Events associated with phagocytolytic activity of PMN cells (Homo sapiens)
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Description
When neutrophils engulf bacteria they enclose them in small vacuoles (phagosomes) into which superoxide is released by activated NADPH oxidase (NOX2) on the internalized neutrophil membrane. The directional nature of NOX2 activity creates a charge imbalance that must be counteracted to prevent depolarization of the membrane and the shutdown of activity (Winterbourn CC et al. 2016). Also, protons are produced in the cytosol and consumed in the external compartment (for example, the phagosome) through the dismutation of superoxide. Both situations are largely overcome by a balancing flow of protons transported by voltage-gated proton channels, primarily VSOP/HV1, which are activated in parallel with the oxidase (Demaurex N & El Chemaly A 2010; El Chemaly A et al. 2010; Petheo GL et al. 2010; Kovacs I et al. 2014; Henderson LM et al. 1987, 1988). The pH of the phagosome is regulated by these activities. In contrast to the phagosomes of macrophages, in which pH drops following particle ingestion, neutrophil phagosomes remain alkaline during the period that the oxidase is active. Until recently, their pH has been accepted to lie between 7.5 and 8. However, in a 2015 study using a probe that is more sensitive at higher pH, an average pH closer to 9 was measured in individual phagosomes (Levine AP et al. 2015).
The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase (MPO) to generate other oxidants, including the highly microbicidal species such as hypochlorous acid (Winterbourn CC et al. 2013, 2016). View original pathway at Reactome.</div>
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phagocytesThe 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.
Annotated Interactions
MPO is a heme enzyme that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. MPO reacts with hydrogen peroxide, which is produced by stimulated neutrophils, to form the redox intermediate compound I (Winterbourn CC et al 2006; Davies MJ 2011; Pattison DI et al. 2012). Compound I is strongly oxidizing and reacts with a variety of substrates such as halide and pseudo-halide ions to produce hypohalous acids (HOX where X = Cl, Br, SCN). Its main physiological substrate is assumed to be chloride, which undergoes a two-electron oxidation to form hypochlorous acid (HOCl) (Winterbourn CC et al 2006; Davies MJ 2001; Pattison DI et al. 2012).
The basic structure of bacterial peptidoglycan (PGN) contains a carbohydrate backbone of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which can be a target for HOCl -mediated oxidation (van Heijenoort J et al. 2001; Davies MJ 2011).
The basic structure of bacterial peptidoglycan (PGN) contains a carbohydrate backbone of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which can be a target for HOCl -mediated oxidation (van Heijenoort J et al. 2001; Davies MJ 2011).
All the members of XPO family catalyze a similar multi-step reaction by oxidizing the heme iron in the catalitic site from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical (Furtmüller PG et al. 2006; Davies MJ et al. 2008; Gumiero A et al. 2011; Bafort F et al. 2014). The classic peroxidases catalytic cycle begins in the presence of H2O2 which reacts rapidly and reversibly with the native state of peroxidases (enzyme:Fe(III) state). Two electrons transfer from the native enzyme:Fe(III) to H2O2 generates a ferryl pi-cation-radical (E-Fe(IV)=O.+pi) intermediate named Compound I and reduces H2O2 into water. In the presence of a halogen (Cl-, Br-, or I-) or a pseudohalogen (SCN-), Compound I is reduced back to its native enzymatic form through a two-electron transfer while the (pseudo)halogen is oxidized into a hypo(pseudo)halide. Hypo(pseudo)halides are powerful oxidants with antimicrobial activity. Alternatively, Compound I is also capable of oxidizing multiple organic and inorganic molecules (AH2) by two successive sequential one-electron-transitions generating their corresponding radicals (.AH) and the peroxidase intermediate Compound II (enzyme:Fe(IV)=O) and the native enzyme:Fe(III), respectively (Furtmüller PG et al. 2006; Zederbauer M et al. 2007; Davies MJ et al. 2008; Gumiero A et al. 2011; Bafort F et al. 2014).
The Reactome event describes the halogenation cycle where LPO-derived Compound I catalyzes the oxidation of thiocyanate ion (SCN-) to hypothiocyanite ion (OSCN-).
In the presence of hydrogen peroxide (H2O2) MPO can catalyze both one- and two-electron oxidations (Davies MJ 2011). Generally, ferric or native MPO reacts with H2O2 forming intemediate compound I (MPO-I). This redox intermediate is known to oxidize halides via a single two-electron reaction to produce the respective hypohalous acids and regenerate the native enzyme. Alternatively, stepwise reduction of compound I by two donor-derived electrons produces compound II (MPO-II) and subsequently the resting ferric state. Mechanistic studies have demonstrated that nitrite acts as an electron donor and reacts with compounds I to yield nitrogen dioxide (NO2) and compaund II. Subsequently, an additional nitrite molecule reduces compound II by one electron to regenerate a native state of MPO and to produce a second NO2 molecule (Burner U et al. 2000; Cape JC & Hurst JK 2009).