Cytosolic phospholipase A2 participates with TNF-α in the induction of apoptosis of human macrophages infected with Mycobacterium tuberculosis H37Ra

Macrophage (M Phi) apoptosis, an important innate microbial defense mechanism induced by Mycobacterium tuberculosis (Mtb) H37Ra, depends on the induction of TNF-a synthesis. When protein synthesis is blocked, both infection with Mtb and addition of TNF-a are required to induce caspase 9 activation, caspase 3 activation and apoptosis. In this study, we show that the second protein synthesis-independent signal involves activation of group IV cytosolic phospholipase A(2) (cPLA(2)). Apoptosis of Mtb-infected M Phi and concomitant arachidonic acid release are abrogated by group IV cPLA(2) inhibitors (methyl arachidonyl fluorophosphate and methyl trifluoromethyl ketone), but not by inhibitors of group VI Ca2+-independent (iPLA(2); bromoenol lactone) or of secretory low molecular mass PLA(2). In M Phi homogenates, the predominant PLA, activity showed the same inhibitor sensitivity pattern and preferred arachidonic acid over palmitic acid in substrates, also indicating the presence of one or more group IV cPLA(2) enzymes. In concordance with these findings, M Phi lysates contained transcripts and protein for group IV cPLA(2)-alpha and cPLA(2)-gamma. Importantly, group IV cPLA(2) inhibitors significantly reduced M Phi antimycobacterial activity and addition of arachidonic acid, the major product of group IV cPLA(2), to infected M Phi treated with cPLA(2), inhibitors completely restored the antimycobacterial activity. Importantly, addition or arachidonic acid alone to infected M Phi significantly reduced the mycobacterial burden. These findings indicate that Mtb induces M Phi apoptosis by independent signaling through at least two pathways, TNF-a and cPLA(2) which are both also critical for antimycobacterial defense of the M Phi.