Role of lipid mediators in host resistance to Mycobacterium tuberculosis

描述（由申请人提供）：结核分枝杆菌（Mtb）生长在人类巨噬细胞（主要宿主细胞）中，在那里它占据一个生态位，允许它在不被宿主识别的情况下复制。减毒的结核分枝杆菌诱导宿主巨噬细胞凋亡，从而抑制杆菌并使其被吞噬细胞消灭。毒性结核分枝杆菌的一个关键特性是诱导坏死，其特征是质膜溶解和杆菌释放到细胞间隙，导致感染的传播。我们发现，诱导坏死是线粒体的结果！内膜扰动导致线粒体通透性转变。我们进一步发现，脂质介质前列腺素E2和脂素A4分别诱导和干扰线粒体内膜的扰动，导致细胞凋亡或坏死。PGE2在被感染的巨噬细胞中启动膜修复机制也是必不可少的，这是建立细胞凋亡所必需的，而细胞凋亡在被毒性Mtb感染的巨噬细胞中被抑制。我们最近可以用小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，因此将在体外和体内对具有前列腺素和类二十烷合成相关基因缺失的小鼠进行研究。PGE2生成(前列腺素合成酶-/-、COX2 -/-、PGE2受体生成（EP1 - 5）和LXA4生成（5-LO和15- LO-/-）不足的小鼠将用于体外和体内实验。我们将在体外研究与坏死相关的线粒体内膜损伤，诱导凋亡所需的细胞色素c释放，以及在WT感染巨噬细胞和上述各种-/-小鼠模型中产生凋亡细胞所需的膜修复机制。这些实验将与WT和各种脂质介质以及EP - / -小鼠的体内研究并行，使用一种新的细菌传递模型来确定细胞凋亡和坏死在先天和克隆防御TB中的作用，评估肺和脾脏的病理和细菌负荷。本应用程序中提出的研究将为结核分枝杆菌与宿主巨噬细胞之间的相互作用以及结核病的早期防御提供重要的新信息。

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.