Stress-induced glucocorticoids at the earliest stages of herpes simplex virus-1 infection suppress subsequent antiviral immunity, implicating impaired dendritic cell function

Abstract

The systemic elevation of psychological stress-induced glucocorticoids strongly suppresses CD8(+) T cell immune responses resulting in diminished antiviral immunity. However, the specific cellular targets of stress/glucocorticoids, the timing of exposure, the chronology of immunological events, and the underlying mechanisms of this impairment are incompletely understood. In this study, we address each of these questions in the context of a murine cutaneous HSV infection. We show that exposure to stress or corticosterone in only the earliest stages of an HSV-1 infection is sufficient to suppress, in a glucocorticoid receptor-dependent manner, the subsequent antiviral immune response after stress/corticosterone has been terminated. This suppression resulted in early onset and delayed resolution of herpetic lesions, reduced viral clearance at the site of infection and draining popliteal lymph nodes (PLNs), and impaired functions of HSV-specific CD8(+) T cells in PLNs, including granzyme B and IFN-gamma production and the ability to degranulate. In knockout mice lacking glucocorticoid receptors only in T cells, we show that these impaired CD8(+) T cell functions are not due to direct effects of stress/corticosterone on the T cells, but the ability of PLN-derived dendritic cells to prime HSV-1-specific CD8(+) T cells is functionally impaired. These findings highlight the susceptibility of critical early events in the generation of an antiviral immune response to neuroendocrine modulation and implicate dendritic cells as targets of stress/glucocorticoids in vivo. These findings also provide insight into the mechanisms by which the clinical use of glucocorticoids contributes to altered immune responses in patients with viral infections or tumors.

FIGURE 1

Early onset and delayed resolution of footpad viral lesions in restraint stressed mice. A, Digital photographs of footpads from control and stressed HSV-infected mice were blind-coded, scrambled, and scored for the severity of lesions. Data are mean ± SEM; n = 15 mice/group. *p <0.05. B, Representative photographs of footpads on day 7 postchallenge. White arrowheads indicate herpetic blisters.

FIGURE 2

Stressed mice have higher viral loads. A, Viral titers from footpads of stressed or control mice at various times postchallenge. Data are mean ± SEM; n = 3 mice/group for days 1, 3, and 9; n = 8–9 mice/group for days 5 and 7. Data were compiled from two independent experiments. B, Quantitative real-time PCR for HSV DNA isolated from PLNs of stressed or control mice 3 d postchallenge. Data are mean ± SEM, compiled from two independent experiments; n = 8 mice/group. *p < 0.05.

FIGURE 3

Stress impairs the CD8⁺ T cell responses against HSV. CD8⁺ T cell responses were assessed in cells from PLNs on day 5 postchallenge. Stress reduced (A) total number of granzyme B-expressing CD8⁺ T cells, (B) the number of HSV-1 gB_498–505-specific CD8 T cells, and numbers of functional HSV-1 gB_498–505-specific CD8⁺ T cells as assessed by (C) degranulation, and (D) IFN-γ production. Data are mean ± SEM; n = 5 mice/group. *p < 0.05.

FIGURE 4

The effects of stress on the CD8⁺ T cell response are mediated through the GR. CD8⁺ T cell responses in RU-486 and vehicle-treated mice were assessed in cells from PLNs on day 5 postchallenge. The administration of GR antagonist (RU-486) prevented stress from reducing the number of (A) granzyme B-expressing CD8⁺ T cells, (B) the number of HSV-1 gB_498–505-specific CD8 T cells, and numbers of functional HSV-1 gB_498–505-specific CD8⁺ T cells as assessed by (C) degranulation, arid (D) IFN-γ production. Data are mean ± SEM, compiled from two independent experiments; n = 4–7 mice/group. *Significant difference from control (p < 0.05).

FIGURE 5

Corticosterone impairs CD8⁺ T cell responses independently of direct effects on T cells. A, Western blot for GR in sorted Thy-I⁺ thymocytes or sorted splenic DCs from Cre⁺ and Cre⁻ mice. CD8⁺ T cell responses in cells from PLNs on day 5 postchallenge in corticosterone- or vehicle-treated mice were measured for (B) granzyme B expression, (C) the number of gB_498–505-specific CD8 T cells, and numbers of functional HSV-1 gB_498–505-specific CD8⁺ T cells as assessed by (D) degranulation, and (E) IFN-γ production. Data are mean ± SEM; n = 2–7 mice/group. *p < 0.05.

FIGURE 6

Stress suppresses CD8⁺ T cell responses and limits control of viral replication independently of direct effects on T cells. CD8⁺ T cell responses in cells from PLNs on day 5 postchallenge in stressed or control (nonstressed) Cre⁺ GR-T_KO mice (that lack GR in their T cells) or Cre⁻ littermates (that possess GR in their T cells) were measured for(A) granzyme B expression, (B) the number of HSV-1 gB_498–505-specific CD8⁺ T cells, and numbers of functional HSV-1 gB_498–505-specific CD8⁺ T cells as assessed by (C) degranulation, and (D) IFN-γ production. E, Infectious virus was quantified by plaque assay on footpads from stressed or control mice on day 5 postchallenge. Data are mean ± SEM; n = 5–8 mice/group. *p < 0.05.

FIGURE 7

Stress impairs the functional ability of DCs from HSV-infected mice to induce proliferation of Ag-specific CD8⁺ T cells. Twenty-four hours postchallenge, equivalent numbers of DCs from the PLNs of HSV-infected stressed or control mice were cocultured with 2.5 × 10⁴ CFSE-labeled HSV-1 gB_498–505 CD8⁺ T cells. Proliferation of the T cells is indicated by a reduction in the CFSE signal as determined by flow cytometric analysis. Data in A–D are flow cytometry plots from one of three experiments. The panels show proliferation (CFSE^dim) of T cells cocultured with excess DCs (8 × 10⁴) from (A) a naive mouse, (B) a naive mouse pulsed with a control peptide (OVA_257–264), or 4 × 10³ DCs from (C) nonstressed HSV-infected mice, and (D) stressed HSV-infected mice. (DC numbers were titrated [not shown], and the various numbers of DC used for controls [A and B] resulted in no proliferation above background, indicating that this lack of proliferation was not due to any potential suppressive effect of excess DCs). E, % CFSE^dim data compiled from three experiments, mean ± SEM. *p < 0.05.