Endogenous glucocorticoids protect against TNF-alpha-induced increases in anxiety-like behavior in virally infected mice

Abstract

Endogenous glucocorticoids restrain proinflammatory cytokine responses to immune challenges such as viral infection. In addition, proinflammatory cytokines induce behavioral alterations including changes in locomotor/exploratory activity. Accordingly, we examined proinflammatory cytokines and open-field behavior in virally infected mice rendered glucocorticoid deficient by adrenalectomy (ADX). Mice were infected with murine cytomegalovirus (MCMV), and open-field behavior (36 h post-infection) and plasma concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 (42 h post-infection) were assessed. Compared to sham-ADX-MCMV-infected animals, ADX-MCMV-infected mice exhibited significant reductions in total distance moved, number of center entries, and time spent in center. These behavioral alterations were accompanied by significantly higher plasma concentrations of TNF-alpha and IL-6, both of which were correlated with degree of behavioral change. To examine the role of TNF-alpha in these behavioral alterations, open-field behavior was compared in wild-type (WT) and TNF-R1-knockout (KO), ADX-MCMV-infected mice. TNF-R1-KO mice exhibited significantly attenuated decreases in number of rearings, number of center entries and time spent in center, but not distance moved, which correlated with plasma IL-6. Given the potential role of brain cytokines in these findings, mRNA expression of TNF-alpha, IL-1 and IL-6 was assessed in various brain regions. Although MCMV induced increases in proinflammatory cytokine mRNA throughout the brain (especially in ADX animals), no remarkable differences were found between WT and TNF-R1-KO mice. These results demonstrate that endogenous glucocorticoids restrain proinflammatory cytokine responses to viral infection and their impact on locomotor/exploratory activity. Moreover, TNF-alpha appears to mediate cytokine-induced changes in open-field behaviors, especially those believed to reflect anxiety.

Figure 1

MCMV-induced plasma TNF-alpha and IL-6 in ADX and non-ADX Mice. Plasma TNF-alpha (a) and IL-6 (b) concentrations after i.p. injection of MCMV (1 × 10⁵ PFU/ mouse) or vehicle (media) were measured in WT mice having undergone a sham surgery or ADX. Trunk blood was collected from mice at 42 h post-infection (n = 5–10 animals per group). Data were analyzed by two-way ANOVA, and group differences were assessed using the Student–Newman–Keuls post hoc test of significance. *P < 0.05 compared to ADX/media group. ^#P < 0.05 compared to sham/MCMV group.

Figure 2

Brain cytokine mRNA expression in adrenal intact and ADX WT and TNF-R1-KO mice treated with MCMV or media. TNF-R1-KO and WT mice received i.p. injections of MCMV (1 × 10⁵ PFU/mouse) or vehicle (media) after having undergone a sham surgery or ADX. Whole brains were collected at 42 h post-infection (n = 3–5 animals per group), and crude dissections of prefrontal cortex, hippocampus and hypothalamus were performed. Total RNA was extracted from brain tissues, and an RNase protection assay including cRNA probes for TNF-alpha, IL-1 alpha, IL-1 beta, IFN-alpha and IL-6 was conducted. A representative experiment is depicted. See Tables 1 and 2 for statistical analyses of autoradiographic results from multiple RNase protection assays.

Figure 3

Effects of MCMV infection and adrenalectomy on open-field behavior. Total distance moved (a), number of rearings (b), number of entries into the center (c), and time spent in the center (d) at 36 h after i.p. injection of MCMV (1 × 10⁵ PFU/mouse) or vehicle (media) were measured over 5 min in WT mice having undergone a sham surgery or ADX (n = 5–10 animals per group). Data were analyzed by two-way ANOVA, and group differences were assessed using the Student–Newman–Keuls post hoc test of significance. *P < 0.05 compared to respective media-treated control group. ^#P < 0.05 compared to sham/MCMV group.

Figure 4

MCMV-induced TNF-alpha and open-field behavior. MCMV-induced TNF-alpha levels in sham-operated and ADX, MCMV-infected animals were negatively correlated with total distance moved (a), number of entries into the center (c), and time spent in the center (d) of an open field, but not number of rearings (b). **P < 0.01, *P < 0.05.

Figure 5

MCMV-induced TNF-alpha and IL-6 in ADX, TNF-R1-KO mice. Plasma TNF-alpha (a) and IL-6 (b) levels after i.p. injection of MCMV (1 × 10⁵ PFU/mouse) or vehicle (media) were measured in ADX WT and TNF-R1-KO mice. Trunk blood was collected from mice at 42 h post-infection (n = 5–10 animals per group). Data were analyzed by a two-way ANOVA, and group differences were evaluated using the Student–Newman–Keuls post hoc test of significance. *P < 0.05 compared to respective media-treated control group. ^#P < 0.05 compared to WT-MCMV-infecetd group.

Figure 6

Effects of MCMV infection and adrenalectomy on open-field behavior in TNF-R1-KO Mice. Total distance moved (a), number of rears (b), number of entries into the center (c), and time spent in the center (d) at 36 h after i.p. injection of MCMV (1 × 10⁵ PFU/mouse) or vehicle (media) were measured over 5 min in ADX WT or TNF-R1-KO mice (n = 5–10 animals per group). Data were analyzed by a two-way ANOVA, and group differences were assessed using the Student–Newman–Keuls post hoc test of significance. *P < 0.05 compared to respective media-treated group. ^#P < 0.05 compared to WT-MCMV-infected group.