Role of lipid mediators in host resistance to Mycobacterium tuberculosis

描述（申请人提供）：结核分枝杆菌（Mtb）生长在人巨噬细胞（主要宿主细胞）中，在那里它占据了一个小生境，使其能够复制而不被宿主识别。减毒的Mtb诱导宿主巨噬细胞的凋亡，导致细菌的遏制和它们被吞噬细胞消除。毒性Mtb的一个关键特性是诱导坏死，其特征在于质膜溶解和杆菌释放到细胞间隙中，导致感染扩散。我们发现，诱导坏死是线粒体的后果!内膜扰动导致线粒体通透性转变。我们进一步检测到，脂质介质前列腺素E2和脂氧素A4诱导和干扰线粒体内膜的扰动，导致细胞凋亡或坏死，分别。PGE2对于启动感染的巨噬细胞中的膜修复机制也是必需的，所述膜修复机制是建立细胞凋亡所需的，所述细胞凋亡在用毒性Mtb感染的巨噬细胞中被抑制。我们最近可以用鼠巨噬细胞显示相同的结果。由于细胞凋亡与抗分枝杆菌活性相关，因此将使用与前列腺素和类花生酸合成相关的基因缺失的小鼠进行体外和体内研究。将在体外和体内实验中使用PGE2产生（前列腺素合酶-/-、COX2-/-、PGE2受体产生（EP 1 - 5））和LXA 4产生（5-LO和15-LO-/-）缺陷的小鼠。我们将在体外研究与坏死相关的线粒体内膜损伤，细胞色素c释放诱导凋亡所必需的，以及在WT和上述各种-/-小鼠模型的感染巨噬细胞中产生凋亡细胞所必需的膜修复机制。这些实验将通过涉及WT和各种脂质介体和EP-/-小鼠的体内研究进行验证，使用新型细菌递送模型来确定细胞凋亡和坏死在针对TB的先天性和克隆防御中的作用，评估肺和脾中的病理学和细菌负荷。本申请中提出的研究将提供有关结核分枝杆菌与宿主巨噬细胞之间相互作用以及结核病早期防御的重要新信息。

DESCRIPTION (provided by applicant): 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.