Increased weight loss with reduced viral replication in interleukin-10 knock-out mice infected with murine cytomegalovirus

Abstract

The anti-inflammatory cytokine interleukin (IL)-10 plays an important role in the regulation of host-immune responses. Here we studied the role IL-10 plays in host responses to cytomegalovirus (CMV) infection. We demonstrate that manifestations of murine CMV (MCMV) disease are more severe in IL-10 knock-out mice, despite significantly reduced levels of viral replication. Cytokine analysis of serum revealed increased levels of interferon (IFN)-gamma, monocyte chemotactic protein 1 (MCP-1) and IL-6, all of which are potent stimulators of inflammatory responses. Depletion of IFN-gamma by monoclonal antibodies in IL-10 knock-out mice failed to improve the physical condition of the mice, while increasing viral replication. In contrast, serum levels of IL-6 in the knock-out animals were unaffected by IFN-gamma depletion and remained significantly elevated early in the course of infection. These data suggest that increased weight loss observed in IL-10 knock-out mice may be attributed to the uncontrolled production of proinflammatory cytokines, including IL-6.

Fig. 1

Serum concentrations of interleukin (IL)-10 (pg/ml ± standard deviation) in C57BL/6 mice after murine cytomegalovirus (MCMV) infection. Each data point represents the results of enzyme-linked immunosorbent assays (ELISAs) performed in duplicate using serum from five to seven mice. Serum levels of IL-10 were significantly greater in MCMV-infected mice at days 1, 2, 3, 4 and 7 after infection compared to uninfected animals (*P < 0·01).

Fig. 2

Percentage change in body weights of C57BL/6 immunocompetent (solid symbols) and interleukin (IL)-10 knock-out (KO) (open symbols) mice after murine cytomegalovirus (MCMV) infection (triangles) and in uninfected controls (circles). Each data point represents the change in body weight from baseline measured in five to 18 mice. Loss of body weight was significantly greater in IL-10 KO mice compared to immunocompetent mice infected with MCMV (*P < 0·05, immunocompetent versus IL-10 KO mice with MCMV infection).

Fig. 3

Serum cytokine concentrations (pg/ml ± standard deviation) in immunocompetent C57BL/6 mice (black bars) and interleukin (IL)-10 knock-out (KO) mice (grey bars) after murine cytomegalovirus (MCMV) infection. Cytokines studied were IL-10 (a), interferon (IFN)-γ (b), IL-6 (c) and tumour necrosis factor (TNF)-α (d). Results shown are from one of three sets of experimental animals; similar results were obtained in two additional experiments. Data represent the results of assays performed in duplicate using serum from five to 10 mice at each time-point. Cytokine levels of IL-10, IFN-γ and IL-6 were significantly different in the two groups (*P < 0·05); n.d.: time-points at where data were not available.

Fig. 4

Cytokine mRNA detection (relative mRNA concentration ± standard deviation) in immunocompetent C57BL/6 mice (black bars) and interleukin (IL)-10 knock-out (KO) mice (grey bars) after murine cytomegalovirus (MCMV) infection. Cytokines studied were IL-10 (a), interferon (IFN)-γ (b), IL-6 (c) and tumour necrosis factor (TNF)-α (d). Results shown are from one of three sets of experimental animals; similar results were obtained in two additional experiments. Data represents the results of assays performed in duplicate using spleens from five to 10 mice at each time-point. Cytokine mRNA levels of IL-10, IFN-γ and IL-6 were significantly different in the two groups (*P < 0·05).

Fig. 5

Detection of intracellular interferon (IFN)-γ in CD4⁺ (a) and CD8⁺ (b) splenocytes (number of cells ± standard deviation) from C57BL/6 immunocompetent (black bars) and interleukin (IL)-10 knock-out (KO) (grey bars) mice after murine cytomegalovirus (MCMV) infection. On day 7 after MCMV infection, the number of CD4⁺/IFN-γ⁺ cells was significantly greater in the IL-10 KO mice compared to immunocompetent animals (*P < 0·05); and on day 14 after MCMV infection, the number of CD8⁺/IFN-γ⁺ cells was significantly greater in the IL-10 KO mice compared to immunocompetent animals (*P < 0·05).

Fig. 7

(a) Percentage change in body weights of C57BL/6 immunocompetent (solid circles), interleukin (IL)-10 knock-out (KO) (open circles) and anti-interferon (IFN)-γ-treated IL-10 KO (solid triangles) mice after murine cytomegalovirus (MCMV) infection. Each data point represents the change in body weight from baseline measured in five to 18 mice. Loss of body weight was significantly greater in IL-10 KO mice at days 4, 5 and 7 post-infection compared to immunocompetent C57BL/6 mice infected with MCMV (*P < 0·05). No differences were noted in anti-IFN-γ-treated mice compared to untreated IL-10 KO mice. (b) Viral titres of spleens from adult C57BL/6 (black filled), IL-10 KO (grey filled) and IL-10 KO mice treated with anti-IFN-γ monoclonal antibody (200 mg) (checkered filled). Mice were injected intraperitoneally with 5 × 10⁴ plaque-forming units of MCMV; 10% w/v spleen suspensions were quantified by the plaque assay method using CCL163 fibroblasts. Each data point represents the mean ± standard deviation of three animals. At all time-points, IL-10 KO mice showed significantly decreased levels of viral replication compared to C57BL/6 mice (*P < 0·05). IL-10 KO mice that received anti-IFN-γ antibody showed significantly increased levels of viral replication compared to non-treated IL-10 KO mice (**P < 0·05) as early as 3 days post-treatment (6 days post-infection). Continued treatment of IL-10 KO mice with anti-IFN-γ antibody led to significantly increased levels of viral replication at days 9 and 12 post-infection.

Fig. 6

Detection of serum interleukin (IL)-6 (a) and monocyte chemotactic protein 1 (MCP-1) (b) concentrations (pg/ml ± standard deviation) in immunocompetent C57BL/6 mice (black filled), IL-10 knock-out (KO) mice (grey filled) and IL-10 KO mice treated with interferon (IFN)-γ neutralizing antibody every third day starting at day 3 (checkered filled). Results shown are means of four to five animals per group. Levels of IL-6 are significantly greater in IL-10 KO mice at days 3 and 5 post-infection compared to normal controls (*P < 0·05). By days 7 and 12 post-infection there was no statistical difference (n.s.) between any of the groups. Treatment of IL-10 KO animals with IFN-γ neutralizing antibody had no effect on IL-6 levels throughout the course of these experiments. Serum MCP-1 levels were increased significantly in IL-10 KO animals; treatment with IFN-γ neutralizing antibody reduced levels to that below detectable levels.