LC3-Associated Phagocytosis

描述(申请人提供)：两个古老的过程，吞噬和巨自噬，作为满足细胞能量需求的方式出现。两者也都进化成了寄主防御机制。在对这笔赠款的前一次支持期间，我们发现了一种我们称之为“LC3相关吞噬细胞作用”(LAP)的过程。在这个过程中，吞噬细胞吞噬颗粒时产生的信号诱导自噬机制的组件与吞噬小体相关联，促进其与溶酶体的融合(吞噬小体成熟)。虽然乳胶珠(例如)的吞噬不会引起LAP，但与TLR1/2、TLR2/6、TLR4、FCR或吞噬死亡细胞的受体结合的颗粒会导致LC3(ATG8)重新聚集到吞噬体膜上。与巨型自噬一样，这种Lc3关联依赖于Beclin1、PI3P生成、ATG5和ATG7，但与自噬不同的是，Lc3关联于单吞噬体膜(而不是自噬小体的双膜)。此外，与宏自噬不同的是，LAP在缺乏自噬预起始复合体ULK1、ATG13和FIP200元件的情况下继续进行。这提出了一种有趣的可能性：现在已经确定，自噬机制的某些组件中的缺陷会促进炎症性疾病，并损害宿主对细胞内感染的防御。LAP作为一种离散现象的存在表明，至少有一些这样的影响可能与LAP特别相关。在这里，我们建议表征LAP，它与吞噬小体成熟的关系，以及它在先天性免疫反应和正常内稳态中的作用。我们的中心假设是，根据伴随吞噬作用的信号，LAP可以促进吞噬小体货物分选到细胞内，以便进一步检测、处理或降解信号。具体地说，我们会问：1.LAP的启动与宏观自噬有什么区别？在这里，我们将探索启动和传播LAP的分子事件，并评估这些事件与传统的宏观自噬有何不同。2.LAP如何促进吞噬小体成熟？在这里，我们将研究LAP的成分如何极大地加速吞噬小体成熟，以及每条途径中发生这种增强的点。我们将进一步研究LAP诱导的吞噬小体成熟对巨噬细胞介导的宿主防御的影响。3.LAP对炎症和动态平衡有何影响？在这里，我们将使用体外和体内系统来询问LAP在体外和体内对死亡细胞的炎症反应中的作用。虽然凋亡细胞被认为是免疫沉默的，但我们的证据表明，这可能至少部分是由于吞噬细胞中的LAP抑制了炎性细胞因子的反应。我们将测试这一令人兴奋的想法，并探索巨噬细胞和其他隔室中LAP缺陷的长期炎症后果。总体而言，我们的项目试图描述LAP作为两条古老途径的连接点如何影响先天免疫，为理解炎症性疾病提供新的途径。

DESCRIPTION (provided by applicant): Two ancient processes, phagocytosis and macroautophagy, arose as ways to meet the energy demands of the cell. Both also evolved into mechanisms of host defense. During the previous support period for this grant, we discovered a process we term "LC3-Associated Phagocytosis" (LAP). In this process, signals that are generated upon engulfment of particles by phagocytic cells induce components of the autophagy machinery to associate with the phagosome, promoting its fusion to lysosomes (phagosome maturation). While engulfment of latex beads (for example) does not induce LAP, particles that engage TLR1/2, TLR2/6, TLR4, FcR, or receptors for engulfment of dying cells, cause recruitment of LC3 (ATG8) to the phagosome membrane. Like macroautophagy, this LC3 association depends on Beclin1, PI3P generation, ATG5, and ATG7, but unlike autophagy, LC3 associates with the single phagosome membrane (rather then the double membrane of autophagosomes). Further, unlike macroautophagy, LAP proceeds in the absence of elements of the autophagic pre-initiation complex, ULK1, ATG13, and FIP200. This raises an intriguing possibility: It is now well established that defects in some components of the autophagy machinery promote inflammatory disease and compromise host defense to intracellular infections. The existence of LAP as a discrete phenomenon suggests that at least some such effects may specifically relate to LAP. Here, we propose to characterize LAP, its relationship to phagosome maturation, and its roles in innate immune responses and normal homeostasis. Our central hypothesis, upon which this application is based, is that depending on signaling that accompany phagocytosis, LAP can be engaged to promote the sorting of the phagosome cargo to intracellular compartments for further signal detection, processing, or degradation. Specifically, we will ask: 1. What distinguishes the initiation of LAP versus macro-autophagy? Here we will explore the molecular events that initiate and propagate LAP and evaluate how these differ from those of conventional macroautophagy. 2. How does LAP promote phagosome maturation? Here we will investigate how the components of LAP greatly accelerate phagosome maturation and the points in each pathway where this enhancement occurs. We will further examine the consequences of LAP-induced phagosome maturation for macrophage-mediated host defense. 3. How does LAP impact on inflammation and homeostasis? Here we will use in vitro and in vivo systems to interrogate the roles of LAP in the inflammatory response to dying cells, in vitro and in vivo. While apoptotic cells are thought to be "immunologically silent" our evidence suggests that this may be, at least in part, due to suppression of the inflammatory cytokine response by LAP in phagocytes. We will test this exciting idea, and explore the long-term inflammatory consequences of defective LAP in macrophages and other compartments. Overall, our project seeks to characterize how LAP, as the conjunction of two ancient pathways, impacts innate immunity, offering new avenues for understanding inflammatory disease.