Role of NADPH oxidase in host defense against aspergillosis

Abstract

NADPH oxidase plays a critical role in antimicrobial host defense, as evident in chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal diseases. Invasive aspergillosis and other moulds are the major cause of mortality in CGD. We also learn from CGD patients that NADPH oxidase plays an important role in regulating inflammation; CGD patients are prone to developing inflammatory diseases such as inflammatory bowel disease, obstructive granulomata of the genitourinary tract, and hypersensitivity pneumonitis. Indeed, the NADPH oxidase plays an essential role in calibrating innate and T-cell responses to control the growth of inhaled fungi while protecting against excessive and injurious inflammation. Knowledge gained on the mechanisms by which NADPH oxidase kills fungi and regulates inflammation may lead to new therapeutics for CGD and will have broad relevance to understanding host-pathogen interactions between mammals and ubiquitous moulds to which we are continually exposed.

Introduction

The lung is an interface where inhaled microbes and microbial products (e.g., bacterial and fungal cell wall constituents) interact with host defense cells. The ability of the mammalian host to evolve in the context of continual exposure to fungi requires a well-calibrated immune response. First, the immune system must kill or at least control the growth of inhaled pathogens, such as fungal spores. Second, counter-regulatory mechanisms must be active to limit the inflammatory response to avert tissue injury and allergy. There is strong evidence that NADPH oxidase is critical for both functions, i.e., antimicrobial and regulation of inflammation.

Pathogen recognition receptors such as toll-like receptors (TLRs) sample microbial motifs and initiate signaling that may result in NADPH oxidase activation. NADPH oxidase activation leads to generation of superoxide anion and downstream reactive oxidant intermediates (ROIs) and activation of neutrophil antimicrobial proteases [1].

Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase characterized by life-threatening bacterial and fungal diseases and by abnormally exuberant inflammatory responses (e.g., inflammatory bowel disease, obstructive granulomata of the genitourinary tract) [2]. Activated NADPH oxidase is responsible for the respiratory burst – a rapid consumption of oxygen – that occurs following neutrophil stimulation. Phagocyte NADPH oxidase activation occurs in response to physiologic stimuli such as opsonized particles, integrin-dependent adhesion to the extracellular matrix [3,4], and ligation of specific pathogen recognition receptors (e.g., dectin-1 [5]). The phagocyte NADPH oxidase functions to rapidly generate superoxide anion by transferring electrons from NADPH to molecular oxygen. The cytochrome, composed of gp91^phox (phox, phagocyte oxidase) and p22^phox, is embedded in membranes. Upon activation of the oxidase, the cytoplasmic subunits p47^phox, p67^phox, and p40^phox appear to translocate en-bloc to the membrane-bound cytochrome. Activation of rac, a member of the low molecular weight GTP-binding proteins, and translocation of rac to the membrane-bound cytochrome are also critical for NADPH oxidase activation [6,7].

Invasive aspergillosis in CGD

CGD patients are susceptible to a broad spectrum of opportunistic filamentous fungi, but Aspergillus infection is the most common. The incidence has been estimated to be 0.1 fungal infections per patient year [8]. Patients with X-linked CGD appear to be at increased risk for invasive aspergillosis compared with the autosomal recessive forms [9,10]. Analysis of clinical data from 429 European patients showed that the most frequently cultured micro-organisms per episode were Staphylococcus aureus (30%) and Aspergillus spp. (26%); Aspergillus species (111 cases) was the most common cause of pneumonia [11]. Very rarely, invasive aspergillosis may occur in a female carrier of X-linked CGD in whom the random process of lyonization (X-chromosome inactivation) has led to a skewing of the circulating normal neutrophil population to less than 10% oxidant competent [12].

Aspergillus fumigatus and Aspergillus nidulans are the most common Aspergillus species in CGD. With the exception of CGD patients, A. nidulans is a rare pathogen. We reviewed all cases in which A. nidulans was isolated from patients at the National Institutes of Health (Bethesda, MD) between 1976 and 1997 [9]. A. nidulans infection occurred in 6 patients with CGD, but was not a pathogen in any other patient group. A. fumigatus was a more common pathogen in CGD (n = 17 cases), but A. nidulans was more virulent. A. nidulans was significantly more likely to result in death compared with A. fumigatus (3 of 6 versus 1 of 17 cases, respectively), to involve adjacent bone, and to cause disseminated disease. Patients with A. nidulans received longer courses of amphotericin B therapy than patients with A. fumigatus, and were treated with surgery more often. In contrast to A. fumigatus, A. nidulans was generally refractory to intensive antifungal therapy, suggesting that early surgery may be important. However, the need for early resection of pulmonary lesions will need to be reevaluated with the availability of extended spectrum azoles (voriconazole and posaconazole).

CGD patients often do not have typical symptoms and signs of infection [13]. Fever and leukocytosis may be absent, and an elevated sedimentation rate may be the only abnormal laboratory test [13]. In a review of aspergillosis in CGD patients at the NIH, one third of patients were asymptomatic at diagnosis and only ~20% were febrile [9]. In many of these patients, a pulmonary infiltrate on routine screening chest x-ray or CT scan was the first indication of an infection. The white blood cell count was ≤10,000/µl in 13/23 cases and the sedimentation rate was ≤40 mm/h in 9/20 cases.

In contrast to patients with chemotherapy-induced neutropenia, hyphal angioinvasion is not a feature of CGD. In CGD mice, pulmonary aspergillosis is characterized by dense pyogranulomatous areas of consolidation in the absence of vascular hyphal invasion [14,15]. These findings suggest that NADPH oxidase-independent pathways are sufficient to protect against hyphal angioinvasion. Serum galactomannan is not elevated in experimental pulmonary aspergillosis in CGD mice [14] and appears to be an insensitive diagnostic marker of aspergillosis in CGD patients [16].

How does NADPH oxidase mediate host defense?

The requirement for NADPH oxidase in host defense against Aspergillus fumigatus is fungal stage-specific. NADPH oxidase appears to be dispensable regarding neutrophil-mediated [17] and macrophage-mediated [18] conidiocidal activity. Neutrophils from normal volunteers and CGD patients were equally effective in limiting the growth of Aspergillus conidia in vitro; the anti-conidial activity was dependent on iron sequestration mediated by lactoferrin, an abundant constituent of neutrophil secondary granules [17]. In contrast, NADPH oxidase is required for neutrophil-mediated damage against Aspergillus hyphae [15,17].

NADPH oxidase activation leads to generation of superoxide anion. Superoxide, a relatively weak microbicidal oxidant, is metabolized to the more toxic hydrogen peroxide by superoxide dismutase. Hydrogen peroxide can in turn be converted to hypohalous acid by myeloperoxidase, and to hydroxyl anion. Superoxide anion can also interact with nitric oxide to generate peroxynitrate anion, a free radical with cytotoxic and antimicrobial properties. Possible molecular targets of these species include genomic DNA, electron transport, and sulfhydryl groups of proteins and nonproteins.

In addition to the direct antimicrobial properties of superoxide anion and its downstream metabolites, NADPH oxidase can also mediate host defense by activation of neutrophil primary granular proteases. Reeves et al. [1] showed that activation of NADPH oxidase in neutrophils leads to an influx of reactive oxidant species into the endocytic vacuole, resulting in an accumulation of anionic charge. To maintain electrogenic neutrality, K+ ions cross the membrane in a pH-dependent manner. The rise in ionic strength leads to the release of cationic granule proteins, including elastase and cathepsin G, which are bound to the anionic proteoglycan matrix in the inactivated state within primary granules. Knockout mice deficient in the cationic granule proteins recapitulate some of the features of the CGD phenotype regarding susceptibility to experimental bacterial and fungal infections [19]; however, work from our laboratory showed that double knockout neutrophil elastase- and cathepsin G-deficient mice have intact host defense against pulmonary challenge with Aspergillus fumigatus, in contrast to CGD mice, which are highly susceptible (unpublished data). Thus, deficiency in these two serine proteases does not fully recapitulate the severity of immune impairment observed in CGD in mice.

Following phagocytosis of bacteria, neutrophil granules fuse with the phagosome to augment intracellular killing. Neutrophils also release granule proteins and chromatin that co-mingle in the extracellular space to form neutrophil extracellular traps (NETs). These NETs bind to and kill bacteria and degrade bacterial virulence factors [20], and target fungi [21]. Release of NETs requires death of neutrophils and breakdown of cell membranes [22]. Neutrophils from CGD patients are deficient in NET formation [21,22]. This deficiency in NET formation was reversed in neutrophils from a CGD patient following gene therapy that led to NADPH oxidase-competent neutrophils [21], supporting the role of NADPH oxidase in NET generation.

The long pentraxin (PTX) 3, a soluble pathogen recognition receptor critical for anti-Aspergillus host defense in mice [23], is stored in neutrophil secondary granules and can localize in NETs following neutrophil activation [24]. Interestingly, administered PTX3 augmented anti-Aspergillus host defense in CGD mice [25]. It is a high priority to understand the relative contributions of NADPH oxidase-derived free radicals versus protease activation and NET formation to antimicrobial host defense.

Invasive aspergillosis in CGD: a disorder of host defense and excessive inflammation

‘Mulch pneumonitis’ is a life-threatening complication in CGD characterized by rapid onset life-threatening pulmonary inflammation following mould exposure, and treated with systemic antifungals and prolonged systemic corticosteroids [26]. Mulch pneumonitis emphasizes the dual role of NADPH oxidase both as a critical mediator of host defense and as a regulator of inflammation.

Knockout mouse models further shed light on the pathogenesis of aspergillosis in CGD. Even low virulent mutant strains of A. nidulans caused mortality in pulmonary aspergillosis in CGD mice due to excessive inflammation [27]. In addition, intratracheal administration of heat-killed A. fumigatus hyphae elicited mild self-limited inflammation in wildtype mice, but robust and persistent inflammation in CGD mice [28]. Consistent with these findings, subcutaneous administration of branched fungal beta-glucan (a major pro-inflammatory component of fungal cell wall) induced increased inflammation and necrosis in CGD versus wildtype mice [29]. These results point to NADPH oxidase functioning to limit inflammation induced by fungal cell wall-derived products.

NADPH oxidase: a regulator of innate immunity

Dectin-1 is a receptor and immunomodulator of beta-glucans, which are ubiquitous cell wall constituents of fungi and plants [30,31]. Dectin-1 signals through the tyrosine kinase Syk and the adaptor protein, caspase recruitment domain (CARD)-9 [27,33,34]. Syk controls both pro-IL-1β synthesis and Nlrp3-dependent inflammasome activation required for host defense against Candida albicans [35]. During germination (transition from spores to hyphae), cell wall beta-glucans of A. fumigatus become unmasked and can ligate dectin-1, leading to NADPH oxidase activation and production of pro-inflammatory cytokines [35–37]. The ability of host cells to recognize fungal cell wall products displayed in a stage-specific fashion likely assists in calibrating the inflammatory response, and to avert excessive inflammation following inhalation of ubiquitous fungal spores [38].

Dectin-1-mediated antifungal host defense likely involves NADPH oxidase activation, and triggering of pro-inflammatory cytokines. Werner et al. showed that dectin-1-deficient mice had increased susceptibility to aspergillosis [39]. At a very high fungal inoculum (~ intratracheal 5 × 10⁷ spores per mouse), greater mortality occurred in dectin-1−/− versus wildtype mice. Dectin-1−/− mice had impaired IL-1α, IL-1β, TNF-α, CCL3/MIP-1α, CCL4/MIP-1β, CXCL1/KC, and IL-17 production, which resulted in diminished lung neutrophil recruitment and increased A. fumigatus growth. Neutrophils from dectin-1−/− mice had impaired NADPH oxidase activity and killing of A. fumigatus in vitro [39].

The IL-17 family of cytokines and receptors consist of six ligands (IL-17A→F) and five receptors (IL-17RA→IL-17RE) in mammals. IL-17A stimulates production of G-CSF, GM-CSF, TNF-α, and chemokines that regulate myelopoiesis and neutrophil recruitment to inflammatory sites [40,41]. The IL-23/Th-17 (IL-23 expands Th-17 cells) axis mediates several experimental autoimmune disorders, and is a promising target for drug development [42,43]. Both positive and negative effects on immune resistance to fungi have been attributed to the IL-23/Th17 axis in mouse models [43]. Depletion of IL-17 impaired A. fumigatus clearance in the lungs of wildtype mice [39], but enhanced anti-Aspergillus host defense in CGD mice [44].

How do dectin-1 and NADPH oxidase interact to mediate anti-Aspergillus host defense and inflammation? Data from mice and humans indicate that these two pathways have inter-related but distinct functions. In humans, deficiency in dectin-1 signaling is linked to familial mucocutaneous candidiasis, but not invasive fungal diseases [45,46]. In contrast, CGD is associated with a high risk of invasive aspergillosis. In mice, a lethal inoculum of A. fumigatus is several log-fold greater in dectin-1−/− mice versus previously published results in CGD mice [14,47].

Data in mice support the notion that dectin-1 and NADPH oxidase have opposing effects in regulating the inflammatory response to fungal constituents. Dectin-1 induces proinflammatory cytokines and chemokines, including IL-17 [32,39,45,46,48,49]. In contrast, NADPH oxidase limited expansion of IL-17-producting lymphocytes and augmented regulatory T-cell responses in murine aspergillosis [44].

We found that intratracheal zymosan (a fungal cell wall product comprised principally of beta-glucans that ligates dectin-1 and toll-like receptor-2) [5,49] elicited progressive pyogranulomata in CGD mice that histologically resembled aspergillosis, whereas wildtype mice developed minimal self-limited inflammation [50]. CGD mice developed increased NF-κB activation, and downstream proinflammatory cytokines (TNF-α, IL-17, and G-CSF) versus wildtype mice. Studies in vivo and in isolated macrophages demonstrated that NADPH oxidase was required for zymosan-induced activation of Nrf2, a redox-sensitive anti-inflammatory transcription factor [51]. Consistent with these finding in mice, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation. These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling limits lung inflammation. In addition, these data also suggest that one of the functions of NADPH oxidase is to counter-regulate pro-inflammatory cytokine responses induced by dectin-1.

NADPH oxidase regulates T-cell phenotypes

In addition to modulating innate immune responses, NADPH oxidase can regulate dendritic cell and T-cell phenotypes [44,52–55]. NADPH oxidase is expressed in dendritic cells and recruited to early phagosomes [3,56,57]. NADPH oxidase activation prevents acidification of phagosomes, limiting antigen degradation and enhancing antigen presentation [3,55,57]. NADPH oxidase can also prime the development of myeloid-derived suppressor cells, which suppress T-cell immunity [54,58].

Romani et al. [44] demonstrated a central role of NADPH oxidase in determining the balance between Th17 and regulatory T-cell (Tregs) development through activation of tryptophan catabolism in mice challenged with A. fumigatus. Naturally occurring Tregs are recruited early in the inflammatory response to Aspergillus in wildtype mice, and are capable of suppressing inflammation [59]. Superoxide is a co-factor of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway. IDO-mediated tryptophan metabolism along the kynurenine pathway is defective in CGD mice [44]. CGD mice developed acute neutrophilic fungal pneumonia after intratracheal Aspergillus fumigatus challenge whereas wildtype mice were resistant to infection. CGD mice had augmented Th17 and diminished Treg responses associated with impaired IDO activation compared to wildtype mice. Administration of natural kynurenine distal to the IDO target enhanced host defense and limited lung inflammation in CGD mice after Aspergillus challenge. A limitation of this study is that all experiments were conducted in mice, and regulation of IDO activation [60] and Th17 differentiation [61] may differ in humans.

In contrast to Aspergillus species, CGD mice are more resistant to pulmonary Cryptococcus neoformans infection than wildtype mice [62]. CGD mice demonstrated increased Th1 responses, improved pathogen containment within pulmonary granulomatous lesions, and decreased dissemination to the brain. These studies illustrate the context-dependent role of NADPH oxidase in shaping the host immune response.

Conclusions and future perspectives

NADPH oxidase is both a critical mediator of antimicrobial host defense and inflammation. Knowledge gained from CGD patients and mice has significantly added to our understanding of antifungal immunity. Understanding the interaction between NADPH oxidase and dectin-1 in regulating innate and T-cell responses will be critical to gaining insight into the balance between controlling the growth of inhaled fungi while averting excessive inflammation. More broadly, the role of NADPH oxidase in regulating myeloid and T-cell responses is relevant to regulation of physiological inflammation and to pathological states, including acute lung injury, autoimmunity and tumor immunology.

A large family of NADPH oxidases has been identified in plants, fungi, invertebrate animals and higher animals. In mammals, NADPH oxidase isoforms are found in a variety of cells and tissues, including kidney [63], colon [64], thyroid gland, lymphoid cells [65], vascular endothelium, smooth muscle cells, and adventitia [66–68], and airways [69]. The role of non-myeloid Nox isoforms in regulating host defense and inflammation is not understood, and is an important area of active research [70].