Peroxynitrite, a potent macrophage-derived oxidizing cytotoxin to combat invading pathogens

Abstract

Macrophages are among the first cellular actors facing the invasion of microorganisms. These cells are able to internalize pathogens and destroy them by means of toxic mediators, many of which are produced enzymatically and have strong oxidizing capacity. Indeed, macrophages count on the NADPH oxidase complex activity, which is triggered during pathogen invasion and leads to the production of superoxide radical inside the phagosome. At the same time, the induction of nitric oxide synthase results in the production of nitric oxide in the cytosol which is able to readily diffuse to the phagocytic vacuole. Superoxide radical and nitric oxide react at diffusion controlled rates with each other inside the phagosome to yield peroxynitrite, a powerful oxidant capable to kill micro-organisms. Peroxynitrite toxicity resides on oxidations and nitrations of biomolecules in the target cell. The central role of peroxynitrite as a key effector molecule in the control of infections has been proven in a wide number of models. However, some microorganisms and virulent strains adapt to survive inside the potentially hostile oxidizing microenvironment of the phagosome by either impeding peroxynitrite formation or rapidly detoxifying it once formed. In this context, the outcome of the infection process is a result of the interplay between the macrophage-derived oxidizing cytotoxins such as peroxynitrite and the antioxidant defense machinery of the invading pathogens.

Keywords: free radicals; nitric oxide; oxidants; peroxynitrite; superoxide.

Figure 1. Regulation of iNOS expression

PAMPs recognition through TLR-4 leads to the activation of NFκB, which is translocated to the nucleus and moderately enhances the iNOS promoter activity. IFNγ signaling depends on the JAK/STAT pathway. Phosphorylated STAT1α binds to the regulatory region of the iNOS gene and also to that of the IFNγ regulatory factor (IRF-1), which in turn promotes iNOS transcription as well. When both IFNγ and a pathogen motif are present, iNOS expression is maximal.

Figure 2. NADPH oxidase assembling

The NADPH oxidase is composed of three cytosolic (p67phox, p47phox, and p40phox) and two membrane-bound subunits (gp91phox and p22phox). Activation during phagocytosis leads to phosphorylation of cytosolic components and their migration to the membrane to form an active complex. Low doses of LPS or other stimuli, such as TNFα, are not sufficient to promote the oxidase activation, but lead to the primed state, increasing the phosphorylation level of cytosolic subunits.

Figure 3. Redox biochemistry in the phagosome

The scheme shows how a parasite (e.g Trypanosoma cruzi) is engulfed and attacked with powerful oxidants in a very narrow space. NADPH oxidase is activated immediately after phagocytosis directing superoxide production towards the interior of the phagosome. Superoxide dismutation leads to the formation of hydrogen peroxide, which has a cytotoxic effect at high doses. If iNOS is active in the cytosol or in small vesicles, nitric oxide will diffuse across membranes exerting cytostatic or cytotoxic effects on the microorganism. When both superoxide and nitric oxide co-exist in the phagosome they result in peroxynitrite formation, with lethal consequences for the pathogen.

Figure 4. Intraphagosomal peroxynitrite formation

T. cruzi trypomastigotes preloaded with DHR were exposed to macrophages, and cells were washed 30 min after incubation. A, RH 123 accumulation after 2 h of infection in unstimulated (T. cruzi) or preactivated macrophages (IFNγ /LPS + T. cruzi) was determined in a fluorescence plate reader. The effect of apocynin (NADPH oxidase inhibitor) was evaluated under both conditions. RFU, relative fluorescence units. B, activated macrophages plated in slides were infected with DHR-loaded trypomastigotes (5:1, parasite:macrophage ratio). Merged DIC and fluorescence images were obtained 2 h after infection; the green fluorescence corresponds to oxidized DHR (magnification, X400). This research was originally published in J Biol Chem, Álvarez MN et al, “Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity”, J Biol Chem 2011, 286: 6627–6640. © the American Society for Biochemistry and Molecular Biology.