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


CONCLUSIONS
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.
Biswas
Debasis
D
Division of Immunity and Infection, The Medical School, University of Birmingham, B15 2TT, UK. dbiswas71@rediffmail.com
Qureshi
Omar S
OS
Lee
Wing-Yiu
WY
Croudace
Joanne E
JE
Mura
Manuela
M
Lammas
David A
DA
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001343:Autophagy; D002118:Calcium / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000502:physiology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000382:microbiology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D009000:Monocytes / Q000187:drug effects / Q000276:immunology* / Q000382:microbiology; D009163:Mycobacterium bovis / Q000187:drug effects / Q000276:immunology*; D010588:Phagosomes / Q000382:microbiology / Q000502:physiology / Q000648:ultrastructure; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7
C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D002118:Calcium