Distinct roles of reactive nitrogen and oxygen species to control infection with the facultative intracellular bacterium Francisella tularensis

Abstract

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are important mediators of the bactericidal host response. We investigated the contribution of these two mediators to the control of infection with the facultative intracellular bacterium Francisella tularensis. When intradermally infected with the live vaccine strain F. tularensis LVS, mice deficient in production of RNS (iNOS(-/-) mice) or in production of ROS by the phagocyte oxidase (p47(phox-/-) mice) showed compromised resistance to infection. The 50% lethal dose (LD(50)) for iNOS(-/-) mice was <20 CFU, and the LD(50) for p47(phox-/-) mice was 4,400 CFU, compared to an LD(50) of >500,000 CFU for wild-type mice. The iNOS(-/-) mice survived for 26.4 +/- 1.8 days, and the p47(phox-/-) mice survived for 10.1 +/- 1.3 days. During the course of infection, the serum levels of gamma interferon (IFN-gamma) and interleukin-6 were higher in iNOS(-/-) and p47(phox-/-) mice than in wild-type mice. Histological examination of livers of iNOS(-/-) mice revealed severe liver pathology. Splenocytes obtained 5 weeks after primary infection from antibiotic-treated iNOS(-/-) mice showed an in vitro recall response that was similar in magnitude and greater secretion of IFN-gamma compared to cells obtained from wild-type mice. In summary, mice lacking expression of RNS or ROS showed extreme susceptibility to infection with F. tularensis LVS. The roles of RNS and ROS seemed to be distinct since mice deficient in production of ROS showed dissemination of infection and died during the early phase of infection, whereas RNS deficiency led to severe liver pathology and a contracted course of infection.

FIG. 1.

Percentage of surviving mice after intradermal injection of F. tularensis LVS. Mice (10 mice per group) were inoculated intradermally with 2 × 10¹ CFU (□), 2 × 10³ CFU (▵), or 2 × 10⁴ CFU (○). p47^phox−/− mice that survived for 28 days were sacrificed, and no bacteria were found in the livers or spleens. (A) iNOS ^−/− mice. (B) p47 ^phox−/− mice.

FIG. 2.

Growth curves for F. tularensis LVS in the skin, spleens, and livers of iNOS^−/− and iNOS^+/+ mice (A) and p47^phox−/− and p47^phox+/+ mice (B). Mice were inoculated intradermally with 5 × 10² CFU of F. tularensis LVS, and the numbers of bacteria (CFU per organ sample) were determined at different times. The means ± standard errors of the means for five mice per group and time are shown. One asterisk indicates that the P value is <0.05, two asterisks indicate that the P value is <0.01, and three asterisks indicate that the P value is <0.001.

FIG. 3.

After inoculation of 5 × 10⁴ CFU of F. tularensis LVS, the serum liver enzymes s-ALT and s-AST were monitored in iNOS^−/− and iNOS^+/+ mice (A) and in p47^phox−/− and p47^phox+/+ mice (B). The means ± standard errors of the means for five mice per group and time are shown. An asterisk indicates that the P value is <0.05.

FIG. 4.

Representative liver histopathology for different strains of mice killed on day 6 after intradermal infection with 5 × 10⁴ CFU of F. tularensis LVS. (A) Liver from an iNOS^+/+ mouse, showing a medium-size focal accumulation of mixed inflammatory cells and hepatic necrosis (the scores were 1, 1, and 1, as shown in Table 3). (B) Liver from an iNOS^−/− mouse, showing the presence of numerous small to medium-size inflammatory infiltrates throughout the entire liver section (the scores were 4, 4, and 4). (C and D) Livers from p47^phox+/+ (C) and p47^phox−/− (D) mice, showing the presence of small to medium-size focal accumulations of mixed inflammatory cells and occasional hepatic necrosis of some severity (in both cases the scores were 2, 2, and 2). Hematoxylin and eosin staining was used. Bars = 20 μm.

FIG. 5.

Growth curves for F. tularensis LVS in the skin, livers, and spleens of leaky iNOS^−/− mice and iNOS^+/+ 129 mice after primary intradermal infection with 5 × 10² F. tularensis LVS cells. The means ± standard errors of the means for five mice per group and time are shown. One asterisk indicates that the P value is <0.05, and three asterisks indicate that the P value is <0.001.

FIG. 6.

iNOS^+/+ mice (solid bars), iNOS^−/− mice (striped bars), p47^phox+/+ mice (gray bars), and p47^phox−/− mice (open bars) were inoculated with 5 × 10⁴ CFU of F. tularensis LVS. On different days after inoculation mice were sacrificed, and splenocytes were prepared and analyzed for NO production (A) and ROS production (B). The values are the means ± standard errors of the means for three mice per group and time. Samples were analyzed in triplicate. No values are given for iNOS^+/+ and iNOS^−/− mice on day 4. One asterisk indicates that the P value is <0.05, two asterisks indicate that the P value is <0.01, and three asterisks indicate that the P value is <0.001. RLU, relative light units.