Cooperation between reactive oxygen and nitrogen intermediates in killing of Rhodococcus equi by activated macrophages

Abstract

Rhodococcus equi is a facultative intracellular bacterium of macrophages which can infect immunocompromised humans and young horses. In the present study, we examine the mechanism of host defense against R. equi by using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-gamma), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO(-)), which kills R. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS(-/-)) or the oxidative burst (gp91(phox-/-)) are more susceptible to lethal R. equi infection and display higher bacterial burdens in their livers, spleens, and lungs than wild-type mice. These in vivo observations, which implicate both nitric oxide (NO) and superoxide (O(2)(-)) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays, R. equi remains fully viable following prolonged exposure to high concentrations of either nitric oxide or superoxide, indicating that neither compound is sufficient to mediate bacterial killing. In contrast, brief exposure of bacteria to ONOO(-) efficiently kills virulent R. equi. The intracellular killing of bacteria in vitro by activated macrophages correlated with the production of ONOO(-) in situ. Inhibition of nitric oxide production by activated macrophages by using N(G)-monomethyl-L-arginine blocks their production of ONOO(-) and weakens their ability to control rhodococcal replication. These studies indicate that peroxynitrite mediates the intracellular killing of R. equi by IFN-gamma-activated macrophages.

FIG. 1

R. equi infection in IFN-γ^−/−, gp91^phox−/−, and iNOS^−/− mice. C57BL/6 (n = 7), IFN-γ^−/− (n = 8), gp91^phox−/− (n = 5), and iNOS^−/− (n = 7) mice were infected intravenously with virulent R. equi (3 × 10⁶ cells). Data are taken from a single experiment in which all groups were included in parallel. The data are representative of three separate experiments including a total of ≥18 mice per group.

FIG. 2

CFU recovered from organs of IFN-γ^−/− and C57BL/6 mice following R. equi infection. IFN-γ^−/− mice (squares) and wild-type littermates (circles) were infected intravenously with virulent R. equi (9 × 10⁶ cells). Bacterial burdens in the liver (top), spleen (middle), and lung (bottom) were determined at 0, 3, and 7 days postinfection. Each data point represents CFU (± standard deviation) of five mice per group. ∗, significantly different from control mice (P ≤ 0.008; Mann-Whitney U test).

FIG. 3

R. equi exposure to NO released by DEA/NO or O₂⁻ generated by X-XO. (A) R. equi (10⁶ cells) was exposed to 1 mM inactive or active DEA/NO or 1 U of xanthine oxidase and 5 mM xanthine (hatched bar). Following a 30-min incubation at 37°C, bacteria were centrifuged, resuspended, and reexposed to X-XO or DEA/NO for a second and third time. Control samples were incubated in phosphate buffer alone. Cell viability was determined by a CFU assay. The data are representative of three separate experiments. (B) The amount of NO released by DEA/NO was determined by using the Griess reagent (9) following the incubation of 1, 2.5, or 5 mM DEA/NO in phosphate buffer at 37°C for 30 min. (C) Increasing amounts of xanthine oxidase were added to phosphate buffer containing 5 mM xanthine in the presence of cytochrome c. The amount of O₂⁻ generated was determined following a 30-min incubation at 37°C by measuring the reduction of cytochrome c (20). Xanthine oxidase which had been boiled for 15 min was used as a control.

FIG. 4

Exposure of R. equi to ONOO⁻. R. equi (10⁶ cells) in 50 mM potassium phosphate buffer was exposed to 1 mM active peroxynitrite. Following a 1-min incubation at 37°C, bacteria were centrifuged, resuspended, and reexposed to ONOO⁻ for a second and third time. Control (untreated) samples were incubated in phosphate buffer alone or with inactive ONOO⁻ which had completely decomposed prior to addition to bacteria. Cell viability was determined by a CFU assay. Data shown are means (± standard deviation) of triplicate samples and are representative of three independent experiments. ∗, significantly different from untreated samples (P < 0.001; Student's t test).

FIG. 5

Quantitation of R. equi growth within activated wild-type, iNOS^−/−, and gp91^phox−/− macrophages. BMMϕ were either untreated or activated overnight with 100 U of IFN-γ/ml plus 50 ng of LPS/ml before infection with R. equi at an MOI of between 5 and 10 bacteria per macrophage. At 1, 24, 48, and 72 h postinfection, parallel macrophage monolayers were washed, fixed, and stained for fluorescence microscopy. (A) R. equi replication inside untreated and activated BALB/c BMMϕ was quantitated. Bacterial growth was expressed as both the total number of bacteria per 200 macrophages (solid lines, left axis) and the number of macrophages containing ≥10 bacteria (dashed lines, right axis). Each data point shown represents the mean (± standard error of the mean) of three separate experiments done in triplicate. At all time points (24 to 72 h), bacterial numbers in activated macrophages were statistically different from those in untreated macrophages (P ≤ 0.001; Mann-Whitney U test). (B) The replication of R. equi inside untreated or activated macrophages from iNOS^−/− or gp91^phox−/− mice was quantitated. Data represent the number of macrophages infected with 10 or more bacteria. Each point represents the mean (± standard error of the mean) of ≥3 experiments counted in triplicate except for 72-h iNOS−/−, which represents a triplicate sample from a single experiment.

FIG. 6

Peroxynitrite formation by activated, R. equi-infected macrophages. (A) Monolayers of BMMϕ were either untreated or activated overnight with 100 U of IFN-γ/ml and 50 ng of LPS/ml in the presence or absence of NMLA. R. equi was added to monolayers at an MOI of 50:1 in the presence of 1 mM DHR for 15 min at 37°C. Uninfected cells were treated similarly but were not infected with R. equi. Monolayers were analyzed for rhodamine fluorescence and photographed on an inverted microscope. (B) The number of cells positive for rhodamine fluorescence was quantitated and expressed as a percent positive. These data are representative of three independent experiments. (Inset) The intracellular growth of R. equi in resident cells (closed circles), activated cells (open circles), or activated cells treated with 500 μM NMLA (squares). Bacterial numbers are statistically different between macrophages activated with or without NMLA at 24 and 48 h (P ≤ 0.04; Student's t test).

FIG. 6

Peroxynitrite formation by activated, R. equi-infected macrophages. (A) Monolayers of BMMϕ were either untreated or activated overnight with 100 U of IFN-γ/ml and 50 ng of LPS/ml in the presence or absence of NMLA. R. equi was added to monolayers at an MOI of 50:1 in the presence of 1 mM DHR for 15 min at 37°C. Uninfected cells were treated similarly but were not infected with R. equi. Monolayers were analyzed for rhodamine fluorescence and photographed on an inverted microscope. (B) The number of cells positive for rhodamine fluorescence was quantitated and expressed as a percent positive. These data are representative of three independent experiments. (Inset) The intracellular growth of R. equi in resident cells (closed circles), activated cells (open circles), or activated cells treated with 500 μM NMLA (squares). Bacterial numbers are statistically different between macrophages activated with or without NMLA at 24 and 48 h (P ≤ 0.04; Student's t test).