilization using saponin, with PE-labeled anti–IFN-γ before analysis by three-color flow cytometry. Shown are the percentages of CD8⁺ (C) or CD4⁺ (D) T cells producing IFN-γ. Values are averaged from two groups (n = 3/group). The frequency of IL-4-producing cells was below the detection limit (<2%) and is not shown. *, P < 0.05, **, P < 0.02.

Figure 6.

Virus-specific CTL activity in the lung of VV-infected mice. Groups of six mice were infected with 10⁴ PFU of vA44L (▪), vΔA44L (○), or vA44L-rev (▴). At days 7 (A and C) and 10 (B and D) mice were killed and lung cell suspensions were prepared. Specific lysis of WR-infected P815 cells was assessed by ⁵¹Cr-release assay. Data are expressed as the mean percent specific lysis ± SEM from two groups of three mice plotted against the lung cell:target ratio (A and B) or the CD8⁺ lung cell:target ratio (C and D). Lysis of uninfected P815 cells by day 7 and day 10 effector cell populations was always <10% at an effector:target ratio of 100:1 (unpublished data).

Figure 7.

Corticosterone levels in plasma and lungs after intranasal infection with VV. Corticosterone levels were measured in (A) plasma and (B) lung extracts collected from BALB/c mice under low stress conditions (samples were obtained within 4 min of handling) at the indicated times after intranasal infection with 10⁵ PFU of vA44L, vΔA44L, or A44L-rev. Lung extracts were prepared as described in Materials and Methods. Data represent mean ± SEM of four or five mice per time point and are expressed as ng/ml of plasma or as ng/g of lung tissue. Columns marked with an asterisk represent corticosterone levels from vΔA44L-infected mice that were significantly different to those from vA44L- and vA44L-rev-infected mice. *, P < 0.05, **, P < 0.02. (C) Titers of infectious virus in the lungs of mice after infection with 10⁵ PFU of VV. Virus titers were determined by plaque assay on BS-C-1 cells and are expressed as PFU/g of lung tissue.