Nitric oxide (NO) generated by inducible NO synthase 2 (NOS2) affects cellular iron homeostasis, but the underlying molecular mechanisms and implications for NOS2-dependent pathogen control are incompletely understood. In this study, we found that NO up-regulated the expression of ferroportin-1 (Fpn1), the major cellular iron exporter, in mouse and human cells. Nos2 −/− macrophages displayed increased iron content due to reduced Fpn1 expression and allowed for an enhanced iron acquisition by the intracellular bacterium Salmonella typhimurium . Nos2 gene disruption or inhibition of NOS2 activity led to an accumulation of iron in the spleen and splenic macrophages. Lack of NO formation resulted in impaired nuclear factor erythroid 2-related factor-2 (Nrf2) expression, resulting in reduced Fpn1 transcription and diminished cellular iron egress. After infection of Nos2 −/− macrophages or mice with S. typhimurium , the increased iron accumulation was paralleled by a reduced cytokine (TNF, IL-12, and IFN-γ) expression and impaired pathogen control, all of which were restored upon administration of the iron chelator deferasirox or hyperexpression of Fpn1 or Nrf2. Thus, the accumulation of iron in Nos2 −/− macrophages counteracts a proinflammatory host immune response, and the protective effect of NO appears to partially result from its ability to prevent iron overload in macrophages

The contribution of the NADPH phagocyte oxidase (phox) and inducible nitric oxide (NO) synthase (iNOS) to the antimicrobial activity of macrophages for Salmonella typhimurium was studied by using peritoneal phagocytes from C57BL/6, congenic gp91 phox −/ −, iNOS −/ −, and doubly immunodeficient phox −/ − iNOS −/ − mice. The respiratory burst and NO radical (NO·) made distinct contributions to the anti- Salmonella activity of macrophages. NADPH oxidase–dependent killing is confined to the first few hours after phagocytosis, whereas iNOS contributes to both early and late phases of antibacterial activity. NO-derived species initially synergize with oxyradicals to kill S . typhimurium , and subsequently exert prolonged oxidase-independent bacteriostatic effects. Biochemical analyses show that early killing of Salmonella by macrophages coincides with an oxidative chemistry characterized by superoxide anion (O 2 · − ), hydrogen peroxide (H 2 O 2 ), and peroxynitrite (ONOO − ) production. However, immunofluorescence microscopy and killing assays using the scavenger uric acid suggest that peroxynitrite is not responsible for macrophage killing of wild-type S . typhimurium . Rapid oxidative bacterial killing is followed by a sustained period of nitrosative chemistry that limits bacterial growth. Interferon γ appears to augment antibacterial activity predominantly by enhancing NO· production, although a small iNOS-independent effect was also observed. These findings demonstrate that macrophages kill Salmonella in a dynamic process that changes over time and requires the generation of both reactive oxidative and nitrosative species.

The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91 phox −/ −, iNOS −/ −, and congenic wild-type mice. Although both gp91 phox −/ − and iNOS −/ − mice demonstrated increased susceptibility to infection with S . typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91 phox −/ − and iNOS −/ − mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91 phox −/ − mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91 phox −/ − mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS −/ − mice only beyond the first week of infection. Influx of inflammatory CD11b + cells, granuloma formation, and serum interferon γ levels were unimpaired in iNOS −/ − mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.