Determining the role of NADPH oxidases in the context of virus infection is an emerging area of research and our knowledge is still sparse. The expression of various isoforms of NOX/DUOX (NADPH oxidase/dual oxidase) in the epithelial cells (ECs) lining the respiratory tract renders them primary sites from which to orchestrate the host defence against respiratory viruses. Accumulating evidence reveals distinct facets of the involvement of NOX/DUOX in host antiviral and pro-inflammatory responses and in the control of the epithelial barrier integrity, with individual isoforms mediating co-operative, but surprisingly also opposing, functions. Although in vivo studies in mice are in line with some of these observations, a complete understanding of the specific functions of epithelial NOX/DUOX awaits lung epithelial-specific conditional knockout mice. The goal of the present review is to summarize our current knowledge of the role of individual NOX/DUOX isoforms expressed in the lung epithelium in the context of respiratory virus infections so as to highlight potential opportunities for therapeutic intervention.

INTRODUCTION

Reactive oxygen species (ROS), mostly derived from phagocytes recruited to the airway mucosa, are well known to be important culprits in the pathophysiology of various acute and chronic lung inflammatory diseases, including acute lung injury, acute respiratory distress syndrome, asthma and chronic obstructive pulmonary disease [1,2]. However, it is now also evident that non-phagocytic cells, including airway and alveolar epithelial cells (ECs), express oxidases capable of generating stimulus-induced intermediate levels of ROS, such as superoxide anion (O2•−) and hydrogen peroxide (H2O2) [3]. In the past decade, the concept of ‘redox biology’ has been refined with an improved characterization of the role of sub-toxic levels of ROS as a cellular switch for signalling cascades in various physiological processes, including cell proliferation, apoptosis and immune and pro-inflammatory responses. Similar to the regulation of cellular functions by the well-known phosphorylation/dephosphorylation processes, ROS actions in the regulation of cellular signalling are generally mediated by reversible oxidative post-translational modifications within specific target proteins [4]. This novel concept has led to re-evaluation of the role of ROS in the lung and their contribution to physiological responses.

An intensive area of investigation has been the identification of the source of ROS in the lung and the family of NADPH oxidase membrane enzymes has emerged as a major player in this regard. In the late 1980s, the identification of gp91phox {now known as NADPH oxidase 2 (NOX2); reviewed in [5]} as the NADPH oxidase responsible for the respiratory burst observed in professional phagocytic cells (neutrophils, macrophages and eosinophils) resulted from the study of the genetic defect causing chronic granulomatous disease (CGD), which is characterized by the failure to mount an effective innate defence against bacteria and fungi resulting in life-threatening recurrent infections and formation of granulomas. The clinical picture is diverse with multiple organs involved, including gastrointestinal (GI) tract, liver and skin, but the most commonly involved organ is the lung [6,7]. Antibiotic-/antifungal-based prophylactic treatment has successfully transformed CGD from a disease of early fatality to a manageable disease with a high survival rate [6,7].

The phagocyte NADPH oxidase consists of a membrane flavocytochrome b558 composed of NOX2 and the associated p22phox protein. Stimulus-dependent activation results in the phosphorylation and subsequent Rac-dependent translocation of cytosolic regulatory subunits, p47phox, p67phox and p40phox, which associate with the membrane subunits allowing electrons to transfer from NADPH to FAD and ultimately reducing O2 to generate O2•− [5]. A number of mutations in this complex have been identified as causative for CGD. X-linked CGD results from mutations in the genes encoding NOX2 (cybb gene). Less frequent autosomal recessive forms of CGD involve mutations in p22phox (cyba gene), p47phox (NCF1 gene), p67phox (NCF2 gene) or p40phox (NCF4 gene) [5,6].

Until the identification of the first NOX2 homologue in 1999, now known as NOX1, the prevailing idea was that NADPH oxidase activity was restricted to professional phagocytes [8]. However, with the discovery of a total of six human homologues of NOX2, NOX1–NOX5 and dual oxidases (DUOX1 and DUOX2), it is now clear that virtually all cell types, including airway and alveolar ECs, express at least one of these isoforms. The NOX family is involved in a large spectrum of functions ranging from host defence, cell growth/death, thyroid hormone synthesis, angiogenesis and blood pressure [3,9]. Although the basic functional principles are similar among the different family members, such that all catalyse the reduction of O2 by electrons transferred from NADPH, each NOX/DUOX exhibits specific molecular features that define their distinct regulatory mechanisms, their association with regulatory subunits and their oxidant subtype production (summarized in Table 1 and reviewed in details in [10,11]).

Table 1
Shared and specific characteristics of NOX/DUOX enzymes
 
 

Over the past decade, increased characterization of NOX/DUOX enzyme expression and function has revealed that distinct non-phagocyte isoforms are also involved in host defence. This function is not restricted to bacteria and fungus but also expands to a number of viruses, including the major respiratory viruses influenza virus (IAV), respiratory syncytial virus (RSV) and rhinovirus (RV). In the present paper, we review the current knowledge of the role of NOX/DUOX enzymes expressed in ECs along the respiratory tract in the host defence against respiratory viruses.

EXPRESSION OF NOX/DUOX IN ECs ALONG THE RESPIRATORY TRACT DURING RESPIRATORY VIRUS INFECTION

Multiple NOX/DUOX isoforms are present at basal levels in ECs

H2O2 has long been shown to be present in exhaled breath condensate and airway/alveolar secretions and the contribution of ECs to the release of H2O2 in the airway/alveolar lumen was shown in ex vivo culture of isolated rat alveolar ECs [3,12]. The demonstration that ECs can produce ROS in response to a specific stimulation came later from the observation that the basal rate of H2O2 produced by guinea pig tracheal ECs cultured at the air–liquid interface (ALI) can be enhanced following ex vivo stimulation with PMA or platelet-activating factor (PAF) [13]. Since then, various studies have demonstrated that ECs along the respiratory tract, including nasal, airways and alveolar epithelium, are capable of producing ROS in response to various cell stimulations [3]. In this regard, the role of NOX/DUOX enzymes has started to attract much attention.

The most prevalent NADPH oxidase transcripts found in ECs in the different sections of the respiratory tract are DUOX1 and DUOX2. DUOX mRNA and protein expression are detected in primary normal human nasal epithelial cells (NHNECs) [14,15], in ECs isolated from human or mouse trachea and bronchi cultured in submerged conditions or differentiated ex vivo by culture in ALI [1618] and in human type 2 alveolar ECs [19]. Although present at lower levels, transcripts for NOX1, NOX2, NOX4 and NOX5 isoforms are expressed in primary human tracheal ECs [16]. NOX1 mRNA has been detected in murine type 2 alveolar ECs [20]. The expression of NOX1 protein in murine alveolar ECs was confirmed by immunofluorescence staining of mouse lung sections [21]. In contrast, analysis of type 1 and type 2 alveolar ECs isolated from male Sprague–Dawley rats showed NOX2 protein expression in both cell types, with a substantially higher expression level in type 1 cells, accompanied by the detection of p22phox and the regulatory subunits p47phox and p67phox [22]. Expression of the NOX2 transcripts and protein was also reported in primary normal human bronchial epithelial cells (NHBECs) [23,24]. Further detailed studies will be required to determine whether NOX/DUOX isoforms expression in ECs along the respiratory tract is dependent on cellular subtype and species.

The expression of NOX/DUOX isoforms has also been widely studied in various cell lines including A549 adenocarcinomic human alveolar basal cells, Calu-3 human sub-bronchial serous gland cells, hTE human tracheal ECs, NCI-H292 human pulmonary mucoepidermoid carcinoma cells and HBE1 immortalized human bronchial ECs. Although these studies confirmed the overall expression of DUOX1/DUOX2 and of NOX1, NOX2, NOX4 and NOX5, they also highlight significant discrepancies between cell lines that should be taken into account for functional NOX analysis [16,23,2528].

Inducible expression of DUOX2 during respiratory virus infection

Analysis of NOX/DUOX expression in airway and alveolar ECs subjected to respiratory virus infection provided the first hint of a role for DUOX2 in the host antiviral response. The initial observation that epithelial DUOX2 transcripts were inducible in conditions of viral infection was made in human primary tracheobronchial ECs stimulated with poly (I:C), a mimetic of viral dsRNA widely used to study the response to virus sensing or infected with the non-segmented positive-sense ssRNA picornavirus RV1B [29]. Further studies have demonstrated that DUOX2 expression, at both transcript and protein levels, is also observed in response to non-segmented negative-sense ssRNA paramyxoviruses, Sendai virus (SeV) and RSV in A549 cells, polarized Calu-3 cells and NHBECs [25]. Infection with negative-sense ssRNA orthomyxovirus IAV also induces DUOX2 transcripts and protein levels in polarized NHBECs, to an extent that depends on the strain. Infection with the IAV Texas/36/1991 (H1N1) strain led to a robust increase in DUOX2 expression, whereas IAV Wyoming/3/2003 (H3N2) induced DUOX2 expression to a lower level [24]. In agreement with the now well-characterized bi-directional promoter that concomitantly transcribes DUOX2 and DUOXA2 [30,31], DUOXA2 mRNA transcript levels were also induced in response to SeV and IAV [24,25]. In contrast with a clear pattern of DUOX2 induction in the various respiratory virus infections assessed to date, the analysis of DUOX1 expression revealed distinct profiles. Although DUOX1 mRNA levels were unchanged following poly (I:C) stimulation or infection by RV1B in human primary tracheobronchial ECs, DUOX1 mRNA and protein expression levels were down-regulated by both IAV Texas/36/1991 (H1N1) and Wyoming/3/2003 (H3N2) IAV strains, with H3N2 virus inducing a more pronounced effect [24,29] (Figure 1).

Regulation of DUOX expression during respiratory viral infection in ECs of the respiratory tract

Figure 1
Regulation of DUOX expression during respiratory viral infection in ECs of the respiratory tract

Infection of ECs by respiratory viruses, such as SeV, RSV, IAV or RV trigger the secretion of antiviral and pro-inflammatory cytokines, including IFNβ and TNFα. Binding of IFNβ to its cognate receptor activates the classical Janus kinase (JAK)/STAT antiviral pathway mediated by the ISGF3 (IFN-stimulated gene factor 3) transcription factor complex that transcribes numerous ISGs encoding proteins with antiviral activities. Additionally, the synergism between IFNβ and TNFα induces late DUOX2 and DUOXA2 transcripts (transcribed from the same bi-directional promoter) through a non-canonical STAT1-independent antiviral signalling pathway. The co-stimulation with IFNβ and IL-1β and the stimulation by IFNγ alone also induce DUOX2/DUOXA2 expression by a pathway that remains to be determined. IFNβ and TNFα secreted upon viral infection in ECs are sufficient to trigger DUOX2/DUOXA2, but all cytokines responsible for DUOX2/DUOXA2 induction in ECs could also be secreted by immune cells attracted to and activated in the lung following infection. Viruses have evolved multiple mechanisms to counteract the host response. IAV infection was shown to down-regulate DUOX1 levels. Additionally, RSV was shown to interfere with DUOX2 induction. Analysis of DUOX localization in pulmonary ECs revealed that DUOX proteins are located at the apical plasma membrane, but also in nuclear speckles.

Figure 1
Regulation of DUOX expression during respiratory viral infection in ECs of the respiratory tract

Infection of ECs by respiratory viruses, such as SeV, RSV, IAV or RV trigger the secretion of antiviral and pro-inflammatory cytokines, including IFNβ and TNFα. Binding of IFNβ to its cognate receptor activates the classical Janus kinase (JAK)/STAT antiviral pathway mediated by the ISGF3 (IFN-stimulated gene factor 3) transcription factor complex that transcribes numerous ISGs encoding proteins with antiviral activities. Additionally, the synergism between IFNβ and TNFα induces late DUOX2 and DUOXA2 transcripts (transcribed from the same bi-directional promoter) through a non-canonical STAT1-independent antiviral signalling pathway. The co-stimulation with IFNβ and IL-1β and the stimulation by IFNγ alone also induce DUOX2/DUOXA2 expression by a pathway that remains to be determined. IFNβ and TNFα secreted upon viral infection in ECs are sufficient to trigger DUOX2/DUOXA2, but all cytokines responsible for DUOX2/DUOXA2 induction in ECs could also be secreted by immune cells attracted to and activated in the lung following infection. Viruses have evolved multiple mechanisms to counteract the host response. IAV infection was shown to down-regulate DUOX1 levels. Additionally, RSV was shown to interfere with DUOX2 induction. Analysis of DUOX localization in pulmonary ECs revealed that DUOX proteins are located at the apical plasma membrane, but also in nuclear speckles.

The observation that DUOX2 induction in response to virus infection is dependent on virus replication [24,25] is consistent with the described induction of DUOX2 by the autocrine/paracrine action of virus-induced cytokines [24,25]. Secretion of antiviral and pro-inflammatory cytokines is a hallmark of the early innate immune response in ECs that contribute to the early elimination and limit the progression of the infection in the airway mucosa. This cytokine response is initiated by the recognition of RNA moieties that contain highly conserved ‘molecular signatures’ produced upon viral replication by a heterogeneous group of pattern recognition receptors, including the Toll-like receptors (TLRs) and the cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) [32,33]. Sensing of ssRNA viruses in airway/alveolar ECs is mainly mediated by RIG-I and melanoma-differentiation-associated gene 5 (MDA5) members of the RLRs family. Upon nucleic-acid sensing, multiple downstream signalling pathways are engaged that ultimately induce the expression of antiviral and pro-inflammatory cytokine and chemokine genes [34,35] (Figure 1). Analysis of the apical and basolateral media of differentiated NHBECs provided a broad profile of the cytokines and chemokines secreted in response to IAV and RSV. The main antiviral cytokines secreted by ECs are type I (α/β) and type III (λ) interferons (IFNs) [36]. Recently, conditional reporter mice that express luciferase exclusively in defined cell types were used to confirm that ECs, together with lung macrophages, are the main source of IFNβ in IAV-infected lung in vivo [37]. ECs also secrete multiple pro-inflammatory cytokines and chemokines including, but not restricted to, tumour necrosis factor (TNF) α, interleukin (IL)-1α, CXC chemokine ligand (CXCL) 8 (IL-8), CC chemokine ligand (CCL) 2 [monocyte chemoattractant protein (MCP)-1], CCL3 [macrophage inflammatory protein (MIP)-1α], CCL5 [regulated upon activation normal T-cell expressed and presumably secreted (RANTES)] and granulocyte/macrophage colony-stimulating factor (GM-CSF) [36]. Molecular analysis revealed that, unlike typical IFN-stimulated genes (ISGs), IFNβ is not sufficient to trigger induction of DUOX2 and DUOXA2 expression in airway/alveolar ECs. Rather, co-stimulation with IFNβ and another pro-inflammatory cytokine is required. IFNβ and TNFα co-stimulation is known to trigger a delayed antiviral response [38] and it was demonstrated that it induces DUOX2/DUOXA2 expression in A549 cells and NHBECs (Figure 1) [25]. The signalling cascade engaged downstream of this specific cytokines co-stimulation is still sparsely characterized, but DUOX2 expression was found to be mediated by a non-canonical signal transducer and activator of transcription (STAT) 2- and IFN-regulatory factor (IRF)-9-dependent, but STAT1-independent, pathway that requires tyrosine kinase 2 (Tyk2)-dependent STAT2 phosphorylation [25]. Later studies revealed that IFNβ/IL-1β co-stimulation also triggers significant levels of DUOX2/DUOXA2 expression in polarized NHBECs, but the pathway responsible for this induction remains unknown [24]. Importantly, silencing of TNFα receptor (TNFR) or type 1 IFN receptor subunit (IFNAR1) in A549 cells impaired paramyxovirus-induced DUOX2/DUOXA2 expression, thus strongly supporting that IFNβ and TNFα derived from infected ECs are responsible for the induction [25]. This shows that the early infection of ECs is sufficient to induce DUOX2/DUOXA2 expression. However, it also supports the possibility that immune cells recruited to the lung while the infection progresses, such as plasmacytoid dendritic cells (pDCs) and macrophages that produce high levels of IFNβ, TNFα and IL-1β, could further enhance the induction of epithelial DUOX2. Cytokines secreted by immune cells also include IFNγ, which was shown to trigger DUOX2 induction in tracheobronchial ECs [29] (Figure 1).

Consistent with the induced expression of DUOX2/DUOXA2, increased extracellular H2O2 release measured using the homovanilic acid (HVA)-based fluorimetric assay, is observed in A549 and polarized NHBECs following virus infection or IFNβ/TNFα cytokine co-stimulation [24,25]. Of note, increased intracellular Ca2+ concentrations via inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump with thapsigargin was required for the induction of detectable levels of H2O2 in polarized NHBECs infected with IAV H1N1 [24]. Whereas siRNA-mediated silencing of DUOX2 in A549 cells demonstrated that DUOX2 was responsible for extracellular H2O2 induction upon IFNβ/TNFα cytokine co-stimulation [25], contribution of DUOX2 to extracellular H2O2 release from IAV H1N1-infected polarized NHBECs remains to be clearly demonstrated. This extracellular release of H2O2 in conditions of viral infection is consistent with the observation that DUOX2 and DUOXA2 are present as stable functional complexes detectable at the apical plasma membrane of ECs [3,16,39,40]. In addition to extracellular H2O2 release, lentivirus-based shRNA-mediated knockdown of DUOX2 revealed that DUOX2, together with mitochondria, significantly contributes to the intracellular production of H2O2 as measured by dihydrodichlorofluorescein diacetate (DCF-DA) fluorescence in NHNECs infected with the IAV A/WSN/33 (H1N1) strain [15]. This might reflect DUOX2 localization in intracellular membranes or in IAV H1N1-induced nuclear speckles adjacent to sites of RNA processing [24,40].

ROLE OF EPITHELIAL NOX/DUOX IN THE MUCOSAL HOST IMMUNE DEFENCE

The pseudostratified epithelium lining the respiratory tract is central to the multiple defence mechanisms that protect the airways and alveoli from the continuous exposure to invading pathogens. The epithelium constitutes the very first physical barrier erected against airborne pathogens shielding the host from the infection. Apical cilia of ECs and secreted mucus are responsible for the mucociliary escalator that facilitates mechanical clearance of the pathogens. Importantly, as mentioned above, ECs are equally equipped for detecting pathogens and mounting the earliest elements of the innate arm of host defence via secretion of antimicrobial compounds, including proteins and peptides, and through the secretion of cytokines and chemokines that not only limit viral replication and spreading, but also alarm the immune system of the infection. The distribution of NOX/DUOX in ECs along the respiratory tract has led to the study of their role in the host defence to virus infections. Current results have led to a picture in which DUOX2, NOX2 and NOX1 are involved in the regulation of various aspects of the antiviral response mounted by ECs, as detailed below.

Antiviral functions of DUOX2

The first demonstration of a role for DUOX2-derived ROS in the antiviral host defence came from the observation that silencing of DUOX2 in A549 cells or NHBECs significantly impairs establishment of an efficient antiviral response induced by SeV or by IFNβ/TNFα co-stimulation [25]. Additional studies showed that shRNA-mediated knockdown of DUOX2 in NHNECs results in highly increased IAV A/WSN/33 (H1N1) titres [15]. Treatment of polarized NHBECs with the previously recognized NOX1/NOX4 inhibitor GKT136901 was shown to abolish thapsigargin-induced H2O2 release, indicative of inhibition of DUOX1/DUOX2 activity. GKT136901 treatment of polarized NHBECs enhanced IAV A/Texas/36/1991 (H1N1) replication [24]. Confirmation of the role of epithelial DUOX isoforms in the antiviral response in vivo is biased since mice deficient in DUOX activities suffer from severe hypothyroidism [41]. Moreover, DUOX expression pattern and virus-induced DUOX isoforms up-regulation differ in the lung of mice compared with humans [24]. However, use of intranasal siRNA to knockdown both DUOX1 and DUOX2 in C57BL/6J mice before infection with the pathogenic clinical isolate of 2009 pandemic HH05 (H1N1) IAV yielded increased virus titre compared with control siRNA-treated mice [24]. Taken together with the in vitro and ex vivo studies described above, this work strongly supports a key role for DUOX2 in the overall airway antiviral defence in vivo. Possible mechanisms of DUOX2-dependent antiviral function have been highlighted as outlined below.

The cytokine/chemokine response is a major determinant of the elimination or progression of respiratory virus infections. Antiviral cytokines restrict virus replication, whereas pro-inflammatory cytokines and chemokines contribute to the recruitment and activation of leucocytes in the airway mucosa and thereby orchestrate the development of an appropriate adaptive immune response. This cytokine response is tightly regulated and evidence supports a role of epithelial DUOX2 in various steps of this regulation. First, analysis of cytokine levels produced by ECs in response to SeV or IAV infection in two independent studies revealed that the role of DUOX2 in restricting virus replication is at least, in part, attributable to the requirement of DUOX2-dependent H2O2 production for the sustained production of IFNβ and IFNλ [15,25]. However, discrepancies have been reported concerning the underlying mechanism. Indeed, DUOX2 was found to regulate IFNλ at the mRNA level during IAV infection of NHNECs, but IFNβ/λ mRNA levels remained steady in A549 cells and NHBECs infected with SeV [15,25] (Figure 2). A second observation supports a role of DUOX2 in the regulation of cytokine production. DUOX2 is required for nuclear factor κB (NF-κB) activation and subsequent IL-8 expression, in response to flagellin-induced TLR5 signalling in NHNECs cells. In this same study, challenge of DUOX2 knockout mice with flagellin revealed that DUOX2 influences the expression of various cytokines and chemokines as well as IFN-related genes culminating in a decreased neutrophil infiltration [42]. Although the latter report strongly supports DUOX2-dependent expression of NF-κB-regulated pro-inflammatory cytokines, this remains to be demonstrated in ECs infected with respiratory viruses. Alternatively, epithelial DUOX2 might also modulate virus-induced inflammation through the regulation of a disintegrin and metalloproteinase 17 (ADAM17), also known as TNFα-converting enzyme (TACE). TACE is a membrane-bound enzyme responsible for the shedding of cytokines or their respective receptors, including TNFα, TNFR1 and IL-6 [43]. Stimulation of NCI-H292 ECs and primary human small airway EC (SAECs) using poly (I:C) to engage TLR3 results in the TACE-dependent release of the 34 kDa soluble TNFR1 (sTNFR1) that binds to and sequesters TNFα to modulate its bioactivity [44] (Figure 2). sTNFR1 shedding is mediated via activation of a TLR3–Toll/IL-1 receptor domain-containing adaptor protein inducing IFNβ-related adaptor molecule (TRIF)–RIP1 signalling cascade and is dependent on DUOX2-mediated H2O2 production [44]. DUOX1 was also attributed an important role in TACE activation following PMA, human neutrophil elastase or lipopolysaccharide (LPS) stimulation of airway ECs [28,45]. However, the contribution of DUOX1-mediated TACE activation in the context of virus infections remains to be evaluated.

Antiviral functions of DUOX2

Figure 2
Antiviral functions of DUOX2

Virus- or cytokine-induced DUOX2-dependent H2O2 production in ECs is associated with different functions that play a role in the antiviral and pro-inflammatory responses to respiratory virus infections. Viral nucleic acid sensing is ensured by the RIG-I and MDA5 cytosolic receptors and the TLR3 that mediate activation of NF-κB and IRF-3 transcription factors that transcribe genes encoding for antiviral IFNβ and IFNλ. First, DUOX2-dependent H2O2 is required to sustain the IFNβ and IFNλ levels at the late stage of infection, but it is unclear whether regulation occurs at the mRNA or protein levels. In addition, activation of TLR3 using the viral mimetic poly (I:C) revealed that DUOX2-dependent H2O2 positively regulates the activity of the TACE that is involved in the shedding of the TNFR1 to generate sTNFR1 thereby down-regulating the inflammatory response. Solid lines represent reported mechanistic links, whereas dotted lines indicate potential mechanistic links that have not yet been fully demonstrated.

Figure 2
Antiviral functions of DUOX2

Virus- or cytokine-induced DUOX2-dependent H2O2 production in ECs is associated with different functions that play a role in the antiviral and pro-inflammatory responses to respiratory virus infections. Viral nucleic acid sensing is ensured by the RIG-I and MDA5 cytosolic receptors and the TLR3 that mediate activation of NF-κB and IRF-3 transcription factors that transcribe genes encoding for antiviral IFNβ and IFNλ. First, DUOX2-dependent H2O2 is required to sustain the IFNβ and IFNλ levels at the late stage of infection, but it is unclear whether regulation occurs at the mRNA or protein levels. In addition, activation of TLR3 using the viral mimetic poly (I:C) revealed that DUOX2-dependent H2O2 positively regulates the activity of the TACE that is involved in the shedding of the TNFR1 to generate sTNFR1 thereby down-regulating the inflammatory response. Solid lines represent reported mechanistic links, whereas dotted lines indicate potential mechanistic links that have not yet been fully demonstrated.

Besides the cytokine response, epithelial DUOX2 might also contribute to the antiviral response through the regulation of the antimicrobial activity in the lumen of the airways. Lactoperoxidase (LPO) secreted in the airway lumen by submucosal glands and surface goblet cells utilizes H2O2 and thiocyanite (SCN) to form the bactericidal compound hypothiocyanate (OSCN) [4648]. DUOX-derived H2O2 contributes to this LPO/OSCN defence mechanism [17,18,49,50]. The importance of DUOX enzymes in this system is also demonstrated by the capacity of Pseudomonas aeruginosa, an important aetiologic agent in cystic fibrosis, to subvert killing by the LPO/OSCN system through inhibition of DUOX up-regulation and activity via the virulence factor pyocyanin [51]. Few studies have approached the question of whether this oxidative defence system is also active against respiratory viruses and participates in the antiviral defence of the airways. In a cell-free assay, OSCN inhibits RSV and the pandemic IAV A/California/2009 (H1N1) strain infectivity [52,53]. Virucidal activity against RSV was found to be pH-dependent, with OSCN being effective at pH 6, but not at neutral pH [53]. However, a more recent study concluded that hypoiodous acid (HOI), an alternative end-product generated by LPO through the use of iodide as a substrate, but not OSCN, possesses antiviral properties against adenovirus or RSV [54]. HOI exhibited significant virucidal activity against adenovirus at pH between 6 and 8, whereas a pH between 6 and 6.5 was required for virucidal activity against RSV. Additionally, supplementation of well-differentiated primary porcine airway ECs with LPO and sodium iodide abrogated infection by adenovirus or RSV [54]. The pH required for effectiveness of this LPO-dependent antiviral activity is consistent with the pH of the airway surface liquid being slightly acidic [55] and in this regard DUOX activity might contribute to H+ secretion into the airways and acidification of the airway lumen [16]. In conclusion, although the afore-mentioned studies indicate a potential antiviral role for DUOX2 through an LPO-dependent mechanism, the physiological relevance of this mucosal oxidative antiviral defence remains to be demonstrated.

Epithelial NOX2 is essential for the capacity of ECs to mount an antiviral host defence

The central importance of NOX2 expressed in phagocytes to innate host defence is clearly illustrated in the CGD condition characterized by severe and recurrent bacteria and fungi infections as a result of defective innate defence (reviewed in [5]). Patients with CGD are considered to have a normal immunity against viruses, although susceptibility to severe RSV infection was reported [56]. As described above, expression of NOX2 in ECs along the respiratory tract has only been recently described, but the limited understanding of its function in these cells points to an important role in antiviral defence.

The role of NOX2 in the host defence against respiratory viruses was first reported in the context of IAV infection of NOX2 knockout mice. Two independent studies concluded that the absence of NOX2 increases IAV A/X-31 (H3N2) clearance reduces lung damage and improves lung function, but they diverge regarding the affect of NOX2 on lung inflammation [57,58] (Table 2). In the study by Snelgrove et al. [57], the lack of functional NOX2 in C57BL/6 Cybb tm1 mice [59] paradoxically resulted in an increased inflammatory infiltrate, consisting of macrophages, neutrophils and Th1-skewed T-cells that is restricted to the airways. In contrast, the lung parenchyma inflammatory infiltration was reduced [57]. On the other hand, Vlahos et al. [58] reported that male NOX2−/y C57BL/6 mice infected with IAV exhibit decreased MCP-1 levels and reduced airway inflammation due to decreased infiltration of macrophages. This decrease in macrophages probably contributes to the observed decreased levels of lung peroxinitrite as IAV was shown to enhance NOX2-dependent ROS production in macrophages [60]. Although Snelgrove et al. [57] proposed that decreased virus titre was due to the increased macrophage numbers in the airways of NOX2 Cybb tm1 mice presumably clearing the virus, the study by Vlahos et al. [58] reported a reduction in macrophages in the NOX2−/y lungs, which is thus unlikely to explain the observed reduction in virus titres in this model [57,58]. These divergences could be due to either the different genetic backgrounds of the mice and/or the viral doses used (Table 2). Mice harbouring a mutation in the NCF1 gene encoding p47phox (B10.Q.Ncf1mut/mut) exhibit highly reduced p47phox expression and undetectable ROS response. These mice exhibit decreased severity of H5N1-mediated acute lung injury, which is associated with an impaired production of oxidized phospholipids that induce cytokine expression through a TLR4-dependent pathway [61] (Table 2). This observation further supports a role for NOX2 in IAV-induced inflammation [61]. A major limitation of the different mouse models used in these studies (Table 2) is that they could not distinguish the function of NOX2 in phagocytic cells compared with ECs. Assessing this aspect in vivo is awaiting lung epithelial-specific NOX2 knockout models. Meanwhile, in vitro and ex vivo infections of A549 cells and NHBECs has started to decipher the specific involvement of epithelial NOX2 in response to virus infection. That silencing of NOX2 in A549 cells yielded an increase in SeV replication underlines the importance of NOX2 in the capacity of ECs to mount an antiviral response [62]. Further mechanistic analysis unveiled a central role of basal NOX2 activity in the potent mRNA and protein expression of the central adaptor MAVS (mitochondrial antiviral signalling protein) that mediates the innate immune response downstream of RIG-I [62]. Thereby, NOX2 regulates SeV- and RSV-dependent inhibitor of NF-κB kinase β (IKKβ) and TNF-associated factor-associated NF-κB activator-binding kinase 1 (TBK1) kinase activities acting upstream of NF-κB and IRF-3 for pro-inflammatory and antiviral cytokines TNFα, CCL5 and IFNβ expression [23,62,63] (Figure 3). Although NOX2 was mainly detected in the cytoplasmic fraction of NHBECs [24], NOX2 was also found located at the luminal surface of alveolar, mainly type 1, ECs [22]. Extracellular O2•− release was found to be essential for basal and epidermal growth factor (EGF)-induced epithelial Na+ channel (ENaC) activity [22,64]. ENaC ensures Na+ absorption in the airways and is thereby critical for controlling fluid homoeostasis for proper luminal space moisture essential for the efficient protection of the airway mucosa [65]. Several viruses, RSV, IAV and severe acute respiratory syndrome coronavirus (SARS-CoV), inhibit ENaC function thus leading to reduced alveolar fluid clearance contributing to lung oedema formation [6668]. Whether this is related to an affect of these viruses on epithelial NOX2 activity remains to be determined.

Table 2
Phenotype of mice deficient in NOX activity subjected to IAV infection
 
 

Function of NOX2 in ECs in relation to respiratory virus infections

Figure 3
Function of NOX2 in ECs in relation to respiratory virus infections

Basal NOX2-dependent O2•− production is essential to regulate the expression of the MAVS, a central adaptor that co-ordinates the innate immune response downstream of nucleic acid sensing by the RIG-I and MDA5 cytosolic pattern recognition receptors. Thus, NOX2-dependent regulation of MAVS expression is essential to trigger the activation of NF-κB and IRF-3 transcription factors that translocate to the nucleus to transcribe genes encoding antiviral and pro-inflammatory cytokines. Furthermore, NOX2 basal activity is also required for the activation of the ENaC, which plays an important role in controlling fluid homoeostasis in the lung. If and how viruses regulate NOX2 activity remains to be determined. However, viruses, including RSV, IAV and SARS-CoV, have been shown to inhibit ENaC function thereby contributing to lung oedema formation. Whether this is related to NOX2 activity modulation remains unknown. Analysis of NOX2 localization in pulmonary ECs revealed that NOX2 is present either at the apical plasma membrane or in yet to be identified cytoplasmic membranes. Solid lines represent reported mechanistic links, whereas dotted lines indicate potential mechanistic links that have not yet been fully demonstrated.

Figure 3
Function of NOX2 in ECs in relation to respiratory virus infections

Basal NOX2-dependent O2•− production is essential to regulate the expression of the MAVS, a central adaptor that co-ordinates the innate immune response downstream of nucleic acid sensing by the RIG-I and MDA5 cytosolic pattern recognition receptors. Thus, NOX2-dependent regulation of MAVS expression is essential to trigger the activation of NF-κB and IRF-3 transcription factors that translocate to the nucleus to transcribe genes encoding antiviral and pro-inflammatory cytokines. Furthermore, NOX2 basal activity is also required for the activation of the ENaC, which plays an important role in controlling fluid homoeostasis in the lung. If and how viruses regulate NOX2 activity remains to be determined. However, viruses, including RSV, IAV and SARS-CoV, have been shown to inhibit ENaC function thereby contributing to lung oedema formation. Whether this is related to NOX2 activity modulation remains unknown. Analysis of NOX2 localization in pulmonary ECs revealed that NOX2 is present either at the apical plasma membrane or in yet to be identified cytoplasmic membranes. Solid lines represent reported mechanistic links, whereas dotted lines indicate potential mechanistic links that have not yet been fully demonstrated.

Role of epithelial NOX1 in the pro-inflammatory response to virus infection

The role of NOX1 in the host response to virus infection has been evaluated in vivo using NOX1−/y mice [21]. Infection of NOX1−/y mice with X-31 IAV strain resulted in a transient increase in the lung pro-inflammatory cytokines GM-CSF, CCL2, CCL3, CXCL2, TNFα, IL-1β and IL-6 mRNA expression at an early stage (day 3) of the infection compared with wild-type (WT) mice. By contrast, at a later stage of infection (day 7), the levels of CCL2, CCL3, CXCL2, IFNγ and IL-10 were significantly lower in NOX1−/y mice compared with WT control, whereas IL-1β, IL-6, TNFα and GM-CSF were comparable. The transient increase in pro-inflammatory cytokines expression at day 3 was associated with enhanced neutrophilia. Moreover, IAV-infected NOX1−/y mice exhibited greater peribronchial inflammation and alveolitis at both day 3 and day 7. However, the viral titre was similar to WT and NOX1−/y mice [21]. Although this study provides evidence of a protective role of NOX1 toward virus-induced inflammation in the early phase of infection, a direct link between NOX1-derived ROS and this phenotype cannot be fully concluded. Indeed, an unexpected increase in lung oxidative stress, together with NOX2-dependent inflammatory cell ROS production, was observed in IAV infected NOX1−/y mice compared with WT mice [21]. NOX1 is expressed in the lung epithelium, but is also detected in endothelial cells [20,21]. Although the specific contribution of epithelial NOX1 in the phenotype observed in vivo was not evaluated, the central role of ECs in the secretion of pro-inflammatory cytokines during IAV infection [37] strongly supports a major role of epithelial NOX1 in the modulation of the early cytokine wave that is increased in NOX−/y mice. A major transcription factor involved in the expression of pro-inflammatory cytokines is NF-κB. However, analysis of SeV-induced NF-κB-dependent promoter activation in A549 cells following knockdown of NOX1 by RNA interference failed to establish a link between NOX1 and NF-κB activation [23].

ROLE OF NOX/DUOX IN EPITHELIAL BARRIER INTEGRITY

In the respiratory mucosa, the intimate EC layer integrity and permeability are ensured by cell–cell junctions, including tight junctions and adherens junctions, which provide strong adhesion to form a physical barrier against invading pathogens while preserving communication between neighbouring cells. Respiratory viral infections can injure the epithelium through cytotoxic effects, disruption of tight junctions or interference with epithelial repair, causing a loss of integrity and protection that can be detrimental for the host [34].

Epithelial DUOX1 has a major implication in the process of wound healing. DUOX1-derived H2O2 is critical for wound closure in human NCI-H292 and HBE1 cell lines and murine tracheal ECs in vitro through the activation of the known mediator of EC migration and wound responses epidermal growth factor receptor (EGFR) [26,6971]. DUOX1 is also critical for epithelium regeneration in vivo in a murine model of epithelial injury induced by naphthalene [70]. Interestingly, a study conducted in a zebrafish model links DUOX-generated H2O2 production at the site of the wound to leucocyte recruitment, a necessary event for wound repair. Wounded epithelium might thus use DUOX-derived H2O2 not only to activate its own repair programme, but also as a second messenger for communication with the haematopoietic compartment further contributing to the repair mechanisms [72]. Although the role of DUOX1 in epithelium repair has not been directly studied in the context of respiratory virus infections, the observation that IAV inhibits DUOX1 expression suggests that this could be a factor contributing to IAV-induced epithelium injury [24] (Figure 2).

In contrast with the positive contribution of epithelial DUOX1 to the airway epithelium integrity, NOX1 has an adverse effect on epithelial barrier function. RV and poly (I:C) were shown to mediate disruption of the epithelial barrier function through dissociation of the tight junctions in polarized cultures of 16HBE14o cells [73]. This virus-induced barrier disruption was blocked by inhibitors of NOX activity, such as the Rac1 inhibitor NSC23766 or the generic flavoprotein inhibitor diphenyleneiodonium. Similar results were obtained following interference with NOX1 expression suggesting a key role of NOX1 in this process [73]. Importantly, NOX1-dependent barrier disruption facilitates bacterial transmigration across polarized airway ECs, a major event in the susceptibility to bacterial infections secondary to virus infection [73].

CONCLUSIONS

The role of NOX/DUOX-derived ROS in the host response to respiratory virus infections is an exciting emerging area of research that has a strong potential to unveil opportunities for therapeutic intervention. Improved therapeutic strategies are required for most respiratory viruses for which universal vaccine is not available or development of resistance to antiviral is a concern. Major advances have been made over the past decade that dramatically increased our knowledge with regards to the specific functions of each NOX/DUOX during respiratory virus infections. From the recent evidence, a multi-faceted picture is emerging in which individual isoforms mediate co-operative, but surprisingly, also opposing roles in the regulation of the antiviral and pro-inflammatory responses and in the control of the epithelial barrier integrity. A key challenge will be to fully determine the cell-specific role of each NOX/DUOX isoforms in vivo. Various isoforms are expressed in the ECs lining the respiratory tract, which renders them primary sites from which to orchestrate the host defence against respiratory viruses. Although animal models do not faithfully reflect every mechanisms occurring in humans, the field would highly benefit from the generation of lung epithelial-specific conditional knockout mice.

The current observations have already built the case for the inefficacy of global antioxidant strategies or generic approaches that interfere with the overall ROS production. Indeed these strategies would inhibit both detrimental and protective ROS-dependent effects. Development of more refined strategies based on the selective targeting of NOX/DUOX (reviewed in [74,75]) to control specific aspects of the host response to virus infection are needed. However, even if the specific targeting of NOX/DUOX is achieved, such a strategy would have to be finely tuned to target the harmful response, such as excessive pro-inflammatory response, while sparing or enhancing the beneficial actions, such as antiviral defence and protection of epithelium integrity. Given the importance of ROS in a number of acute and chronic lung diseases such as acute lung injury, asthma and chronic obstructive pulmonary disease that are all exacerbated by virus infections, a therapeutic strategy aimed at targeting specific NOX/DUOX may prove efficient in a broad range of human respiratory health conditions. However, the current knowledge of the role of NOX/DUOX in the host defence against respiratory virus infections strongly suggest that the patients under such therapy will need to be evaluated to ensure that they retain a sufficient capacity to mount a protective innate antiviral defence.

Abbreviations

     
  • ALI

    air–liquid interface

  •  
  • CCL

    CC chemokine ligand

  •  
  • CGD

    chronic granulomatous disease

  •  
  • CXCL

    CXC chemokine ligand

  •  
  • DUOX

    dual oxidase

  •  
  • EC

    epithelial cell

  •  
  • ENaC

    epithelial Na+ channel

  •  
  • GM-CSF

    granulocyte macrophage-colony stimulating factor

  •  
  • HOI

    hypoiodous acid

  •  
  • IAV

    influenza virus

  •  
  • IFN

    interferon

  •  
  • IL

    interleukin

  •  
  • IRF

    interferon-regulatory factor

  •  
  • ISG

    interferon-stimulated gene

  •  
  • LPO

    lactoperoxidase

  •  
  • MAVS

    mitochondrial antiviral signalling protein

  •  
  • MCP

    monocyte chemoattractant protein

  •  
  • MDA5

    melanoma-differentiation-associated gene 5

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NHBEC

    normal human bronchial epithelial cell

  •  
  • NHNEC

    normal human nasal epithelial cell

  •  
  • NOX

    NADPH oxidase

  •  
  • OSCN

    hypothiocyanate

  •  
  • RIG-I

    retinoic acid-inducible gene I

  •  
  • RLR

    RIG-I-like receptor

  •  
  • ROS

    reactive oxygen species

  •  
  • RSV

    respiratory syncytial virus

  •  
  • RV

    rhinovirus

  •  
  • SARS-CoV

    severe acute respiratory syndrome coronavirus

  •  
  • SeV

    Sendai virus

  •  
  • STAT

    signal transducer and activator of transcription

  •  
  • TLR

    Toll-like receptor

  •  
  • TNF

    tumour necrosis factor

  •  
  • TNFR

    TNFα receptor

  •  
  • sTNFR1

    soluble TNFR1

  •  
  • TACE

    TNFα-converting enzyme

  •  
  • WT

    wild-type

We thank Dr C. Vande Velde (CRCHUM, Montréal) for critical reading of the manuscript before submission.

FUNDING

This work was supported by the Canadian Institutes of Health Research (CIHR) [grant number MOP-130527]; and the Institute of Infection and Immunity (III) of the CIHR [grant number III-134054 (to N.G.)]. N.G. is a recipient of a Tier II Canada Research Chair.

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