Induction of Antimicrobial Pathways during Early-Phase Immune Response to Salmonella spp. in Murine Macrophages: Gamma Interferon (IFN-γ) and Upregulation of IFN-γ Receptor Alpha Expression Are Required for NADPH Phagocytic Oxidase gp91-Stimulated Oxidative Burst and Control of Virulent Salmonella spp.

Over a number of years, Salmonella enterica serovar Typhimurium infection of mice has been used as a model of human typhoid, which remains a disease of global importance. Mutants of S. enterica serovar Typhimurium that are unable to survive in murine macrophages are avirulent (10), and this has stimulated a great deal of interest in the nature of the killing pathways employed by murine macrophages, including the generation of reactive nitrogen species via the inducible nitric oxide synthase pathway and reactive oxygen species (ROS) via the NADPH phagocytic oxidase system. Previous studies have suggested that the oxidative burst is an important killing pathway employed by murine macrophages infected with salmonellae. Mutations in the S. enterica serovar Typhimurium recombination genes induce avirulence due to an inability of the salmonellae to repair DNA following exposure to the oxidative burst in J774 cells (2). Other studies have suggested that S. enterica serovar Typhimurium utilizes superoxide dismutase to protect periplasmic membranes against the effect of peroxynitrite formation and that sodC mutants are susceptible to peroxynitrite killing and are attenuated (5). Recent reports have indicated that reactive oxygen species (ROS) precede inducible nitric oxide synthase when knockout mice are challenged with S. enterica serovar Typhimurium, and it has been suggested that the timing of these responses is augmented by gamma interferon (IFN-γ) (24, 32). The generation of ROS occurs via a membrane-bound flavocytochrome b558, consisting of two phagocytic oxidase components (gp91phox and p22phox) and four cytosolic components, p40phox, 47phox, p67phox, and a GTP-binding Rac protein. During activation, the cytosolic components translocate to the site of gp91phox/p22phox on the phagosomal membrane, forming a functional enzyme complex which generates ROS by catalyzing electron transfer from NADPH to molecular oxygen (21, 31). NADPH phagocytic oxidase (NADPHphox) is active in murine macrophages infected with S. enterica serovar Typhimurium (5), and greater numbers of S. enterica serovar Typhimurium are recovered from the livers and spleens of gp91phox−/− C57BL/6 mice than from wild-type mice (24). Furthermore, a more recent study suggested that Salmonella pathogenicity island 2 genes are required for evasion of the murine macrophage oxidative burst by preventing oxidase trafficking (32). Therefore, wild-type salmonellae may survive longer in macrophages due to their ability to evade ROS via Salmonella pathogenicity island 2 genes, but the NADPHphox system ultimately controls the early phase of infection and possibly prevents it from overwhelming the host immune response. In the animal studies mentioned above, murine macrophages would have been stimulated by cytokines produced by infected cells at the site of invasion while ex vivo macrophages were stimulated with 20 U of IFN-γ per ml (24). Lower levels of IFN-γ are produced by natural killer cells early in infection, while the most productive source of IFN-γ comes from T lymphocytes active later in infection (19). Lipopolysaccharide and IFN-γ synergistically stimulate the signal-transducing activator of transcription 1 (STAT1) signaling pathway in murine macrophage-like RAW 264.7 cells (14). In vivo studies have also shown the importance of IFN-γ in mediating responses to lipopolysaccharide in mice, especially in the lethal shock reaction (Schwartzman reaction) (15). However, none of these studies elucidated the full effect of IFN-γ in terms of the generation of ROS during the early phase of infection with virulent or avirulent salmonellae. The aim of the work described here was to investigate the oxidative burst in Salmonella-infected J774.2 cells and to determine the effect of IFN-γ on this killing pathway in the absence of an effective inducible nitric oxide synthase pathway during infection of J774.2 cells with virulent and avirulent S. enterica serovar Typhimurium. Furthermore, the study was designed to ascertain why ROS are important in Salmonella control early in infection compared to later control by inducible nitric oxide synthase.