Unlocking the power of NOX2: A comprehensive review on its role in immune regulation

Abstract

Reactive oxygen species (ROS) are a family of highly reactive molecules with numerous, often pleiotropic functions within the cell and the organism. Due to their potential to destroy biological structures such as membranes, enzymes and organelles, ROS have long been recognized as harmful yet unavoidable by-products of cellular metabolism leading to "oxidative stress" unless counterbalanced by cellular anti-oxidative defense mechanisms. Phagocytes utilize this destructive potential of ROS released in high amounts to defend against invading pathogens. In contrast, a regulated and fine-tuned release of "signaling ROS" (sROS) provides essential intracellular second messengers to modulate central aspects of immunity, including antigen presentation, activation of antigen presenting cells (APC) as well as the APC:T cell interaction during T cell activation. This regulated release of sROS is foremost attributed to the specialized enzyme NADPH-oxidase (NOX) 2 expressed mainly in myeloid cells such as neutrophils, macrophages and dendritic cells (DC). NOX-2-derived sROS are primarily involved in immune regulation and mediate protection against autoimmunity as well as maintenance of self-tolerance. Consequently, deficiencies in NOX2 not only result in primary immune-deficiencies such as Chronic Granulomatous Disease (CGD) but also lead to auto-inflammatory diseases and autoimmunity. A comprehensive understanding of NOX2 activation and regulation will be key for successful pharmaceutical interventions of such ROS-related diseases in the future. In this review, we summarize recent progress regarding immune regulation by NOX2-derived ROS and the consequences of its deregulation on the development of immune disorders.

Keywords: Antigen presentation; Autoimmunity; Immune regulation; NOX2.

Graphical abstract

Fig. 1

Cellular sources of sROS generation in leukocytes a) Mitochondrial sROS originate in the course of normal respiration from the electron transport chain located at the mitochondrial inner membrane. During this electron transport process, electrons are transferred to molecular oxygen (O₂). However, single electrons may be transferred to oxygen generating the radical O₂^•-. Spontaneous or enzymatic dismutation into H₂O₂ enables transmembrane transfer into the cytosol. Complex I (NADH dehydrogenase) or complex III (Ubiquinone-cytochrome c reductase) of the electron transport chain have been described to be the main sources of this "electron escape". b) Structure of the activated NOX2 complex, comprising the integral membrane proteins NOX2/gp91^phox, p22^phox and associated components p40^phox, p47^phox, p67^phox and GTP-bound Rac1 or Rac2. Here, 2 electrons are transferred from cytosolic NADPH to extra-cytosolic oxygen (O₂), generating O₂^•- radicals which may dismutate into H₂O₂ or give rise to further ROS specimen as indicated. Figure created with BioRender.com.

Fig. 2

Receptor-mediated Activation of the NOX2 complex The catalytic core of the NOX2 complex consists of the integral membrane proteins NOX2/gp91^phox and p22^phox which reside in the cell membrane and in membranes of endo-/lysosomal compartments. Upon activation events like phosphorylation - most prominently mediated by members of the PKC-family - the cytosolic subunits p40^phox, p47^phox and p67^phox associate and translocate to the catalytic core to form the functional NOX2 complex. Independently, and mediated by exchange of GDP for GTP, the small GTPases Rac1 or Rac2 translocate to the membrane and associate with the NOX2 complex. Receptor classes indicated on the left are able to activate the NOX2 complex directly. Several other receptor classes as indicated on the right lead to priming of the cell, which needs to be fully activated by a second stimulus. Priming events include transcriptional upregulation, partial phosphorylation and cellular redistribution of subunits of the NOX2 complex. Figure created with BioRender.com.

Fig. 3

NOX2 modulates activation of T cells by antigen presenting cells Priming of naïve T cells by antigen presenting cells (APC) relies on the presence of 3 signals: The peptide:MHC complex (pMHC) recognized by the T cell receptor (TCR) directly (signal 1), additional costimulatory ligands of the B7-family recognized by the CD28 co-receptor (signal 2) and the cytokine milieu secreted by the APC during this interaction (signal 3). Endo- or phagocytosis of exogenous antigens is typically associated with phagocytic receptor-mediated activation of the NOX2 complex at endo-/lysosomal compartments. NOX2-generated sROS retard the proteolysis of antigens by inhibiting proteases (either directly or by preventing endo-/lysosomal acidification) (1) as well as by stabilizing disulfide bridges within the antigens (2). By activating the receptor DNGR1 (3), NOX2-derived sROS facilitate endo-/lysosomal rupture and escape of antigens into the cytosol, promoting antigen presentation on MHC I complexes (cross presentation). While signal 1 is increased, NOX2 does not induce or even decreases expression of costimulatory surface molecules (e.g., of the B7 family) (4) and inhibits the secretion of pro-inflammatory and T cell polarizing cytokines (e.g. IL-1ß, TNF, IL-12) (5). In sum, NOX2-derived sROS promote antigen presentation in the context of a tolerogenic APC phenotype due to a reduction in signals 2 and 3. Figure created with BioRender.com.

Fig. 4

Activating and inhibitory signaling of Dectin-1 Pathogen-derived β-glucans and the conserved annexin core domain bind to distinct sites on the Dectin-1 receptor. Upon binding, β-glucans induce phosphorylation at 4 specific tyrosines (Y348, Y352, Y525 & Y526) within the adaptor molecule SYK. Phosphorylation of Y525 and Y526 leads to downstream activation of the transcription factor NF-κB followed by induction of inflammatory cytokines (e.g. TNF, IL-6, IL-12) and co-stimulatory surface markers (e.g. CD40, CD80, CD86), while concomitant phosphorylation of Y348 and Y352 initiates a negative feedback loop by activation of the NOX-2 complex, generation of sROS and subsequent inhibition of NF-κB. In contrast to inflammatory β-glucan-signaling, the annexin core domain induces solely phosphorylation of Y348 and Y352. This way, only the NOX-2-mediated inhibitory signaling is initiated, and the cell develops a tolerogenic phenotype, characterized by low expression of co-stimulatory surface molecules and inflammatory cytokines as well as resistance to activation. Figure created with BioRender.com.