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ATP-induced autophagy is associated with rapid killing of intracellular mycobacteria within human monocytes/macrophages.

Biswas D, Qureshi OS, Lee WY, Croudace JE, Mura M, Lammas DA - BMC Immunol. (2008)

Bottom Line: The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes.In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy.Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP).

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunity and Infection, The Medical School, University of Birmingham, B15 2TT, UK. dbiswas71@rediffmail.com

ABSTRACT

Background: We have previously reported that ATP treatment of M bovis-BCG infected human macrophages induces P2X7 receptor-dependent killing of intracellular mycobacteria. The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes. In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy.

Results: We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocyte-derived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP).

Conclusion: We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections.

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ATP-induced autophagy triggers phagosome-lysosome fusion. A. Confocal images of MDMs treated with 3 mM ATP for 30 minutes and immuno stained for intracellular LC3. B. Confocal images of live MDMs infected with GFP-BCG (green) and pre-pulsed with Lysotracker (red) to stain acidic lysosomes. Cells were pre-incubated with anti-P2X7 (3 μg/ml) or wortmannin (100 nM) for 1 hr and then treated with 3 mM ATP for 30 minutes. Note scale bars = 10 um. C. Graph showing the percentage of Lysotracker positive, GFP-BCG containing phagosomes. Histogram shows means ± s.e.m. (n = 25–60 phagosomes). *** = P < 0.01.
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Figure 2: ATP-induced autophagy triggers phagosome-lysosome fusion. A. Confocal images of MDMs treated with 3 mM ATP for 30 minutes and immuno stained for intracellular LC3. B. Confocal images of live MDMs infected with GFP-BCG (green) and pre-pulsed with Lysotracker (red) to stain acidic lysosomes. Cells were pre-incubated with anti-P2X7 (3 μg/ml) or wortmannin (100 nM) for 1 hr and then treated with 3 mM ATP for 30 minutes. Note scale bars = 10 um. C. Graph showing the percentage of Lysotracker positive, GFP-BCG containing phagosomes. Histogram shows means ± s.e.m. (n = 25–60 phagosomes). *** = P < 0.01.

Mentions: Another commonly used assay for monitoring for the occurrence of cell autophagy relies on demonstrating by microscopy the transitional shift of the 18 kDa cytosolic (microtubule-associated) form of LC3 (LC3-1) to its membrane-associated, 16 kDa lipidated form, LC3-II on autophagosomes [9,10]. Using an anti-LC3 antibody, we observed, by confocal microscopy, an increased transition of the diffuse cytosolic staining pattern, typical of LC3-I, (Figure 2A; left panel) to the punctuate staining pattern, characteristic of LC3-II, within ATP-treated MDMs (Figure 2A; right panel).


ATP-induced autophagy is associated with rapid killing of intracellular mycobacteria within human monocytes/macrophages.

Biswas D, Qureshi OS, Lee WY, Croudace JE, Mura M, Lammas DA - BMC Immunol. (2008)

ATP-induced autophagy triggers phagosome-lysosome fusion. A. Confocal images of MDMs treated with 3 mM ATP for 30 minutes and immuno stained for intracellular LC3. B. Confocal images of live MDMs infected with GFP-BCG (green) and pre-pulsed with Lysotracker (red) to stain acidic lysosomes. Cells were pre-incubated with anti-P2X7 (3 μg/ml) or wortmannin (100 nM) for 1 hr and then treated with 3 mM ATP for 30 minutes. Note scale bars = 10 um. C. Graph showing the percentage of Lysotracker positive, GFP-BCG containing phagosomes. Histogram shows means ± s.e.m. (n = 25–60 phagosomes). *** = P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2483257&req=5

Figure 2: ATP-induced autophagy triggers phagosome-lysosome fusion. A. Confocal images of MDMs treated with 3 mM ATP for 30 minutes and immuno stained for intracellular LC3. B. Confocal images of live MDMs infected with GFP-BCG (green) and pre-pulsed with Lysotracker (red) to stain acidic lysosomes. Cells were pre-incubated with anti-P2X7 (3 μg/ml) or wortmannin (100 nM) for 1 hr and then treated with 3 mM ATP for 30 minutes. Note scale bars = 10 um. C. Graph showing the percentage of Lysotracker positive, GFP-BCG containing phagosomes. Histogram shows means ± s.e.m. (n = 25–60 phagosomes). *** = P < 0.01.
Mentions: Another commonly used assay for monitoring for the occurrence of cell autophagy relies on demonstrating by microscopy the transitional shift of the 18 kDa cytosolic (microtubule-associated) form of LC3 (LC3-1) to its membrane-associated, 16 kDa lipidated form, LC3-II on autophagosomes [9,10]. Using an anti-LC3 antibody, we observed, by confocal microscopy, an increased transition of the diffuse cytosolic staining pattern, typical of LC3-I, (Figure 2A; left panel) to the punctuate staining pattern, characteristic of LC3-II, within ATP-treated MDMs (Figure 2A; right panel).

Bottom Line: The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes.In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy.Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP).

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunity and Infection, The Medical School, University of Birmingham, B15 2TT, UK. dbiswas71@rediffmail.com

ABSTRACT

Background: We have previously reported that ATP treatment of M bovis-BCG infected human macrophages induces P2X7 receptor-dependent killing of intracellular mycobacteria. The mechanism mediating this bactericidal effect has not been full characterized but is known to be Ca2+-dependent and to promote the maturation and acidification of mycobacteria-containing phagosomes. In this study we demonstrate that the ATP/P2X7-mediated, mycobactericidal effect also involves the induction of cell autophagy.

Results: We report that 3 mM ATP induces rapid cell autophagy in THP1 cells and monocyte-derived macrophages within 30 minutes post-treatment, as revealed by the expression of LC3-II bands on western blot analysis. Using Ca2+-free media and selective P2X7 agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X7 receptor-dependent. Electron microscopy of ATP-treated, BCG-infected MDMs revealed the presence of the bacteria within characteristic double-membraned autophagosomes. Confocal analysis further confirmed that pharmacological inhibition of autophagy by wortmannin or pre-treatment of macrophages with anti-P2X7 antibody blocked ATP-induced phago-lysosomal fusion. Induction of cell autophagy with ATP was also temporally associated with a fall in intracellular mycobacterial viability, which was suppressed by treatment with wortmannin or the selective P2X7 antagonist, oxidized ATP (oATP).

Conclusion: We provide the first evidence that ATP/P2X7-mediated killing of intracellular mycobacteria involves the induction of cell autophagy. The findings support the hypothesis that autophagy plays a key role in the control of mycobacterial infections.

Show MeSH
Related in: MedlinePlus