Background
Tuberculosis continues to be a leading cause of human mortality and morbidity, with WHO figures estimating a global prevalence exceeding 14 million and mortality of approximately 1.6 million in the year 2005 [
1]. The continuing global crisis has become further complicated by the emergence of multi-drug resistant strains of the bacteria and an increasing population of HIV-infected patients around the world. In view of this escalating clinical challenge, there is a growing need to develop novel therapeutic alternatives employing potent mycobactericidal mechanisms. Hence, there has been a lot of interest in cell autophagy as a potential immune defense mechanism against a number of bacterial pathogens, including mycobacteria [
2‐
5]. Physiological or pharmacological induction of autophagy via cell starvation or treatment with rapamycin, have been reported to suppress mycobacterial survival within RAW cells through increased acidification and maturation of mycobacterial phagosomes [
4].
It has been previously reported from our laboratory that treatment of infected human macrophages with adenosine 5'-triphosphate (ATP) was capable of killing mycobacteria by subverting the mycobacterium-induced block in phago-lysosomal fusion [
6,
7]. In the light of recent findings, the present study was undertaken to look for evidence of induction of cell autophagy within ATP-treated macrophages and of a role for this process in mediating its associated mycobactericidal activity. In this work we demonstrate, for the first time, that ATP induces autophagy in human macrophages within 30 minutes of exposure, which was associated with a subsequent decrease in
Mycobacterium bovis BCG viability within infected cells. We further confirmed a role for extracellular Ca
2+ and delineated the identity of the purinergic receptor involved in this process by using Ca
2+-free media and selective purinergic receptor agonists and antagonists. We were thus able to confirm that ATP induces rapid cell autophagy and killing of intracellular mycobacteria within infected human macrophages via a Ca
2+-dependent process mediated by activation of P2X
7 receptors. The results support the importance of autophagy as a mycobactericidal mechanism utilized by human macrophages and provide a possible rationale for the recently reported P2X
7 polymorphisms associated with extra-pulmonary TB [
8]. The findings also suggest a potential therapeutic role for P2X
7 agonists in treating mycobacterial infections.
Discussion
In this study we have shown that ATP-treated, BCG-infected THP-1 cells and MDMs undergo rapid cell autophagy, which involves the activation of P2X7 receptors and mobilization of extracellular calcium. Electron microscopy of ATP-treated, BCG-infected MDMs, revealed the presence of the bacteria within characteristic double-membraned autophagosomes. In addition, ATP-induced cell autophagy was accompanied by rapid phago-lysosomal fusion and loss of mycobacterial viability within infected cells suggesting that autophagy contributes to this mycobactericidal process.
Autophagy has recently been highlighted as an innate immune defense mechanism against bacterial pathogens such as Group A Streptococcus, Shigella and Mycobacterium tuberculosis [
2‐
5]. Bacterial agents like Listeria monocytogenes and Staphylococcus aureus have also been reported to evade host immunity by subverting autophagy, thereby underscoring its importance as a potent mycobactericidal mechanism [
12,
13]. Earlier studies in rat hepatocytes had shown an association between reduction in the volume density of autophagic vacuoles and a fall in intracellular ATP levels [
14]. In this study we sought to obtain more direct confirmation that extracellular ATP could induce autophagy in human macrophages. We demonstrated that LC3 lipidation and LC3-II association with autophagosomal membranes, which are both commonly used read-outs of autophagy [
9,
10], were induced by ATP, as revealed by immunoblotting and the transition of LC3 from its diffuse cytosolic appearance to a punctate intracellular distribution (Figure
1A &
1B, Figure
2A).
Exploring the identity of the purinergic receptor involved in the process, we observed that ATP-induced autophagy was mediated via activation of P2X
7, as revealed by the use of specific agonists and antagonists of this receptor. Autophagy was not induced in the absence of extracellular calcium (Fig
1c), which was consistent with the known property of P2X
7 to promote influx of extracellular calcium into the cytosol in contrast to P2Y, which mobilize intracellular calcium.
The highly polymorphic structure of P2X
7 has recently been described and five polymorphisms have been described that lead to reduction or loss of P2X
7 function [
15]. It has recently been shown that P2X
7 polymorphisms were associated with impaired ATP-induced mycobacterial killing [
16] and increased susceptibility to extra-pulmonary TB [
8]. The present study is the first to report on the involvement of P2X
7 in inducing cell autophagy and provides a possible rationale for the impaired mycobactericidal activity and increased predilection for extra-pulmonary TB associated with certain polymorphic forms of P2X
7.
Our laboratory and others have previously reported that elevation of cytosolic calcium levels in ATP-treated, BCG-infected macrophages is linked to increased phago-lysosomal fusion [
7,
25]. Since induction of autophagy was dependent on mobilization of extracellular calcium, we explored its effect on maturation of mycobacterial phagosomes. Induction of autophagy by ATP coincided with increased acidification of mycobacterial phagosomes, which was reversed on pre-treatment of MDMs with wortmannin. In ATP-treated cells, the bacteria were found to localize within characteristic double-membraned autophagosomes. A difference in appearance and positioning of both lysostraker red labeled vesicles and GFP-BCG within wortmannin treated cells was observed as compared to anti-P2X
7 and non-treated macrophages (Figure
2B). This was attributed to the previously reported effect of wortmannin to induce swelling of late endosomal compartments associated with PI3-K inhibition involved in membrane trafficking between late endosomes and lysosomes [
26,
27]. We further observed that addition of ATP had a bactericidal effect on BCG, which was reversed partially by wortmannin and almost completely by oATP (Figure
4). These findings are consistent with recent observations by Gutierrez et al who demonstrated that induction of autophagy, by starvation or rapamycin, in macrophages infected with
M. tuberculosis (Mtb), leads to maturation of mycobacteria-containing phagosomes into phagolysosomes and suppression of mycobacterial viability [
4]. Hence, induction of autophagy with ATP, as with the classical inducers like starvation or rapamycin, can override the mycobacterial block on phagosomal maturation. This ability of autophagy to induce phago-lysosomal fusion and subsequent bactericidal activity could have potential therapeutic implications. In view of the involvement of P2X
7 receptors in this phenomenon, the development of selective, potent and safe P2X
7 agonists as therapeutic alternatives in the treatment of multi drug resistant (MDR)-TB should be explored.
The bactericidal effect of ATP on BCG or Mtb in human and bovine MDMs has been reported previously by our laboratory and other groups [
6,
17,
18], though the mechanism of such action has not been clearly elucidated. It has been recently shown that Mtb infection of macrophages leads to intracellular accumulation and extracellular release of ATP, which in turn activates P2X
7 receptors to mediate cellular apoptosis and bacterial killing [
19]. Apoptosis was induced in this latter study in 26.5 ± 2.5% of macrophages in response to stimulation with 5 mM ATP at an MOI of 10:1, following 48 hours of infection [
19]. However, the evidence for ATP-induced apoptosis is still equivocal, as we have found that caspase inhibitors do not block ATP-mediated killing of intracellular mycobacteria although they do block IL-1β release [
20]. Moreover, in contrast to ATP, classical inducers of cell apoptosis such as FasL or anti-Fas antibody are relatively ineffective at reducing intracellular BCG viability within infected human macrophages [
6,
21]. In addition, using a lower dose of ATP (3 mM), a lower MOI (5:1) and a lower duration of culture, we failed to observe any difference in apoptosis levels between ATP-treated and control cells, as revealed by flow cytometric determination of active caspase-3 expression (data not shown). Though it is probable that macrophage apoptosis plays a role in ATP-mediated mycobacterial killing, the rapid decline in intracellular BCG viability immediately after ATP exposure appears to be primarily mediated by autophagy. This is consistent with our observation that wortmannin, a classical inhibitor of autophagy, achieved only partial reversal of bactericidal activity while the P2X
7-antagonist, oATP, almost completely inhibited ATP-induced bacterial killing (Figure
4). Similarly, in our confocal studies neither anti-P2X
7 or wortmannin treatment completely ablated the co-localisation observed following ATP stimulation (Figure
2B &
2C), implying that other mechanisms are involved in mediating this effect. The results suggest that ATP-induced mycobactericidal activity is dependent on both autophagy as well as apoptosis. The use of a more specific P2X
7 inhibitor such as KN62 or combinations of inhibitors such as anti-P2X
7 and wortmannin, which may exhibit synergistic activity may help to clarify the relative roles of each process. That autophagy is involved in ATP-mediated mycobactericidal activity is supported by previous reports describing profound changes in the architecture of the intracellular vacuolar system of ATP-treated infected macrophages [[
17], our unpublished observations]. However, these changes were also accompanied by membrane blebbing and nuclear condensation (results not shown), which are morphological changes associated with early stages of apoptosis supporting a role for both processes in the associated mycobactericidal effect. Multiple, large cytosolic vacuoles were also formed within non-BCG infected cells following ATP treatment (Figure
3B). Although no detailed assessment was made of their structure in terms of whether they represented autophagosome formation, it was noted that cellular debris was apparent in a high percentage of them. Our laboratory is currently undertaking a detailed assessment of such structures to determine the degree of autophagosome formation within ATP-treated macrophages.
That autophagy may prove a critical mycobactericidal effector mechanism utilized by phagocytes is also suggested by the fact that we have previously reported, using macrophages derived from p47Phox-/- and iNOS-/- mice, that neither oxygen or nitrogen radical generation are involved in mediating ATP-induced, mycobactericidal activity [
22]. Moreover, parallel studies performed in BCG-infected macrophages, derived from both NRAMP resistant and susceptible strains of mice, also failed to reveal any influence of this additional macrophage-associated, anti-bacterial mechanism on the mycobactericidal effects of ATP [
22]. Thus many of the classical, cell-mediated, antibacterial effector mechanisms of oxygen and nitrogen radical generation, or Nramp protein expression do not appear to be involved in, and hence as effective as induction of autophagy in the ATP-mediated killing of intracellular mycobacteria.
Work is currently underway in our laboratory to further analyze the relative roles played by autophagy and apoptosis and their inter-dependence in the mycobactericidal activity mediated by ATP.
Methods
Reagents
ATP, bzATP, oATP, UTP, wortmannin, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and calcium chloride (CaCl2) were purchased from Sigma (Poole, UK). The rabbit anti-LC3 polyclonal antibody and anti-rabbit secondary antibody were obtained from Novus Biologicals and Pierce Biotechnology respectively. The anti-P2X7 antibody was a kind gift from Dr Gary Buell (Geneva Biomedical Research Institute). Lysotracker Red was purchased from Cambrex Biosciences.
Cells and bacteria
Peripheral blood mononuclear cells (PBMC) were isolated from EDTA blood taken from healthy individuals by standard Ficoll-Hypaque (GE Healthcare Biosciences) gradient separation. Monocytes were then isolated from the PBMC by overnight adherence to 75 ml plastic tissue culture flasks (Corning), washed thrice to remove any non-adherent cells and resuspended by chilling on ice for 30 mins and pipetting with pre-chilled (4°C) PBS. The monocytes were washed and re-suspended (2.5 × 106 cells/ml) in RPMI 1640 medium (Life Technologies, Paisley, U.K.) containing 5% pooled AB+ male human serum (First Link, West Midlands, U.K.), 2 mM L-glutamine (Life Technologies) (complete medium) and rh-GM-CSF (90 IU/ml) (Berlex USA).
THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 medium + 10% fetal calf serum + 2 mM L-glutamine. The cells were used in a non-activated/differentiated state to maintain their early monocytic phenotype.
For experiments using calcium-free media, the cells were grown in standard Hank's Balanced Salt Solution (HBSS) or in calcium free HBSS + 2 mM EGTA. The latter was added to chelate any residual Ca2+.
Stock cultures of M bovis-BCG (Evans strain) were maintained in log phase growth in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with 10% Middlebrook ADC enrichment media (Difco) and 0.02% Tween (Difco) at 37°C. The concentration of the BCG stock was determined by direct counting using a Thoma counting chamber (Weber Scientific UK) under dark ground illumination.
GFP-BCG was obtained as a kind gift from Prof D. Young (Imperial College of London, London, U.K). The BCG contained the gene encoding a FACS-optimized GFP protein [
23] constitutively expressed under the control of the mycobacterial heat shock protein 60 promoter in a pSMT3 shuttle vector construct [
24]. Stock aliquots, stored in glycerol at -70°C, were grown to log phase in 7H9 broth supplemented with 10% ADC enrichment medium, 0.2% Tween 80, and 50 μg/ml hygromycin (Sigma, St. Louis, MO).
Western blot assay
Following exposure for 30 min to 3 mM ATP, THP1 cells and blood-derived monocytes were washed once in PBS and resuspended in RIPA buffer + protease inhibitor (Sigma, St. Louis, MO) and the cell lysate stored at -80°C prior to assay. SDS-PAGE was performed using a 15% discontinuous gel at 120 V. Following electrophoresis the resolved proteins were transferred onto a PVDF membrane at 80 V for 60 minutes, using a Biorad transfer apparatus. The blot was removed from the transfer apparatus and blocked overnight at 4°C in TBS-T, containing 5% nonfat dried milk. The blot was washed thrice in TBS-T following overnight incubation and then probed with a rabbit polyclonal anti-LC3 antibody (diluted 1:1000 in TBS-T/milk) for 60 minutes. After 3 rinses in TBS-T, peroxidase-conjugated anti-rabbit secondary antibody was applied for 1 hour and the excess antibody removed by washing thrice in TBS-T and the reaction developed using Supersignal West Pico ECL reagents.
In experiments incorporating purinergic receptor agonist and antagonists, oATP (0.3 mM) or anti-P2X7 antibody (3 μg/ml) were added to the cells for 2 hr/37°C and 1 hr/37°C respectively, prior to addition of ATP (3 mM) for 30 minutes/37°C. UTP (3 mM), and bzATP (3 mM) were similarly added to cells for 30 minutes/37°C before processing them for western blot analysis for LC3 protein expression.
Confocal fluorescent microscopy
MDMs were grown to sub-confluence on poly-L-lysine coated glass-bottom dishes (MatTek) at approximately 5 × 105 cells/dish for 5 days. GFP-BCG were then added to the macrophages at an MOI of 5:1 and incubated overnight. Following removal of extracellular bacteria by multiple washing, LysoTracker red (75 nM) was added to the cells for 1 hour at 37°C to stain lysosomes. Wortmannin (100 nM) and anti-P2X7 antibody (3 μg/ml) was then added for 1 hr at 37°C before addition of ATP (3 mM) in appropriate wells. Following exposure to ATP for 30 minutes, the cells were washed in RPMI. Cells were imaged live using a Zeiss LSM510 confocal microscope using excitation wavelengths of 488 and 546 nm to visualize GFP and Lysotracker Red respectively. For LC3 staining, MDMs were grown on poly-L-lysine coated glass coverslips for 5 days. Following treatment with ATP, cells were fixed with methanol at -20°C for 30 minutes. Cells were then washed with PBS and incubated in PBS containing 5% donkey serum to block non-specific binding. Cells were incubated with anti- LC3 antibody (1:500) for 1 hour, followed by three washes in PBS and treatment with anti-rabbit secondary antibody, conjugated with AlexaFluor 555 (Invitrogen), for 1 hour. Cells were washed 5 times in PBS and mounted onto slides with Vectashield (Vector Laboratories, CA) as a mounting medium. Fluorescence was visualized by confocal microscopy.
Electron microscopy
Cells were prepared for transmission electron microscopy (TEM) by pelleting the various cell preparations in 1.5 ml eppendorfs following centrifugation for 1 min/6000 rpm in an eppendorf (Hettich) centrifuge. The cell pellets were then fixed in freshly prepared gluteraldhyde fixative and stored at 4°C until ready for processing. TEM processing of the cell samples was performed at the EM microscopy suite, University of Birmingham according to standard fixation protocols.
BCG viability assay
Macrophages were grown to sub-confluence (approx 5 × 104cells/well) in 96-well round-bottom microtiter plates (Corning) at 200 μl/well. A total of 2.5 × 105 BCG per well was added (MOI 5:1) and incubated at 37°C/5%CO2 overnight. Excess BCG was removed by washing thrice in RPMI. Where appropriate, wortmannin (100 nM) and oATP (0.3 mM) were added 1 and 2 hr respectively prior to addition of ATP (3 mM). Intracellular BCG viability following exposure of infected cells to ATP for 30 min, was assessed by 3H-uridine incorporation assay as this provided a much more rapid assessment (within 72 hrs) of the mycobacterstatic effect of ATP as compared to colony forming unit (CFU) assay assessment which takes > 14 days to form countable colonies. The BCG-infected cells were washed and cultured in RPMI +5%AB+ serum and at appropriate time points, a total of 150 μl cell supernatant was removed and transferred to a separate 96 well plate. Cell lysis was performed on the remaining cell pellets by addition of 50 ul of a 0.2% saponin solution for 30 min/37°C. After 30 min, 100 μl of Middlebrook 7H9 (+10% ADC supplement) was added to each well of both the cell supernatant and pellet lysates followed by addition of 20 μl/well of a 100 μCi/ml stock of 3H-uridine (Amersham UK). The wells were harvested after 72 hours on a '1205 betaplate scintillation counter' (Wallac) and the combined counts/min of the matched cell supernatant and pellet wells were taken as an indicator of relative bacterial viability.
Statistics
The Student's t-test was used for all statistical comparisons.
Authors' contributions
DB carried out the majority of the experiments. OSQ performed the confocal microscopy studies, W–YL performed the flow cytometry studies analysing active caspase 3 expression, DAL performed the electron microscopy studies. Experimental design and manuscript preparation were carried out by DB and DAL. All authors have read and approved the manuscript.