NO in Host Defense Against Microbial Pathogens

Top Abstract NO in Host Defense... Role of NO in... Role of NO in... Role of Epithelial Cells... Role of Dendritic Cells... Role of T Cells... Inducers of NO Expression... Mycobacterial Responses to NO Summary References

In immunocompetent individuals, the innate and adaptive arms of the immune system are relatively efficient in containing and killing Mycobacterium tuberculosis. It is estimated that of 100 people newly infected with the tubercle bacilli, only about 5-10 individuals will develop tuberculosis (TB) over their lifetime (1). Host cells that are protective against TB include macrophages, dendritic cells (DC), T-lymphocytes, and airway epithelial cells. The production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) by innate immune cells is considered to be a relatively effective host-defense mechanism against microbial pathogens. Catalytic action of the respiratory burst by the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex produces ROI such as hydrogen peroxide (H₂O₂), superoxide anion (O₂), and hydroxyl radical (OH^·). The importance of ROI in antimicrobial defense is exemplified in patients with chronic granulomatous disease (CGD), a disorder due to a mutation in any one of the four subunits of NADPH-oxidase complex, resulting in an inability to generate ROI. Humans with CGD are unusually susceptible to pyogenic infections with Staphylococcus aureus, Aspergillus species, and Nocardia species (2). Although the incidence of mycobacterial infections is not considered to be increased in CGD patients (2), TB appears to be a problematic issue in these patients in areas endemic for TB (3). For example, Lau and colleagues (3) estimated that in Hong Kong, the annual incidence of TB in CGD patients was > 170 times that for the general population. In three neonates with CGD who received bacillus Calmette-Guerin (BCG) as an immunogen, disseminated disease due to this mycobacteria developed (4). In the X-linked CGD mice (X-CGD), a strain with a genetic disruption to the gp91^phox subunit of NADPH-oxidase, M. tuberculosis growth was markedly enhanced in the lungs compared with the background B6 mice (5). However, when infected X-CGD macrophages were stimulated with interferon (IFN)-, the RNI produced was able to inhibit M. tuberculosis growth. In contrast, other studies found ROI to be relatively ineffective in killing M. tuberculosis (6).

The high output expression of nitric oxide (NO) in response to cytokines or to pathogen-derived molecules is an important component in the host defense against intracellular microorganisms as diverse as Toxoplasma gondii, Leishmania major, Listeria monocytogenes, Plasmodium species, Ectromelia virus, Coxsackie B3 virus, M. leprae, and M. tuberculosis (7). NO is formed when the guanidino nitrogen of L-arginine is oxidized by a family of isoenzymes known as NO synthases (NOSs). Exposure to NO at low concentrations, e.g., < 100 ppm, killed more than 99% of M. tuberculosis in culture (11).

The potential mechanisms by which NO may affect antimicrobial activity are protean. NO and other RNI can modify bacterial DNA, protein, and lipids at both the microbial surface and intracellularly. NO can also deaminate as well as directly damage bacterial DNA, generating abasic sites and strand breaks. Other potential mechanisms of killing by NO include interaction with accessory protein targets such as iron-sulfur groups, heme group, thiols, aromatic or phenolic residues, tyrosyl radicals, and amines, resulting in enzymatic inactivation or other protein malfunctions (12). Peroxynitrite, ONOO, can also mediate nitrosylation of tyrosine residues, and therefore has the potential to disrupt tyrosine phosphorylation-dependent signaling pathways (8).

Since M. tuberculosis may find a haven in macrophages, macrophage apoptosis is considered by many to be necessary to initiate mycobacterial killing; NO may induce such apoptosis. The mechanism by which macrophage apoptosis induced killing of M. bovis BCG was by facilitating the fusion of the mycobacterial-containing vacuoles to lysosomes, thereby subverting the mycobacterial control to prevent such a lethal fusion (13). Another potential mechanism for the apoptosis-induced mycobacterial killing is that, as with host nuclear fragmentation, mycobacterial DNA may also be destroyed during apoptosis (13). The mechanism by which the natural resistance associated macrophage protein (Nramp) locus confers resistance to M. tuberculosis may be related, in part, to NO. Macrophage cell lines have been derived with either the wildtype Nramp (B10R), a strain that is resistant to M. tuberculosis, or the Nramp mutation (B10S), a strain that is susceptible to the tubercle bacillus. In B10R macrophages infected with H37Rv M. tuberculosis, the induction of apoptosis and subsequent killing of M. tuberculosis directly correlated with NO production (14). This induction of apoptosis appeared to be dependent on metabolically active mycobacteria because killed M. tuberculosis rescued macrophages from apoptosis (14). Furthermore, this process is TNF--dependent because neutralization of TNF- diminished both NO production and apoptosis (15). Although B10R macrophages also produced greater amounts of O₂ than the susceptible B10S macrophages after infection with M. tuberculosis, ROI scavengers did not inhibit apoptosis or alter mycobacterial viability (16). Despite the accumulation of these potential effects of NO, the prime mechanism(s) by which NO or other RNI kill M. tuberculosis is still not fully understood.

In addition to mammalian host-cell factors, various M. tuberculosis strains also have differential susceptibility to the different species of RNIs. O'Brien and colleagues (17) found the relative in vitro resistance of various M. tuberculosis strains to sodium nitrite directly correlated with the virulence of the strains in guinea pigs. Rhoades and Orme (18) studied virulent strains of M. tuberculosis and found that intracellular NO₂ production by macrophages was more likely to be bacteriostatic than bactericidal. In mycobacteria exposed to various RNIs, NO and NO₂ exhibited antimycobactericidal activity against either BCG or a virulent strain of M. tuberculosis, with NO₂ significantly more potent than NO (19). Interestingly, whereas BCG was susceptible to ONOO, the Erdman strain of M. tuberculosis and a clinical isolate M160 were resistant to it. A possible mechanism for the ONOO resistance is by the ability of M. tuberculosis peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) to catalytically detoxify ONOO (20). These findings further illustrate the specificity of RNI in regard to their effects on different species or strains of mycobacteria. Nathan and Shiloh (8) reported a preliminary observation that drugs that appear to cure M. tuberculosis infection in immunocompetent mice failed to do so in iNOS-deficient mice, suggesting that tuberculocidal drugs may be effective in vivo only with help from iNOS-derived NO.

	Role of NO in Rodent Host-Defense against TB

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In the murine model of TB, NO plays an essential role in the killing of M. tuberculosis by mononuclear phagocytes (7, 10, 21). Intratracheal administration of virulent M. tuberculosis to rats stimulated iNOS and NO production in alveolar macrophages (22). Moreover, administration of the NOS inhibitor L-NG-monomethylarginine (L-NMMA) intraperitoneally attenuated the M. tuberculosis-induced increase in RNI in lung homogenates and bronchoalveolar fluid. One of the best examples of the protective role of NO in murine TB is illustrated by the genetically disrupted iNOS mouse strain (iNOS/), where infection with M. tuberculosis was associated with a significantly higher risk of dissemination and mortality compared with the wild-type C57BL/6 mice (5, 21). In mice that express the Bcg/ Nramp-1 resistance gene to M. tuberculosis, NO also mediated this resistance (23). Recent work indicates that preventing reactivation of latent infection appears to be under the control of both RNI and non-RNI pathways (24, 25). In murine models of latent infection, administration of the NOS inhibitor aminoguanidine led to the development of reactivation TB, although a non-RNI pathway also seemed to be involved. In a more recent study, depletion of CD4(+) T cells in a mouse model of latent infection revealed that although CD4 was required for preventing reactivation disease, it was by an iNOS- and IFN--independent antimycobacterial mechanism (24). These experimental findings indicate both NO-dependent and NO-independent mechanisms are operative to maintain the latent state, although the applicability of these results to humans is uncertain.

	Role of NO in Human Host-Defense against TB

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In contrast to the murine models of TB, there is a greater controversy on the role of NO in killing or limiting the growth of M. tuberculosis in humans (8). Aston and colleagues (26) showed that in vitro, the early mycobacteriostatic activity of human alveolar macrophages following M. tuberculosis infection was NO-independent. Interestingly, they also noted that exogenous IFN- failed to inhibit mycobacterial growth by human alveolar macrophages. Nevertheless, there is a growing body of evidence that NO produced by TB-infected human macrophages and by epithelial cells is also antimycobacterial against M. tuberculosis (27). Previously, it was thought that human macrophages did not produce NO in response to inflammatory stimuli. However, three experimental issues have shed light on this apparent paradox and it is now abundantly clear that human macrophages do make NO from increased iNOS activity. First, the difficulty in detecting NO in human macrophages may be due to the lack of tetrahydrobiopterin with in vitro cultures, a necessary co-factor for iNOS catalytic activity and a co-enzyme not constitutively synthesized by human monocytes or macrophages (32). However, the circumstances are different in vivo because human macrophages may obtain tetrahydrobiopterin from other neighboring cells capable of synthesizing it, such as activated lymphocytes and endothelial cells (32). Second, Jagannath and coworkers (31) recently demonstrated that the difficulty in detecting NO in human macrophages may, in part, be due to the inability of the standard colorimetric assay to detect relatively low levels of NO₂. Using a more sensitive fluorometric assay, they showed that NO was detectable in peripheral blood monocyte-derived macrophages infected with M. tuberculosis (31). Furthermore, they demonstrated the significance of this NO production by showing that iNOS inhibition with L-NMMA resulted in enhanced M. tuberculosis growth in human macrophages (31). Recently, Wang and coworkers (33) showed that cultured peripheral blood monocyte isolated from patients with active TB not only had increased iNOS expression compared with cells from normal subjects, but also had increased spontaneously released NO₂ in culture medium (7,513 ± 4,868 nmol/10⁶ cells versus 45.3 ± 13.1 nmol/10⁶ cells). Third, as noted by Nathan and Shiloh (8), it may have been overlooked that few, if any, have reported the induction of NO in macrophages derived from murine blood monocytes. In other words, the primary anatomic source of the macrophages used in in vitro experiments may have a profound impact on whether certain genes such as iNOS are expressed or active due to differences in the state of differentiation of the macrophages. This concept is supported by studies of Weinberg and colleagues (34), who demonstrated that human peripheral blood monocytes stimulated with LPS or various proinflammatory cytokines have no increase in NO production over basal levels, whereas human peritoneal macrophages have significantly enhanced NOS activity and NO₂/NO₃ production after LPS and/or IFN- stimulation.

Another example of this "site-specific" phenotype of macrophages is that bone marrow-derived macrophages from the C3H/HeJ mice, which has natural mutation of Toll-like receptor 4, do not respond to LPS, whereas alveolar macrophages from the same mice are LPS-responsive (35). Rich and coworkers (27) also showed that in alveolar macrophages from normal volunteers, iNOS and NO were inducible after M. tuberculosis infection and that there was an inverse correlation between the magnitude of intracellular growth and the amount of NO produced. Bonecini-Almeida and colleagues (36) showed that iNOS was inducible in IFN--primed monocyte-derived macrophages that are infected with M. tuberculosis. There was greater iNOS induction when the infected macrophages were cocultured with M. tuberculosis lysate/IFN--primed peripheral blood lymphocytes. Nicholson and coworkers (29) demonstrated increased iNOS protein expression in alveolar macrophages from TB patients. Moreover, they showed by a diaphorase cytochemistry assay that the iNOS was catalytically active, providing proof that there was high-output NO production in TB-infected macrophages (29). An immunofluorescence assay showed increased production of iNOS and ONOO in M. bovis-inoculated human alveolar macrophages and inhibition of NOS activity with L-NMMA treatment markedly reduced killing efficiency of mycobacteria (37). Kim and coworkers (30) observed that peripheral blood mononuclear cells (PBMC) infected with M. tuberculosis produced NO, and that the avirulent H37Ra strain induced significantly higher levels than the virulent H37Rv strain. In the human promyelocytic cell line HL-60, vitamin D3, known to have some therapeutic effect against TB, suppressed the growth of M. tuberculosis via the production of NO (28). Kuo and colleagues (38) showed that alveolar macrophages from TB patients produced increased amounts of NO compared to healthy control subjects. Furthermore, NO played an autoregulatory role in amplifying the synthesis of TNF- and IL-1 (38). Wang and coworkers (39) demonstrated that the increased exhale NO in patients with TB was due to upregulation of iNOS in alveolar macrophages. In addition, the amount of exhaled NO correlated with the capacity of the alveolar macrophages in vitro to produce NO. NO is not only mycobactericidal but may also participate in the formation of protective tissue granulomas (40). In a preliminary experiment, Raju and coworkers (41) showed that aerosolized IFN- increased NO levels in the bronchoalveolar lavage fluids in patients with active TB. In an intriguing epidemiologic study, Friedman and colleagues (42) reported that the drug-susceptible but more virulent C-strain of M. tuberculosis, which caused widespread dissemination in an at-risk population and accounted for a large proportion of new TB cases in New York City, was resistant to RNI, whereas thirteen other unrelated isolates were susceptible to RNI. The investigators speculated that the RNI-resistant C-strain had a biologic advantage over the RNI-susceptible isolates, resulting in a greater likelihood of progressively active disease with the C-strain. Collectively, these studies provide evidence that NO likely plays a contributory role in human host defense against M. tuberculosis.

	Role of Epithelial Cells in NO Expression

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High output NO production by iNOS may also occur in human lung epithelial cells (43). Airway epithelial cells exposed to M. tuberculosis have been shown to produce chemokines such as IL-8 and regulated on activation, normal T-cells expressed and secreted (RANTES) (43). The co-addition of culture supernatant fluid from M. tuberculosis-infected PBMC plus exogenous IFN- to A549 airway epithelial cells induced NO in a TNF-- and IL-1-dependent fashion (44). Pasula and colleagues (45) showed that surfactant protein A (SP-A), although able to enhance the attachment of M. tuberculosis to macrophages by acting as a ligand, inhibited M. tuberculosis-induced RNI expression in macrophages.

	Role of Dendritic Cells in NO Production

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Lung dendritic cells (DC) are key antigen-presenting cells capable of triggering specific T-cell responses to inhaled pathogens, including mycobacteria. In turn, the effector T cells produce cytokines that activate alveolar macrophages and lyse target cells in an effort to eliminate the pathogen. M. bovis-infected DC, when instilled into mice trachea, protected the mice from subsequent aerosol M. tuberculosis infection (46). Due to their antigen presentation, DC play an essential role in the initiation of primary T-cell responses to foreign antigens. Human or mouse DC are also capable of phagocytosing M. tuberculosis with subsequent increases in the cell surface expression of several inflammatory cytokines (IL-6, IL-1, and IL-12), co-stimulatory molecules (CD54, CD40, and B7.1), and class I MHC molecules (47, 48). One important experimental caveat to bear in mind with DC is that their isolation requires elaborate negative selection with monoclonal antibody and prolonged culture in cytokine-enriched milieu, which may ultimately alter their functions in vitro compared with the in vivo state.

The literature is replete with studies showing that DC have the capacity to produce NO in the context of a variety of biologic functions. In the rat thymus, DC-induced NO expression mediates thymic cell selection by inducing apoptosis in thymocytes that encounter self-antigens (49). Mouse bone marrow-derived DC were also shown to produce NO in response to IFN- + LPS (50). Kradin and coworkers (51) showed that in response to heat-killed Listeria, rat alveolar macrophages with DC characteristics produced NO. Stenger and colleagues (52) showed that C57BL/6 mice with acute cutaneous leishmaniasis had increased iNOS activity present in both macrophages and DC, cell types important in controlling the infection. Although Chambers and coworkers (53) showed that the expression of iNOS, IFN-, and the number of DC and monocytes were all increased in lymph nodes draining the site of live BCG vaccination, the significance of the iNOS and whether or not the increased iNOS was due to the DC were not known. In comparing the ability of DC and macrophages generated from the bone marrow cells of C57BL/6 mice to kill M. tuberculosis, Bodnar and colleagues (54) showed that both activated DC and macrophages inhibited the growth of intracellular mycobacteria in an iNOS-dependent fashion. In contrast, however, while this activation enabled macrophages to kill the mycobacteria, the tubercle bacilli within activated DC were not killed. This difference occurred despite the fact that there was essentially similar levels of RNI produced in DC and in macrophages. The mechanism for this inability of DC to kill M. tuberculosis is not fully understood, but may be due to persistence of the bacilli in special vacuoles in DC. The implication is that DC may serve as a reservoir for M. tuberculosis in tissues and use this cell as a vehicle for dissemination from the lung to the lymph nodes and other organs (54).

	Role of T Cells in NO Expression and the Effects of M. tuberculosis-Induced NO on T-Cells

Top Abstract NO in Host Defense... Role of NO in... Role of NO in... Role of Epithelial Cells... Role of Dendritic Cells... Role of T Cells... Inducers of NO Expression... Mycobacterial Responses to NO Summary References

The antigen-presenting cell-T-cell interaction is a fundamental process that links the innate and adaptive arms of the immune system in a synergistic fashion. Upon such interaction, mutual induction of cytokines occurs, such as IL-12 and IL-18 from macrophages and IFN- from activated CD4(+) T cells and possibly from macrophages (55). IFN- may also be produced by CD8(+) cytotoxic T-cells. IFN- is an essential cytokine in iNOS-NO induction by LPS, TNF-, IL-1, and lipoglycans of M. tuberculosis. Cooper and colleagues (56) suggested in a murine lung infection model that IFN- expression by CD8(+) T-cells may be the primary mechanism for the protective role of these cells rather than via cell lysis molecules such as perforin or granzyme.

Interaction of CD40 ligand present on activated T cells and CD40 present on B cells, macrophages, and other antigen-presenting cells is important in the control of intracellular pathogens (e.g., Leishmania spp and Toxoplasma gondii) through enhanced expression of IFN-, TNF-, IL-12, and NO (57). In contrast, CD40 ligand is not essential for the development of murine cell-mediated immunity and resistance to M. tuberculosis or H. capsulatum as evinced by mice with genetic disruption for CD40 ligand (CD40L^/) (60, 61). In fact, spleen cells from CD40L^/ mice stimulated with M. tuberculosis produced IL-12, TNF-, and NO levels comparable to control mice cells. These findings would suggest that redundant pathways exist for the expression of these host-defense mediators. In a human correlate, individuals with a defective CD40 ligand gene may develop a high-IgM syndrome and display increased susceptibility to infection with Cryptococcus neoformans, Pneumocystis carinii, and Histoplasma capsulatum (62). However, the role of CD40-CD40 ligand in human host-defense against TB remains unknown.

Sciorati and colleagues (63) showed that NO produced by M. tuberculosis-infected macrophages following CD95- CD95 ligand interaction inhibited apoptosis of gamma-delta () T cells. Thus, NO may prolong the life span of activated T cells, strengthening the link between the innate and adaptive immunity against TB. In contrast, macrophages from mice chronically infected with M. tuberculosis suppressed the number of CD4 (+) T cells and their nonspecific and PPD-specific proliferative responses through production of NO (64). The mechanism of the NO production was shown to be IFN--dependent because in BCG-infected mice with genetic disruption of IFN-, the activated CD4 (+) T cells did not undergo apoptosis. However, reconstitution with exogenous IFN- to cultured splenocytes from BCG-infected IFN- knockout mice induced NO-mediated apoptosis of activated CD4 (+) T cells (65).

	Inducers of NO Expression in the Context of M. tuberculosis

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iNOS and NO expression is induced by a wide variety of cytokines and inflammatory mediators such as TNF-, IFN-, LPS, IL-1, hypoxia, and picolinic acid (reviewed in [66]). However, mycobacterial cell wall components are also capable of eliciting host-inflammatory responses. Surrounding the plasma membrane of M. tuberculosis is a layer of cross-linked peptidoglycan. Protruding from the peptidoglycan layer are macromolecules that include mycolic acid-arabinogalactan peptidoglycan complexes and lipoarabinomannan (LAM). LAM is comprised of a linear series of ringed mannose sugar residues, with periodic branches of single mannoses. At the proximal end of LAM, a phosphatidylinositol group anchors it to the plasma membrane. Distal to the mannose residues are attached a linear series of arabinoses. In M. tuberculosis, these arabinose residues are further "capped" to various degrees with mannoses. Adams and coworkers (67) showed that the LAM derived from the Erdman strain of M. tuberculosis, in conjunction with IFN-, induced NO expression in mouse peritoneal macrophages. LAM is also known to induce TNF- and IL-1, cytokines capable of inducing NO expression (68, 69). In addition, M. tuberculosis LAM is also directly capable of inducing iNOS-NO expression in a mouse macrophagic cell line RAW 264.7 that is poorly responsive to TNF- or IL-1 (70). Also anchored to the plasma membrane are simpler precursors of LAM such as the disaccharide dimannosylphosphatidylinositides (PIM₂) and lipomannan (LM), the latter comprised of a phosphatidylinositol group linked to a mannan core. Barnes and coworkers (71) demonstrated that PIM₂ or LM were able to induce inflammatory and antiinflammatory molecules such as TNF, GM-CSF, IL-1, IL-1, IL-6, IL-8, and IL-10. Recently, Brightbill and colleagues (72) showed that the 19-kDa lipoprotein of M. tuberculosis, also a mycobacterial cell wall component, induced iNOS and NO expression. In an autocrine or paracrine fashion, extracellular nucleotides such as ATP released from M. tuberculosis-infected macrophages may also induce NO production by engagement of P2 purinergic receptors (73). Teleologically, this unique mechanism of NO induction may limit the expression of NO locally to sites of infection, sparing unaffected tissues the potentially injurious effects of NO.

	Mycobacterial Responses to NO

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M. tuberculosis has also evolved clever ways to evade the toxic effects of RNI. Ehrt and coworkers (74) showed that a novel M. tuberculosis gene, noxR1, conferred resistance to the toxic effects of RNI, although the precise mechanism is not known. This same group later showed that the M. tuberculosis noxR3 gene also protected bacteria from the toxic effects of ROI and RNI (75). Similarly, the M. tuberculosis-derived peroxiredoxin gene Ahpc (alkyl hydroperoxide reductase subunit C) prevented RNI-induced necrosis and apoptosis in human cells (76). Ahpc has also been shown to detoxify ONOO to NO₂ (20). NO may also induce the expression of a M. tuberculosis-derived 16-kDa heat shock protein, a molecule that promotes stationary phase of growth of mycobacteria (77). In hypoxic conditions, nitrate (NO₃), a degradation product of NO, is reduced by the tubercle bacilli to nitrite (NO₂) at a rate that is significantly greater than in aerobic conditions. This induction of nitrate reductase under hypoxic conditions may serve a respiratory function in supporting the shift of the tubercle bacilli from aerobic growth to a state of dormancy (78).

	Summary

Top Abstract NO in Host Defense... Role of NO in... Role of NO in... Role of Epithelial Cells... Role of Dendritic Cells... Role of T Cells... Inducers of NO Expression... Mycobacterial Responses to NO Summary References

Unlike the adaptive arm of the immune system, NO is a nonspecific, chemically reactive molecule that is important in host defense against a wide variety of microbial pathogens. However, it is also becoming increasingly clear that no one killing mechanism or cell type is sufficient to kill mycobacteria in vivo. For example, cell lytic molecules such as granzyme, granulysin, and perforin may also contribute in the killing of M. tuberculosis (79). Although O₂ appears not to be required in rodents, the lack of a role for O₂ or its products (such as ONOO) has not been definitively proven in humans. Nevertheless, a substantial body of evidence now exists that implicates a participatory role for NO in human host defense against M. tuberculosis. Defining the relative importance of NO in host defense as compared with other molecules is difficult, although it appears that certain strains of M. tuberculosis have evolved strategies to combat the toxic effects of NO. It is paramount that future work should not only further define the role of NO in relevant human cells, such as alveolar macrophages and airway epithelial cells, but also under conditions that best mimic the in vivo environment, such as co-culture of the relevant cells. Such studies, which refine our understanding of the importance and exact role of NO in defense against TB, may lead to innovative vaccination or treatment strategies.