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 P2X(7) 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/P2X(7)-mediated, mycobactericidal effect also involves the induction of cell autophagy. Results: We report that 3 mM ATP induces rapid cell autophagy in THPI 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 P2X(7) agonists and antagonists, ATP-induced cell autophagy was shown to be Ca2+ and P2X(7) 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-P2X(7) 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 P2X(7) antagonist, oxidized ATP (oATP). Conclusion: We provide the first evidence that ATP/P2X(7)-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.