The increasing incidence of drug-resistant M. tuberculosis
strains has compromised efforts toward both the treatment and prevention of TB (4
). The ability of the WHO and its member countries to control the enormous burden of TB will require the development of not only new-generation vaccines but also novel anti-TB drugs and/or immunomodulatory therapeutics and compounds that can enhance the antituberculosis activity of human macrophages.
Based on the demonstration that DCP induces protective effects against tumor growth and hepatitis B virus in vivo
), we tested the hypothesis that DCP could exert microbicidal activity against mycobacteria within infected human monocytes/macrophages. Our study demonstrates that DCP induces a potent inhibitory effect against mycobacteria, resulting in a 65 to 75% decrease in viability of bacilli within human monocytes at 72 h posttreatment ( and ). Though the activity of DCP was lower than that of RIF, DCP and PZA exhibited similar in vitro
activities against intracellular M. bovis
BCG ( and ), further demonstrating the potential clinical relevance of our work. PZA is a first-line agent used in combination with isoniazid, RIF, and ethambutol for short-course (6-month) treatment of drug-sensitive TB and frequently for MDR-TB (35
DCP had no lytic effects on mycobacterium-infected macrophages () and no direct inhibitory effects on mycobacterial growth (). Similar numbers of viable monocytes were recovered after 3 days of culture in the absence or presence of the highest doses (1.6 mM) of DCP. Moreover, a possible direct effect of DCP on mycobacteria was ruled out by culturing extracellular M. bovis BCG with DCP. These observations demonstrated that DCP activates the antimycobacterial effector functions of human macrophages.
The antimicrobial effects of RIF are attributed to its interaction with and inhibition of the bacterial DNA-dependent β-subunit of RNA polymerase (59
). RIF has bactericidal activity in vitro
against slow-growing and intermittently growing M. tuberculosis
. PZA, an analog of nicotinamide, is a prodrug requiring conversion into its active form, pyrazinoic acid (POA), by the bacterial pyrazinamidase (61
). It is preferentially active against nonreplicating persisting bacilli in vivo
during active inflammation. The biochemical basis for antituberculosis activity of PZA was recently reported to be the inhibition of trans
-translation in M. tuberculosis
). Shi and colleagues showed that POA binds ribosomal protein S1, which is involved in protein translation and the ribosome-sparing process of trans
-translation. Interestingly, PZA does not act directly on M. bovis
because of the lack of a pncA
gene in this species. Therefore, our results further suggest that PZA activates human macrophages to better control intracellular mycobacteria, which may explain the clinical observation that PZA seems more important for the efficacy of the first-line multidrug treatment regimens than would be predicted based on PZA MICs alone.
Mononuclear phagocytes, including macrophages, play an important role in innate immunity against M. tuberculosis
and therefore are considered the first and essential line of defense against TB (38
). Whereas resting monocytes/macrophages fail to control replication of mycobacteria and other intracellular pathogens, activated macrophages can suppress growth of intracellular bacilli. The suppression of growth is accomplished via various effector mechanisms, such as biosynthesis of inflammatory cytokines (e.g., TNF-α and IL-1β), chemokine-mediated recruitment of neutrophils/lymphocytes, activation of intracellular microbicidal activities, or direct activation of the production of reactive oxygen species and/or reactive nitrogen intermediates (NO and/or its derivatives) (18
Our results indicate that the impairment of intracellular mycobacterial growth observed in human monocytes treated with DCP can be attributed at least in part to the production of MIP-1β by infected cells. Neutralization of MIP-1β reversed the DCP-mediated inhibitory effects on intracellular mycobacteria (). As illustrated in and , the microbicidal role of MIP-1β we observed is in agreement with the findings reported by Saukkonen and colleagues, who demonstrated that MIP-1β suppresses growth of mycobacteria within macrophages (42
The detailed intracellular signaling pathways by which DCP induces production of MIP-1β by infected monocytes are unknown. There are lines of evidence indicating that many of the processes involved in killing of intracellular mycobacteria are regulated by Ca2+
. For instance, activation of cytokine production and preformed granule secretion is regulated by Ca2+
). Even more relevant to our work, Méndez-Samperio and colleagues demonstrated that in vitro
, M. bovis
BCG stimulates human monocytes to produce C-C chemokines through mobilization of intracellular Ca2+
and influx of extracellular Ca2+
). Therefore, the mechanism of DCP effects observed in our work could at least partially involve Ca2+
fluxes directly altered by the Ca2+
complexed within DCP. However, further investigations are required to determine the specific signaling events triggered by DCP that result in MIP-1β production.
The influence and biological activities of chemokines are wide-ranging and involve more than simple recruitment of circulating leukocytes. In addition to their chemotatic role, chemokines can stimulate a cascade of proinflammatory events, lead to macrophage activation, and enhance T-cell activation and proliferation (67
). In human macrophages, C-C chemokines were reported to induce NO production and killing of Trypanosoma cruzi
, an intracellular parasite and the causative agent of Chagas' disease (46
). Taking into account the importance of NO as an effector molecule involved in the control of mycobacteria both in vitro
and in vivo
), it was of relevance to investigate whether DCP-induced MIP-1β and/or DCP alone could induce iNOS and thus production of NO. Quantitative RT-PCR results demonstrated that DCP can stimulate increased production of iNOS mRNA in mycobacterium-infected monocytes (). Additional experiments with murine macrophages confirmed that DCP induced NO production in mycobacterium-infected macrophages (). We then established that DCP-induced MIP-1β production was required for DCP-induced upregulation of the monocyte/macrophage iNOS pathway by showing that the addition of anti-MIP-1β antibody prevented induction of iNOS mRNA by DCP. As shown in , neutralization of MIP-1β significantly reduced iNOS mRNA in infected cells treated with DCP, demonstrating that MIP-1β is involved in the induction of iNOS.
We confirmed the importance of the iNOS-NO pathway in DCP-mediated inhibition of intracellular mycobacteria by adding the l
-arginine analogue that serves as an iNOS inhibitor, l
-NMMA monoacetate (50
), to DCP-treated infected monocytes. clearly demonstrates that inhibition of iNOS activity by l
-NMMA prevented the DCP-mediated suppression of intracellular mycobacterial growth. These data verify that DCP induction of the iNOS-NO effector pathway in human monocytes significantly contributes to suppression of intracellular mycobacteria. The mechanism(s) by which NO and/or RNI kill mycobacteria is unclear but has been suggested to involve disruption of bacterial DNA, proteins, signaling, and/or induction of monocyte/macrophage apoptosis (15
). Furthermore, given that previously reported activities of DCP include the induction of apoptosis (20
), there may be a connection between DCP-mediated NO production and apoptosis of infected cells. Since M. tuberculosis
promotes its replication by inhibiting the apoptosis of infected macrophages (reviewed in reference 72
), it would be worth investigating in future studies whether apoptosis is restored in mycobacterium-infected cells by DCP treatment.
iNOS is regulated at both the transcriptional and posttranscriptional levels by a number of signal transduction pathways and molecules, such as Jak-1/Stat-α/IRF-1; IκB/nuclear factor kappa B (NF-κB); enzyme activity cofactors, protein kinases, and phosphatases; and tyrosine phosphatases (11
). The induction of iNOS mRNA expression is the main regulatory step controlling iNOS activity, and the iNOS promoter is activated by multiple different transcription factors (reviewed in reference 73
). Bacterial LPS, IL-1β, TNF-α, and oxidative stress have been shown to induce iNOS expression by activating the transcription factor NF-κB. Furthermore, the interaction of C-C chemokines with their cognate Gαi
-protein-coupled receptors can activate phosphatidylinositol 3-kinase (PI3K), which, in turn, stimulates NF-κB production and thus can lead to activation of the iNOS promoter (reviewed in references 73
). Therefore, DCP induction of MIP-1β may result in both NF-κB and iNOS promoter activation, important for the observed intracellular inhibitory effects on mycobacteria. We have shown that MIP-1β is necessary for the inhibitory effects of DCP and that neutralization of MIP-1β alone inhibited iNOS induction (). These results suggest that the inhibitory effects of DCP involve in part the induction of iNOS via the MIP-1β signaling pathway. However, the exact molecular details require further detailed dissection.
Various laboratories have shown that different human cell types are capable of controlling growth of parasites (55
), bacteria (76
), and viruses (78
) via IDO1-dependent antimicrobial effects. In fact, IDO1 induction was thought to be at least partially responsible for the anticancer and antiviral effects of DCP (19
). Catabolism of local l
-tryptophan into kynurenine and its metabolites has been implicated as the mechanism of IDO1-mediated inhibition of microbial replication (55
). However, in recent years evidence has emerged indicating that degradation of l
-tryptophan by IDO1 also can downregulate T cell responses in a wide range of disease states (52
). Müller and coworkers addressed these seemingly contradictory roles of IDO1 by determining the relative concentrations of l
-tryptophan required for bacterial and T cell growth (81
). The levels of l
-tryptophan required to support bacterial growth were 10- to 40-fold higher than the levels necessary for optimal T cell activation and proliferation (81
). Hence, IDO1 can exert predominant antimicrobial effects during the acute phase of a rapidly replicating infection. In contrast, at later time points of infection, especially with a chronic, slow-growing pathogen, the immunoregulatory effects of IDO1 may predominate. In our model system, accumulation of IDO1 mRNA in mycobacterium-infected monocytes was suppressed by DCP treatment (). Therefore, IDO1 does not appear to be involved in the DCP-mediated inhibitory effects on intracellular mycobacteria. However, DCP may be able to reduce the immunoregulatory effects of IDO1 during TB infection, providing further benefits during treatment of TB disease. The latter possibility should be tested in future experiments.
In conclusion, this study shows that DCP has significant effects on intracellular mycobacterial growth. The findings presented herein favor the concept that DCP mediates its effects via the C-C chemokine MIP-1β and iNOS-NO effector pathways. It will be interesting to investigate whether macrophage activation with DCP would enhance the killing of M. tuberculosis
with current anti-TB drugs, such as PZA, which is preferentially used with several new drug candidates for optimal efficiency (82
). Further studies are warranted to evaluate DCP effects on drug-resistant and drug-sensitive virulent strains of M. tuberculosis
, used alone and in combination with PZA. In addition, further molecular dissection of the pathways involved in DCP-mediated enhancement of intracellular control of mycobacteria is important to pursue.