REVIEW
published: 12 October 2017
doi: 10.3389/fmicb.2017.02008
Nitric Oxide in the Pathogenesis and
Treatment of Tuberculosis
Hamidreza Jamaati 1 , Esmaeil Mortaz 2, 3, 4*, Zeinab Pajouhi 1 , Gert Folkerts 4 ,
Mehrnaz Movassaghi 4 , Milad Moloudizargari 3 , Ian M. Adcock 5, 6 and Johan Garssen 4, 7
1
Chronic Respiratory Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti
University of Medical Sciences, Tehran, Iran, 2 Clinical Tuberculosis and Epidemiology Research Center, National Research
Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 3 Department of
Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 4 Division of Pharmacology,
Faculty of Science, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands, 5 Cell and
Molecular Biology Group, Airways Disease Section, Faculty of Medicine, National Heart and Lung Institute, Imperial College
London, London, United Kingdom, 6 Priority Research Centre for Asthma and Respiratory Disease, Hunter Medical Research
Institute, University of Newcastle, Newcastle, NSW, Australia, 7 Nutricia Research Centre for Specialized Nutrition, Utrecht,
Netherlands
Edited by:
Yuji Morita,
Aichi Gakuin University, Japan
Reviewed by:
Chris Sassetti,
University of Massachusetts Medical
School, United States
Adrian Lee Smith,
University of Oxford, United Kingdom
Diana Gassó Garcia,
Universitat Autònoma de Barcelona,
Spain
*Correspondence:
Esmaeil Mortaz
emortaz@gmail.com
Specialty section:
This article was submitted to
Infectious Diseases,
a section of the journal
Frontiers in Microbiology
Received: 17 May 2017
Accepted: 29 September 2017
Published: 12 October 2017
Citation:
Jamaati H, Mortaz E, Pajouhi Z,
Folkerts G, Movassaghi M,
Moloudizargari M, Adcock IM and
Garssen J (2017) Nitric Oxide in the
Pathogenesis and Treatment of
Tuberculosis. Front. Microbiol. 8:2008.
doi: 10.3389/fmicb.2017.02008
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is globally
known as one of the most important human pathogens. Mtb is estimated to infect
nearly one third of the world’s population with many subjects having a latent infection.
Thus, from an estimated 2 billion people infected with Mtb, less than 10% may develop
symptomatic TB. This indicates that the host immune system may constrain pathogen
replication in most infected individuals. On entering the lungs of the host, Mtb initially
encounters resident alveolar macrophages which can engulf and subsequently eliminate
intracellular microbes via a plethora of bactericidal mechanisms including the generation
of free radicals such as reactive oxygen and nitrogen species. Nitric oxide (NO), a key
anti-mycobacterial molecule, is detected in the exhaled breath of patients infected with
Mtb. Recent knowledge regarding the regulatory role of NO in airway function and Mtb
proliferation paves the way of exploiting the beneficial effects of this molecule for the
treatment of airway diseases. Here, we discuss the importance of NO in the pathogenesis
of TB, the diagnostic use of exhaled and urinary NO in Mtb infection and the potential of
NO-based treatments.
Keywords: nitric oxide, non-tuberculous mycobacteria, Mycobacterium, macrophages, drug-resistance, nitric
oxide donors
INTRODUCTION
The formation of the gaseous mediator nitric oxide (NO) from L-arginine by NO synthases (NOS)
has been traditionally considered as the first-line defense against parasitic infections in all species
including metazoans (Schmidt and Walter, 1994). The importance of NO production and its release
in pathogen-directed defense mechanisms has been confirmed by the effect of NOS inhibitors
(Boom, 1996; Flynn et al., 1998; Sciorati et al., 1999) and NOS knock-out mice on enhancing the
severity of infection and of exacerbations in-vivo and ex-vivo (Cooper et al., 2000; Kuo et al., 2000).
However, there is uncertainty regarding the magnitude of the response and which strains of bacilli
are the most susceptible to NO-induced killing (Denis, 1991; Flesch and Kaufmann, 1991; Chan
et al., 1992; Appelberg and Orme, 1993; Doi et al., 1993; Rhoades and Orme, 1997; Garbe et al.,
1999).
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NO also plays an important role in bacteriostatic and
bactericidal processes as part of the host defense mechanisms
against pulmonary infections (Flesch and Kaufmann, 1991;
Appelberg and Orme, 1993). For example, inflammatory stimuli
can enhance NO release via the up-regulation of the inducible
form of NOS (iNOS or NOS2) within inflammatory macrophages
(Liew and Cox, 1991; Nathan and Hibbs, 1991; Wang et al.,
1998). NO is converted into highly Reactive Nitrogen Species
(RNS) such as NO3– and NO2– within infected macrophages to
drive bacterial death. The term, Reactive Oxygen Intermediates
(ROI) refers to the reduction products of oxygen and include
superoxide (•O2), hydrogen peroxide (H2O2), and the hydroxyl
radical (•OH). These reactive products also form reactive
conjugates with halides and amines, as well as with NO, giving
rise to the production of peroxynitrite (ONOO–) (Nathan and
Shiloh, 2000) (Figure 1).
The bactericidal effect of NO in human tissue macrophages
may be direct or indirect via RNS (Rich et al., 1997; Scanga et al.,
2001). However, there is evidence that the bactericidal effects
of RNS may be an artifact of in vitro laboratory conditions as
a reduced effect is seen under less harsh, more physiological
conditions (Garbe et al., 1999). Bacillus Calmette–Guérin (BCG)inoculated alveolar macrophages (AM) from patients with
pulmonary fibrosis express increased levels of NOS2 protein and
mRNA as well as peroxynitrite (Nozaki et al., 1997). Furthermore,
human AM-induced killing of cytoplasmic BCG is attenuated
by the NOS inhibitor NG-monomethyl-L-arginine monoacetate
implicating both NO and peroxynitrite in this process (Nozaki
et al., 1997). The importance of NO and an altered immune
system in the control of tuberculosis (TB) infection was further
shown with the altered levels of fractional exhaled NO (FeNO)
and urinary NO metabolites reported in TB patients particularly
those immunocompromised by human immunodeficiency virus
(HIV) infection (Idh et al., 2008). These data highlight the
critical role of NOS2 and of reactive nitrogen intermediates (RNI)
in controlling mycobacterium bacilli infection of macrophages
(Denis, 1991; Flesch and Kaufmann, 1991; Chan et al., 1992).
Phagocytosis of Mycobacterium tuberculosis (Mtb) bacilli by
mononuclear cells is the primary immunological mechanism
in the face of TB infection (Edwards and Kirkpatrick, 1986;
Rook et al., 1986; Wang et al., 2001). When exposed to
Mtb, individuals who are healthy and immune-competent will
mount an effective early and late immune response and do
not develop the disease. In those who do develop disease, the
initial response is mediated mainly by phagocytes while the late
response is characterized by the action of CD4+ T-cells (Dunn
and North, 1995). This ultimately leads to the formation of
granulomas consisting of epithelioid and multinucleated giant
cells (Kaufmann, 1993) (Figure 2). The severity of the disease
is determined by phagocytic cells including polymorphonuclear
cells (PMN), monocytes and AM (Aston et al., 1998). Indeed, the
diagnosis of acute Mtb infection is often aided by the observation
of abundant PMNs in the bronchoalveolar lavage (BAL) fluid
(Zhang et al., 1995). Building on the above-mentioned ex vivo and
in vivo data highlighting the importance of NO in TB infection
and the altered levels of urinary and exhaled NO levels in infected
patients, we review here the mechanisms by which NO regulates
TB pathogenesis, the potential use of NO as a diagnostic of early
infection and the future of NO-based therapeutic interventions.
THE ROLE OF NO AND ONOO IN
ANTI-MTB IMMUNITY
NO is an endogenous molecule produced at different sites
throughout the body (Mikaili et al., 2014). This molecule is
chemically active and is effective against a variety of pathogens
including Mtb. Different mechanisms are used for killing Mtb
in vivo, such as acidification of the phagosomes and phagosomelysosome membrane fusion along with granzyme, granulysin,
and perforin production (Lewinsohn et al., 1998; Stenger et al.,
1998; Ernst et al., 2000; Serbina et al., 2000). These, together
with ROI-mediated antimicrobial mechanisms, help in killing
Mtb (Figure 1). The precise role of ROI in Mtb killing is
difficult to accurately discern as peroxynitrite is ineffective in
rodents and different strains of Mtb have differing sensitivity
to NO (Cunningham-Bussel et al., 2013). However, studies
in rodent cells may not give accurate insight into human
disease as they generally produce greater quantities of NO
compared to human cells although this may also relate to the
culture conditions used (Cunningham-Bussel et al., 2013). It
is important, therefore, that future studies investigating the
role of NO and ROI in Mtb killing should be performed
in primary human AMs in addition to experiments being
performed in vivo (Yu et al., 1999; Chan et al., 2001a; Yang
FIGURE 1 | Nitric oxide (NO) production pathway inside a macrophage.
Interferon (IFN)-γ as well as other inflammatory stimuli increase NO production
by stimulating inducible nitric oxide synthase (iNOS). Elevated levels of the NO
precursor, L-arginine (L-arg) also enhances NO production. NO may either act
directly, or in combination with superoxide (•O2–) to form peroxynitrite
(ONOO•), to kill mycobacteria (Mtb) within the phagosome.
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NO and Tuberculosis
FIGURE 2 | Schematic illustration of granuloma components in a TB-infected lung. Mtb bacilli are ingested by macrophages within the lung. This produces a
profound inflammatory and immune response which ultimately leads to the formation of granulomas consisting of epithelioid and multinucleated giant cells. Mtb bacilli
within the granuloma-associated macrophages are killed by nitric oxide (NO) generated from inducible NO synthase (iNOS)—see expanded macrophage. NO
production also stimulates NF-κB activation leading to the production of inflammatory cytokines such as TNFα and IL-1β. Epithelial cells surrounding the granuloma
further support bacterial killing by producing more amounts of NO. Mtb infection results in inhibition of macrophage apoptosis as a means of increasing its survival
which is prevented by altered levels of the serine/threonine protein kinase, PknE. NO, nitric oxide; PknE, Protein Kinase E; Mtb, Mycobacterium tuberculosis.
et al., 2009). In addition, the measurement of the intracellular
survival of the bacilli should be undertaken (Yu et al., 1999).
NO production is not only increased in macrophages but NOS2
mRNA expression and the production of NO is increased in A549
lung epithelial cells following infection with Mtb bacilli. A549
cells produced greater amounts of NO compared to macrophages
which implicates the active involvement of lung epithelial cells
in the anti-mycobacterial host defense process (Kwon et al.,
1998).
The pivotal role of NO in protecting against infection
by mycobacterial species is well-established. For example,
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NO production is the major determinant of macrophage
resistance to Mycobacterium bovis (M. bovis)-induced
infection, at least in comparison to apoptosis (EsquivelSolís et al., 2013). Macrophages from cattle infected with,
but protected against, M. bovis have a two-fold lower
bacterial load and produce more NO than macrophages
from cattle infected and not resistant to infection (AlcarazLópez et al., 2016). In addition the presence of inhibitors
of arginine-dependent NO production results in a more
rapid growth of M. bovis in infected cattle (Nozaki et al.,
1997).
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FACTORS AFFECTING NOS EXPRESSION
AND NO PRODUCTION
et al., 1995; Hausladen et al., 1996; Nicholson et al., 1996) may
regulate infectivity and disease latency.
The expression of the solute carrier family 11 member 1
(SLC11A1) gene determines the susceptibility or resistance
to in vitro infection with the H37Rvt strain of Mtb. This
is a consequence of the differential capability of resistant
and susceptible macrophages to produce NO in response to
Mtb (Arias et al., 1997). In addition, interferon (IFN)-γ and
lipopolysaccharide treatment enhanced the expression of the
arginine permease, MCAT2B, but this could not account for
the observed increase in L-[3 H] arginine uptake. Together this
indicates that the activity of the L-arginine transporter(s) may
also alter in response to macrophage activation (Peteroy-Kelly
et al., 2001) (Figure 1).
NOS2 is not homogenously distributed within macrophages
but is preferentially distributed in newly formed phagosomes
following receptor-mediated uptake of latex beads opsonized
with either complement products or IgG (Miller et al., 2004).
However, the intraphagosomal NOS2 localization is not seen
following infection with M. bovis (BCG-associated var.) or the
H37R strain of Mtb (Miller et al., 2004).
Mtb infection results in inhibition of macrophage apoptosis
as a means of increasing its survival (Velmurugan et al., 2007)
(Figure 2). The serine/threonine protein kinase, PknE, interferes
with the signaling pathways involved in apoptosis following NO
stress (Jayakumar et al., 2008) and, thereby, modulates Mtbinduced macrophage survival.
Interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and NOS2
are up regulated concomitantly in AM following exposure to
Mtb (Bhatt and Salgame, 2007) (Figure 1). The production of
NO by AMs in TB patients may have an auto-regulatory role
which, through the activation of the transcription factor nuclear
factor (NF)-κB, potentiates the generation of pro-inflammatory
cytokines (Dlugovitzky et al., 2000; Kuo et al., 2000; Chan
et al., 2001b). This hypothesis is supported by the presence of
significantly higher levels of IFN-γ in milder cases of TB than
in more advanced disease (Sahiratmadja et al., 2007). In contrast,
patients with severe TB had greater levels of IL-12, transforming
growth factor-β and TNF-α in comparison to those with less
severe TB (Sahiratmadja et al., 2007). Moreover, nitrite levels
were significantly increased in advanced TB patients compared
with controls (Dlugovitzky et al., 2000). Over expression of
the inhibitor of NF-κB, IκBα, confirmed that the IκBα kinase
(IKK)–NF-κB signaling pathway enhanced IFN-γ- and Mtb
lipoarabinomannan-induced NOS2 promoter activity and NO
expression (Chan et al., 2001b).
Since Mtb affects one third of world’s population and NOS2
seems to play an important role in growth of this bacilli
(Raviglione et al., 1995), it is important to understand how
genetic factors that may influence the susceptibility in disease
of infected individuals. Thus, a combination of polymorphisms
within the host NOS2 locus and the balance of NOS2- inducing or
NOS2-inhibiting cytokines (MacMicking et al., 1997) may affect
susceptibility to disease. In contrast, the expression of microbial
genes that confer resistance to nitroxergic products (Nunoshiba
MECHANISMS OF GRANULOMA
FORMATION AND THE ROLE OF NO
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Granuloma formation following exposure to Mtb is correlated
with strong inflammatory and protective responses. The
expression of NOS2, NOS3 and nitrotyrosine (N-tyr) are all
increased in the granuloma-associated inflammatory cells and in
pneumonitis regions of human tuberculous lungs (Jung et al.,
2013). In addition, granuloma-associated macrophages from
untreated patients with pleuropulmonary and pulmonary TB
demonstrate high levels of NOS2-mediated NO production and
of N-tyr (Schön et al., 2004). The elevated expression of NOS
isoforms and N-tyr is predominantly within AM, epithelioid
macrophages and multinucleated giant cells (Choi et al., 2002)
(Figure 2).
The progressive granulomatous response to TB can be
tissue damaging and contribute to chronic infection, at least
in immunocompetent hosts. NO can however, have a yingyang effect on the clearance of infection and the inflammatory
response depending upon the mycobacterial strain. IFN-γ and
NOS2 knockout mice highlight the critical role of these mediators
in protective immunity against Mtb (Cooper et al., 2002).
Results suggest that they are important in the resolution of
inflammation resulting from an increased lymphocytic response
and can also decrease tissue damage as measured by granuloma
regression (Cooper et al., 2002). However, during infection with
Mycobacterium avium, which is less dependent upon IFN-γ and
NO for preventing the growth of bacilli, a lack of NO results
in a shift in the pattern of immunological response leading to
increased bacterial clearance and enhanced the inflammatory
response (Cooper et al., 2002). Thus, the effects of NO on
mycobacterial growth and on the inflammatory and immune
response are complex and strain-dependent.
NO AND DRUG RESISTANT
TUBERCULOSIS
A reduced ability of NO to kill disease-causing strains of Mtb
was found to be associated with first-line anti-TB drug resistance
(Idh et al., 2012). Earlier studies indicated that, certain strains
of Mtb including M. intracellulare 31F093T, KUMS 9007 (Doi
et al., 1993), M. tuberculosis CDC1551, CB3.3 (Firmani and Riley,
2002), M. bovis, M. tuberculosis 79499 (O’Brien et al., 1994), a
C strain cluster defined by IS6110-based strain-typing (Friedman
et al., 1997) and the genotypes G1, G2, S2, and U (Idh et al., 2012)
can all to some extent resist killing by acidified nitrite, a RNS,
generated in vitro.
The resistance of the CDC1551 and CB3.3 strains of Mtb to
H2O2 and acidified sodium nitrite is significantly higher than
that of other strains which may account for why these two species
may be responsible for large outbreaks of TB (Firmani and
Riley, 2002). In isoniazid-resistant strains, H2O2 susceptibility
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NO and Tuberculosis
NO production allowing Mtb infection to occur. Importantly,
intracellular Mtb replication is attenuated by the loss of STAT3
expression with the resultant increase in NOS2 (Queval et al.,
2016).
However, alternative mechanisms for the control of Mtb
infection have been proposed that center on the control
of inflammation per se rather than on direct NO-induced
inhibition of mycobacterial growth. Protective immunity to
TB requires the release of IFN-γ from T-cells which induces
host cell NOS2 expression and enhances NO production
(Figure 1) (Mishra et al., 2013). NO prevents Mtb growth
and the subsequent inflammatory response. NO can also
directly modulate inflammation to impact upon Mtb growth
and function (Mishra et al., 2017). NO acts to prevent
the growth and immunopathology caused by TB via Snitrosylation and inhibition of the Nod-like receptor (NLR)
Family Pyrin Domain Containing 3 (NLRP3) inflammasome
factor (Mishra et al., 2013). NLRP3 inhibition results in a
reduction in IL-1β expression and in neutrophil recruitment.
Neutrophilic inflammation generates a local niche that supports
M. tuberculosis growth (Mishra et al., 2017). The presence of
12/15-lipoxygenase (ALOX12) products in cavitary tuberculosis
lesions is correlated with airway neutrophilia and bacterial
burden (Mishra et al., 2017). A genetic polymorphism associated
with elevated ALOX12 expression is associated with an enhanced
risk of tuberculosis. The data suggests that preventing the
NLRP3/IL-1β/neutrophilic axis will prevent Mtb replication
independent of NO (Mishra et al., 2017).
The importance of inflammation in the control of Mtb
infection is further demonstrated by the ability of thymoquinone
(TQ), an essential compound of Nigella sativa (black cumin)
(Mikaili et al., 2013), to suppress Mtb-induced bacterial
replication and inflammation in human and murine macrophage
cell lines (Mahmud et al., 2017). Importantly, TQ acts despite
markedly suppressing NOS2 expression and attenuating the host
cell production of NO (Mahmud et al., 2017).
correlated well with the presence of small amounts of catalase
but this does not account for low-virulence, isoniazid-sensitive,
catalase-positive strains (Firmani and Riley, 2002).
Under physiological conditions of 10% rather than 21%
oxygen, Mtb within infected primary human macrophages utilize
nitrate and generating large quantities of nitrite (CunninghamBussel et al., 2013). Mtb lacking a functioning nitrate reductase,
narG, are more susceptible to isoniazid and are more resistant
to H2O2 and these phenotypes can be reversed by exogenous
nitrite (Cunningham-Bussel et al., 2013). This suggests that
nitrite production by Mtb under normal conditions may induce
isonicotinic acid hydrazide (INH) insensitivity (CunninghamBussel et al., 2013).
This indicates that a common mechanism for both peroxide
and RNS resistance may exist. Since the mechanism of action
for anti-TB immunity is via the production of free radicals
(Figure 1), strain variations in repair systems related to ROI and
RNS, comparable to that seen in DNA repair, might be important
(O’Brien et al., 1994).
Multi-drug-resistant tuberculosis (MDR) is a major threat
to global health (Matteelli et al., 2007). There are currently
two promising new drugs, the bicyclic nitroimidazoles, PA-824
and OPC-67683, which are currently undergoing human clinical
trials (Li et al., 2008; Stop TB Initiative and World Health
Organization, 2008). These agents are both active against actively
replicating bacteria, as well as, bacteria that are non-replicating
by virtue of hypoxia (Stover et al., 2000). Non-replicating cells
are particularly difficult to eradicate and may be a major cause of
relative treatment insensitivity and the need for long treatment
periods (6–8 month) and disease relapses (Boshoff and Barry,
2005). Moreover, these non-replicating bacteria are thought to be
associated with latent tuberculosis (Gomez and McKinney, 2004;
Singh et al., 2008).
MECHANISMS OF NO-MEDIATED MTB
KILLING
The mechanism(s) by which pathogens such as Mtb suppress
NO production are varied and have been recently elucidated.
Initial studies indicated that IFN-γ-induced NOS2 expression
was mediated via the transcription factor IFN regulatory factor1 (Kamijo et al., 1994). More recent evidence indicates that
although this process may not be critical for the control of early
bacterial infection, it clearly plays a role in the granulomatous
response (Cooper et al., 2000) (Figure 2).
The Proline-Proline-Glutamate (PPE) family of proteins are
particularly abundant in pathogenic Mycobacterial species such
as Mtb (Bhat et al., 2017). Recent evidence indicates that
these proteins can act as repressive transcription factors in
the nucleus of host macrophages to suppress NOS2 expression
and thereby reduce the release of the anti-mycobacterial NO
molecule (Bhat et al., 2017). In addition, soon after Mtb infection,
macrophages produce IL-10 which induces the phosphorylation
and activation of the transcription factor signal transducer and
activator of transcription (STAT3) (Queval et al., 2016). STAT3
activation results in repression of NOS2 expression and reduced
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MTB PROTECTION AGAINST
NO-MEDIATED KILLING
In addition to the densely mycolylated cell wall, Mtb use
other mechanisms to circumvent the host defense system and
prevent killing by the host including those induced by pattern
recognition receptors (PRRs) (Mortaz et al., 2017). PRRs are
critically important in the host response to Mtb infection and
their roles are summarized in several recent reviews (Mortaz
et al., 2017). Full activation of murine macrophages depends
upon IFN-γ, PRR activation, and/or TNF whereas vitamin D2
is required as a cofactor for maximal activation of human
macrophages. This full activation results in enhanced expression
of antimicrobial peptides/proteins (AMPs), such as cathelicidin
and other antimicrobial moieties including ROS and RNS
generation (Awuh and Flo, 2017). NO and ROS interact within
the phagosome to generate highly reactive intermediates that
destroy microbial membrane lipids, DNA, and thiol- and tyrosine
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residues by oxidation. NO also directly targets the iron sulfur
clusters of bacterial enzymes (Awuh and Flo, 2017).
Mycobacteria have evolved over time to develop systems that
reduce the antimicrobial activity of ROS (Awuh and Flo, 2017).
For example, Mtb express many anti-oxidant enzymes such
as superoxide dismutase, catalase, alkyl hydroperoxidase, and
peroxiredoxins, to neutralize the free radicals generated by the
host (Awuh and Flo, 2017). Furthermore, elevated expression of
the Mtb protein, enhanced intracellular survival (Eis), increases
intracellular Mtb survival. Eis has ROS-dependent effects on
autophagy and inflammatory responses including the expression
of TNFα and IL-6 to prevent cell death. Generally, Mtb can
detect ROS/RNS-induced changes in the host environment and
respond by producing proteins that limit the toxic effects of these
changes (Awuh and Flo, 2017). This enables survival of Mtb in
the nutrient-deficient, hypoxic and ROS/RNS-high environment
present within granulomas (Awuh and Flo, 2017).
In addition, the Mtb proteasome protects the microbe
from the damaging effects of NO and its derivatives. NO
and RNI may modify or irreversibly damage Mtb proteins,
possibly by nitrosylation, and this is counteracted by the
removal of the damaged or modified proteins (Rhee et al.,
2005). The key Mtb proteins involved in this protective
response are the proteasomal accessory subunits proteasome
accessory factor A (Paf) and Mycobaterium proteasomal ATPase
(Mpa) (Wang et al., 2009). These proteins either recognize
damaged proteins and deliver them to the proteasome or
repair the damaged proteins. Mice challenged with Mpa or
Paf mutant mycobacteria could combat infection because
the mutants had reduced virulence (Ehrt and Schnappinger,
2009). Finally, the Mtb proteasome substrates, malonyl
Co-A acyl carrier protein transacylase and ketopantoate
hydroxymethyltransferase, are essential for Mtb pathogenesis.
This suggests that targeting the Mtb proteasome may represent a
novel anti-TB therapeutic approach (Darwin et al., 2003; Pearce
et al., 2006).
macrophages in men and women with active pulmonary TB
(Wang et al., 1998).
The urinary levels of the NO metabolites nitrite and nitrate
are elevated in patients with active tuberculosis and these levels
are reduced with anti-TB treatment (Chan et al., 1992). The
endogenous generation of NO, as a defense mechanism against
Mtb, is the most probable explanation for this (Chan et al.,
1995; Nicholson et al., 1996). These two NO metabolites have
frequently used as an indirect measurement of the production
of NO in vivo (Anstey et al., 1996; Dykhuizen et al., 1996;
Ellis et al., 1998; Sundqvist et al., 1998; Schön et al., 1999).
In an interesting study that compared TB infected patients
with and without HIV co-infection; the patients without HIV
infection showed significantly higher amounts of FeNO (>25
ppb), compared to the patients with HIV and TB co-infection
(Idh et al., 2008). In contrast, the amounts of urinary NO
were greater in HIV/TB co-infected patients. FeNO or urinary
NO levels did not significantly correlate with clinical signs, the
grade of chest X-ray or inflammatory cytokine levels (Idh et al.,
2008). In both HIV negative and HIV co-infected TB patients,
there were low levels of FeNO compared to blood donors and
household contacts. It would be an interesting topic for future
studies to confirm whether low levels of FeNO could be used as a
risk factor for acquiring TB (Idh et al., 2008). A corollary to this is
that immunosuppressed patients, such as those with HIV, mount
a less effective T-cell response to infection which may result in a
reduced FeNO level (Idh et al., 2008).
The expression of NOS enzymes or levels of NO in various
compartments may also represent a good biomarker for disease
(Nicholson et al., 1996). BAL macrophages from patients with
Mtb express higher levels of NOS2 mRNA and this has been
linked to higher FeNO levels in the patient (Nicholson et al.,
1996). Changes in serum levels of nitrites and nitrates as well as
NOS2 activity in blood neutrophils may be another prognostic
tool to predict the treatment outcome of TB infection (Butov
et al., 2014).
DIAGNOSTIC ANALYSIS OF NO AND NO
METABOLITES
THERAPEUTIC USE OF NO AND
NO-DONORS
Gustafsson et al. (1991) first described the presence of NO in
exhaled breath (Gustafsson et al., 1991). Thereafter, numerous
reports showed that the concentration of exhaled NO is increased
in patients with several lung diseases (Kharitonov, 2004). FeNO
can be measured both in real-time (online) and off line (collected
and then sent to a remotely located analyser) but it is now
generally measured online by having the subject blow directly
into the analyser (Kissoon et al., 2000; Olivieri et al., 2006).
Interestingly, there is a significant difference in FeNO values
between men and women; with a higher level in men (range
2.6–28.8 ppb) compared to women (range 1.6–21.5 ppb) at
expiratory flows of 50 ml/s (Olivieri et al., 2006). The mechanisms
underlying this difference may reflect an effect of estrogen on
NOS2 expression but more research in this field is required
(Olivieri et al., 2006). In addition, it is unclear whether there
are differences in FeNO and NOS2 expression in alveolar
NO plays an important role in the host defense against
intracellular pathogens, but different cells in human body
generate different amounts of NO. For example, murine
macrophages produce sufficient levels of NO to act as a
bactericidal effector molecule (Singh et al., 2008) and this may
also occur in human macrophages (Fang and Vazquez-Torres,
2002). Many invading organisms compromise host macrophages
by impairing host NOS2 activity resulting in reduced NO
production. Decreased host NO production will, therefore, result
in a more sustainable niche for host infection. It is posited
therefore, that NO donors given to TB patients will be able to
compensate for the lack of endogenous NO by the compromised
macrophages (Seabra et al., 2015; Seabra and Duran, 2017).
The importance of NO in the control of Mtb infection is also
indicated by evidence in other species. NOS2 expression and NO
production is extremely limited in macrophages of the European
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a significant early bactericidal activity as measured by a reduction
in the rate of decline of sputum Mtb numbers particularly in
patients with MDR or drug-intolerant disease (Long et al., 2005).
The design of NO-donor moieties has developed significantly
over the past decade although there are still major steps
required to translate these in vitro technologies into clinical
drugs (Seabra et al., 2015). There is an increasing range of
these NO-donors that have extended beyond traditional low
molecular weight NO donors such as S-nitrothiols (RSNOs) and
NONOates toward novel biomaterials including NO-releasing
nanomaterials such as polymeric nanoparticles, dendrimers and
liposomes/micelles. These NO-releasing nano-materials may
enable the correct spatio-temporal production of NO within the
airway macrophages to target Mtb infection (Seabra et al., 2015).
Polymeric nanoparticles are biocompatible whilst the
dendrimer scaffold has the ability to store large amounts of NO,
due to their highly branched structure. However, dendrimers
are difficult to manufacture and the process involves toxic
organic solvents. In addition, polymeric micelles have a low
thermodynamic stability in biological fluids resulting in doses
which are usually too low to be effective (Seabra et al., 2015).
Improved medicinal, computational and click chemistry
approaches should result in the development of more heatstable drugs with enhanced sustained NO release profiles
preferably at the disease site. Drug efficacy may be improved
for pulmonary TB by using inhaled delivery. As with all new
drugs, there is a need to determine the effect of chronic
dosing in vivo matched to improved biodistribution and
pharmacokinetics/pharmacodynamics for each compound. With
improved features, it is likely that the scale-up costs for
development will be markedly reduced (Seabra et al., 2015).
The identification of volatile organic compound (VOC)
signatures in response to active infection with Mtb in macaques
raises the possibility of the rapid determination of clinical efficacy
of treatments such as NO-donors. Three compounds, dodecane,
hexylcyclohexane, and tridecane, may be the most promising as
they are also seen in humans infected with Mtb (Mellors et al.,
2017). Thus, measures of FeNO or of VOCs in exhaled breath
may enable dosing to be calibrated according to the dose required
(Mellors et al., 2017). It is likely that these novel NO-donating
polymeric nanomaterials will be used in concert with current
low molecular weight NO donors to achieve maximal therapeutic
effect.
As highlighted above, IFN-γ is important in the human
immune response to Mtb. Delivery of aerosolized IFN-γ given in
conjunction with standard anti-TB therapy enhanced expression
of NOS2 and IFN-inducible protein 10 (IP-10) mRNA expression
in AM of TB patients (Raju et al., 2004). Despite other studies
providing strong evidence for in vitro anti-mycobacterial activity
of IFN-γ in mouse macrophages (Rook et al., 1986; Flesch and
Kaufmann, 1987; Denis, 1991), another study reported that IFNγ is relatively ineffective in restricting intracellular Mtb growth
in human macrophages. The anti-mycobacterial effect of IFN-γ
was enhanced by adding retinoic acid and vitamin D3 (Douvas
et al., 1985; Cholo et al., 2005). However, due to the potential of
side-effects and the costs it is unlikely that this approach will be
pursued as an anti-TB therapy.
badger (Meles meles) and this may account for the species acting
as a reservoir for the bovine tuberculosis (Bilham et al., 2017).
Further support of the importance of NO in the pathogenesis
of TB is that these patients are deficient in both L-arginine,
the NO precursor, and in vitamin D (Ralph et al., 2008). These
analytes both have anti-TB and immunomodulatory actions
against TB in vitro (Ralph et al., 2008). Furthermore, the levels
of FeNO were significantly lower in patients with pulmonary
TB than in controls, particularly in those with more severe
disease, possibly reflecting reduced NO bioavailability (Ralph
et al., 2013). Of interest, subjects whose FeNO levels were elevated
or remain unchained after 2 months of anti-TB treatment had
better mycobacterial clearance (Ralph et al., 2013).
Therefore, low molecular weight NO-donors should enhance
Mtb killing and/or prevent intracellular replication. Indeed,
phenylsulfonyl furoxan derivatives which are effective NOdonors have low micromolar efficacy against Mtb H37Rv (ATCC
27294) and a clinical isolate MDR-TB strain in vitro (Fernandes
et al., 2016). Interestingly, clinical isolates of Mtb with reduced
survival after exposure to the NO donor DETA/NO had a
reduced response to first-line anti-TB drugs (Idh et al., 2012).
Altogether, this data suggests that increased NO delivered to
the lung of patients with pulmonary TB might reduce infectivity
and improve the response in patients with drug-resistant TB
(Ralph et al., 2008). However, a 4-arm randomized, doubleblind, placebo-controlled factorial trial in adults with smearpositive pulmonary TB in Indonesia, showed no clinical benefit
of combined oral vitamin D and L-arginine over 8 weeks
(NCT00677339)(Ralph et al., 2013). The failure to achieve an
improved clinical outcome may reflect the inability of L-arginine
to enhance NO production in the airways as reflected by a
failure to increase FeNO in patients on the active treatment.
This highlights the need to develop better, more effective NOdonor platforms to deliver therapeutic doses of NO to the correct
biological site and several approaches are being utilized toward
this end (Seabra and Duran, 2017). The therapeutic use of
gaseous NO itself may not be practical due to the cumbersome
nature of the machine, the expertise intensive procedure and
the long duration of intermittent exposure required to show
efficacy would be difficult to implement on ambulatory patients.
In addition, NO may cause tissue injury within the lung and
long-term exposure to NO may causes cardiovascular and other
pharmacological side effects and should not be given to patients
diagnosed with end stage renal disease or severe left ventricular
dysfunction for example. Therefore, inhaled NO-donors which
deliver high levels of intracellular NO to macrophages may give
better results (Verma et al., 2012).
INH is an important anti-TB agent and can produce NO
following oxidation in cells infected by Mtb (Long et al.,
2005). The importance of this process in INH function was
emphasized by the ability of NO scavengers to attenuate the antimycobacterial activity of INH in cell culture (Long et al., 2005).
Furthermore, inhaled NO (80 ppm) was safely administered
to patients with pulmonary TB (Long et al., 2005) but this
had no effect on the mycobacteriologic response achieved with
conventional therapy. There is also a need for more studies to
determine whether inhaled NO, delivered over the first 48 h, has
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October 2017 | Volume 8 | Article 2008
Jamaati et al.
NO and Tuberculosis
CONCLUSION
pulmonary TB, show promise and may be improved by structurebased design to produce agent(s) that not only treat but also
have the potential to cure active and latent tuberculosis (Singh
et al., 2008). However, the importance of inflammation in Mtb
pathophysiology must also be considered when treating these
patients. Overall, increased understanding the role of NO in Mtb
pathophysiology has provided great insight into many aspects of
disease mechanisms and elucidated potential novel treatments.
In conclusion, there have been significant increases in our
understanding of the mechanisms by which NO regulates
Mtb growth and emphasize this as a potential target for
anti-TB therapy. Indeed, NO-donating drugs have therapeutic
potential in a number of human diseases including TB (Rigas
and Williams, 2008). Analysis of the effect of these novel
agents, and of other modifiers of Mtb proliferation including
immunomodulators and Mtb nitrate reductase inhibitors, should
be undertaken in primary human cells under physiological
conditions. It is also important that sufficient NO is delivered
to the target cell within the airway and that the effect can be
monitored effectively to provide a rapid readout of drug action.
Thus, measurements of FeNO or of specific VOCs are essential
to monitor drug efficacy and enable variable dosing to minimize
any potential side-effect issues. Novel NO-donors, particularly
polymeric nanoparticles ideally delivered by the inhaled route for
AUTHOR CONTRIBUTIONS
HJ, EM, and ZP wrote the original draft of manuscript. GF
and MeM helped with literature collating and referencing. MiM,
IMA, and JG revised and edited the manuscript.
FUNDING
IMA is supported by Wellcome Trust grant 093080/Z/10/Z.
REFERENCES
Butov, D. O., Kuzhko, M. M., Kalmykova, I. M., Kuznetsova, I. M., Butova, T. S.,
Grinishina, O. O., et al. (2014). Changes in nitric oxide synthase and nitrite
and nitrate serum levels in patients with or without MDR-TB undergoing the
intensive phase of anti-tuberculosis therapy. Int. J. Mycobacteriol. 3, 139–143.
doi: 10.1016/j.ijmyco.2014.02.003
Chan, E. D., Chan, J., and Schluger, N. W. (2001a). What is the role of nitric
oxide in murine and human host defense against tuberculosis? Current
knowledge. Am. J. Respir. Cell Mol. Biol. 25, 606–612. doi: 10.1165/ajrcmb.25.
5.4487
Chan, E. D., Morris, K. R., Belisle, J. T., Hill, P., Remigio, L. K., Brennan,
P. J., et al. (2001b). Induction of inducible nitric oxide synthase-NO by
Lipoarabinomannan of Mycobacterium tuberculosis is mediated by MEK1ERK, MKK7-JNK, and NF-κB signaling pathways. Infect. Immun. 69,
2001–2010. doi: 10.1128/IAI.69.4.2001-2010.2001
Chan, J., Tanaka, K., Carroll, D., Flynn, J., and Bloom, B. (1995). Effects of nitric
oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis.
Infect. Immun. 63, 736–740.
Chan, J., Xing, Y., Magliozzo, R. S., and Bloom, B. R. (1992). Killing
of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates
produced by activated murine macrophages. J. Exp. Med. 175, 1111–1122.
doi: 10.1084/jem.175.4.1111
Choi, H. S., Rai, P. R., Chu, H. W., Cool, C., and Chan, E. D. (2002).
Analysis of nitric oxide synthase and nitrotyrosine expression in human
pulmonary tuberculosis. Am. J. Respir. Crit. Care Med. 166, 178–186.
doi: 10.1164/rccm.2201023
Cholo, M. C., Boshoff, H. I., Steel, H. C., Cockeran, R., Matlola, N. M., Downing,
K. J., et al. (2005). Effects of clofazimine on potassium uptake by a Trk-deletion
mutant of Mycobacterium tuberculosis. J. Antimicrob. Chemother. 57, 79–84.
doi: 10.1093/jac/dki409
Cooper, A. M., Adams, L. B., Dalton, D. K., Appelberg, R., and Ehlers, S. (2002).
IFN-γ and NO in mycobacterial disease: new jobs for old hands. Trends
Microbiol. 10, 221–226. doi: 10.1016/S0966-842X(02)02344-2
Cooper, A. M., Pearl, J. E., Brooks, J. V., Ehlers, S., and Orme, I. M. (2000).
Expression of the nitric oxide synthase 2 gene is not essential for early control of
Mycobacterium tuberculosis in the murine lung. Infect. Immun. 68, 6879–6882.
doi: 10.1128/IAI.68.12.6879-6882.2000
Cunningham-Bussel, A., Bange, F. C., and Nathan, C. F. (2013). Nitrite impacts
the survival of Mycobacterium tuberculosis in response to isoniazid
and hydrogen peroxide. Microbiologyopen 2, 901–911. doi: 10.1002/
mbo3.126
Darwin, K. H., Ehrt, S., Gutierrez-Ramos, J. C., Weich, N., and Nathan, C.
F. (2003). The proteasome of Mycobacterium tuberculosis is required for
resistance to nitric oxide. Science 302, 1963–1966. doi: 10.1126/science.1091176
Alcaraz-López, O. A., García-Gil, C., Morales-Martínez, C., López-Rincón,
G., Estrada-Chávez, C., Gutiérrez-Pabello, J. A., et al. (2016). Divergent
macrophage responses to Mycobacterium bovis among naturally exposed
uninfected and infected cattle. Immunol. Cell Biol. 95, 436–442.
doi: 10.1038/icb.2016.114
Mahmud, H. A., Seo, H., Kim, S., Islam, M. I., Nam, K. W., Cho, H. D., et al. (2017).
Thymoquinone (TQ) inhibits the replication of intracellular Mycobacterium
tuberculosis in macrophages and modulates nitric oxide production. BMC
Complement. Altern. Med. 17:279. doi: 10.1186/s12906-017-1786-0
Anstey, N. M., Weinberg, J. B., Hassanali, M. Y., Mwaikambo, E. D., Manyenga,
D., Misukonis, M. A., et al. (1996). Nitric oxide in Tanzanian children
with malaria: inverse relationship between malaria severity and nitric oxide
production/nitric oxide synthase type 2 expression. J. Exp. Med. 184, 557–567.
doi: 10.1084/jem.184.2.557
Appelberg, R., and Orme, I. (1993). Effector mechanisms involved in cytokinemediated bacteriostasis of Mycobacterium avium infections in murine
macrophages. Immunology 80:352.
Arias, M., Rojas, M., Zabaleta, J., Rodríguez, J. I., París, S. C., Barrera, L. F.,
et al. (1997). Inhibition of virulent Mycobacterium tuberculosis by Bcg r and
Bcg s macrophages correlates with nitric oxide production. J. Infect. Dis. 176,
1552–1558. doi: 10.1086/514154
Aston, C., Rom, W. N., Talbot, A. T., and Reibman, J. (1998). Early
inhibition of mycobacterial growth by human alveolar macrophages is
not due to nitric oxide. Am. J. Respir. Crit. Care Med. 157, 1943–1950.
doi: 10.1164/ajrccm.157.6.9705028
Awuh, J. A., and Flo, T. H. (2017). Molecular basis of mycobacterial
survival in macrophages. Cell. Mol. Life Sci. 74, 1625–1648.
doi: 10.1007/s00018-016-2422-8
Bhat, K. H., Srivastava, S., Kotturu, S. K., Ghosh, S., and Mukhopadhyay, S. (2017).
The PPE2 protein of Mycobacterium tuberculosis translocates to host nucleus
and inhibits nitric oxide production. Sci. Rep. 7:39706. doi: 10.1038/srep39706
Bhatt, K., and Salgame, P. (2007). Host innate immune response to Mycobacterium
tuberculosis. J. Clin. Immunol. 27, 347–362. doi: 10.1007/s10875-007-9084-0
Bilham, K., Boyd, A. C., Preston, S. G., Buesching, C. D., Newman, C.,
and Macdonald, D. W., et al. (2017). Badger macrophages fail to produce
nitric oxide, a key anti-mycobacterial effector molecule. Sci. Rep. 7:45470.
doi: 10.1038/srep45470
Boom, W. H. (1996). The role of T-cell subsets in Mycobacterium tuberculosis
infection. Infect. Agents Dis. 5, 73–81.
Boshoff, H. I., and Barry, C. E. (2005). Tuberculosis–metabolism and respiration
in the absence of growth. Nat. Rev. Microbiol. 3:70. doi: 10.1038/nrmicro1065
Frontiers in Microbiology | www.frontiersin.org
8
October 2017 | Volume 8 | Article 2008
Jamaati et al.
NO and Tuberculosis
Gustafsson, L. E., Leone, A. M., Persson, M. G., Wiklund, N. P., and Moncada,
S. (1991). Endogenous nitric oxide is present in the exhaled air of rabbits,
guinea pigs and humans. Biochem. Biophys. Res. Commun. 181, 852–857.
doi: 10.1016/0006-291X(91)91268-H
Hausladen, A., Privalle, C. T., Keng, T., DeAngelo, J., and Stamler, J. S. (1996).
Nitrosative stress: activation of the transcription factor OxyR. Cell 86, 719–729.
doi: 10.1016/S0092-8674(00)80147-6
Idh, J., Mekonnen, M., Abate, E., Wedajo, W., Werngren, J., Ängeby, K., et al.
(2012). Resistance to first-line anti-TB drugs is associated with reduced
nitric oxide susceptibility in Mycobacterium tuberculosis. PLoS ONE 7:e39891.
doi: 10.1371/journal.pone.0039891
Idh, J., Westman, A., Elias, D., Moges, F., Getachew, A., Gelaw, A., et al.
(2008). Nitric oxide production in the exhaled air of patients with pulmonary
tuberculosis in relation to HIV co-infection. BMC Infect. Dis. 8:146.
doi: 10.1186/1471-2334-8-146
Jayakumar, D., Jacobs, W. R., and Narayanan, S. (2008). Protein kinase E of
Mycobacterium tuberculosis has a role in the nitric oxide stress response and
apoptosis in a human macrophage model of infection. Cell. Microbiol. 10,
365–374. doi: 10.1111/j.1462-5822.2007.01049.x
Jung, J. Y., Madan-Lala, R., Georgieva, M., Rengarajan, J., Sohaskey, C. D., Bange,
F. C., et al. (2013). The intracellular environment of human macrophages that
produce nitric oxide promotes growth of mycobacteria. Infect. Immun. 81,
3198–3209. doi: 10.1128/IAI.00611-13
Kamijo, R., Harada, H., Matsuyama, T., Bosland, M., Gerecitano, J.,
Shapiro, D., et al. (1994). Requirement for transcription factor IRF1 in NO synthase induction in macrophages. Science 263, 1612–1616.
doi: 10.1126/science.7510419
Kaufmann, S. H. (1993). Immunity to intracellular bacteria. Annu. Rev. Immunol.
11, 129–163. doi: 10.1146/annurev.iy.11.040193.001021
Kharitonov, S. A. (2004). Exhaled markers of inflammatory lung diseases: ready for
routine monitoring? Swiss Med. Wkly. 134, 175–192.
Kissoon, N., Duckworth, L. J., Blake, K. V., Murphy, S. P., Taylor, C. L., and Silkoff,
P. E. (2000). FE NO: relationship to exhalation rates and online versus bag
collection in healthy adolescents. Am. J. Respir. Crit. Care Med. 162, 539–545.
doi: 10.1164/ajrccm.162.2.9909124
Kuo, H.-P., Wang, C.-H., Huang, K.-S., Lin, H.-C., Yu, C.-T., Liu, C.-Y.,
et al. (2000). Nitric oxide modulates interleukin-1 β and tumor necrosis
factor-α synthesis by alveolar macrophages in pulmonary tuberculosis.
Am. J. Respir. Crit. Care Med. 161, 192–199. doi: 10.1164/ajrccm.161.1.
9902113
Kwon, O. J., Kim, J. H., Kim, H. C., Suh, G. Y., Park, J. W., Chung,
M. P., et al. (1998). Nitric oxide expression in airway epithelial cells
in response to tubercle bacilli stimulation. Respirology 3, 119–124.
doi: 10.1111/j.1440-1843.1998.tb00109.x
Lewinsohn, D. M., Bement, T. T., Xu, J., Lynch, D. H., Grabstein, K. H., Reed, S. G.,
et al. (1998). Human purified protein derivative-specific CD4+ T cells use both
CD95-dependent and CD95-independent cytolytic mechanisms. J. Immunol.
160, 2374–2379.
Li, X., Manjunatha, U. H., Goodwin, M. B., Knox, J. E., Lipinski, C. A., Keller, T. H.,
et al. (2008). Synthesis and antitubercular activity of 7-(R)-and 7-(S)-methyl-2nitro-6-(S)-(4-(trifluoromethoxy) benzyloxy)-6, 7-dihydro-5H-imidazo [2, 1b][1, 3] oxazines, analogues of PA-824. Bioorg. Med. Chem. Lett. 18, 2256–2262.
doi: 10.1016/j.bmcl.2008.03.011
Liew, F., and Cox, F. (1991). Nonspecific defence the role of nitric oxide. Parasitol.
Today 7, 17–21. doi: 10.1016/0169-4758(91)90023-H
Long, R., Jones, R., Talbot, J., Mayers, I., Barrie, J., and Hoskinson, M., et al.
(2005). Inhaled nitric oxide treatment of patients with pulmonary tuberculosis
evidenced by positive sputum smears. Antimicrob. Agents Chemother. 49,
1209–1212. doi: 10.1128/AAC.49.3.1209-1212.2005
MacMicking, J., Xie, Q.-W., and Nathan, C. (1997). Nitric oxide
and macrophage function. Annu. Rev. Immunol. 15, 323–350.
doi: 10.1146/annurev.immunol.15.1.323
Matteelli, A., Migliori, G. B., Cirillo, D., Centis, R., Girard, E., and Raviglion,
M. (2007). Multidrug-resistant and extensively drug-resistant Mycobacterium
tuberculosis: epidemiology and control. Expert Rev. Anti Infect. Ther. 5,
857–871. doi: 10.1586/14787210.5.5.857
Mellors, T. R., Blanchet, L., Flynn, J. L., Tomko, J., O’Malley, M., Scanga, C. A.,
et al. (2017). A new method to evaluate macaque health using exhaled breath: a
Denis, M. (1991). Interferon-gamma-treated murine macrophages inhibit growth
of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell.
Immunol. 132, 150–157. doi: 10.1016/0008-8749(91)90014-3
Dlugovitzky, D., Bay, M. L, Rateni, L., Fiorenza, G., Vietti, L., Farroni, M.
A, et al. (2000). Influence of disease severity on nitrite and cytokine
production by peripheral blood mononuclear cells (PBMC) from patients
with pulmonary tuberculosis (TB). Clin. Exp. Immunol. 122, 343–349.
doi: 10.1046/j.1365-2249.2000.01394.x
Doi, T., Ando, M., Akaike, T., Suga, M., Sato, K., and Maeda, H. (1993). Resistance
to nitric oxide in Mycobacterium avium complex and its implication in
pathogenesis. Infect. Immun. 61, 1980–1989.
Fernandes, G. F. D. S., de Souza, P. C., Marino, L. B., Chegaev, K., Guglielmo,
S., Lazzarato, L., et al. (2016). Synthesis and biological activity of furoxan
derivatives against Mycobacterium tuberculosis. Eur. J. Med. Chem. 123,
523–531. doi: 10.1016/j.ejmech.2016.07.039
Douvas, G. S., Looker, D. L., Vatter, A. E., and Crowle, A. J. (1985).
Gamma interferon activates human macrophages to become tumoricidal
and leishmanicidal but enhances replication of macrophage-associated
mycobacteria. Infect. Immun. 50, 1–8.
Dunn, P. L., and North, R. J. (1995). Virulence ranking of some Mycobacterium
tuberculosis and Mycobacterium bovis strains according to their ability to
multiply in the lungs, induce lung pathology, and cause mortality in mice.
Infect. Immun. 63, 3428–3437.
Dykhuizen, R. S., Masson, J., McKnight, G., Mowat, A. N., Smith, C. C., Smith,
L., et al. (1996). Plasma nitrate concentration in infective gastroenteritis and
inflammatory bowel disease. Gut. 39, 393–395. doi: 10.1136/gut.39.3.393
Edwards, D., and Kirkpatrick, C. H. (1986). The immunology of
Mycobacterial diseases 1, 2. Am. Rev. Respir. Dis. 134, 1062–1071.
doi: 10.1164/arrd.1986.134.5.1062
Ehrt, S., and Schnappinger, D. (2009). Mycobacterial survival strategies in the
phagosome: defence against host stresses. Cell. Microbiol. 11, 1170–1178.
doi: 10.1111/j.1462-5822.2009.01335.x
Ellis, G., Adatia, I., Yazdanpanah, M., and Makela, S. K. (1998). Nitrite and
nitrate analyses: a clinical biochemistry perspective. Clin. Biochem. 31, 195–220.
doi: 10.1016/S0009-9120(98)00015-0
Ernst, W. A., Thoma-Uszynski, S., Teitelbaum, R., Ko, C., Hanson, D.
A., Clayberger, C., et al. (2000). Granulysin, a T cell product, kills
bacteria by altering membrane permeability. J. Immunol. 165, 7102–7108.
doi: 10.4049/jimmunol.165.12.7102
Esquivel-Solís, H., Vallecillo, A. J., Benítez-Guzmán, A., Adams, L. G., López-Vidal,
Y., and Gutiérrez-Pabello, J. A. (2013). Nitric oxide not apoptosis mediates
differential killing of Mycobacterium bovis in bovine macrophages. PLoS ONE
8:e63464. doi: 10.1371/journal.pone.0063464
Fang, F. C., and Vazquez-Torres, A. (2002). Nitric oxide production by human
macrophages: there9s NO doubt about it. Am. J. Physiol. Lung Cell. Mol. Physiol.
282, L941–L943. doi: 10.1152/ajplung.00017.2002
Firmani, M. A., and Riley, L. W. (2002). Mycobacterium tuberculosis CDC1551 is
resistant to reactive nitrogen and oxygen intermediates in vitro. Infect. Immun.
70, 3965–3968. doi: 10.1128/IAI.70.7.3965-3968.2002
Flesch, I., and Kaufmann, S. (1987). Mycobacterial growth inhibition by
interferon-gamma-activated bone marrow macrophages and differential
susceptibility among strains of Mycobacterium tuberculosis. J. Immunol. 138,
4408–4413.
Flesch, I. E., and Kaufmann, S. H. (1991). Mechanisms involved in mycobacterial
growth inhibition by gamma interferon-activated bone marrow macrophages:
role of reactive nitrogen intermediates. Infect. Immun. 59, 3213–3218.
Flynn, J. L., Scanga, C. A., Tanaka, K. E., and Chan, J. (1998). Effects of
aminoguanidine on latent murine tuberculosis. J. Immunol. 160, 1796–1803.
Friedman, C. R., Quinn, G. C., Kreiswirth, B. N., Perlman, D. C., Salomon,
N., Schluger, N., et al. (1997). Widespread dissemination of a drugsusceptible strain of Mycobacterium tuberculosis. J. Infect. Dis. 176, 478–484.
doi: 10.1086/514067
Garbe, T., Hibler, N., and Deretic, V. (1999). Response to reactive nitrogen
intermediates in Mycobacterium tuberculosis: induction of the 16-Kilodalton
α-Crystallin Homolog by Exposure to Nitric Oxide Donors. Infect. Immun. 67,
460–465.
Gomez, J. E., and McKinney, J. D. (2004). M. tuberculosis persistence, latency, and
drug tolerance. Tuberculosis 84, 29–44. doi: 10.1016/j.tube.2003.08.003
Frontiers in Microbiology | www.frontiersin.org
9
October 2017 | Volume 8 | Article 2008
Jamaati et al.
NO and Tuberculosis
Raviglione, M. C., Snider, D. E., and Kochi, A. (1995). Global epidemiology of
tuberculosis: morbidity and mortality of a worldwide epidemic. JAMA 273,
220–226. doi: 10.1001/jama.1995.03520270054031
Rhee, K. Y., Erdjument-Bromage, H., Tempst, P., and Nathan, C. F. (2005). Snitroso proteome of Mycobacterium tuberculosis: enzymes of intermediary
metabolism and antioxidant defense. Proc. Natl. Acad. Sci. U.S.A. 102, 467–472.
doi: 10.1073/pnas.0406133102
Rhoades, E. R., and Orme, I. M. (1997). Susceptibility of a panel of virulent
strains of Mycobacterium tuberculosis to reactive nitrogen intermediates. Infect.
Immun. 65, 1189–1195.
Rich, E. A., Torres, M., Sada, E., Finegan, C. K., Hamilton, B. D., and Toossi,
Z. (1997). Mycobacterium tuberculosis (MTB)-stimulated production of nitric
oxide by human alveolar macrophages and relationship of nitric oxide
production to growth inhibition of MTB. Tubercle Lung Dis. 78, 247–255.
doi: 10.1016/S0962-8479(97)90005-8
Rigas, B., and Williams, J. L. (2008). NO-donating NSAIDs and cancer: an
overview with a note on whether NO is required for their action. Nitric Oxide
19, 199–204. doi: 10.1016/j.niox.2008.04.022
Rook, G. A., Steele, J., Ainsworth, M., and Champion, B. R. (1986). Activation
of macrophages to inhibit proliferation of Mycobacterium tuberculosis:
comparison of the effects of recombinant gamma-interferon on human
monocytes and murine peritoneal macrophages. Immunology 59:333.
Sahiratmadja, E., Alisjahbana, B., de Boer, T., Adnan, I., Maya, A., Danusantoso,
H., et al. (2007). Dynamic changes in pro-and anti-inflammatory cytokine
profiles and gamma interferon receptor signaling integrity correlate with
tuberculosis disease activity and response to curative treatment. Infect. Immun.
75, 820–829. doi: 10.1128/IAI.00602-06
Scanga, C. A., Mohan, V. P., Tanaka, K., Alland, D., Flynn, J. L., and Chan, J. (2001).
The inducible nitric oxide synthase locus confers protection against aerogenic
challenge of both clinical and laboratory strains of Mycobacterium tuberculosis
in mice. Infect. Immun. 69, 7711–7717. doi: 10.1128/IAI.69.12.7711-7717.2001
Schmidt, H. H., and Walter, U. (1994). NO at work. Cell 78, 919–925.
doi: 10.1016/0092-8674(94)90267-4
Schön, T., Elmberger, G., Negesse, Y., Hernandez Pando, R., Sundqvist, T., and
Britton, S. (2004). Local production of nitric oxide in patients with tuberculosis.
Int. J. Tubercul. Lung Dis. 8, 1134–1137.
Schön, T., Gebre, N., Sundqvist, T., Aderaye, G., and Britton, S. (1999). Effects
of HIV co-infection and chemotherapy on the urinary levels of nitric oxide
metabolites in patients with pulmonary tuberculosis. Scand. J. Infect. Dis. 31,
123–126. doi: 10.1080/003655499750006137
Sciorati, C., Rovere, P., Ferrarini, M., Paolucci, C., Heltai, S., Vaiani, R., et al. (1999).
Generation of nitric oxide by the inducible nitric oxide synthase protects γδ
T cells from Mycobacterium tuberculosis-induced apoptosis. J. Immunol. 163,
1570–1576.
Seabra, A. B., and Duran, N. (2017). Nanoparticulated nitric oxide donors
and their biomedical applications. Mini Rev. Med. Chem. 17, 216–223.
doi: 10.2174/1389557516666160808124624
Seabra, A. B., Justo, G. Z., and Haddad, P. S. (2015). State of the art,
challenges and perspectives in the design of nitric oxide-releasing polymeric
nanomaterials for biomedical applications. Biotechnol. Adv. 33, 1370–1379.
doi: 10.1016/j.biotechadv.2015.01.005
Serbina, N. V., Liu, C. C., Scanga, C. A., and Flynn, J. L. (2000). CD8+
CTL from lungs of Mycobacterium tuberculosis-infected mice express
perforin in vivo and lyse infected macrophages. J. Immunol. 165, 353–363.
doi: 10.4049/jimmunol.165.1.353
Singh, R., Manjunatha, U., Boshoff, H. I., Ha, Y. H., Niyomrattanakit, P., Ledwidge,
R., et al. (2008). PA-824 kills nonreplicating Mycobacterium tuberculosis
by intracellular NO release. Science 322, 1392–1395. doi: 10.1126/science.
1164571
Stenger, S., Hanson, D. A., Teitelbaum, R., Dewan, P., Niazi, K. R., Froelich,
C. J., et al. (1998). An antimicrobial activity of cytolytic T cells mediated by
granulysin. Science 282, 121–125. doi: 10.1126/science.282.5386.121
Stop TB Initiative and World Health Organization (2008). Stop TB Partnership
Annual Report 2007: Gaining Global Momentum. Available online at: http://
apps.who.int/iris/handle/10665/69867
Stover, C. K., Warrener, P., VanDevanter, D. R., and Sherman, D. R. (2000).
A small-molecule nitroimidazopyran drug candidate for the treatment of
tuberculosis. Nature 405:962. doi: 10.1038/35016103
case study of M. tuberculosis in a BSL-3 setting. J. Appl. Physiol. 122, 695–701.
doi: 10.1152/japplphysiol.00888.2016
Mikaili, P., Maadirad, S., Moloudizargari, M., Aghajanshakeri, S., and Sarahroodi,
S. (2013). Therapeutic uses and pharmacological properties of garlic, shallot,
and their biologically active compounds. Iran. J. Basic Med. Sci. 16:1031.
Mikaili, P., Moloudizargari, M., and Aghajanshakeri, S. (2014). Treatment with
topical nitroglycerine may promote the healing process of diabetic foot ulcers.
Med. Hypotheses. 83, 172–174. doi: 10.1016/j.mehy.2014.05.002
Miller, B. H., Fratti, R. A., Poschet, J. F., Timmins, G. S., Master, S. S., Burgos,
M., et al. (2004). Mycobacteria inhibit nitric oxide synthase recruitment to
phagosomes during macrophage infection. Infect. Immun. 72, 2872–2878.
doi: 10.1128/IAI.72.5.2872-2878.2004
Mishra, B. B., Lovewell, R. R., Olive, A. J., Zhang, G., Wang, W., Eugenin, E., et al.
(2017). Nitric oxide prevents a pathogen-permissive granulocytic inflammation
during tuberculosis. Nat. Microbiol. 2:17072. doi: 10.1038/nmicrobiol.2017.72
Mishra, B. B., Rathinam, V. A., Martens, G. W., Martinot, A. J., Kornfeld,
H., Fitzgerald, K. A., et al. (2013). Nitric oxide controls tuberculosis
immunopathology by inhibiting NLRP3 inflammasome-dependent IL-1β
processing. Nat. Immunol. 14:52. doi: 10.1038/ni.2474
Mortaz, E., Adcock, I. M., Tabarsi, P., Darazam, I. A., Movassaghi, M., Garssen,
J., et al. (2017). Pattern recognitions receptors in immunodeficiency disorders.
Eur. J. Pharmacol. 808, 49–56. doi: 10.1016/j.ejphar.2017.01.014
Nathan, C. F., and Hibbs, J. B. (1991). Role of nitric oxide synthesis
in macrophage antimicrobial activity. Curr. Opin. Immunol. 3, 65–70.
doi: 10.1016/0952-7915(91)90079-G
Nathan, C., and Shiloh, M. U. (2000). Reactive oxygen and nitrogen intermediates
in the relationship between mammalian hosts and microbial pathogens. Proc.
Natl. Acad. Sci. U.S.A. 97, 8841–8848. doi: 10.1073/pnas.97.16.8841
Nicholson, S., Bonecini-Almeida Mda, G., Lapa e Silva, J. R., Nathan, C., Xie, Q.
W., Mumford, R., et al. (1996). Inducible nitric oxide synthase in pulmonary
alveolar macrophages from patients with tuberculosis. J. Exp. Med. 183,
2293–2302. doi: 10.1084/jem.183.5.2293
Nozaki, Y., Hasegawa, Y., Ichiyama, S., Nakashima, I., and Shimokata, K. (1997).
Mechanism of nitric oxide-dependent killing of Mycobacterium bovis BCG in
human alveolar macrophages. Infect. Immun. 65, 3644–3647.
Nunoshiba, T., DeRojas-Walker, T., Tannenbaum, S. R., and Demple, B. (1995).
Roles of nitric oxide in inducible resistance of Escherichia coli to activated
murine macrophages. Infect. Immun. 63, 794–798.
O’Brien, L., Carmichael, J., Lowrie, D. B., and Andrew, P. W. (1994). Strains
of Mycobacterium tuberculosis differ in susceptibility to reactive nitrogen
intermediates in vitro. Infect. Immun. 62, 5187–5190.
Olivieri, M., Talamini, G., Corradi, M., Perbellini, L., Mutti, A., Tantucci, C., et al.
(2006). Reference values for exhaled nitric oxide (reveno) study. Respir. Res.
7:94. doi: 10.1186/1465-9921-7-94
Pearce, M. J., Arora, P., Festa, R. A., Butler-Wu, S. M., Gokhale, R. S., and Darwin,
K. H. (2006). Identification of substrates of the Mycobacterium tuberculosis
proteasome. EMBO J. 25, 5423–5432. doi: 10.1038/sj.emboj.7601405
Peteroy-Kelly, M., Venketaraman, V., and Connell, N. D. (2001). Effects of
Mycobacterium bovis BCG infection on regulation of L-arginine uptake and
synthesis of reactive nitrogen intermediates in J774. 1 murine macrophages.
Infect. Immun. 69, 5823–5831. doi: 10.1128/IAI.69.9.5823-5831.2001
Queval, C. J., Song, O. R., Deboosère, N., Delorme, V., Debrie, A. S.,
Iantomasi, R., et al. (2016). STAT3 represses nitric oxide synthesis in human
macrophages upon Mycobacterium tuberculosis infection. Sci. Rep. 6:29297.
doi: 10.1038/srep29297
Raju, B., Hoshino, Y., Kuwabara, K., Belitskaya, I., Prabhakar, S., Canova, A., et al.
(2004). Aerosolized gamma interferon (IFN-γ) induces expression of the genes
encoding the IFN-γ-inducible 10-kilodalton protein but not inducible nitric
oxide synthase in the lung during tuberculosis. Infect. Immun. 72, 1275–1283.
doi: 10.1128/IAI.72.3.1275-1283.2004
Ralph, A. P., Kelly, P. M., and Anstey, N. M. (2008). L-arginine and vitamin
D: novel adjunctive immunotherapies in tuberculosis. Trends Microbiol. 16,
336–344. doi: 10.1016/j.tim.2008.04.003
Ralph, A. P., Yeo, T. W., Salome, C. M., Waramori, G., Pontororing,
G. J., Kenangalem, E., et al. (2013). Impaired pulmonary nitric oxide
bioavailability in pulmonary tuberculosis: association with disease severity and
delayed mycobacterial clearance with treatment. J. Infect. Dis. 208, 616–626.
doi: 10.1093/infdis/jit248
Frontiers in Microbiology | www.frontiersin.org
10
October 2017 | Volume 8 | Article 2008
Jamaati et al.
NO and Tuberculosis
Yang, C. S., Yuk, J. M., and Jo, E. K. (2009). The role of nitric oxide
in mycobacterial infections. Immune Netw. 9, 46–52. doi: 10.4110/in.2009.
9.2.46
Yu, K., Mitchell, C., Xing, Y., Magliozzo, R. S., Bloom, B. R., and Chan, J.
(1999). Toxicity of nitrogen oxides and related oxidants on mycobacteria:
M. tuberculosis is resistant to peroxynitrite anion. Tubercle Lung Dis. 79,
191–198. doi: 10.1054/tuld.1998.0203
Zhang, Y., Broser, M., Cohen, H., Bodkin, M., Law, K., Reibman, J., et al. (1995).
Enhanced interleukin-8 release and gene expression in macrophages after
exposure to Mycobacterium tuberculosis and its components. J. Clin. Invest.
95:586. doi: 10.1172/JCI117702
Sundqvist, T., Laurin, P., Fälth-Magnusson, K. E., Magnusson, K.-E., and
Stenhammar, L. (1998). Significantly increased levels of nitric oxide products in
urine of children with celiac disease. J. Pediatr. Gastroenterol. Nutr. 27, 196–198.
doi: 10.1097/00005176-199808000-00013
Velmurugan, K., Chen, B., Miller, J. L., Azogue, S., Gurses, S., and Hsu,
T., et al. (2007). Mycobacterium tuberculosis nuoG is a virulence
gene that inhibits apoptosis of infected host cells. PLoS Pathog. 3:e110.
doi: 10.1371/journal.ppat.0030110
Verma, R. K., Singh, A. K., Mohan, M., Agrawal, A. K., Verma, P. R., Gupta, A.,
et al. (2012). Inhalable microparticles containing nitric oxide donors: saying
NO to intracellular Mycobacterium tuberculosis. Mol. Pharm. 9, 3183–3189.
doi: 10.1021/mp300269g
Wang, C. H., Lin, H. C., Liu, C. Y., Huang, K. H., Huang, T. T., Yu, C. T., et al.
(2001). Upregulation of inducible nitric oxide synthase and cytokine secretion
in peripheral blood monocytes from pulmonary tuberculosis patients. Int. J.
Tubercul. Lung Dis. 5, 283–291.
Wang, C. H., Liu, C. Y., Lin, H. C., Yu, C. T., Chung, K. F., and Kuo, H. P.
(1998). Increased exhaled nitric oxide in active pulmonary tuberculosis due to
inducible NO synthase upregulation in alveolar macrophages. Eur. Respir. J. 11,
809–815. doi: 10.1183/09031936.98.11040809
Wang, T., Li, H., Lin, G., Tang, C., Li, D., Nathan, C., et al. (2009). Structural
insights on the Mycobacterium tuberculosis proteasomal ATPase Mpa.
Structure 17, 1377–1385. doi: 10.1016/j.str.2009.08.010
Frontiers in Microbiology | www.frontiersin.org
Conflict of Interest Statement: The authors declare that the research was
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