ROS and RNS production in phagocytes

The presence of RONS characterized by a relatively low reactivity, such as H2O2, O2˙− or NO, has no deleterious effect on biological environment (Attia SM 2010; Weidinger A & and Kozlov AV 2015). Their activity is controlled by endogenous antioxidants (both enzymatic and non-enzymatic) that are induced by oxidative stress. However the relatively low reactive species can initiate a cascade of reactions to generate more damaging “secondary” species such as hydroxyl radical (•OH), singlet oxygen or peroxinitrite (Robinson JM 2008; Fang FC et al. 2004). These "secondary" RONS are extremely toxic causing irreversible damage to all classes of biomolecules (Weidinger A & and Kozlov AV 2015; Fang FC et al. 2004; Kohchi C et al. 2009; Gostner JM et al. 2013; Vatansever F et al. 2013).

Although macrophages and neutrophils use similar mechanisms for the internalization of targets, there are differences in how they perform phagocytosis and in the final outcome of the process (Tapper H & Grinstein S 1997; Vierira OV et al. 2002). Once formed, the phagosome undergoes an extensive maturation process whereby it develops into a microbicidal organelle able to eliminate the invading pathogen. Maturation involves re-modeling both the membrane of the phagosome and its luminal contents (Vierira OV et al. 2002). In macrophages, phagosome formation and maturation follows a series of strictly coordinated membrane fission/fusion events between the phagosome and compartments of the endo/lysosomal network gradually transforming the nascent phagosome into a phagolysosome, a degradative organelle endowed with potent microbicidal properties (Zimmerli S et al. 1996; Vierira OV et al. 2002). Neutrophils instead contain a large number of preformed granules such as azurophilic and specific granules that can rapidly fuse with phagosomes delivering antimicrobial substances (Karlsson A & Dahlgren C 2002; Naucler C et al. 2002; Nordenfelt P and Tapper H 2011). Phagosomal pH dynamics may also contribute to the maturation process by regulating membrane traffic events. The microbicidal activity of macrophages is characterized by progressive acidification of the lumen (down to pH 4–5) by the proton pumping vATPase. A low pH is a prerequisite for optimal enzymatic activity of most late endosomal/lysosomal hydrolases reported in macrophages. Neutrophil phagosome pH regulation differs significantly from what is observed in macrophages (Nordenfelt P and Tapper H 2011; Winterbourn CC et al. 2016). The massive activation of the oxidative burst is thought to result in early alkalization of neutrophil phagosomes which is linked to proton consumption during the generation of hydrogen peroxide (Segal AW et al. 1981; Levine AP et al. 2015). Other studies showed that neutrophil phagosome maintained neutral pH values before the pH gradually decreased (Jankowski A et al. 2002). Neutrophil phagosomes also exhibited a high proton leak, which was initiated upon activation of the NADPH oxidase, and this activation counteracted phagosomal acidification (Jankowski A et al. 2002).

The Reactome module describes ROS and RNS production by phagocytic cells. The module includes cell-type specific events, for example, myeloperoxidase (MPO)-mediated production of hypochlorous acid in neutrophils. It also highlights differences between phagosomal pH dynamics in neutrophils and macrophages. The module describes microbicidal activity of selective RONS such as hydroxyl radical or peroxynitrite. However, detection of any of these species in the phagosomal environment is subject to many uncertainties (Nüsse O 2011; Erard M et al. 2018). The mechanisms by which reactive oxygen/nitrogen species kill pathogens in phagocytic immune cells are still not fully understood.