Oxidative innate immune defenses by Nox/Duox family NADPH oxidases

Abstract

The importance of reactive oxygen species (ROS) in innate immunity was first recognized in professional phagocytes undergoing a 'respiratory burst'upon activation. This robust oxygen consumption is related to a superoxide-generating enzyme, the phagocytic NADPH oxidase (Nox2-based or phox). The oxidase is essential for microbial killing, since patients lacking a functional oxidase suffer from enhanced susceptibility to microbial infections. ROS derived from superoxide attack bacteria in the isolated niche of the neutrophil phagosome. The oxidase is electrogenic, alters ion currents across membranes, induces apoptosis, regulates cytokine production, influences gene expression, and promotes formation of extracellular traps. Recently, new homologues of Nox2 were discovered establishing the Nox family of NADPH oxidases that encompasses seven members. Nox1 is highly expressed in the colon epithelium, and can be induced by LPS or IFN- gamma. Nox4 was implicated in innate immunity since LPS induces Nox4-dependent ROS generation. Duox1 and Duox2 localize to the apical plasma membrane of epithelial cells in major airways, salivary glands, and the gastrointestinal tract, and provide extracellular hydrogen peroxide to lactoperoxidase to produce antimicrobial hypothiocyanite ions. Th1 and Th2 cytokines regulate expression of dual oxidases in human airways and may thereby act in host defense or in proinflammatory responses.

Figure 1. Structures of Nox/Duox family NADPH oxidases

All NADPH oxidase catalytic components have similar structural elements required for transmembrane electron transfer from the cytosol to molecular oxygen: the C-terminal intracellular (IC) tails containing NADPH and FAD-binding sites and six transmembrane segments anchoring two heme groups. In addition, Nox5 has an N-terminal extension containing four EF-hands responsible for calcium binding. The dual oxidases have an additional transmembrane helix, an extracellular (EC) N-terminal domain with peroxidase homology, and two EF-hands within their first intracellular loop.

Figure 2. Activated complexes of multi-component Nox1- and Nox2-based NADPH oxidases

The Nox1 and Nox2(gp91phox) flavocytochromes form heterodimeric complexes with a common p22phox chain. Both oxidases are regulated by homologous organizer (Noxo1 or p47phox) and activator (Noxa1 or p67phox) proteins, and require GTP-bound Rac. The cytosolic subunits of Nox2 (p47phox, p67phox and p40phox) are preassembled in the cytosol and translocate to the flavocytochrome upon activation. In resting cells, Rac is found in a GDP-bound state stabilized by RhoGDI. When activated, both oxidases produce superoxide anions. Nox1 is localized to the plasma membrane of colon epithelial cells and produces superoxide into the extracellular space, whereas Nox2 is assembled and activated on phagosomes of phagocytic cells.

Figure 3. Oxygen metabolites of phagocytes

A variety of reactive oxygen species are formed inside the phagosome. The phagocyitic NADPH oxidase (Nox2-based) produces intraphagosomal superoxide by consuming cytosolic NADPH and transporting electrons across the membrane. The superoxide anions can be further dismutated into hydrogen peroxide by superoxide dismutase or converted into peroxynitrite by nitric oxide. Catalase dismantles H2O2 into water and oxygen. Myeloperoxidase catalyzes formation of hypochlorous acid (HOCl) from chloride and H2O2. See text for further details.

Figure 4. The neutrophil phagosome

Upon engulfment of bacteria into the neutrophil phagosome, granules fuse with the phagosome and the phagocytic NADPH oxidase is assembled and activated on the phagosomal membrane. By transporting electrons from cytosolic NADPH, the phagosomal membrane depolarizes as superoxide is produced in the phagosome lumen. Superoxide gives rise to the whole spectrum of reactive oxygen species that are highly reactive and attack bacteria. The Nox2-generated depolarization drives protons and potassium ions into the phagosome. The protons maintain a neutral pH in the phagosome required for optimal protease activity and sustained oxidase function. Potassium ions liberate and activate latent proteases from the granule matrix, allowing them to attack and destroy bacteria.

Figure 5. The Duox/Lactoperoxidase/Thiocyanate antimicrobial system in human airways

Microbicidal hypothiocyanite anions (OSCN-) are formed in the airway surface liquid (ASL) by lactoperoxidase (LPO) through hydrogen peroxide-mediated oxidation of thiocyanate (SCN-). Lactoperoxidase is produced in serous acini of submucosal glands of the airways and transported into the ASL. Dual oxidases (Duox) are localized to the apical plasma membrane of airway epithelial cells, releasing hydrogen peroxide into the ASL, thereby providing the most labile component of the H2O2/LPO/SCN system. The sodium/iodide symporter (NIS) transports thiocyanate into the epithelial cells, while at least three different transporters can deliver thiocyanate into the ASL: calcium-dependent chloride channels (CaCC), pendrin, and the cystic fibrosis conductance regulator (CFTR). Under inflammatory conditions Th1 (IFN-γ) and Th2 (IL-4, IL-13) cytokines induce higher expression of the transporters and Duox. Goblet cells found in the airway epithelium produce and secrete mucin, the major component of mucus.