Cellular Microbiology (2014) 16(8), 1168–1178

doi:10.1111/cmi.12322 First published online 7 July 2014

Microreview Phagocyte NADPH oxidase, chronic granulomatous disease and mycobacterial infections Christine Deffert,1,2* Julien Cachat,2 and Karl-Heinz Krause2 1 Laboratory for Biological Fluids, University Hospitals and Faculty of Medicine of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Geneva 14, Switzerland. 2 Department of Pathology and Immunology, Medical Faculty and University of Geneva; 1211 Geneva 4, Switzerland. Summary Infection of humans with Mycobacterium tuberculosis remains frequent and may still lead to death. After primary infection, the immune system is often able to control M. tuberculosis infection over a prolonged latency period, but a decrease in immune function (from HIV to immunosenescence) leads to active disease. Available vaccines against tuberculosis are restricted to BCG, a live vaccine with an attenuated strain of M. bovis. Immunodeficiency may not only be associated with an increased risk of tuberculosis, but also with local or disseminated BCG infection. Genetic deficiency in the reactive oxygen species (ROS)-producing phagocyte NADPH oxidase NOX2 is called chronic granulomatous disease (CGD). CGD is among the most common primary immune deficiencies. Here we review our knowledge on the importance of NOX2-derived ROS in mycobacterial infection. A literature review suggests that human CGD patient frequently have an increased susceptibility to BCG and to M. tuberculosis. In vitro studies and experiments with CGD mice are incomplete and yielded – at least in part – contradictory results. Thus, although observations in human CGD patients leave little doubt about the role of NOX2 in the control of mycobacteria, further studies will be necessary to unequivocally define and understand the role of ROS.

Received 25 April, 2014; revised 3 June, 2014; accepted 6 June, 2014. *For correspondence. E-mail [email protected]; Tel. (+41) 22 372 7333; Fax (+41) 22 372 73 39.

Introduction Tuberculosis remains a scourge of humanity. It is estimated that about one third of the human population carry the pathogen and that the worldwide death toll is over one million per year. Ancient descriptions of tuberculosis go back over several thousand years, and victims of tuberculosis have been immortalized in many pieces of art, including the opera La Boheme by Giacomo Puccini, the novel ‘Der Zauberberg’ by Thomas Mann, and the painting ‘Camille Monet at her death bed’ by Claude Monet. Recent insights into the genome of various mycobacteria suggest that Mycobacterium tuberculosis evolved in Africa in humans (or possibly pre-human hominides) from water and soil dwelling mycobacteria (Galagan, 2014). Thus, most likely tuberculosis has accompanied human evolution from the beginning and its life style is a compromise between efficient transmission and the need for survival of its principal host, i.e. Homo sapiens. Accordingly most cases of M. tuberculosis infection result in latency, characterized by the survival of a small number of mycobacteria within a primary disease complex. Transmission is assured by a small percentage of clinically overt infections occurring either directly after exposure to the pathogen or as reactivation after a latency period. Clinically overt infections occur most commonly in patients suffering from some degree of immunodeficiency. Such immunodeficiencies may either be acquired, for example HIV, malnutrition, immunosenescence, or congenital, for example Mendelian susceptibility to mycobacterial diseases (MSMD) or severe combined immune deficiency (SCID). Given the wide distribution of M. tuberculosis in the human population, vaccination appears the most promising approach towards a control of the disease. However, the only vaccine with a documented (albeit limited) efficacy is BCG (Bacille de Calmette et Guérin). BCG is a live vaccine derived through attenuation of Mycobacterium bovis, a bovine-adapted mycobacterial species belonging to the M. tuberculosis complex. The molecular mechanisms underlying BCG attenuation are complex and not yet entirely understood. However, there is clear evidence that circulating vaccine BCG strains have marked differences in their genome (Brosch et al., 2007), which might explain differences in efficacy, but possibly also

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cellular microbiology

NADPH oxidase and BCG infection differences in virulence of different vaccine strains. Clinically apparent infection with the vaccine BCG strain is relatively rare and mostly occurs in immunocompromised patients. Chronic granulomatous disease (CGD) is a genetic deficiency in the phagocyte NADPH oxidase NOX2. Mutations leading to the CGD phenotype may occur in the main electron transport subunit NOX2 (also referred to as gp91phox or CYBB), or one of its subunits (p22phox, p67phox, p47phox, or p40phox). In the majority of cases (∼ 65%), mutations leading to the CGD phenotype occur in the main electron transport subunit NOX2 (also referred to as gp91phox). This protein is coded for by the CYBB gene (OMIM#306400) localized on the X-chromosome. Mutations may also found in the CYBA (OMIM#233690), NCF1 (OMIM#233700), NCF2 (OMIM#233710) and NCF4 (OMIM#601488) genes (Holland, 2013). These genes code for the p22phox, p67phox, p47phox or p40phox NOX2 subunits respectively (Bedard and Krause, 2007). The assembled NOX2 complex is a reactive oxygen species (ROS)-generating enzyme, crucial for the oxygen-dependent killing of microorganisms. CGD, i.e. the lack of a functional NOX2 complex, is among the most frequent primary immunodeficiencies (estimated frequency between 1:200 000 and 1:500 000). Killing of microorganisms through ROS production by neutrophils is thought to be one of the key mechanisms how the NOX2 complex contributes to the host defence (Klebanoff and Clark, 1978). However, more recent data also suggest that ROS generation macrophages contribute to bacterial killing (Pizzolla et al., 2012; Cifani et al., 2013). Here we review the present knowledge on the role of NOX2 in antimycobacterial defence.

Mycobacterial infections in CGD patients Mycobacterium bovis BCG BCG is an attenuated live vaccine, generally considered as a safe. Yet, in rare instances it may cause infectious complications. ‘BCG-itis’ is characterized by persistent ulcers, abscesses, fistulas, or lymphadenopathy limited to the region of inoculation. ‘BCG-osis’ in contrast refers to disseminated infection, typically affecting several organs. It is usually admitted that mild complications are found in less than 1 per 1000, and serious events (BCG-osis) in less than 1 per million vaccinations (Lotte et al., 1984). However, in immunodeficient individuals, BCG complications may be frequent. Indeed, given their rarity in the immunocompetent host, BCG complications often are suggestive of compromised immunity. The classically described primary immunodeficiencies with increased susceptibility to BCG comprise ‘severe combined immunodeficiency’ (SCID) and ‘Mendelian susceptibility to © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1168–1178

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mycobacterial diseases’ (MSMD). However, the knowledge is rapidly evolving. Of relevance for this review are the following observations. • There are numerous case reports of BCG complications in CGD patients. It appears that such cases were previously overlooked: Before the years 2000, only 24 cases of CGD patients who develop BCG complications have been described; since 2000, this number has risen to 196. Note that BCG vaccination, as other attenuated live vaccines, is contraindicated in immunedeficient patients, such as CGD. However, as the vaccine is generally administrated shortly after birth, patients with CGD are vaccinated before other clinical manifestations evoke the diagnosis of an immune deficiency. • Percentage of CGD patients among BCG complications: when systematic search for immunodeficiency in individuals with BCG complications is performed, generally a high proportion of CGD patients (9–58%; Scoazec et al., 1984; Gonzalez et al., 1989; Casanova et al., 1995; Jacob et al., 1996; Afshar Paiman et al., 2006; Toida and Nakata, 2007; Lee et al., 2009; Sadeghi-Shanbestari et al., 2009; Li et al., 2010; Shahmohammadi et al., 2014) is found. Indeed, there are only few studies that did not detect CGD patients among individuals with BCG complications. • Risk of BCG complications in CGD patients: retrospective studies estimate that between 6% and 57% of CGD patients will develop BCG complications if vaccinated (Mouy et al., 1989; Movahedi et al., 2004; Stasia et al., 2005; von Goessel et al., 2006; Lee et al., 2008; Teimourian et al., 2008; Bakri et al., 2009; van den Berg et al., 2009; Fattahi et al., 2011). In comparison, the incidence of BCG complications in SCID and MSMD patients was estimated to be in the range of ∼ 50% and ∼ 20% respectively (Norouzi et al., 2012). BCG-itis. In a majority of CGD patients, BCG disease will present as local or regional complication, also referred to as BCG-itis (Bustamante et al., 2007; van den Berg et al., 2009; Koker et al., 2009; Fattahi et al., 2011; Ying et al., 2012). Cutaneous complications include longlasting swelling, ulcerations and fistulization. BCG lymphadenitis may be restricted to local lymph nodes or show a more locoregional pattern. In CGD patients, BCG-itis is treated with anti-mycobacterial, such as isoniazid, rifampicin (Vieira et al., 2004; Kawashima et al., 2007; Bakri et al., 2009). Medical treatment often leads to an apparent full clinical recovery, however recurrence is often observed (Movahedi et al., 2004; Bustamante et al., 2007). Cases of BCG lymphadenitis sometimes require surgical excision of involved lymph nodes (Bustamante et al., 2007).

1170 C. Deffert, J. Cachat and K.-H. Krause BCG-osis. The BCG can disseminate to lung, liver, bone or brain. This disseminated BCG disease is less frequently encountered in CGD patients (∼ 15%), however appear to be associated with a substantial mortality (Deffert et al., 2014). For comparison, in SCID patients 67% of disseminated and 33% of localized BCG complications are observed (Marciano et al., 2014). The vaccine-associated factors, such as BCG strain and doses might also influence the outcome of BCG complications. The following factors do not account for the risk to develop disseminated BCG-osis: (i) co-infection with other pathogens; (ii) the genotype of the CGD patients (i.e. X-linked versus autosomal recessive) (Bustamante et al., 2007). Disseminated BCG disease has been associated with a fatal outcome in 50% of patients (Afshar Paiman et al., 2006; Lee et al., 2008; Sadeghi-Shanbestari et al., 2009). For this reason, generally a prolonged duration of the anti-mycobacterial treatment is recommended and IFN-γ treatment is often added (Afshar Paiman et al., 2006).

Mycobacterium tuberculosis Until recently, M. tuberculosis has not described as a major pathogen of CGD patients. Recent publications have changed that perception. An exhaustive analysis of the published literature suggests that approximately 20% of published cases of mycobacteria infection in CGD patients are due to M. tuberculosis (Deffert et al., 2014). Seven such cases had been described before the year 2000, as compared with 65 cases between 2001 and 2014. These recently described cases come from regions endemicity for tuberculosis, such as Iran, China and Argentina. In these countries, M. tuberculosis has become one of the most prominent bacteria found in lung, bone or skin lesions of CGD patients. The incidence of tuberculosis in cohort of CGD patients in endemic area ranged from 10% to 54%. In Hong Kong, the incidence of tuberculosis in CGD patients was calculated to be approximately 170 times higher than the incidence in the general population. The principal manifestation of M. tuberculosis in CGD patients is lung disease but also meningitis and disseminated disease. According to some authors, tuberculosis is more severe in CGD compared with immunocompetent patients (Lee et al., 2008; Fattahi et al., 2011; Koker et al., 2013). Also, treatment failure and recurrence is observed more frequently. Recommended treatment is not fundamentally different for tuberculosis in CGD patients, most likely a prolonged duration of anti-mycobacterial therapy should be given. In some cases, infectious foci did not respond to medical therapy and surgical removal was necessary (Lau et al., 1998).

Environmental mycobacteria Reports of infection of CGD patients with non-tuberculous mycobacteria remain anecdotal. Four cases (Chusid et al., 1975; Allen and Chng, 1993; Ohga et al., 1997; Weening et al., 2000) have been reported in the literature: 2 with M. avium (Ohga et al., 1997; Weening et al., 2000), 1 with M. flavescens (Allen and Chng, 1993) and 1 with M. fortuitum (Chusid et al., 1975) infection. Mycobacterial infections and ROS production: in vitro studies During mycobacterial infection, interaction between host and pathogen is key to determine whether mycobacteria will lead to (i) clinically apparent infection (which is the exception, rather than the rule); (ii) latency, where surviving mycobacteria are controlled by the immune system (which is the most common outcome for M. tuberculosis); or (iii) clearance of mycobacteria by the immune system (which is probably the most common outcome for BCG and for non-tuberculous mycobacteria). Humans and M. tuberculosis have evolved in parallel and therefore developed mechanism to counteract each other. In this chapter, in vitro data on the interaction of host phagocytes with mycobacteria are discussed, with a particular focus on ROS/NOX2-dependent mechanisms. Mycobacterial defence responses are also discussed. Mechanisms of mycobacterial killing and control by host cells Killing via oxidative stress: RNS and ROS Reactive nitrogen species (RNS) and ROS are two major antibacterial effectors. RNS are secreted by iNOS into the phagolysosome upon macrophage activation. NO is the first RNS produced. ROS are produced by NOX2 in phagocytes (neutrophils, monocytes, macrophages and dendritic cells) upon phagocytosis of pathogens. Superoxide anion (O2−) is the first ROS produced and subsequently converts into other oxygen derivatives, such as H2O2. RNS and ROS can react together to form new and potent oxidative molecules: for example peroxynitrite is the result of superoxide and nitric oxide reaction. It is thought that RNS and ROS exert their bactericidal effect mainly by direct oxidation of mycobacterial elements (e.g. DNA, proteins, lipids) and thereby damages mycobacteria. Based on in vitro studies of mycobacteria treated with different RNS and ROS, it seems that RNS have a more potent direct bactericidal effect than ROS (Chan et al., 1992; Voskuil et al., 2011). This is corroborated by in vivo data which suggest that iNOS-deficient mice are more susceptible to mycobacterial infection than NOX2-deficient mice (Adams et al., 1997; Ng et al., © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1168–1178

NADPH oxidase and BCG infection 2004). There is evidence that peroxynitrite is more toxic for BCG than for M. tuberculosis (Yu et al., 1999). The role of ROS in antimycobacterial defence was studied in different cell types. While M. tuberculosis induces ROS production by neutrophils, no direct killing effect of neutrophil-produced ROS has been demonstrated (Fazal, 1997). There are however some indications that myeloperoxidase, highly abundant in neutrophils, might exert some microbicidal activity against mycobacteria (Borelli et al., 1999). Along these lines is the recent discovery of a family with a peculiar variant of CGD with a lack ROS production in macrophages, but not in neutrophils (Bustamante et al., 2011a). These variant CGD patients showed a high susceptibility to mycobacterial infection. Yet, there are no strong in vitro data implicating ROS produced by macrophages in the killing of mycobacteria: in one study no difference in mycobacterial killing by CGD phagocytes was found (Fazal, 1997; Bustamante et al., 2011a). Note however that beside a direct role of ROS in pathogen killing, these molecules have a role as signalling molecules in many cell types of the immune mechanism. Thus, rather than acting as bactericidal effectors, ROS might be important as signalling molecules co-ordinating the antimycobacterial host defence. Apoptosis Apoptosis of infected cells is believed to be a protective mechanism against intracellular pathogen. Two mechanisms have been suggest to explain how apoptosis might control of infection: (i) at least under certain experimental conditions, death of infected macrophages stops mycobacterial replication (Placido et al., 2006) and (ii) uptake of mycobacteria-containing apoptotic bodies by dendritic cells might facilitate presentation of mycobacterial antigens and subsequent activation of adaptive immunity (Schaible et al., 2003). Mycobacteriainduced apoptosis of neutrophils seem to be independent of NOX2 and rely on caspase-3 activation (Gonzalez-Cortes et al., 2009). A change in expression of pro- and anti-apoptotic gene was also observed (Perskvist et al., 2002). Thus, it is possible that induction of apoptosis of infected cells by NOX2-derived ROS participates in host defence against mycobacteria. However, this concept is not definitively proven and depending on the experimental conditions, NOX2-deficient neutrophils may be actually more likely to go into apoptosis (Schappi et al., 2008). NETosis NETosis is a more recently described mechanism how phagocytes control microorganisms. Neutrophil © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1168–1178

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extracellular traps (NETs) describe the extracellular release of cellular DNA (Neeli et al., 2008) as a tool of host defence. NETs can ‘trap’ pathogens and participate in the killing. Mechanism by which NET exert their killing activity is not clear and a direct effect is still debated. NOX2-derived ROS have been reported to be an important mediator of NETosis, and neutrophils from CGD patients are deficient in NET formation (Bianchi et al., 2011). However, the importance of ROS in NET formation appears to depend on the stimulus (Parker et al., 2012). Concerning mycobacteria, in in vitro studies, NET formation has been described upon interaction of M. tuberculosis with neutrophils (Ramos-Kichik et al., 2009) and with macrophages (Wong and Jacobs, 2013), however the importance of ROS and NOX2 for mycobacteria-induced NET formation has not been studied. Also, it is not clear whether mycobacteria are indeed controlled by NETs, or whether macrophage cell death during NET formation rather facilitates mycobacterial infection through cell lysis which ultimately might enhance dissemination of mycobacteria. Phagolysosome fusion and autophagy IFN-γ and TNF are key cytokines that lead to cell activation and enhanced phagosome–lysosome fusion in mycobacteria-infected macrophages (Schaible et al., 1998; Via et al., 1998). Phagolysosomes are formed upon fusion of phagosomes with intracellular organelles, in particular lysosomes. These fusion events result in the formation of an acidic compartment containing abundant hydrolytic enzymes. ATP-dependent proton pumps ensure the formation of a hostile acidic environment within the phagosome and full activation of the acid hydrolases. Autophagy is a mechanism leading to the clearance of the cytoplasm from damaged macroproteins and organelles. It also has a role in intracellular pathogen sensing and killing (Jo, 2010). In the context of mycobacterial infection, it has been shown that autophagy also increases antigen presentation and cross-presentation (Deretic, 2005). It appears that autophagy, which is also driven by IFN-γ and TNF, is a mechanism to bypass the phagosome–lysosome block exerted by phagocytosed mycobacteria (Gutierrez et al., 2004). Furthermore, autophagy could lead to production of TNF and other protective mechanisms, thereby exerting a positive feedback on mycobacterial control (Crisan et al., 2011). In contrast, Th2-derived cytokines, IL-4 and IL-13 suppress autophagy (Harris et al., 2007). NOX2-produced ROS seem to be implicated in autophagy of pathogen-infected organelle/cytoplasm by recruiting certain autophagy proteins to the phagosome (Huang and Brumell, 2009; Mitroulis et al., 2011).

1172 C. Deffert, J. Cachat and K.-H. Krause NADPH oxidase in mycobacteria-induced cellular activation and cytokine production A variety of cellular receptors, including TLR2 (toll like receptor 2) and dectin-1, have been implicated in the recognition of mycobacteria by phagocytes and the subsequent cellular activation. Several studies demonstrate that mycobacteria induce NADPH oxidase activation and ROS production (Yang et al., 2009; 2012; Deffert et al., 2014; Liu et al., 2014). In TLR2-deficient phagocytes, no mycobacterial ROS production was observed, suggesting that the TLR2 pathway is crucial for NOX2 activation. The role of the phagocyte NADPH oxidase in cytokine release is a complex issue and more specifically, the role of NOX2 in mycobacteria-induced cytokine production has received only little attention. Globally speaking, there are two, apparently conflicting, observations: i. Several cytokines, in particular TNF-α, IFN-γ and IL-12, have been implicated in the antimycobacterial host defence (Sasindran and Torrelles, 2011). Indeed, genetic defects (Bustamante et al., 2011b) or pharmacological inhibition (Perlmutter et al., 2009) of these cytokine pathways is associated with increased mycobacterial susceptibility. Given the role of ROS in cytokine release, a decreased cytokine production in NOX2 deficiency has been forwarded as an explanation for increase mycobacterial susceptibility in CGD (Bustamante et al., 2011a). And indeed several in vitro studies show a decreased cytokine production in the presence of antioxidants or in the absence of NOX2 activity (Yang et al., 2009; Liu et al., 2014). ii. However, there is also abundant evidence that in CGD patients and CGD mice, there is overshooting production of pro-inflammatory cytokines (Hatanaka et al., 2004; Deffert et al., 2011; 2012). There are relatively few published studies on this topic in the context of mycobacterial infection (Segal et al., 1999; Cooper et al., 2000). Recent work from our laboratory demonstrate in CGD mice, a massive overproduction of pro-inflammatory cytokines in response to systemic mycobacterial infection (Deffert et al., 2014). It has also been suggested that overshooting IL-1β production in CGD patients is linked to decreased autophagy and that IL-1β inhibitor might therefore be beneficial to block inflammatory CGD complications (de Luca et al., 2014). Taken together, we think that a decrease in cytokine production is not the cause of increased mycobacterial susceptibility in CGD patients. Yet, as the interplay between mycobacteria and the host response depends on cytokine fine-tuning, it is possible that an overproduction of cytokines contributes to the inefficient response of CGD patients to mycobacterial infection.

Granuloma formation The final step of mycobacterial control by immune system is the development of granulomas. In general, granuloma formation is thought to be the response of the immune system to a structure which is unable to eliminate. Such reactions may occur in response to inert material (foreignbody granuloma), but also in response to pathogens, where the apparent aim of granuloma formation is the generation of a delimitated structure avoiding further disease dissemination. However, at least for M. tuberculosis, mycobacteria survive for decades within granulomas. Thus, the control of M. tuberculosis infection through granuloma formation is a fragile equilibrium: a decrease in the immune response may lead to disease reactivation and secondary tuberculosis (Sasindran and Torrelles, 2011). Mechanisms leading to granuloma formation involve a fine-tuned interaction of several cell types and the release of various cytokines and chemokines. A role of NOX2 in granuloma formation is indirectly suggested by the fact that NOX2-deficiency leads to chronic granulomatous disease, i.e. exuberant formation of large size and poorly structured granulomas. Recent data demonstrate that granuloma formation in BCG-infected CGD mice is abnormal: granuloma are larger, however atypically infiltrated with neutrophils and unable to sequestrate mycobacteria (Deffert et al., 2014). A recent study suggests that M2 macrophages have an important role in development of granulomas (Arkhipov et al., 2013), which is interesting given evidence that the M1 to M2 switch of macrophages is ROS-dependent (Zhang et al., 2013). Thus, NOX2 might play several roles in granulomas, including fine-tuning of granuloma structure through ROS-dependent modulation of chemokine and cytokine release, and potentially also determination of macrophage subtypes. Mycobacterial defence mechanism Antioxidant defence As macrophages produce ROS in response to mycobacterial infection, mycobacteria have developed strategies to cope with an oxidative environment, detoxifying ROS through various anti-oxidant mechanisms (Ehrt and Schnappinger, 2009). For example, M. tuberculosis expresses a catalase-peroxidase called KatG, two superoxide dismutases SodC and SodA, a low-molecularweight thiol, mycothiol, an NADH-dependent peroxidase, as well as a peroxynitrite reductase. Defect in all of these proteins have been associated with a higher susceptibility of M. tuberculosis to ROS toxicity (Edwards et al., 2001; Piddington et al., 2001; Ng et al., 2004; Shi and Ehrt, 2006). Thus, M. tuberculosis has developed complex and probably redundant systems to survive within the © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1168–1178

NADPH oxidase and BCG infection phagosome and to detoxify ROS. Mycobacteria have also developed indirect mechanism to cope with oxidative stress, mainly by repair of oxidized protein and by protecting DNA from oxidation. For example, methionine sulfoxide reductase (Mst) is a protein implicated in repairing oxidized protein. As far as DNA protection is concerned, a histone-like protein Lsr2 has been described to shield and physically protect mycobacterial DNA from ROS (Colangeli et al., 2009).

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(Cybb) knock-out mice in a C57Bl/6 background or p47phox (Ncf1) knock-out mice with a partial backcross into C57Bl/6 background were used. Inoculated doses were 107 cfu ml−1. One study did not detect differences between wild-type and p47phox-deficient mice (Segal et al., 1999) However, the other study (Fujita et al., 2010) found an increased susceptibility of NOX2-deficient mice, including a marked increase in mortality and severity of lung damage.

Enhanced intracellular survival protein Eis M. bovis BCG M. tuberculosis possesses the enhanced intracellular survival protein Eis. Eis-deficient M. tuberculosis induce more autophagy formation, suggesting that this protein acts – at least in part – through downregulation of autophagy. However, macrophages exposed to Eisdeficient M. tuberculosis are also capable to induced high production of TNF and IL-6, as well as increase generation of ROS (Shin et al., 2010). Thus, it appears that Eis is – directly or indirectly – involved in the mycobacterial antioxidant defence. Mycobacterial infections in CGD mouse models Only relatively few studies have addressed the relationship between mycobacteria and the phagocyte NADPH oxidase in mouse models of CGD. Four studies used M. tuberculosis (Adams et al., 1997; Cooper et al., 2000; Jung et al., 2002; Ng et al., 2004) and two studies used M. avium (Segal et al., 1999; Fujita et al., 2010), and one M. bovis BCG (see Table 1). M. tuberculosis Mode of infection with M. tuberculosis was intravenous (Adams et al., 1997; Ng et al., 2004) or aerosol (Cooper et al., 2000; Jung et al., 2002). NOX2 (Cybb) knock-out mice in a C57Bl/6 background or p47phox (Ncf1) knock-out mice with a partial backcross into C57Bl/6 background were used. Inoculated doses ranged from 102 to 107 cfu ml−1. Two of the four studies did not find a difference between wild type and CGD mice (Jung et al., 2002; Ng et al., 2004). In contrast the two other studies reported differences (Adams et al., 1997; Cooper et al., 2000). They consistently reported a moderately increased bacterial load (∼ 1 log), as well as increased neutrophil infiltrates and/or aggregates within infected areas. In addition, one of the studies reported large granulomas with an atypical histological structure (Adams et al., 1997). M. avium Mode of infection with M. avium was intravenous (Segal et al., 1999) or intratracheal (Fujita et al., 2010). NOX2 © 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1168–1178

CGD mice are highly susceptible to systemic infection with BCG (Deffert et al., 2014) mimicking the situation in CGD patients. Taken together, the mouse studies are still incomplete. There is no doubt that CGD mice are highly susceptible to BCG. However, the situation with M. tuberculosis remains contradictory, with some studies finding an impact of NOX2 deficiency, while others do not. Neither type of CGD mutation, nor mycobacterial strain, mode of inoculation, or inoculum size can account for the observed differences. The discrepancy between the increasing evidence for M. tuberculosis infection in CGD patients and the little convincing results in CGD mouse models might be accounted for by one or several of the following considerations: • Specificities of human tuberculosis infection might preclude reliable results in mouse models of CGD. • Only few mouse studies have been performed and most of these studies were underpowered. Indeed, most studies investigated only 4–5 CGD mice. Note that the only study which was reasonably well powered (n = 10; Fujita et al., 2010) found a marked difference in the susceptibility of wild type and CGD mice to M. avium infection (see Table 1). Thus, it is possible that the erratic results observed with M. tuberculosis are due to a too small number of mice in the respective studies. • Other factors that might contribute to the observed discrepancies include health status, specificities of the animal facilities, in particular animal microbiota. Taken together, results for the mycobacterial strain that causes most frequently infections in CGD patients, namely BCG, are consistent between human and mice. Concerning M. tuberculosis, clearly more and better powered mouse studies are needed to fully understand the relationship between NOX2 and this pathogen. As human data strongly suggest an increased susceptibility of CGD patients to M. tuberculosis, the use of mice with a humanized immune system should also be considered.

C57Bl/6 n.c. No

No visible difference/WT

Increased (∼ 8-fold) Increased size Neutrophil infiltrate n.d.

Background Number Mortality

Lung pathology

Bacterial load in lung (cfu ml−1) Granuloma

n.d.

n.d.

No difference/WT

No difference/WT

Increase IFN-γ response (in vitro)

Moderate increase of infiltrates; neutrophil aggregates Increased (∼ 10-fold) Neutrophil infiltrate

C57Bl/6 4 n.d.

aerosol; 106 p47phoxKO (Ncf1)

M. tuberculosis

Cooper et al. 2000

n.d.

n.d.

No difference/WT

n.d.

No change/WT

No alteration

Decreased

No difference/WT

C57Bl/6 4–5 No

i.v.; 107 p47phoxKO (Ncf1)

aerosol; ∼ 102 NOX2KO (Cybb) C57Bl/6 5 No

M. avium

Segal et al. 1999

M. tuberculosis

Jung et al. 2002

i.v., intravenous; i.t., intra-tracheal; n.d., not determined; WT, wild-type; n.c., not communicated.

Cytokine profile

i.v.; 106 NOX2 KO (Cybb)

i.v.; 106 NOX2KO (Cybb) C57Bl/6 5 No

M. tuberculosis

M. tuberculosis

Ng et al. 2004

Mycobacterial species Route; dose Mouse strain

Adams et al. 1997

Table 1. Summary of published results on mycobacterial infection in CGD mice.

n.d.

Increased (∼ 10-fold) n.d.

C57Bl/6 10 ∼ 20% (4 weeks) 50% (8 weeks) Severe lung damage, increase neutrophil

i.t.; 107 NOX2KO (Cybb)

M. avium

Fujita et al. 2010

Increased (∼ 8-fold) Disorganized No clear boundaries Increase pro-inflammatory cytokines

Severe lung damage, increase neutrophil

i.v.; 107 p47phoxmutated (Ncf1) C57Bl/10Q 15 ∼ 60% (4 weeks)

M. bovis BCG

Deffert et al. 2014

Disorganized No clear boundaries n.d.

n.d.

Severe lung damage, increase neutrophil

C57Bl/6 9 ∼ 50% (4 weeks)

i.v.; 107 NOX2KO (Cybb)

M. bovis BCG

Deffert et al. 2014

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© 2014 John Wiley & Sons Ltd, Cellular Microbiology, 16, 1168–1178

NADPH oxidase and BCG infection Conclusion Research on the role of the phagocyte NADPH oxidase NOX2 in the defence against mycobacteria has gone a long way. Twenty years ago, there was still the general impression that NOX2 was not relevant for the control of mycobacteria. Research of the last decades has changed this perception and there is now little doubt that NOX2 plays a role in the antimycobacterial defence. Through which mechanisms do NOX2-derived ROS contribute to the defence against mycobacteria? A direct role of ROS in mycobacterial killing is probably not the key mechanism. It appears more likely that ROS-dependent signalling controls crucial cellular host defence mechanisms, such as cytokines production, autophagy, and granuloma formation. The understanding of these aspects of NOX2 biology might be key, not only to understand the pathophysiology of CGD, but also to increase our understanding how the immune system controls mycobacteria. More specifically, the following lines of research need to be explored in the future: • Which are ROS targets that allow the co-ordination of anti-mycobacterial responses? Kinases and phosphatases obviously are prime suspects, but redoxsensitive transcription factors and ion channels should also be taken into consideration. The precise identification of such target proteins might open new avenues for treatment of mycobacterial disease. Note also that NOX2 might not only act on proteins. Redox modifications of lipids and small molecule mediators might be of relevance. • Could NOX2 activators be used for the treatment of mycobacterial disease? Indeed, NOX2 activators have been described, but so far their use has been rather focused on prevention and treatment of autoimmunity associated with the CGD phenotype, in particular Ncf1 mutant (Hultqvist et al., 2006; 2014). These compounds appear well supported in animals and should be tested for mycobacterial disease. • Should a prescreening for CGD performed prior to BCG vaccination? Obviously a simple, rapid and cheap test would be needed, as these vaccinations usually take place in resource-limited countries. Conflict of interest The authors declare no biomedical financial interests or potential conflicts of interest relevant to the subject matter of this work.

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