Molecular mechanisms underlying the promotion of antibacterial immunity by LC3-associated phagocytosis

中性粒细胞和巨噬细胞吞噬入侵细菌是抗菌免疫的重要组成部分。在吞噬小体中，细菌被灭活、杀死和降解。然而，一些病原体，如结核分枝杆菌或单核细胞增生性李斯特菌，已经进化出阻止它们在传统吞噬体内被杀死的机制。我们最近发现了一种名为LC3相关吞噬细胞作用(LAP)的非规范自噬途径，它允许巨噬细胞有效地杀死吞噬的单核细胞增多性乳杆菌(Gluschko等人，Cell host&Microbe 2018)。LAP对消除其他微生物病原体也是至关重要的，如结核分枝杆菌或烟曲霉，这表明它通常代表着一种特别的杀菌途径。在LAP过程中，吞噬小体被LC3蛋白修饰，从而加强了与溶酶体的融合，从而杀灭了吞噬细胞中的病原体。然而，只有一小部分含有病原体的吞噬小体被LAP靶向并用LC3装饰。因此，LAP的高抗菌潜力没有得到充分利用。因此，基于提高LAP对吞噬的病原体的靶向性的治疗方法将有可能显著增强细胞介导的抗菌防御。本项目的目的是进一步阐明LAP诱导的分子机制，从而确定利用这种细胞机制的优越杀菌活性来治疗微生物感染的潜在起点。我们的初步数据表明，促进LAP可能是一种有前景的增强细胞介导的抗菌防御的策略，LAP对单核细胞的靶向和杀灭显著增加了促炎细胞因子肿瘤坏死因子(TNF)的作用。因此，肿瘤坏死因子是LAP的内源性促进剂，从而起到抗菌防御作用。这个项目的第一个具体目标是破译肿瘤坏死因子促进LAP诱导的分子机制。关于LAP的诱导，我们和其他人已经证明，NADPH氧化酶NOX2产生的活性氧物种(ROS)是至关重要的。除了众所周知的直接杀灭吞噬病原体的功能外，ROS还发挥着调节功能。然而，ROS如何通过LAP诱导LC3募集到吞噬小体仍然是完全不清楚的。因此，本项目的第二个具体目标是揭开ROS诱导LAP的分子机制。本项目对巨噬细胞如何利用LAP的特殊杀菌机制所获得的见解将极大地加深我们对细胞介导的抗微生物免疫的理解，并确定基于增加LAP对吞噬细胞病原体的靶向的新治疗方法的潜在起点。

Phagocytosis of invading bacteria by neutrophils and macrophages is a key component of antibacterial immunity. In the phagosome, the bacteria are inactivated, killed and degraded. Some pathogens, such as Mycobacterium tuberculosis or Listeria monocytogenes, however, have evolved mechanisms that prevent their killing in conventional phagosomes. We have recently identified a non-canonical autophagy pathway called LC3-associated phagocytosis (LAP) that allows macrophages to effectively kill phagocytosed L. monocytogenes (Gluschko et al., Cell Host & Microbe 2018). LAP also is crucial for elimination of other microbial pathogens, such as M. tuberculosis or Aspergillus fumigatus, indicating that it generally represents a particularly microbicidal pathway. During LAP, phagosomes are decorated by the protein LC3, which enhances the fusion with lysosomes and thus the killing of the phagocytosed pathogen. However, only a subpopulation of pathogen-containing phagosomes is targeted by LAP and decorated with LC3. Hence, the high antimicrobial potential of LAP is not fully retrieved. Therapeutic approaches based on increasing the targeting of phagocytosed pathogens by LAP would therefore potentially markedly enhance cell-mediated antibacterial defense.The objective of this project is to further elucidate the molecular mechanisms of LAP induction and thereby identify potential starting-points for using the superior microbicidal activity of this cellular mechanism in the treatment of microbial infections.Illustrating that promoting LAP may be a promising strategy for enhancing cell-mediated antibacterial defense, our preliminary data show that the targeting and killing of L. monocytogenes by LAP is substantially increased by the proinflammatory cytokine tumor necrosis factor (TNF). Thus, TNF is an endogenous promotor of LAP and, thereby, antibacterial defense. The first specific aim of this project is to decipher the molecular mechanisms through which TNF promotes LAP induction.Regarding the induction of LAP, we and others have shown that reactive oxygen species (ROS) produced by the NADPH oxidase Nox2 are critically required. In addition to their well-known direct function in killing of phagocytosed pathogens, ROS also exert regulatory functions. How the ROS induce the recruitment of LC3 to phagosomes by LAP, however, remains completely elusive. The second specific aim of this project therefore is to unravel the molecular mechanism through which ROS induce LAP.The insights gained by this project into how macrophages bring to use the particularly microbicidal mechanism of LAP will substantially further our understanding of cell-mediated antimicrobial immunity and identify potential starting-points for novel therapeutic approaches based on increasing the targeting of phagocytosed pathogens by LAP.