Mycobacterium tuberculosis (Mtb) grows in human macrophages, the principal host cell, where it occupies a niche allowing it to replicate without being recognized by the host. Attenuated Mtb induce apoptosis of the host macrophages resulting in containment of the bacilli and to their elimination by phagocytes. A critical property of virulent Mtb is the induction of necrosis characterized by plasma membrane lysis and release of the bacilli into the intercellular space leading to spread of the infection. We found that induction of necrosis is the consequence of mitochondria! inner membrane perturbation leading to mitochondrial permeability transition. We detected further that the lipid mediators prostaglandin E2 and lipoxin A4 induce and interfere with the perturbation of the mitochondrial inner membrane resulting either in apoptosis or necrosis, respectively. PGE2 is also essential for the initiation of membrane repair mechanisms in the infected macrophages required for the establishment of apoptosis which is suppressed in macrophages infected with virulent Mtb. We could recently show identical results using murine macrophages. Because apoptosis is associated with anti-mycobacterial activity, in vitro and in vivo studies using mice with gene deletions relating to prostanoid and eicosanoid synthesis will be performed. Mice deficient in PGE2 production (prostaglandin synthase -/-, COX2 -/-, PGE2 receptor production (EP1 - 5), and in the production of LXA4 (5-LO and 15- LO-/-) will be used in the in vitro and in vivo experiments. We will investigate in vitro mitochondrial inner membrane damage related to necrosis, cytochrome c release necessary for the induction of apoptosis, and membrane repair mechanisms necessary for the generation of apoptotic cells in infected macrophages of WT and of the various -/- murine models mentioned above. These experiments will be paralleled by in vivo studies involving WT and the various lipid mediator and EP - / - mice using a novel model of bacterial delivery to pinpoint the role of apoptosis and necrosis in innate and clonal defenses against TB assessing pathology and bacterial burden in lung and spleen. The studies proposed in this application will provide critical new information about the interactions between Mycobacterium tuberculosis and the host macrophage, and about the early defense against TB.
