Coactivating signals for the hepatic lymphocyte gamma interferon response to Francisella tularensis

Abstract

The facultative intracellular bacterium Francisella tularensis is capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site of F. tularensis infection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-gamma), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-gamma when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-gamma induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-gamma production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-gamma responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type A Francisella tularensis subsp. tularensis organisms were comparable to the live attenuated vaccine strain of Francisella tularensis subsp. holarctica in their ability to induce IL-12 and IFN-gamma expression. These findings demonstrate that cells capable of mounting IFN-gamma responses to F. tularensis are resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.

FIG. 1.

Effects of recombinant coactivating cytokines on IFN-γ production by F. tularensis LVS-stimulated HMC in vitro. (A) Hepatic mononuclear cells from B6 mice were cultured with F. tularensis LVS at the indicated MOI in the presence or absence of recombinant IL-12 (50 pg/ml), and IFN-γ production was measured 24 h later by ELISA. Cells incubated in the absence of bacteria produced 68 pg of IFN-γ per ml. (B) HMC activated in vitro at an MOI of 66 were stained with APC-conjugated anti-NK1.1 and PE-conjugated anti-IFN-γ. (C) Comparison of the effects of rIL-12 and rIL-18 in F. tularensis LVS-challenged HMC cultures (MOI, 30). (D) Synergistic effects of rIL-12 and rIL-18 on IFN-γ production by HMC challenged at an MOI of 30.

FIG. 2.

Effects of neutralizing anti-cytokine antibodies on IFN-γ production by F. tularensis LVS-challenged HMC in vitro. Antibodies were added to HMC cultures just before the addition of F. tularensis LVS (MOI, 48). Antibody-treated cultures were significantly different from the IgG controls at all concentrations (P < 0.05).

FIG. 3.

Lack of IFN-γ production in vitro by HMC from mice deficient in IL-12p35. HMC from wild-type B6 mice (p35^+/+) or IL-12p35-deficient mice (p35^−/−) were challenged in vitro with F. tularensis LVS at the indicated MOI. Cells from the IL-12p35-deficient mice were also challenged with bacteria and rIL-12 (50 pg/ml). Cell culture supernatant fluids were collected after 24 h, and IFN-γ concentrations were measured by ELISA. At MOI greater than 40, the values for the p35^−/− cells were significantly different from those for the other two groups (P < 0.05).

FIG. 4.

Supplementation of HMC cultures with BMMφ. (A) A total of 150,000 HMC were added to cultures containing 30,000 BMMφ, and the cultures were challenged with F. tularensis LVS in vitro at the indicated MOI. (B and C) Neutralizing anti-IL-12 or anti-IL-18 was added to cultures just prior to challenge with F. tularensis LVS at an MOI of 61. (B) The IFN-γ values for antibody-treated cultures were significantly different from those for the IgG controls at all antibody concentrations (P < 0.05). (C) The IL-12p70 values for cultures treated with each concentration of anti-IL-12 were significantly different from those for the IgG controls (P < 0.05).

FIG. 5.

Effects of rIL-12 on IFN-γ responses to F. tularensis LVS in vivo. (A) Wild-type B6 mice were injected i.p. with the indicated doses of F. tularensis LVS, and their sera were collected 16 h later for analysis of IFN-γ or IL-12p70. (B and C) At two different F. tularensis LVS doses, one group of mice was injected with 0.5 μg of rIL-12 just prior to bacterial challenge. Either 5 × 10⁴ CFU was given to 4 mice per group (B) or 4 × 10⁵ CFU was given to 8 mice per group (C). In both cases, the result for each animal is shown, with horizontal lines representing the mean of each group. The groups receiving F. tularensis LVS plus rIL-12 (open circles) were significantly different from the groups receiving the same dose of F. tularensis LVS alone (P < 0.05).

FIG. 6.

Requirement of IL-12p35 for the induction of the IFN-γ response in F. tularensis LVS-infected mice. Wild-type (IL-12p35^+/+) B6 and IL-12p35^−/− mice were injected i.p. with 10⁶ CFU of F. tularensis LVS. Sixteen hours later, HMC were isolated, stained with fluorochrome-labeled antibodies against NK1.1, CD3, and IFN-γ, and analyzed by flow cytometry. Only cells expressing intracellular IFN-γ are shown here.

FIG. 7.

IFN-γ and IL-12 production by HMC challenged in vitro with F. tularensis subsp. tularensis clinical isolates KU49 and KU54. Cell culture supernatant fluids were collected after 24 h, and IFN-γ (A), IL-12p70 (B), and IL-12p40 (C) concentrations were measured by ELISA. Cells incubated in the absence of bacteria produced 110 pg of IFN-γ per ml.