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. 2003 May 19;197(10):1269-78.
doi: 10.1084/jem.20022201.

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

Patrick C Reading  1 , Jeffrey B Moore, Geoffrey L Smith
Affiliations

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

Patrick C Reading et al. J Exp Med. .

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.

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Figures

Figure 1.
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.
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.
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.
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.
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.
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.
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.
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