NRF2 is a transcription factor that regulates the cellular defense against toxic and oxidative insults through the expression of genes involved in the oxidative stress response and drug detoxification. NRF2 activation makes cells resistant to chemical carcinogens and inflammatory challenges. The mechanisms of NRF2 activation by oxidative stress have been understood at a molecular level.
The incidence of pulmonary nontuberculous mycobacterial (NTM) infection is increasing worldwide , and its clinical outcomes with current chemotherapies are unsatisfactory. The incidence of tuberculosis (TB) is still high in Africa, and the existence of drug-resistant TB is also an important issue for treatment . To discover and develop new efficacious anti-mycobacterial treatments, it is important to understand the host-defense mechanisms against mycobacterial infection. During infection with microorganisms, phagocytic cells produce an excess of oxidants that contribute to the clearance of pathogens, but they sometimes cause tissue injury . Therefore, the regulation of oxidative stress may be one of the important host-defense mechanisms during infection. Nuclear erythroid 2 p45-related factor-2 (NRF2) is a redox-sensitive transcription factor that plays an important role in the antioxidant and detoxification activities of the body .
2. The Role of NRF2 in Tuberculous Infection
TB patients show high levels of oxidative stress markers and depletion of antioxidants, including vitamin C, vitamin E, and glutathione 
. A previous study of a TB guinea pig model showed the presence of oxidative stress markers and increased oxidant-mediated lung and spleen lesions. The expression level of NRF2 in M. tuberculosis
-infected cells was increased, but NRF2 was mainly localized in the cytoplasm. These findings suggest that NRF2 is not fully activated in the infected cells, which may account for the decreased expression levels of ARE-mediated antioxidant and phase-II enzymes 
. The administration of the antioxidant drug N-acetyl cysteine (NAC) to M. tuberculosis
-infected guinea pigs partially restored the serum total antioxidant capacity, with decreases in the lung and spleen bacterial counts and the severity of lesion necrosis. In summary, progressive oxidative stress during experimental TB in guinea pigs is partly caused by a defect in host antioxidant defenses, which can be partially restored by the administration of antioxidants. Rockwood et al. found that heme oxygenase 1 (HO-1) expression was markedly increased in rabbits, mice, and non-human primates during M. tuberculosis
infection and decreased gradually during tuberculosis treatment. Interestingly, they also showed that ESAT-6-dependent stimulation of ROS results in HO-1 production by inducing nuclear translocation of NRF2 
. Many pathological effects observed in cells infected with M. tuberculosis
are associated with bacterial production of the virulence factor ESAT-6, which is secreted into the host cell cytosol 
. Actually, ESAT-6 is involved in the inhibition of phagolysosome fusion in M. tuberculosis
-infected phagocytes 
. It is thought that ESAT-6 disrupts phagosome membranes, thereby inhibiting phagolysosome fusion and allowing the bacillus to escape into the cytoplasm 
. Since ESAT-6 is not restricted to M. tuberculosis
, but is also present in some environmental strains including M. kansasii
, M. marinum
, and M. szulgai 
, these three other NTM strains may also induce ROS in the same way by ESAT-6.
HO-1, which is a cytoprotective enzyme, is directly modulated by Nrf2 
. There have been some reports on the relationship between HO-1 and M. tuberculosis
. Using freshly resected human lung tissue and HO-1-deficient mice, Chinta et al. showed that HO-1 in myeloid cells was important for the regulation of inflammation and free-radical-mediated tissue damage in TB 
. In contrast, Costa et al. demonstrated that pharmacological inhibition of HO-1 rather suppressed M. tuberculosis
infection in vivo by a mechanism dependent on T lymphocytes 
. Moreover, Wu et al. showed that HO-1 polymorphism was associated with TB susceptibility in the Chinese Han population 
. These reports indicate that HO-1 is associated with TB.
It has been reported that NRF2 plays an important role in TB infection. To elucidate the genes potentially regulated by NRF2 in TB, a meta-analysis of published gene expression datasets was conducted 
. In this meta-analysis, an NRF2-mediated 17-gene signature (GBP1
) was identified. These genes reflected a cluster of gene ontology terms highly related to TB physiology. They reported that the 17-gene signature can be used to distinguish TB patients from healthy controls and patients with latent TB infection, pneumonia, or lung cancer. Furthermore, the NRF2-mediated gene signature can be used as an indicator of the efficacy in anti-TB therapy. Thus, they confirmed the central role of NRF2 in TB pathogenesis 
. In fact, to elucidate the role of NRF2 in tuberculous infection, experiments in which NRF2-deficient mice were infected with M. tuberculosis
aerially were performed 
. In this study, significant reductions in granuloma formation and tubercle bacilli in granulomas were noted in the NRF2-deficient mice 27 weeks after infection, concurrently with higher expressions of IL-2 and IL-13 mRNA. This finding suggests that NRF2 acts protectively against TB infection. In contrast, Rothchild et al. demonstrated that NRF2 drives expression of the cell-protective signature in alveolar macrophages and impairs the control of early bacterial growth 10 days after M. tuberculosis
infection using NRF2-deficient mice 
Taken together, the role of NRF2 in the pathogenesis of M. tuberculosis infection appears to differ between the early and late stages of infection.
3. The Role of NRF2 in NTM Infection
There are not many reports regarding the role of NRF2 in NTM infection compared to those in TB infection. Nevertheless, Bonay et al. showed that human THP-1-derived macrophages infected with M. abscessus
showed increased ROS production and cell necrosis, whereas M. abscessus
infection triggered activation of the NRF2 signaling pathway. In addition, pretreatment of macrophages with sulforaphane (SFN), an activator of NRF2, followed by M. abscessus
infection significantly decreased mycobacterial burden due to apoptosis 
. Interestingly, this macrophage apoptosis was caspase 3/7-independent, but p38 MAPK-dependent. In this study, they showed that NRF2 stimulators may be interesting as future therapeutic options as a supplement to the standard multi-drug therapies used in pulmonary M. abscessus
As for M. avium
infection, the role of the oxidative stress sensor KEAP1 was reported. Awuh et al. proposed that KEAP1 negatively regulates inflammatory responses in M. avium
-infected human primary macrophages 
. Although this might be important to avoid excessive inflammation, they suggested that a negative effect could facilitate intracellular growth of M. avium 
There have been a few reports on the relationship between HO-1 and M. avium
. HO-1-deficient mice were used in all of these studies. Then, HO-1-deficient mice were more susceptible to M. avium
infection than WT mice. In particular, Regev et al. reported that HO-1 promoted granuloma development and protected against the dissemination of M. avium 
. These reports indicate that HO-1 is also associated with M. avium
Recently, Nakajima et al. reported that NRF2-deficient mice were highly susceptible to M. avium
bacteria compared to wild-type mice 
. Surprisingly, NRF2 did not affect the level of oxidative stress or Th1 cytokine production in this M. avium
-infection model. Comprehensive transcriptome analysis and subsequent ex vivo analysis showed that natural resistance-associated macrophage protein 1 (NRAMP1) and HO-1, regulated by NRF2, were essential in defending against M. avium
infection due to the promotion of phagolysosome fusion and granuloma formation, respectively. In addition, treatment with SFN increased resistance to M. avium
with increased lung expressions of NRAMP1 and HO-1 in wild-type mice. Since the NRAMP1
gene is one of the important disease susceptibility genes of pulmonary NTM disease in humans 
, this NRF2-NRAMP1 pathway is fascinating in terms of elucidating the pathophysiology of pulmonary NTM infection. Thus, NRF2 was thought to be a critical determinant of host resistance to M. avium
infection by a mechanism other than controlling oxidative stress. Consistent with reports of a wide variety of functions of NRF2, their study showed that NRF2 plays a role in the regulation of NRAMP1.
4. Aging and NRF2 in Mycobacterial Infection
Increased oxidative stress, which is a major feature of aging, has been implicated in a variety of age-related diseases. In aging, oxidative stress is increased, whereas antioxidant enzymes decrease, and the adaptive response to oxidative stress decreases. It has been reported that the ability of NRF2 to respond to oxidative stress decreases with aging 
. Decreased antioxidant capacity due to a decrease in NRF2 functions might contribute to several age-related diseases.
In developing countries, TB rates are highest among the young, reflecting primary transmission in this age group. On the other hand, in the United States and other developed countries, the rate of TB among the elderly is higher than among the young and children, reflecting reactivation of the disease due to age-related decline in immunity 
As for the relationship between aging and NTM infection, a total of 1445 patients with treatment-naïve pulmonary NTM who were newly diagnosed between July 1997 and December 2013 were examined 
. Upon multivariable analysis, the LASSO method demonstrated that old age (≥65 years); male sex; low body mass index (<18.5 kg·m2
); underlying diseases including chronic pulmonary aspergillosis, malignancy, and chronic heart or chronic liver disease; and the erythrocyte sedimentation rate (ESR) were significantly associated with mortality in pulmonary NTM disease. This study showed that old age (≥65 years) is an independent factor associated with a poor prognosis in pulmonary NTM disease.
These epidemiological studies clearly demonstrated that mycobacterial infection is increasingly found in the elderly, but the underlying mechanisms are unclear. It has been reported that the expression of the SOCS3
gene is increased and the expression of Bcl2
gene is decreased with age, resulting in inhibition of Th1 cell differentiation 
. A study in which aged and young mice were infected with NTM bacteria was also reported 
. In this study, an attenuated HO-1 response with diffuse inflammation, a high burden of mycobacteria, poor granuloma formation, and decreased survival were observed in aged mice after infection, whereas young mice showed tight, well-defined granuloma, increased HO-1 expression, and increased survival. Taken together, higher susceptibility of the elderly to pulmonary NTM infection is partly caused by attenuated HO-1 responses, subsequent upregulation of SOCS3
, and inhibition of Bcl2
, leading to programmed cell death of macrophages and sustained infection. Since HO-1 is induced under the influence of NRF2 in young mice in response to NTM infection 
, it is likely that increased susceptibility to mycobacterial infection in the elderly is related to the decline of NRF2 functions with advancing age.