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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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Induction of autophagy in THP1 cells and MDMs, following treatment with ATP, is both calcium and P2X7 – dependent. A. THP1 cells undergo autophagy (lane 1) following 30 minutes exposure to ATP (3 mM), as revealed by the presence of LC3-II (16 kDa) by Western blot. Lane 2 represents untreated control THP-1 cells. B. ATP (3 mM) treated MDMs also express LC3-II (lane 2), which is absent in non-treated control cells (lane 1). C. THP1 cells treated with 3 mM ATP for 30 minutes cultured in calcium-free medium (lane 1) and calcium-replete medium (lane 2). LC3-II expression was observed in ATP-treated cells cultured in calcium-replete medium (lane 2) but was absent in cells cultured in calcium-free media (lane 1). D. THP1 cells were treated with different P2 agonists and antagonists. Lane 1 represents untreated control cells, while lanes 2 and 3 represent cells pre-treated with oATP (0.3 mM) and anti-P2X7 antibody (3 μg/ml) for 2 hours and 1 hour respectively, prior to treatment with ATP (3 mM for 30 minutes). Lanes 4, 5 and 6 represent cells treated with ATP (3 mM), UTP (3 mM) and bzATP (3 mM) for 30 minutes. The presence of an LC3-II band, indicating cell autophagy, was observed only in lanes 4 and 6.
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Figure 1: Induction of autophagy in THP1 cells and MDMs, following treatment with ATP, is both calcium and P2X7 – dependent. A. THP1 cells undergo autophagy (lane 1) following 30 minutes exposure to ATP (3 mM), as revealed by the presence of LC3-II (16 kDa) by Western blot. Lane 2 represents untreated control THP-1 cells. B. ATP (3 mM) treated MDMs also express LC3-II (lane 2), which is absent in non-treated control cells (lane 1). C. THP1 cells treated with 3 mM ATP for 30 minutes cultured in calcium-free medium (lane 1) and calcium-replete medium (lane 2). LC3-II expression was observed in ATP-treated cells cultured in calcium-replete medium (lane 2) but was absent in cells cultured in calcium-free media (lane 1). D. THP1 cells were treated with different P2 agonists and antagonists. Lane 1 represents untreated control cells, while lanes 2 and 3 represent cells pre-treated with oATP (0.3 mM) and anti-P2X7 antibody (3 μg/ml) for 2 hours and 1 hour respectively, prior to treatment with ATP (3 mM for 30 minutes). Lanes 4, 5 and 6 represent cells treated with ATP (3 mM), UTP (3 mM) and bzATP (3 mM) for 30 minutes. The presence of an LC3-II band, indicating cell autophagy, was observed only in lanes 4 and 6.

Mentions: The processing of the 18 kDa microtubule-associated form of LC3 protein (LC3-I) to its 16 kDa, truncated form (LC3-II), is accepted as one of the standard read-outs of cell autophagy [9,10]. In Western blot studies, we observed expression of both LC3-I and LC3-II forms of LC3 protein in ATP-treated THP1 cells (Figure 1A) and MDMs, (Figure 1B) as revealed by bands with electrophoretic mobility corresponding to molecular masses of 18 kDa and 16 kDa respectively. In contrast, only the 18 kDa LC3-I band was expressed in control non-ATP treated THP1 or MDM cells (Figures 1A and 1B).


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)

Induction of autophagy in THP1 cells and MDMs, following treatment with ATP, is both calcium and P2X7 – dependent. A. THP1 cells undergo autophagy (lane 1) following 30 minutes exposure to ATP (3 mM), as revealed by the presence of LC3-II (16 kDa) by Western blot. Lane 2 represents untreated control THP-1 cells. B. ATP (3 mM) treated MDMs also express LC3-II (lane 2), which is absent in non-treated control cells (lane 1). C. THP1 cells treated with 3 mM ATP for 30 minutes cultured in calcium-free medium (lane 1) and calcium-replete medium (lane 2). LC3-II expression was observed in ATP-treated cells cultured in calcium-replete medium (lane 2) but was absent in cells cultured in calcium-free media (lane 1). D. THP1 cells were treated with different P2 agonists and antagonists. Lane 1 represents untreated control cells, while lanes 2 and 3 represent cells pre-treated with oATP (0.3 mM) and anti-P2X7 antibody (3 μg/ml) for 2 hours and 1 hour respectively, prior to treatment with ATP (3 mM for 30 minutes). Lanes 4, 5 and 6 represent cells treated with ATP (3 mM), UTP (3 mM) and bzATP (3 mM) for 30 minutes. The presence of an LC3-II band, indicating cell autophagy, was observed only in lanes 4 and 6.
© Copyright Policy - open-access
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Figure 1: Induction of autophagy in THP1 cells and MDMs, following treatment with ATP, is both calcium and P2X7 – dependent. A. THP1 cells undergo autophagy (lane 1) following 30 minutes exposure to ATP (3 mM), as revealed by the presence of LC3-II (16 kDa) by Western blot. Lane 2 represents untreated control THP-1 cells. B. ATP (3 mM) treated MDMs also express LC3-II (lane 2), which is absent in non-treated control cells (lane 1). C. THP1 cells treated with 3 mM ATP for 30 minutes cultured in calcium-free medium (lane 1) and calcium-replete medium (lane 2). LC3-II expression was observed in ATP-treated cells cultured in calcium-replete medium (lane 2) but was absent in cells cultured in calcium-free media (lane 1). D. THP1 cells were treated with different P2 agonists and antagonists. Lane 1 represents untreated control cells, while lanes 2 and 3 represent cells pre-treated with oATP (0.3 mM) and anti-P2X7 antibody (3 μg/ml) for 2 hours and 1 hour respectively, prior to treatment with ATP (3 mM for 30 minutes). Lanes 4, 5 and 6 represent cells treated with ATP (3 mM), UTP (3 mM) and bzATP (3 mM) for 30 minutes. The presence of an LC3-II band, indicating cell autophagy, was observed only in lanes 4 and 6.
Mentions: The processing of the 18 kDa microtubule-associated form of LC3 protein (LC3-I) to its 16 kDa, truncated form (LC3-II), is accepted as one of the standard read-outs of cell autophagy [9,10]. In Western blot studies, we observed expression of both LC3-I and LC3-II forms of LC3 protein in ATP-treated THP1 cells (Figure 1A) and MDMs, (Figure 1B) as revealed by bands with electrophoretic mobility corresponding to molecular masses of 18 kDa and 16 kDa respectively. In contrast, only the 18 kDa LC3-I band was expressed in control non-ATP treated THP1 or MDM cells (Figures 1A and 1B).

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