Structure, Activation, and Regulation of NOX2: At the Crossroad between the Innate Immunity and Oxidative Stress-Mediated Pathologies

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multisubunit enzyme complex that participates in the generation of superoxide or hydrogen peroxide (H2O2) and plays a key role in several biological functions. Among seven known NOX isoforms, NOX2 was the first identified in phagocytes but is also expressed in several other cell types including endothelial cells, platelets, microglia, neurons, and muscle cells. NOX2 has been assigned multiple roles in regulating many aspects of innate and adaptive immunity, and human and mouse models of NOX2 genetic deletion highlighted this key role. On the other side, NOX2 hyperactivation is involved in the pathogenesis of several diseases with different etiologies but all are characterized by an increase in oxidative stress and inflammatory process. From this point of view, the modulation of NOX2 represents an important therapeutic strategy aimed at reducing the damage associated with its hyperactivation. Although pharmacological strategies to selectively modulate NOX2 are implemented thanks to new biotechnologies, this field of research remains to be explored. Therefore, in this review, we analyzed the role of NOX2 at the crossroads between immunity and pathologies mediated by its hyperactivation. We described (1) the mechanisms of activation and regulation, (2) human, mouse, and cellular models studied to understand the role of NOX2 as an enzyme of innate immunity, (3) some of the pathologies associated with its hyperactivation, and (4) the inhibitory strategies, with reference to the most recent discoveries.


NOX2 Structure
The Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase complex (NOX) is a family of reactive oxygen species (ROS)-producing enzymes, first identified in the membrane of neutrophils, where it participates in non-specific host defense against microbes ingested during phagocytosis. In fact, NADPH oxidase and ROS production play a key role in host defense against microbial pathogens.
During human neutrophil stimulation, assembly of the NOX2 complex requires that p47 phox is heavily phosphorylated. The phosphorylation of p47 phox uncovers its N-terminal SH3 domain that then binds the proline-rich region in p22 phox whereas the PX (phox homology) domain of p47 phox binds 3′phosphoinositides, the products of phosphatidylinositol 3-Kinase (PI3K).
The vital role of this phosphoprotein was first described and identified in CGD patients that demonstrated a selective lack of enhanced phosphorylation in neutrophils after activation of the oxidase with phorbol myristate acetate (PMA) [9]. Several pathways are implicated in p47 phox phosphorylation, but a primary pathway involves protein kinase C (PKC) isoforms (δ, β, α, ζ) that, when activated, specifically phosphorylates key serine residues (Ser304, Ser315, Ser320, and Ser328) of p47 phox . Recently, p67 phox , alone or in combination with p40 phox , has been found to be able to potentiate the process of phosphorylation induced by several isoforms of PKC [10]. Thus, p67 phox has been suggested as (1) When activated, PKC phosphorylates Ser304, Ser315, Ser320, and Ser328 of p47 phox ; (2) the phosphorylation of p47 phox uncovers its N-terminal SH3 domain that then binds the proline-rich region (PRR) in p22 phox . The PX domain of p47 phox binds the products of PI3K; (3) the activation of GTPase Rac2 mediates the translocation of p67 phox , which associates with p47 phox to the cytochrome; (4) Rac2 directly binds to the flavocytochrome favoring the initial steps of the electron transfer reaction.
During human neutrophil stimulation, assembly of the NOX2 complex requires that p47 phox is heavily phosphorylated. The phosphorylation of p47 phox uncovers its N-terminal SH3 domain that then binds the proline-rich region in p22 phox whereas the PX (phox homology) domain of p47 phox binds 3 phosphoinositides, the products of phosphatidylinositol 3-Kinase (PI3K).
The vital role of this phosphoprotein was first described and identified in CGD patients that demonstrated a selective lack of enhanced phosphorylation in neutrophils after activation of the oxidase with phorbol myristate acetate (PMA) [9]. Several pathways are implicated in p47 phox phosphorylation, but a primary pathway involves protein kinase C (PKC) isoforms (δ, β, α, ζ) that, when activated, specifically phosphorylates key serine residues (Ser304, Ser315, Ser320, and Ser328) of p47 phox . Recently, p67 phox , alone or in combination with p40 phox , has been found to be able to potentiate the process of phosphorylation induced by several isoforms of PKC [10]. Thus, p67 phox has been suggested as a novel regulator of p47 phox phosphorylation, as corroborated by a dramatic reduction in p47 phox serine residues phosphorylation in lymphocytes from p67 phox−/− CGD patients [10].
After this first event of phosphorylation, the activation of the small GTPase Rac2 mediated the translocation of p67 phox , which associates with p47 phox to the cytochrome. Finally, Rac2 directly binds to the flavocytochrome, favoring the initial steps of the electron transfer reaction.
Data from loss-of-function studies in human primary immunodeficiency and knockout mice highlighted a great deal of mechanistic information on the positive regulation of the NOX2 complex; however, few negative regulators have been identified [11][12][13][14][15].
Among negative regulators of NOX2, Noubade et al. identified a new protein, termed negative regulator of ROS (NRROS), which limits ROS generation [12]. Specifically, NRROS directly interacts with nascent NOX2 in the endoplasmic reticulum (ER) and favors NOX2 degradation through the pathway associated with the ER. Indeed, in NRROS-deficient phagocytes, ROS production is increased upon inflammatory challenges; mice lacking NRROS in their phagocytes showed enhanced bactericidal activity but also developed severe experimental autoimmune encephalomyelitis induced by oxidative stress tissue damage in the central nervous system [12].
By using genomic technology, several negative regulatory nodes controlling phagocyte oxidative bursts were identified [13]. The authors described mechanisms controlling oxidative burst (1) at the level of NOX2 subunit transcription by the Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (Rbpj) that negatively regulates expression of the NOX2 complex components NCF1, NCF4, CYBB, and CYBA; (2) at level of catalytic function by phosphofructokinase, liver type (Pfkl), a rate-limiting glycolytic enzyme that regulates NADPH oxidase activity by affecting the pentose phosphate pathway (PPP) and NADPH levels; (3) at steady-state protein levels (rates of translation and degradation balanced) by Ring Finger Protein 145 (Rnf145) that controls steady-state levels of the NOX2 complex by ubiquitination of lysine residues in gp91 phox and p22 phox [13].
More recently, cAMP-Protein kinase A (PKA)-NOX2-axis has been proposed as a critical gatekeeper of neutrophil ROS production. Indeed, PKA phosphorylates the cytosolic fragment of NOX2 (291-570) that contains the flavoprotein domain, which exhibits NADPH diaphorase activity. Phosphorylation by PKA induces a decrease in diaphorase activity of the cytosolic protein tail by a conformational change in the NOX2 and downregulates the assembly of the complex by the inhibition of PKC-induced interaction with the cytosolic proteins (Rac2, p67 phox , and p47 phox ) [14].
Finally, our group also described a mechanism of negative NOX2 regulation by metalloproteinases (MMP2), which induces the shedding of sNOX2dp, a small peptide [16,17] that corresponds to the third extracellular domain, the E-loop sequence, of NOX2. As a consequence, NOX2 activity and ultimately ROS formation are downregulated [15].
The study of the mechanisms of NOX2 activation and in particular the fine-tuned molecular regulatory systems is of crucial importance since NOX2 can be considered an enzyme at the crossroads between pathologies mediated by NOX2 absence or reduction or NOX2 hyperactivation.

The Immune Function of NOX2: Reactive Oxygen Species and Antimicrobial Activity
NOX2 has an important role in immunity and its structure is clearly linked with its function; as a matter of fact, it is unassembled when inactive. Functionally, the subunit with a considerable role is the gp91 phox subunit. Gp91 phox is highly glycosylated in the extracytoplasmic portion; the transmembrane portion contains six transmembrane alphahelices that bind two hemes, whereas in the cytoplasmic portion are located the N-terminal tail and the C-terminal tail which contain NADPH and the binding sites for the FAD [1]. This structure, therefore, contains every characteristic to transport electrons from the cytoplasm to the extracytoplasmic side to produce ROS, which play an important role in killing bacteria.
Phagocytes are dedicated to microbicidal action thanks to non-oxidants and oxidant systems. Over the years, researchers understood that the two systems cooperate and synergize against bacteria killing. Phagocytes produce different types of ROS that perform this critical function. One of the most important ROS is superoxide ion (O2 •-), a short-lived molecule with a half-life in the range of microseconds as it is rapidly and normally converted by spontaneous dismutation or enzymatic conversion in H 2 O 2 [18].
H 2 O 2 generated by NOX2 must cross phagosome or plasma membranes using aquaporins, integral membrane protein channels that exchange H 2 O 2 and H 2 O; the molecules rapidly bond with catalases, the main pathways to decompose H 2 O 2 at higher concentrations [19], whereas peroxiredoxins and glutathione peroxidases are the main routes for H 2 O 2 metabolism when present at relatively low levels. These enzymes readily react with and manage H 2 O 2 catabolism and protect critical and vulnerable intracellular proteins from the oxidative stress that could be related to [20]. H 2 O 2 is often converted into hypochlorous acid (HClO) by the myeloperoxidase (MPO) enzyme, which is abundant in azurophilic granules of neutrophils and in the lysosomes of monocytes. MPO converts H 2 O 2 to HClO, playing a key role in amplifying the toxicity of H 2 O 2 generated by the respiratory burst. Indeed, HClO is both a stronger oxidant and antimicrobial agent; in fact, the normal amount generated is adequate to eliminate ingested bacteria. This important role is confirmed by a markedly lesser efficiency at killing bacteria in MPO-deficient neutrophils [21].
Neutrophils are usually in a quiescent state and are pre-activated by a process called "priming" that can be established by a range of signaling pathways and intracellular processes causing phenotypic and molecular changes [22]. Even if neutrophils are not able to pre-assemble the NADPH-oxidase complex only with the priming process, they express many receptors on their membrane to facilitate microbial ligands recognition and among them there are toll-like receptors (TLRs), Fc receptors (FcR), G-protein-coupled receptors (GPCR), and integrin receptors that are active in NADPH-oxidase-producing ROS [23]. At the same time as the massive production of oxidants, antimicrobial granule proteins (lactoferrin, myeloperoxidase, defensins, proteases, lysozyme, and calprotectin) are directed to phagosomes or to the extracellular environment to provide for the killing of microbes.
3. Deficit of NOX2: Human, Murine, and Cellular Models 3.1. NOX2 Deficiency: Human Model NOX2 deficiency, or mutations in one of the genes encoding the components of the NADPH oxidase complex, could lead to the development of chronic granulomatous disease (CGD). CGD is a primary immunodeficiency (incidence 1:200,000-1:500,000) that is due to a defect in the NOX2 complex [24]. According to the mode of inheritance, two classical forms of the CGD are known: an autosomal form, with mutations in genes encoding for p22 phox (CYBA), p47 phox (NCF1), p67 phox (NCF2), and p40 phox (NCF4) proteins and the X-linked form of CGD with mutations present in CYBB encoding NOX2, which accounts for more than 60% of all CGD cases.
The most common form of autosomal CGD (about 24% of all cases) is caused by mutations in the gene NCF1 for p47 phox where a two-nucleotide GT deletion (∆GT) occurs as a single defect at the beginning of exon 2 of NCF1.
X-CGD patients with mutations in CYBB can be classified into three groups according to the level of cytb 558 expression and NADPH oxidase activity: X91 0 , characterized by an absence of cytb 558 expression and NADPH oxidase activity; X91 − , characterized by low levels of cytb 558 expression and proportionally decreased NADPH oxidase activity; X91 + , characterized by normally cytb 558 expression but no NADPH oxidase activity [26]. Recently, 11 new mutations were discovered in 16 CGD male patients classified according to the degree of NOX2 expression and activity. These mutations include a new and extremely rare double missense mutation, a deletion, and a substitution [26].
Moreover, a homozygous loss-of-function mutation in cytochrome b558 chaperone-1 (Cybc1) (previously known as C17orf62) was identified in patients diagnosed with CGD [27,28]. Cybc1 encodes for the ER-resident protein EROS (essential for reactive oxygen species), a recently identified protein that acts as a chaperone necessary for the gp91 phox -p22 phox heterodimer expression and controls phagocyte respiratory burst [29]. PLB-985 cells with the deletion of CYBC1/EROS by CRISPR-Cas9 did not express EROS protein and detectable gp91 phox , whereas p22 phox expression was also much lower than in control cells [28].
The investigation of X-linked CGD patients has provided a clinical model that can be used to verify the role of the phagocytic NOX2. First manifestations of CGD occur typically during infancy, before the age of 2 years; however, cases of later onset of the symptoms have also been observed in childhood or even adult life [30]. Clinically, CGD is characterized by severe recurrent bacteria, mainly by Staphylococcus aureus [31] or Mycobacterium tuberculosis [32], and invasive fungal infections [31].
In addition to this immunodeficiency, patients suffer from hyperinflammatory reactions. Indeed, NOX2 can serve to resolve inflammation playing a critical role in limiting lung inflammation and injury in response to pathogens, microbial products, and direct tissue injury [33]. Even if key drivers of hyperinflammation are still incompletely defined, some pathomechanisms can include reduced neutrophil apoptosis, dysbalanced innate immune receptors, induction of T helper 17 (Th17) cells, impaired NF-E2-related factor 2 (Nrf2) activity, and increased inflammasome activation [34].
However, in association with loss of NOX2 function, CGD patients showed enhanced carotid artery dilation, impaired platelet-related thrombosis, and reduced carotid atherosclerotic burden [35]. In particular, CGD patients have decreased platelet activation as suggested by reduced plasma levels of soluble sCD40 L and soluble P (sP)-selectin, two markers of in vivo platelet activation [36], increased vascular production of nitric oxide (NO) [36] and flow-mediated dilation (FMD) [37], and an index of endothelial function. These data provide a direct link between NOX2, platelet activation, and endothelial function.

NOX2 Deficiency: Mouse Models
As the deficit of the NOX2 can have important clinical effects, experiments with mouse knockouts lacking NOX2 provide new insights into the mechanisms accounting for CGD development and progression.
In 1995, Pollock and colleagues generated the first mouse model of X-linked chronic granulomatous disease [38]. In these mice, with a null allele of the gene involved in Xlinked CGD, the respiratory burst oxidase activity was completely absent in neutrophils and macrophages; moreover, they manifested typical X-linked CGD phenotypes with increased susceptibility to infection with S. aureus and A. fumigatus and altered inflammatory response [38].
After this first description, genetic deletion is widely used to verify the impact of NOX2-derived reactive ROS [39][40][41][42][43]. More recent reports confirmed that, in mice, the lack of activity of NOX2 in neutrophils, but not in macrophages, markedly increased susceptibility to infectious A. fumigatus compared with immunocompetent mice [44]. Moreover, bone marrow-derived macrophages (BMDM) from NOX2-deficient mice failed to clear intracellular S. aureus, and showed significantly improved survival and aggravated dissemination of S. aureus infection [39]. As above described, besides recurrent bacterial and fungal infections, CGD suffers from hyperinflammation. In a murine model of generalized inflammation induced by zymosan, reproducing systemic inflammatory response syndrome (SIRS), NOX2-deficient mice developed a robust SIRS response, with an enhanced inflammatory phenotype of polymorphonuclear leukocytes and a persistent inflammatory environment resulting from continued chemokine production [40]. Among possible specific drivers of inflammation, leukotriene B4 (LTB4) was found produced at higher amounts by neutrophils from NOX2-deficient mice, also promoting excessive neutrophilic lung inflammation [45]. Moreover, neutrophil-produced interleukin (IL)-1β and downstream granulocyte-colony stimulating factor (G-CSF) were identified as critical amplifying signals that act sequentially with LTB4 to amplify inflammation in CGD mice [46].
Abnormal inflammatory responses are closely associated with many chronic diseases, especially autoimmune diseases. Indeed, effects related to a compromised NOX2 system include the development of autoimmune diseases such as rheumatoid arthritis (RA) [41,42,47], systemic lupus erythematosus [43,48], or psoriasis and psoriasis arthritis [49]. Dark Agouti rats, a model for acute and chronic arthritis, expressed a polymorphic Ncf1 allele, which led to differences in enzyme activity and expression, resulting in the activation of arthritogenic T cells in lymphoid organs and finally in severe arthritis [41]. NOX2 KO mice spontaneously developed arthritis and the severity was proportionally increased with age. NOX2 deficiency is linked to changes in the development and the modulation of the Th17/Treg immune cell population and promoted inflammatory cytokine production, such as Tumor Necrosis Factor-α (TNF-α), and IL-1β [47]. In a mouse model of pristaneinduced lupus, NOX2 deficiency (Ncf1-mutated) aggravated and promoted experimental lupus-like autoimmunity by reducing NETs formation and increasing inflammation [48].

NOX2 Deficiency: Cellular Models
Advances in genome sequencing have identified many mutations, opening the door to new and attractive gene therapies that enable a genetic defect to be corrected in the patient's own cells. However, evaluating these new therapies requires cellular models. The first described in 1993 [50], and the only cell-based model mimicking the X-CGD form, available for this purpose, was the knockout CYBB PLB-985 cell [50,51]. These cells are bi-potential and can differentiate into either granulocytic or monocytic forms. In these cells, the X chromosome-linked gp91 phox gene was disrupted by homologous recombination to generate cells that, after differentiation to granulocytes, did not generate O 2 − , reproducing the phenotype of the X 0 -CGD patients with this mutation [50]. A second method to generate mutated NOX2 PLB-985 cells was described by Beaumel et al. [51]. PLB-985 cells were transfected with various NOX2 mutations, cultured, and differentiated into neutrophils or monocytes/macrophages to evaluate the impact of these mutations and to study the relationships between NOX2 structure function [51].
By using the CRISPR-Cas9 technology, a cellular model of CGD was developed in a monocyte/macrophage THP-1 cell line [52]. The three THP-1 clones generated, harboring different CYBB mutations, displayed phenotypic and functional characteristics of macrophages/phagocytes from CGD patients: reduced CYBB mRNA level, absence of NOX2 expression, and hyper-inflammation state [52].
More recently, a method to obtain an ex vivo model of X-CGD has been proposed using induced pluripotent stem cells (iPSCs) [53]. iPSCs, introduced in the year 2006, are artificial stem cells produced from somatic cells and provide a great opportunity as they can be generated from patient cells and differentiated to a desired cell type for disease modeling. X0-CGD cells, reprogrammed from human dermal fibroblasts using episomal vectors, are important to evaluate new therapeutic approaches in preclinical studies. Braut et al. demonstrated the therapeutic potential of NOX2/p22 phox liposomes to transport and deliver recombinant cytochrome b558 to the membrane of X 0 -linked CGD (X 0 -CGD) iPSC-derived macrophages and restore the NOX2 activity [54].

NOX2-Derived Reactive Oxygen Species-Mediated Diseases
NOX2 has dual functions. As clearly demonstrated by the genetic loss of NOX2 in CGD patients and CGD mouse model, NOX2 is essential for host defense from bacterial and fungal pathogens and calibrates neutrophilic inflammatory response to protect the host from injury associated with excessive or persistent inflammation. On the other side, the hyperactivation of NOX2, as the main source of ROS, is also proinflammatory and injurious, contributing to several diseases, as described in this paragraph ( Figure 2).

NOX2-Derived Reactive Oxygen Species-Mediated Diseases
NOX2 has dual functions. As clearly demonstrated by the genetic loss of NOX2 in CGD patients and CGD mouse model, NOX2 is essential for host defense from bacterial and fungal pathogens and calibrates neutrophilic inflammatory response to protect the host from injury associated with excessive or persistent inflammation. On the other side, the hyperactivation of NOX2, as the main source of ROS, is also proinflammatory and injurious, contributing to several diseases, as described in this paragraph ( Figure 2).

Figure 2.
(A) NOX2 activation leads to ROS production and contributes to several diseases including (B) carcinogenesis, through genotoxic and non-genotoxic ways which may cause direct and indirect damage to DNA, cancer cell proliferation, increased angiogenesis, and inhibition of apoptosis; (C) neurodegenerative diseases, through different mechanisms such as apoptosis of neuronal cells in PD via AMPK and Akt/mTOR signaling pathways, the compromising of autophagic flux, neuroinflammation of microglia, and consequent release of neurotoxic factors; (D) cardiovascular diseases, such as atherosclerosis and thrombosis through platelet activation, aggregation, and recruitment.
A compromised antioxidant status and oxidative stress, as characteristic in pathological conditions, such as those described in this paragraph, could be a consequence of a A compromised antioxidant status and oxidative stress, as characteristic in pathological conditions, such as those described in this paragraph, could be a consequence of a hormonal imbalance [55]. Indeed, changes in the hormonal milieu may have impacts on the production of ROS both directly, as hormones like melatonin, estrogen, or progesterone exhibit antioxidant properties, or indirectly by regulating metabolic activities [55]. Several hormones can influence NOX2. For example, chronic glucocorticoid exposure, secreted by the adrenal cortex, activated NOX2 accelerating neuronal damage in Alzheimer's transgenic animal models [56]. Additionally, thyroxin can induce NOX2-p47 phox expression, finally favoring cardiac hypertrophy in rats [57]. Conversely, melatonin reduced oxidative stress by regulating NOX2 expression in rat lung tissues following whole radiotherapy [58].
Oxidative stress and its related diseases could also be influenced by sex hormones [59]. Sex differences have been observed in oxidative stress generation between males and females. Indeed, women seem to be less susceptible to oxidative stress with lower levels of oxidative stress biomarkers and higher antioxidant power [59]. These differences could be explained by the antioxidant effect of estrogen [60,61]. However, at the molecular level, some studies consistently show that NOX2 protein expression did not differ between males and females [59].

NOX2 and Carcinogenesis
In general, ROS formed from NOX enzymes could be a trigger for carcinogenesis in genotoxic and non-genotoxic ways. Genotoxicity refers to direct DNA damage, which may cause proto-oncogene activation, tumor suppressor gene inactivation, genomic instability, and epigenetic modifications, further leading to mutations. Nongenotoxicity describes an indirect effect on DNA through the activation of related signaling pathways [62].
Several NOX enzymes are expressed in malignant tissue and may contribute both to cancer progression and the spread of malignant cells. Specifically, NOX2-derived ROS can contribute to carcinogenesis stages, from cell proliferation [63,64] to tumor progression and, finally, to metastasis [63,[65][66][67].
A recent study analyzed the effect of NOX2 on cell proliferation, cell cycle, cell motility, and cell survival in human esophageal squamous cell carcinoma [63]. Results showed that NOX2 expression favors cell cycle progression; indeed, NOX2 depletion significantly inhibited cell proliferation with the G 0 /G 1 arrest and resulted in apoptosis. In addition, in osteosarcoma cell lines, high-level NOX2 mRNA expression was observed, and this expression was associated with ROS generation promoting cell survival [64]. When compared with normal tissue, the expression of NOX2 was also significantly increased in primary prostate cancer tissue, in endosomes, promoting cell proliferation and prostate tumor development [66]. The role of NOX2-produced ROS was also confirmed both by the genetic deletion of NOX2 in the mouse model of prostate cancer, resulting in reduced angiogenesis and an almost complete failure in tumor development, and by NOX2 pharmacological inhibition that suppressed established prostate tumors in mice [66].
Studies with genetically NOX2-deficient mice and pharmacologic inhibition of NOX2 also elucidated the role of NOX2 in metastasis [67,68]. Mice deficient in the NOX2 subunit CYBB showed reduced lung metastasis of melanoma cells by downmodulating natural killer (NK)-cell function. The treatment with the NOX2-inhibitor histamine dihydrochloride (HDC) reduced melanoma metastasis and enhanced the infiltration of IFNγ-producing NK cells into the lungs [67]. Moreover, CYBA-or NCF2-deficient mice displayed reduced lung metastatic colonization, the presence of large granulomas of galectin-3 (Mac-2)-positive macrophages and eosinophilic deposits, and altered immune cell populations [68].

NOX2 and Neurodegenerative Diseases
Neurodegenerative diseases (ND) such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), among others, are becoming a wide cause of disability. Growing evidence supports the role of oxidative stress in the initiation and progression of ND [69]. The biochemical integrity of the brain is essential for the central nervous system's normal function. The brain is highly susceptible to oxidative stress because of its large amount of oxygen consumption and because the neuronal membrane is highly rich in polyunsaturated fatty acids [70].
At the cellular level, NOX2 has been reported to be expressed in neurons and astrocytes, [71] and is heavily expressed in microglia, the resident brain phagocytes, where it is involved in immune and inflammatory responses [71][72][73]. Indeed, under normal conditions, microglia serve a crucial role in immune surveillance. However, the overactivation of microglia has been associated with neurodegeneration through the production of neurotoxic factors, such as proinflammatory cytokines and large amounts of ROS [74,75].
Alzheimer's disease, one of the main human dementias in the elderly, manifests as progressive cognitive decline and profound neuronal loss. Senile plaques of amyloid-β (Aβ) peptides and neurofibrillary tangles are the principal neuropathological hallmarks of AD. The role of NOX2 in AD is now well described [76]. In addition, recent studies further supported and reinforced the critical role of NOX2 in the pathogenesis of AD [77,78].
In an AD-like model of dementia by the intracerebroventricular (ICV) administration of streptozotocin (STZ), advanced glycation end-products (AGEs), derived from methylglyoxal, activate receptors for AGEs (RAGE) that in turn activate NOX2, that ultimately results in being persistently activated [77]. Methylglyoxal/RAGE/NOX-2 pathway could explain the inflammatory status and oxidative stress in this mouse model and in human disease [77]. Moreover, NOX2-derived ROS-mediated Aβ 1-42 -induced glucose hypometabolism, a symptomatic marker of AD implicated in the initiation of sporadic forms [78].
PD is a neurodegenerative disease caused by the loss of dopaminergic neurons and NOX2 plays a critical role in its pathogenesis [79][80][81][82][83][84][85]. First of all, NOX2 is expressed and is activated in dopamine neurons and in microglia both in human brain tissue and animal models of PD [79]. Several mechanisms of the NOX2-mediated effect of oxidative stress in PD have been suggested. NOX2 activation and H 2 O 2 production are induced by PD toxins, including 6-hydroxydopamine (6-OHDA), 1-Methyl-4-phenylpyridin-1-ium (MPP + ), and rotenone, favoring AMPK and Akt/mTOR signaling pathways and apoptosis in neuronal cells [80]. NOX2-mediated oxidative stress also induced the expression of nucleotide-binding oligomerization domain-containing protein (NOD)2 and the inflammatory response induced by the neurotoxin 6-OHDA, ultimately promoting DA degeneration [83]. Finally, another mechanism proposed is the impairment of autophagy flux that is implicated in the elimination of misfolded and aggregated proteins such as α-synuclein (α-Syn). Yan et al. found that in a mouse model of PD induced by MPTP, a nigrostriatal dopaminergic neurotoxin, the protein expression of NOX2 increased coincidentally with increased α-Syn Ser129 and reduced autophagy flux, suggesting a role for NOX2-mediated oxidative stress and autophagy in PD pathogenesis [81].
The key stages of the atherosclerosis process include the accumulation and oxidation of low-density lipoproteins (LDL) by ROS within the arterial wall and the perpetuation of the inflammatory process occurs through the infiltration of monocyte-macrophages, which become foam cells on the absorption of oxidized LDL. NOX2, by activated platelets, oxidizes LDL, which in turn amplifies platelet activation via ox-LDL receptors CD36 or LOX1 [88]. ox-LDL production was more pronounced in hypercholesterolemic adult patients [88] and also in children with obesity and/or hypercholesterolemia associated with NOX2 activation and reduced flow-mediated dilation [89]. Data from hypercholesterolemic mice models also confirmed the role of NOX2 [91,92]. Apolipoprotein E-deficient (ApoE(−/−)) mice, fed a high-fat diet, showed greater superoxide production by NOX2 and reduced basal nitric oxide-mediated relaxation compared to wild-type mice [92]. Moreover, in hypercholesterolemia mice, NOX2 impairs neovascularization and blood flow recuperation after surgically-induced hindlimb ischemia [91].
Several studies have consistently demonstrated a key role for NOX2 in eliciting platelet activation and aggregation [100,101]. Human platelets express NOX2, which is functionally relevant as indicated by the fact that ROS influence platelet recruitment and thrombus growth [102].
Atherosclerosis is a main cause of myocardial infarction (MI), establishing a vicious circle that increases atherosclerosis and the risk of more infarctions [103]. NOX2 is notably upregulated in peripheral blood samples of patients with acute MI [95]. Moreover, in the brain tissue obtained at autopsy from patients with MI, NOX2 is significantly increased in brain microvasculature compared to controls, suggesting also a role for NOX2 in mental health disorders associated with MI [96].
Finally, interestingly, the offspring of patients with early MI had higher NOX2 activation suggesting a key role of NOX2-mediated oxidative stress in the offspring of patients with premature MI [94].
These data support the role of NOX2 as a key source of ROS in the artery wall in conditions that underlie atherogenesis contributing to endothelial dysfunction and vascular inflammation. Therefore, for clinical purposes, NOX2 represents a potential target to develop novel isoform-selective drugs to prevent or treat cardiovascular diseases [104].

NOX2 as a Therapeutic Target: Pharmacological Approaches from Natural to Synthetic Small Molecules
As described above, NOX2 is implicated in many diseases, where both acute and chronic inflammation plays a key role in terms of pathogenesis. For these reasons, NOX2 could be a target for drug development. Indeed, researchers are looking for inhibitors of NOX2 as a novel therapeutic class of drugs to treat diseases such as AD, PD, cardiovascular diseases, and many other different diseases where oxidative stress and inflammation are key drivers.
However, as clearly shown by X-CGD patients, side effects could arise from targeting NOX2, including the possibility that such inhibition can contribute to increased infections and/or autoimmune disorders. Therefore, the development of specific and not toxic inhibitors of NOX2 represents to date a still open challenge in the search for the ideal inhibitor. Ideally, this inhibitor should possess specificity and selectivity for the NOX2 isoform, without interfering with its expression or modifying the upstream pathway. Furthermore, the inhibitor should not have antioxidant activities as a ROS scavenger [105].
The first molecules to be investigated as NOX inhibitors and the most frequently used in in vitro and in vivo experiments are diphenyleneiodonium chloride (DPI) and apocynin (4 -hydroxy-3 -methoxyacetophenone/acetovanillone). Apocynin is frequently used as a NOX2 inhibitor even if several studies demonstrated that apocynin acts as an antioxidant [106] and was inactive for NOX2 or any other NOX isoform, even at high concentrations (300 µM) [107]. DPI is an effective low-micromolar or nanomolar inhibitor widely employed in cell studies as a negative control to assess NOX activity; it is a general blocker of flavoproteins, and for this reason, it is not specific for the NOX2. Indeed, DPI is reported to interfere with xanthine oxidase, cytochrome P-450 reductase, and proteins of the mitochondrial electron transport chain [108]. Moreover, the DPI mode of inhibition implies that it will act only on activated NOXs [108].
To date, several other small molecules have been identified and tested as NOX2 inhibitors.
In this paragraph, we reviewed inhibitors tested for NOX2 as a target in pathologies, mainly those described in the previous paragraph (vascular, cancerous, neurological). These inhibitors have been divided into compounds with a direct or indirect mechanism of inhibition. Inhibitors work directly to regulate NOX2 activity by competing for the NADPH binding site of NOX2, or by blocking the assembly of cytosolic subunits to the cytochrome. Indirect inhibitors work as general antioxidants, or by inhibiting the upstream pathway of NOX2 activation (Table 1).     Moreover, NOX2 inhibitors have been classified as peptide-based inhibitors, drug-like small molecules and drugs, and compounds of natural origin.

Peptide-Based Inhibitors
Among strategies to target NOX2, peptide-based inhibitors still represent a class of promising candidates because they possess enormous potential and offer specific advantages [154]. By their nature, peptide sequences and peptidomimetics have high similarity to endogenous ligands, high affinity, and low toxicity. Thus, therapeutic peptides effectively and selectively disrupt intrinsic protein-protein interactions [154]. Despite these properties, peptide-based inhibitors showed low stability because of their degradation in the gut and limited oral bioavailability [155].

Direct Inhibitor
As a NOX2 inhibitor peptide, the most used is the NOX2ds-tat (NOX2 docking sequence-tat). NOX2ds-tat was, for the first time, designed and described in 2001 [156] as a 9 amino acid sequence from the cytosolic B-loop of NOX2 that interacts with p47 phox , linked to a 9-amino acid sequence derived from HIV-tat transport region protein, facilitating cellular internalization. NOX2ds-tat is a selective inhibitor of NOX2 as it specifically inhibits O 2 •− production by NOX2 without affecting NOX1-or NOX4-mediated ROS production [157].

Indirect Inhibitor
New peptide-based inhibitors have been described by Fischer et al., who identified a 9-aa sequence called PLA 2 -inhibitory peptide (PIP) [158]. This peptide derived from the lung surfactant protein A (SP-A) that binds to peroxiredoxin 6 (Prdx6), inhibits its phospholipase A 2 (PLA 2 ) activity, thus preventing the cellular generation of Rac and NOX2 activation [159,160]. PIP-2, the peptide with human sequences, incorporated into liposomes as a delivery vehicle, reduces the phospholipase A 2 (PLA 2 ) activity of peroxiredoxin 6 (Prdx6), called aiPLA 2 , and NOX2 activation in lungs [158]. Moreover, PIP-2 was tested in a mouse model of acute lung injury induced by LPS administration [110]. Pre-treatment of mice with PIP-2 markedly decreased lung injury and mouse mortality [110], suggesting PIP-2 as a useful NOX2 inhibitor to prevent or treat human acute lung injury.

Drug-Like Small Molecules and Drugs
As new therapeutic alternatives, small-molecule inhibitors have been successfully used since they offer several key advantages for a targeted approach and are expected to have fewer adverse side effects. Small-molecule inhibitors are compounds with a low molecular weight, less than 500 Da in size [161], that, therefore, can enter whole cell populations easily. They can target any portion of a molecule, both extracellular and intracellular proteins, and can often inactivate their targets rapidly [162].

Direct Inhibitor
Among NOX2 small molecule inhibitors, GSK2795039 was the first inhibitor to exhibit promising results in vitro and in vivo [111]. GSK2795039 is a novel 7-azaindole structure with the unique feature of a sulfonamide functionality critical for its activity. GSK2795039 has been reported as a potent NOX2 inhibitor by inhibiting NOX2-mediated activation in cell-free assays (pIC50 6.57 ± 0.17), ROS production in whole-cell assays with human peripheral blood mononucleated cells (PBMCs) (pIC50 6.60 ± 0.075), and in vivo by intravenous infusion in mice [111]. Moreover, GSK2795039 can dose-dependently block NOX2 in models of paw inflammation, and has a protective effect in the mouse model of ceruleaninduced pancreatitis [111]. More recently, Xue et al. [112] demonstrated that GSK2795039 may be a potential therapeutic drug for influenza A virus infection. Established the critical role for NOX2 in the H1N1 infection and subsequent inflammatory reactions, GSK2795039 reduced H1N1-induced NOX2 activity and ROS production in human lung epithelial cells coincidentally with decreased expression of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-β and interleukin (IL)-6 [112].
Several other small molecules were tested as NOX2 inhibitors [113][114][115][116][117][118][119]121]. LMH001 is a small chemical compound, recently described as a competitive inhibitor to block phosphorylated p47 phox binding to p22 phox with an IC50 = 0.149 µM. At a small dose (IC50 = 0.25 µM), LMH001 inhibited angiotensin II (AngII)-induced endothelial NOX2 activation and ROS production by blocking the interaction between phosphorylated p47 phox and p22 phox . Importantly, this small molecule did not have effects on peripheral leucocyte oxidative response to pathogens [113]. In addition, in a mouse model of AngII-induced vascular oxidative stress, hypertension, and aortic aneurysm, LMH001 treatment reduced hypertension, aortic wall inflammation, and incidences of aortic aneurysm, confirming its therapeutic potential [113]. However, a very recent study questions the activity of LMH001 as a NOX2 inhibitor [163]. Indeed, the results of this study showed that LMH001 was chemically unstable in standard aqueous buffer and it was not able to inhibit the p47 phox /p22 phox interaction [163].
Among promising small molecule inhibitors, Phox-I1 is of interest as it specifically binds to the interactive site of p67 phox with Rac1 interfering with Rac1-GTP interaction with p67 phox [116]. Platelets treated with Phox-I1 prevented NOX2 activation, ROS generation, and platelet activation in terms of release of P-selectin, secretion of ATP, and platelet aggregation, suggesting Phox-I1 as a possible approach for antithrombotic therapy [117].
As NOX2 inhibitors, drugs with a pleiotropic effect also have a role. Indeed, some drugs normally prescribed for the treatment of several pathologies showed their beneficial effect also through the inhibition of NOX2 [90,122,133].
Among FDA-approved statins, rosuvastatin and atorvastatin showed antioxidant activity by direct NOX2 inhibition [81,90,122,129]. Indeed, rosuvastatin in vitro inhibited platelet NOX2 activation, PKC phosphorylation and p47 phox translocation from cytosol to membranes, reducing platelet ROS production and platelet activation [122]. Additionally, in vivo, hypercholesterolemic patients treated with rosuvastatin or atorvastatin showed reduced platelet activation via the inhibition of NOX2-derived oxidative stress [90,122].
BJ-1301 is an aminopyridinol derivative of α-tocopherol with strong antioxidant activity [167]. BJ-1301 is able to block the translocation of cytosolic subunits to the cell membrane, thereby inhibiting NOX2 activation. The subsequent reduction in ROS-mediated receptor tyrosine kinase (RTK) signaling results in the regression of tumor growth in mouse models of lung tumors [124], suggesting BJ-1301-mediated NOX2 inhibition as a promising anti-cancer therapeutics approach.
Dexmedetomidine is a highly selective α-2 adrenergic receptor agonist used in the perioperative period and intensive care units to sedate patients who are under intensive medical care and needs a mechanical ventilator [168]. Dexmedetomidine also exhibits neuroprotective effects in numerous neurological disorders.
Among statins, in the experimental study of coronary micro embolism (CME)-induced cardiac injury, rosuvastatin improved the left ventricular function in these mice, reduced inflammatory cell infiltration and fibrin deposition in the myocardium by inhibiting the expression of NOX2 [129], and also alleviated p53/Bax/Bcl-2-dependent cardiomyocyte apoptosis [129]. Finally, by targeting NOX2, atorvastatin may be identified as a new drug in Parkinson's disease (PD) treatment [81]. Indeed, atorvastatin treatment improves muscle capacity, anxiety, and depression in MPTP-lesioned mice by inhibiting NOX2 and by promoting autophagy flow [81].

Direct Inhibitors
Among naturally occurring polyphenols, myricitrin is a flavanol that exists in several foods such as tea and different vegetables, and possesses, among others, anti-cancer activities as demonstrated by the induction of pancreatic cancer cell death, attenuation of both DNA strand breakage and mouse skin tumor formation [169]. In a model of acute lung injury induced by LPS inhalation, pre-treatment with myricitrin attenuated the inflammatory process into the airway and alveolar space [135]. Moreover, in the RAW264.7 macrophage cell line, the treatment with myricitrin inhibited the assembly of components of the gp91 phox and p47 phox thus reducing the intracellular generation of ROS [135].
Ginsenosides are the major, unique, and active components of ginseng and are classified into two categories according to their chemical structure [170]. Among the known ginsenosides, Rb1, and Rg1 have been tested as NOX2 inhibitors.
Rb1 showed specific inhibitory properties by targeting NOX2 for the treatment of atherosclerosis. Indeed, in endothelial cells, Rb1 disrupted the assembly of the NOX2 complex by binding to p47 phox and reducing its phosphorylation and membrane translocation. Moreover, in streptozotocin (STZ)-induced ApoE −/− mice, Rb1 reduced aortic atherosclerotic plaque formation and oxidative stress [136].
Celastrol is a natural bioactive ingredient derived from the Chinese herb Tripterygium wilfordii Hook. f., an important drug in traditional Chinese medicine with anti-inflammatory and immune-modulating properties [171].
Celastrol potently and effectively inhibited H 2 O 2 production by several NOXs including NOX2, without inhibiting PKC and the translocation or the binding of p47 phox to the stimulated neutrophil membrane [134]. At the molecular levels, celastrol directly binds to p47 phox and disrupts the binding of the PRR of p22 phox to the tandem SH3 domain of p47 phox [134].
Celastrol has been studied for its effects on cardiovascular disorders such as vascular calcification endothelial and endothelial dysfunction [145,146]. Indeed, celastrol is able to alleviate calcific aortic valve disease (CAVD) in a mouse model through the reduction in ROS generation and NOX2-mediated glycogen synthase kinase 3 beta/β-catenin pathway in cultured aortic valvular interstitial cells [146]. Moreover, in endothelial cells, celastrol effectively inhibited the NOX2/Angiotensin II(AngII) type 1 receptor (AT 1 ) pathway, improving endothelial cell activity and ameliorating Ang II-mediated HUVEC injury [145].

Indirect Inhibitors
Among polyphenol-rich extracts, wild Alaska bog blueberries (Vaccinium uliginosum) were tested as NOX2 inhibitors. At a concentration that exhibited full potency with no apparent cytotoxicity, blueberry fractions disrupted NOX2 assembly by modulating the lipid raft platform and then the association of p67 phox to the plasma membrane and abolished TNF-α-mediated ROS production [137].
Rosmarinic acid is a natural polyphenol antioxidant found in many Lamiaceae herbs evaluated as a NOX2 inhibitor in the ovalbumin and H 2 O 2 -induced asthma mouse model. The treatment with rosmarinic acid significantly reduced the expression of NOX2, upregulated the activities of antioxidant systems such as SOD, GPx, and catalase, and finally diminished inflammation [140].
Rg1 has been studied for its effects on neurodegenerative diseases [141,142]. Indeed, in a mouse model of AD, amyloid precursor protein/presenilin1 (APP/PS1) AD mice, Rg1 treatment significantly decreased NOX2 expression in the hippocampus and cortex of APP/PS1 mice and then ROS production, finally ameliorating cognitive impairments and neuronal damage [142]. The effect of Rg1 on NOX2 was also confirmed in a mouse model of cerebral ischemia-reperfusion injury, where Rg1 treatment downregulated NOX2 expression and ROS production, attenuating neuroinflammation [141].
Curcumin was tested as an antidepressant in chronic unpredictable mild stress (CUMS)induced depression models in rats [149]. In these rats, curcumin administration relieves depressive-like states by decreasing the protein expression of NOX2 and other biomarkers of oxidative stress such as 4-Hydroxynonenal (4-HNE) and Malondialdehyde (MDA), and increasing the activity of catalase [149].
The described mechanism of action of NOX2 inhibitors and their biological effects highlighted important features that must be taken into account.
Studies on small molecules reported fundamental, pharmacokinetic, or pharmacodynamics profiles highlighting their efficacy. Moreover, most of them work with a direct mechanism of inhibition. Conversely, natural-origin compounds have an indirect effect with limited pharmacokinetic and pharmacodynamic studies. In general, despite the wide distribution and diverse biological properties of naturally occurring compounds, their use is hindered by several limitations. Importantly, these compounds, mainly polyphenols, possess strong functional promiscuity [173]. Promiscuity refers to the ability of confirmed bioactive compounds to specifically interact with multiple targets [174]. Among hundreds of chemical classes, several compounds such as quinones, catechols, and curcumin occur as PAINS (pan-assay interference compound) [175], a term that described an approach introduced by Baell et al. combining expert knowledge of chemical patterns with systematic empirical validation [176]. Therefore, a wider perspective is needed to elucidate whether their use as bioactive components may be valid for the management of diseases.

Conclusions
NOX2 has a key role in the production of ROS with multiple roles in regulating many aspects of innate and adaptive immunity but also in the pathogenesis of several diseases characterized by increased oxidative stress and inflammatory process.
NOX2 could represent a key target for biological science research. Indeed, researchers are looking for inhibitors of NOX2 as a novel therapeutic class of drugs to treat diseases such as AD, PD, cardiovascular diseases, and many other diseases where oxidative stress and inflammation are key drivers. However, as clearly shown by X-CGD patients, possible side effects could arise from targeting NOX2, including the possibility that such inhibition can contribute to increased infections and/or autoimmune disorders.
The development of suitable molecules to treat these conditions primarily requires a thorough understanding of the regulatory processes of NOX2 activation mechanisms through functional and structural studies. Although numerous inhibitors are tested to date, additional approaches are needed to overcome some issues including (1) the sequence homology between different NOX isoforms which complicates to identify new isoformselective small-molecule inhibitors; (2) the redox and ROS-scavenging activities of many chemical compounds and (3) the interference with NOX2 upstream pathways.
Therefore, the development of the ideal inhibitor, specific and not toxic, of NOX2 represents, to date, a still open challenge.