Interleukin-18 (IL-18) enhances innate IL-12-mediated resistance to Toxoplasma gondii

Abstract

Innate resistance to Toxoplasma gondii is dependent on the ability of interleukin-12 (IL-12) to stimulate natural killer (NK) cell production of gamma interferon (IFN-gamma). Since IL-18 is a potent enhancer of IL-12-induced production of IFN-gamma by NK cells, SCID mice (which lack an adaptive immune response) were used to assess the role of IL-18 in innate resistance to T. gondii. Administration of anti-IL-18 to SCID mice infected with T. gondii resulted in an early reduction in serum levels of IFN-gamma but did not significantly decrease resistance to this infection. In contrast, administration of exogenous IL-18 to infected SCID mice resulted in increased production of IFN-gamma, reduced parasite burden, and a delay in time to death. The protective effects of IL-18 treatment correlated with increased NK cell numbers and cytotoxic activity at the local site of administration and with elevated levels of inducible nitrous oxide synthose in the spleens of treated mice. In addition, in vivo depletion studies demonstrated that the ability of exogenous IL-18 to enhance resistance to T. gondii was dependent on IL-12, IFN-gamma, and NK cells. Together, these studies demonstrate that although endogenous IL-18 appears to have a limited role in innate resistance to T. gondii, treatment with IL-18 can augment NK cell-mediated immunity to this pathogen.

FIG. 1

Expression of IL-18 during toxoplasmosis. (A) Total RNA from the spleens of uninfected SCID mice or SCID mice infected for 7 days was extracted, and RPA analysis was performed as described in Materials and Methods. Similar results were observed in two experiments. (B) Densitometric analysis of RNA levels and expression relative to the housekeeping L32 gene. The results shown are the means ± the SD of the data presented. (C) Levels of IL-18 and IL-12 were measured by using ELISA in the serum from uninfected mice or mice infected for 7 days. The data shown are the means ± the SD from four independent experiments with three to six mice per group.

FIG. 2

Effect of administration of anti-IL-18 during toxoplasmosis. SCID mice were infected i.p. with 20 ME-49 cysts and treated with a rabbit isotype control antibody or rabbit anti-IL-18 as described in Materials and Methods. The levels of IFN-γ in serum were measured by ELISA (A), and the percentage of PECs infected was estimated by using cytospins (B). The data shown are the means ± the SD of three pooled experiments with three to six mice per group (∗, P < 0.05). (C) NK cell (spleen) cytolytic activity against YAC-1 cells on day 7 postinfection were measured. The data shown are representative of three independent experiments.

FIG. 3

IL-18 induced production of IFN-γ by splenocytes from infected SCID mice is dependent on IL-12. Splenocytes from SCID mice infected for 7 days were stimulated with 30 μg of TLA, 10 ng of IL-12, or 10 ng of IL-18 per ml in combination with 30 μg of rat IgG or anti-IL-12 (C17.8) per ml for 24 h, and the production of IFN-γ in the supernatants was measured by ELISA. The data shown are the means ± the SD from a single experiment done in triplicate. Similar results were observed in two additional experiments.

FIG. 4

Administration of IL-18 to SCID mice enhances resistance to T. gondii. SCID mice infected with T. gondii were treated with IL-18 (200 ng per mouse) or PBS beginning 1 day before infection and daily thereafter. (A) The percentages of PECs infected were calculated as described in Materials and Methods. Results shown are the means ± the SD of four pooled experiments with three to six mice per group (∗, P < 0.05). (B) SCID mice were infected with T. gondii and treated daily with PBS or IL-18, and the survival was monitored. The results presented are the pooled data from five independent experiments with a total of 15 mice per experimental group. (C) Immunohistochemical detection of T. gondii and iNOS in the spleens of SCID mice infected for 16 days and treated with PBS or IL-18. Similar results were observed in three other mice.

FIG. 5

Administration of IL-18 to SCID mice enhances production of IFN-γ and NK cell activity during toxoplasmosis. (A) Serum levels of IFN-γ from SCID mice treated with PBS or IL-18 were measured by ELISA. The results shown are the means ± the SD of four pooled experiments with three to six mice per group (∗, P < 0.05). (B) Administration of IL-18 to SCID mice enhances NK activity (effector/target ratio = 25) on day 16 postinfection of T. gondii. The data shown are representative of three independent experiments with three mice per group. (C) Effect of administration of IL-18 on NK cell numbers during toxoplasmosis. On day 16 postinfection, PECs and splenocytes from SCID mice treated with IL-18 or PBS were prepared and counted, and FACS analysis was performed to measure the percentage of NK cells. The data shown are the means ± the SD of three pooled experiments with three mice per group (∗, IL-18-treated mice had significantly more NK cells in the peritoneal cavity than PBS control mice; P < 0.05).

FIG. 6

IL-18-mediated resistance is dependent on endogenous IL-12. (A) SCID mice were infected with T. gondii and treated with IL-18 or PBS alone or in combination with anti-IL-12 or rat IgG. The treatment with anti-IL-12 resulted in complete abrogation of serum levels of IL-12p40 on days 3, 5, and 7 postinfection. The serum levels of IFN-γ on day 5 postinfection were measured by ELISA (A), and the percentage of infected PECs on day 7 postinfection calculated (B) as described in Materials and Methods. The data shown are the means ± the SD of the pooled data from three experiments with three to five mice per group. (C) Effect of depletion of endogenous IL-12 on the survival of SCID mice treated with IL-18. Similar results were observed in a repeat experiment with four mice per group.

FIG. 7

IL-18-mediated resistance to T. gondii is dependent on NK cells and IFN-γ. (A) SCID mice (n = 8) were infected T. gondii and treated with IL-18 or PBS alone or in combination with rabbit IgG or rabbit anti-asialoGM1 as described in Materials and Methods, and survival was monitored. Similar results were observed in a repeat experiment. (B) SCID mice were infected with T. gondii and treated with IL-18 or PBS alone or in combination with anti-IFN-γ or rat IgG as described in Materials and Methods, and the percentage of infected PECs on day 7 postinfection was estimated. The data shown are the means ± the SD of three pooled experiments with three mice per group.