Cooperation between Reactive Oxygen and Nitrogen Intermediates in Killing ofRhodococcus equiby Activated Macrophages

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Abstract
Rhodococcus equiis 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 againstR. equiby using a murine model. We show that bacterial killing is dependent upon the presence of gamma interferon (IFN-γ), which activates macrophages to produce reactive nitrogen and oxygen intermediates. These two radicals combine to form peroxynitrite (ONOO−), which killsR. equi. Mice deficient in the production of either the high-output nitric oxide pathway (iNOS−/−) or the oxidative burst (gp91phox−/−) are more susceptible to lethalR. equiinfection 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 (O2−) in bacterial killing, were reexamined in cell-free radical-generating assays. In these assays,R. equiremains 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 virulentR. 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 usingNG-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 ofR. equiby IFN-γ-activated macrophages.