Role of lipid mediators in host resistance to Mycobacterium tuberculosis

描述(申请人提供)：结核分枝杆菌(Mtb)生长在人类巨噬细胞中，这是主要的宿主细胞，在那里它占据了一个利基，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可诱导宿主巨噬细胞的凋亡，从而抑制细菌并使其被吞噬细胞清除。强毒力结核分枝杆菌的一个关键特征是诱导坏死，其特征是质膜溶解并将细菌释放到细胞间隙，导致感染的传播。我们发现，导致坏死是线粒体的结果！内膜扰动导致线粒体通透性转变。我们进一步检测到，脂质介质前列腺素E_2和脂氧素A_4分别诱导和干扰线粒体内膜的扰动，导致细胞凋亡或坏死。在感染的巨噬细胞中，PGE2也是启动膜修复机制所必需的，这是建立凋亡所必需的，而在感染毒力Mtb的巨噬细胞中，凋亡是被抑制的。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有关，因此将使用与前列腺素和二十烷类化合物合成相关的基因缺失的小鼠进行体外和体内研究。PGE2产生(前列腺素合成酶-/-，COX2-/-，PGE2受体产生(EP1-5)和LXA4产生(5-LO和15-LO-/-))缺陷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，诱导细胞凋亡所必需的细胞色素c的释放，以及在感染WT和上述各种-/-小鼠模型的巨噬细胞中产生凋亡细胞所必需的膜修复机制。这些实验将与体内研究平行，涉及WT和各种脂质介体以及EP-/-小鼠，使用一种新的细菌传递模型，以确定细胞凋亡和坏死在抗结核病的天然和克隆防御中的作用，评估肺和脾的病理和细菌负荷。这项申请中提出的研究将提供关于结核分枝杆菌和宿主巨噬细胞之间相互作用的关键新信息，以及关于结核病的早期防御。

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.