Role of endogenous interleukin-18 in resolving wild-type and attenuated Salmonella typhimurium infections

Abstract

The stimulation of gamma interferon (IFN-gamma) has been shown to be essential in resolving infections by intracellular pathogens. As such, several different cytokines including, interleukin-12 (IL-12) and IL-18, can induce IFN-gamma. To resolve Salmonella infections, the stimulation of IL-12 and IFN-gamma are important for mediating its clearance. In this present study, the relevance of IL-18 in protection against oral challenge with Salmonella typhimurium was investigated to determine the role of this IFN-gamma-promoting cytokine. Rabbit anti-murine IL-18 antisera was generated and administered prior to the oral challenge of BALB/c and IL-12p40-deficient knockout (IL-12KO) mice with a wild-type S. typhimurium strain. The median survival time was reduced by 2 days for the anti-IL-18-treated BALB/c mice, while no significant reduction in survival rate for the anti-IL-18-treated IL-12KO mice was observed compared to vehicle-treated mice. To investigate the contribution of IL-18 to resolving Salmonella infections, an attenuated aro-negative mutant (H647) was orally administered to BALB/c mice. This Salmonella infection induced both IL-12 and IFN-gamma in both the Peyer's patches and the spleens. In vehicle-treated mice, Peyer's patch IL-12 peaked by 24 h, while IL-18 levels peaked at 3 days, suggesting sequential support by these cytokines for IFN-gamma. Anti-IL-18 treatment exerted its greatest effect upon the mucosal compartment, limiting early IFN-gamma production. However, anti-IL-18 treatment had little effect upon splenic IFN-gamma levels until late in the response. Infection of IL-12KO mice with H647 strain induced IFN-gamma, but it was not supported by IL-18, although IL-18 levels were reduced by this treatment. These results suggest that IL-18 does contribute to the clearance of S. typhimurium and that endogenously induced IL-18 could not substitute for IL-12.

FIG. 1

Anti-IL-18 treatment diminishes survival rates after oral challenge of BALB/c, but not IL-12p40 KO, mice with wild-type S. typhimurium. Five- to eight-week-old age-matched mice were given vehicle (PBS or normal rabbit serum) or rabbit anti-mouse IL-18 sera by the i.p. route 1 day prior to and on the day of oral challenge with 5 × 10⁷ wild-type S. typhimurium H71. Deaths were recorded daily. The anti-IL-18-treated BALB/c mice (n = 19) showed a median survival rate of 6 days, while vehicle-treated BALB/c mice (n = 21) showed a median survival of 8 days. This difference in survival rates was statistically significant by the Mantel-Haenszel log-rank test (P < 0.005). No statistical differences in survival rates were evident between vehicle-treated (n = 10) and anti-IL-18-treated (n = 10) IL-12KO mice (P = 0.48). Symbols: ●, BALB/c; ○, anti-IL-18-treated BALB/c; ⧫, IL-12KO; and ◊, anti-IL-18-treated IL-12KO.

FIG. 2

In vivo treatment with anti-IL-18 sera alters Peyer's patch IFN-γ, IL-12, and IL-12p40 levels after oral infection with an avirulent, aro-negative S. typhimurium strain. Five- to eight-week-old age-matched mice were given up to four doses of either PBS or normal rabbit serum (n = 9) or rabbit anti-murine IL-18 serum (n = 9) i.p. on days −1, 2, 4, and 6 p.i. with 5 × 10⁹ CFU of attenuated S. typhimurium H647. Peyer's patches were harvested, weighed, and homogenized in PBS on the indicated days. Values represent the mean ± the standard error of the mean (SEM) per group per time period. (A) Peyer's patch weights expressed as a percent increase from noninfected BALB/c age-matched mice. Peyer's patches from H647-infected mice were collected on the indicated days p.i. and were assessed for IFN-γ (B), IL-12p70 (C), and IL-12p40 (D) levels by using cytokine-specific ELISA methods. The two groups were compared against each other within each time period by one-way analysis of variance. ∗, P < 0.001; ∗∗, P = 0.015; ∗∗∗, P = 0.021.

FIG. 3

In vivo treatment with anti-IL-18 serum alters IFN-γ, IL-12, and IL-12p40 levels after oral infection with attenuated, aro-negative S. typhimurium H647. Five- to eight-week-old age-matched mice were given up to four doses of either PBS or normal rabbit serum (n = 9) or rabbit anti-murine IL-18 serum (n = 9) i.p. on days −1, 2, 4, and 6 p.i. with 5 × 10⁹ CFU of attenuated S. typhimurium H647. Spleens were harvested, weighed, and homogenized in PBS. Values represent the mean ± the SEM per group per time period. (A) Splenic weights expressed as the percent increase from noninfected BALB/c age-matched mice. Spleens from H647-infected mice were collected on the indicated days p.i. and were assessed for IFN-γ (B), IL-12p70 (C), and IL-12p40 (D) levels by using cytokine-specific ELISA methods. The two groups were compared against each other within each time period by one-way analysis of variance. ∗, P ≤ 0.001; ∗∗, P = 0.011.

FIG. 4

Anti-IL-18 treatment reduces IL-18 levels in both Peyer's patches (A) and spleens (B). BALB/c mice received up to four doses of either PBS or normal rabbit serum (n = 9) or rabbit anti-murine IL-18 serum (n = 9) i.p. on days −1, 2, 4, and 6 p.i. with 5 × 10⁹ CFU of attenuated, aro-negative S. typhimurium H647. Spleens were harvested, weighed, and homogenized in PBS on the indicated days. Values represent the mean ± the SEM per group per time period. IL-18 was measured in samples from individual mice by using an IL-18-specific ELISA. Antibodies used for detection were different from the rabbit anti-IL-18 antibody used in the treatment groups. Values represent the mean ± the SEM. ∗, P < 0.001; ∗∗, P < 0.007; ∗∗∗, P = 0.045.

FIG. 5

Anti-IL-18 treatment does not alter Peyer's patch and splenic IFN-γ levels, but it does reduce IL-18 levels in IL-12KO mice. Five- to eight-week-old age-matched IL-12KO mice were given vehicle (n = 6) or rabbit anti-mouse IL-18 antisera (n = 6) i.p. on days −1 and 2 p.i. with S. typhimurium H647. Treated IL-12KO mice were orally gavaged with attenuated, aro-negative S. typhimurium H647 on day 0. At 3 days after oral infection, Peyer's patches (PP) and spleens (Sp1) were harvested, weighed, and homogenized. (A) Tissue weights represent the mean ± the standard deviation per group at 3 days p.i. Only spleens showed a significant increase in size. (B) No statistical difference in IFN-γ cytokine levels between Peyer's patches and spleens were observed. (C) However, IL-18 levels were greatly reduced by the anti-IL-18 treatment. ∗, P < 0.001; ∗∗, P = 0.011; ∗∗∗, P = 0.02.