LC3相关吞噬作用在真菌感染中的作用机制研究进展

LC3相关的吞噬作用(LC3-associated phagocytosis, LAP)是由LC3及吞噬了病原体的单层膜吞噬泡所介导的巨噬细胞的吞噬作用。近年来越来越多的研究表明,LAP在清除真菌感染过程中具有非常重要的作用,其作用机制不同于经典的细胞吞噬和自噬。该文旨在比较LAP与经典自噬的区别,回顾LAP与真菌感染相关的新近研究进展,总结LAP在真菌感染中的作用。     

细胞自噬与病毒复制

细胞自噬是真核生物中一种高度保守的细胞内容物降解过程,在维持细胞的内环境稳定中起着重要作用。同时,自噬参与固有免疫系统对病原微生物的识别,以帮助吞噬细胞进行有效的吞噬作用并清除细胞内外的病原体。而病毒,尤其是RNA病毒,具有快速进化以应对宿主细胞中的变化的能力,能通过利用或抑制宿主细胞的自噬作用来为自身的复制服务。因此,针对自噬途径的药物筛选和治疗策略越来越成为抗病毒研究的热点。     

细胞自噬与病毒复制

细胞自噬是真核生物中一种高度保守的细胞内容物降解过程,在维持细胞的内环境稳定中起着重要作用。同时,自噬参与固有免疫系统对病原微生物的识别,以帮助吞噬细胞进行有效的吞噬作用并清除细胞内外的病原体。而病毒,尤其是RNA病毒,具有快速进化以应对宿主细胞中的变化的能力,能通过利用或抑制宿主细胞的自噬作用来为自身的复制服务。因此,针对自噬途径的药物筛选和治疗策略越来越成为抗病毒研究的热点。     

细胞自噬和人类疱疹病毒相互作用的研究进展

自噬是普遍存在于真核生物中的自我保护机制之一,在自噬相关蛋白的参与下形成了完整而复杂的吞噬和自身调节机制,使其防御过程有条不紊地进行。除维持细胞内成分更新外,自噬还可选择性识别并清除入侵的病原微生物,如人类疱疹病毒。人类疱疹病毒在全球范围内普遍存在,感染人群广泛。然而,在与宿主长期共存的过程中,人疱疹病毒已经进化出相当强的抗细胞自噬机制来破坏、阻滞宿主的这一清除作用。     

自噬与微生物感染

自噬作为一种维持胞内代谢稳态的机制,与机体微生物感染有着紧密的关系。一方面,自噬能够协助宿主清除病原体;而另一方面也有细菌通过进化,利用自噬体为其增殖提供必要条件,甚至在其中潜伏增殖。同时,病原体也可诱导过度自噬,促进细胞死亡。总之,自噬与微生物感染的关系,可能远比我们知道的复杂。本篇综述即是从自噬分子机制出发,寻找多种病原体与宿主细胞自噬之间的最新进展,深入探讨了自噬之于微生物感染的作用和意义。     

自噬作用:机体抵御病原入侵的第2道防线

机体的固有免疫系统和获得性免疫系统可有效地抵御病原体的入侵,其中固有免疫系统的皮肤和黏膜屏障是机体抵御病原体入侵的第1道防线,由吞噬细胞、NK细胞等介导的吞噬和杀伤作用构成了机体固有免疫的第2道防线。最近自噬体及自噬现象的研究发现,自噬作用参与了机体对病原体入侵的免疫防御过程,是主要的机体抵御病原入侵的第2道防线。     

磷脂酶D与病原微生物感染

磷脂酶D(phospholipase D,PLD)普遍存在于细菌,真菌以及哺乳动物中.在病原微生物中,PLD作为毒力决定因子在减数分裂、孢子形成等过程中起作用;在哺乳动物细胞中,PLD主要在胞膜转运、调节有丝分裂和细胞肌动蛋白骨架等一些信号转导中起作用.在病原菌感染宿主细胞的过程中,病原体和宿主细胞的PLD都被激活并发生级联反应,病原菌PLD可调节自身肌动蛋白丝的聚合和重排,并引起宿主细胞局部肌动蛋白丝的集聚,诱导宿主细胞对其吞噬.深入探讨PLD激活对感染发生的调控作用对透彻理解病原菌感染宿主细胞的分子机制具有重要意义.     

细胞病变型牛病毒性腹泻病毒对宿主细胞自噬作用的研究

细胞自噬(Autophagy)是一种真核生物细胞内损伤的细胞器和长寿命蛋白通过双层膜结构的自噬小泡包裹后,送入溶酶体或液泡中进行降解并得以循环利用的高度保守的分解代谢过程。本研究旨在了解细胞病变型(CEP)牛病毒性腹泻病毒(BVDV NM)对宿主细胞MDBK细胞自噬作用的影响。实验使用BVDV NM株感染宿主细胞MDBK,在不同时间点分别通过透射电镜观察自噬体形成、报告荧光GFP-LC3自噬底物检测以及Western blot方法鉴定细胞自噬标记物LC3-Ⅰ/LC3-Ⅱ和P62的表达等试验方法对自噬进行检测。结果显示,感染BVDV NM株的MDBK细胞出现了明显的细胞病变;透射电镜能观察到细胞中存在大量的双层膜结构的自噬泡;转染GFP-LC3荧光质粒后,在感染病毒的细胞内出现增多的聚集绿色荧光自噬小体;此外,随着BVDV NM株感染MDBK时间的延长可以发现LC3-Ⅰ/Ⅱ蛋白的量逐渐增加以及P62蛋白的降解。试验表明BVDV NM株感染MDBK可以促进细胞自噬的发生。     

鱼类细胞自噬与炎症反应

细胞自噬和炎症反应是先天性免疫的重要组成部分。细胞自噬是通过溶酶体降解自身组分以维持细胞稳态的一种高度保守的代谢过程，在降解受损的细胞器、抵抗病原感染、调节炎症反应等方面具有举足轻重的地位。在过去的几十年里，对酵母和哺乳动物自噬的研究显著增加了人们对自噬及其与人类疾病关系的理解：调节自噬水平可用于预防或治疗神经退行性疾病、炎症性疾病、肿瘤以及各种病原微生物感染。炎症反应是一个高度复杂的生物过程，是机体在受到紫外线、病原体感染、氧化应激以及机械性损伤等刺激下的一种自然防御反应。鱼类作为低等的脊椎动物，其获得性免疫功能较为低下，先天性免疫是其抵御病原体感染的主要防线。相较于高等动物，鱼类细胞自噬研究虽起步较晚，但近些年围绕病原感染引起的自噬现象及机制、自噬相关基因的表达调控等方面已取得较多进展。作为先天免疫的重要组成部分，自噬参与多种鱼类病原感染，而鱼类疾病通常伴随炎症反应的发生。基于此，本文对于鱼类病原感染引起的细胞自噬、炎症反应以及二者之间相关性研究进行系统阐述，以期深入理解鱼类细胞自噬的发生机制及其与炎症反应的相关关系，为全面解析鱼类的免疫机制提供指导，为制定鱼类疾病防控策略提供依据...     

细胞自噬发生的表观遗传调节

细胞自噬是一种进化上保守的, 通过吞噬降解自身大分子物质或细胞器来维持细胞生存的活动。自噬与多种生命活动息息相关, 其功能的紊乱往往会导致肿瘤发生、神经退行性疾病、微生物感染等疾病。研究表明, 表观遗传修饰可以调控细胞自噬的发生, 并在细胞自噬的生物学功能调节过程中发挥重要作用, 但具体调控机制尚需进一步探究。文章综述了细胞自噬发生过程中存在的表观遗传效应, 包括组蛋白乙酰化对细胞自噬激活或抑制的负反馈调控, 通过DNA甲基化调节自噬相关基因活性来影响细胞自噬的发生, miRNA通过靶向调节自噬相关基因表达来影响组蛋白修饰, 从而调控细胞自噬的发生及作用过程等, 旨在为人们进一步研究细胞自噬发生过程中的表观遗传修饰及其机制提供信息依据。     

Safely removing cell debris with LC3‐associated phagocytosis

Phagocytosis and autophagy are two distinct pathways that degrade external and internal unwanted particles. Both pathways lead to lysosomal degradation inside the cell, and over the last decade, the line between them has blurred; autophagy proteins were discovered on phagosomes engulfing foreign bacteria, leading to the proposal of LC3‐associated phagocytosis (LAP). Many proteins involved in macroautophagy are used for phagosome degradation, although Atg8/LC3 family proteins only decorate the outer membrane of LC3‐associated phagosomes, in contrast to both autophagosome membranes. A few proteins distinguish LAP from autophagy, such as components of the autophagy pre‐initiation complex. However, most LAP cargo is wrapped in multiple layers of membranes, making them similar in structure to autophagosomes. Recent evidence suggests that LC3 is important for the degradation of internal membranes, explaining why LC3 would be a vital part of both macroautophagy and LAP. In addition to removing invading pathogens, multicellular organisms also use LAP to degrade cell debris, including cell corpses and photoreceptor outer segments. The post‐mitotic midbody remnant is another cell fragment, which results from each cell division, that was recently added to the growing list of LAP cargoes. Thus, LAP plays an important role during the normal physiology and homoeostasis of animals.     

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.     

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H⁺-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.     

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H⁺-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.     

Melanin targets LC3-associated phagocytosis (LAP): A novel pathogenetic mechanism in fungal disease

Intracellular swelling of conidia of the major human airborne fungal pathogen Aspergillus fumigatus results in surface exposure of immunostimulatory pathogen-associated molecular patterns (PAMPs) and triggers activation of a specialized autophagy pathway called LC3-associated phagocytosis (LAP) to promote fungal killing. We have recently discovered that, apart from PAMPs exposure, cell wall melanin removal during germination of A. fumigatus is a prerequisite for activation of LAP. Importantly, melanin promotes fungal pathogenicity via targeting LAP, as a melanin-deficient A. fumigatus mutant restores its virulence upon conditional inactivation of Atg5 in hematopoietic cells of mice. Mechanistically, fungal cell wall melanin selectively excludes the CYBA/p22phox subunit of NADPH oxidase from the phagosome to inhibit LAP, without interfering with signaling regulating cytokine responses. Notably, inhibition of LAP is a general property of melanin pigments, a finding with broad physiological implications.     

Interactions of pathogenic bacteria with autophagy systems

Autophagy is a conserved cellular degradative pathway that is now established to be a vital part of the host immune response to microbial infection. Autophagy can directly eliminate intracellular pathogens by mediating their delivery to lysosomes. Canonical autophagy is characterized by the formation of a double-membrane autophagosome and the involvement of over 35 autophagy-related proteins (Atgs), including a commonly used autophagosome marker in mammalian cells, LC3. Recent studies have shown that a subset of autophagy components can lead to LC3 conjugation onto phagosomes. This process of LC3-associated phagocytosis (LAP) results in the degradation of the cargo by promoting phagosome fusion with lysosomes. Other components of the autophagy machinery also play roles in immunity that are distinct from the canonical autophagy and LAP pathways. This minireview highlights the complicated relationship between autophagy components and intracellular bacteria, including bacterial targeting mechanisms and the interaction between autophagy and effectors/toxins secreted by bacteria.     

LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to Listeria monocytogenes

The macroautophagic/autophagic machinery cannot only target cell-endogenous components but also intracellular pathogenic bacteria such as Listeria monocytogenes. Listeria are targeted both by canonical autophagy and by a noncanonical form of autophagy referred to as LC3-associated phagocytosis (LAP). The molecular mechanisms involved and whether these processes contribute to anti-listerial immunity or rather provide Listeria with a replicative niche for persistent infection, however, remained unknown. Recently, using an in vivo mouse infection model, we have been able to demonstrate that Listeria in tissue macrophages are targeted exclusively by LAP. Furthermore, our data show that LAP is required for killing of Listeria by macrophages and thereby contributes to anti-listerial immunity of mice, whereas canonical autophagy is completely dispensable. Moreover, we have elucidated the molecular mechanisms that trigger LAP of Listeria and identified the integrin ITGAM-ITGB2/Mac-1/CR3/integrin α_Mß₂ as the receptor that initiates LAP in response to Listeria infection.     

Autophagy and phagocytosis converge for better vision

The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.     

LC3-associated phagocytosis in myeloid cells,a fireman that restrains inflammation and liver fibrosis,via immunoreceptor inhibitory signaling

ABSTRACT

Control of systemic and hepatic inflammation, in particular originating from monocytes/macrophages, is crucial to prevent liver fibrosis and its progression to end-stage cirrhosis. LC3-associated phagocytosis (LAP) is a non-canonical form of autophagy that shifts the monocyte/macrophage phenotype to an anti-inflammatory phenotype. In a recent study, we uncovered LAP as a protective mechanism against inflammation-driven liver fibrosis and systemic inflammation in the context of cirrhosis. We observed that LAP is enhanced in blood and liver monocytes from patients with liver fibrosis or those who progress to cirrhosis. Combining studies in which LAP was pharmacologically or genetically inactivated, we found that LAP limits inflammation in monocytes from cirrhotic patients, and the hepatic inflammatory profile in mice with chronic liver injury, resulting in anti-fibrogenic effects. Mechanistically, LAP-induced anti-inflammatory and antifibrogenic signaling results from enhanced expression of the Fc immunoreceptor FCGR2A/FcγRIIA and activation of an FCGR2A-mediated PTPN6/SHP-1 anti-inflammatory pathway, leading to increased engulfment of IgG into LC3 ⁺ phagosomes. In patients with cirrhosis progressing to multi-organ failure (acute-on chronic liver failure), LAP is lost in monocytes, and can be restored by targeting FCGR2A-mediated PTPN6/SHP-1 signaling. These data suggest that sustaining LAP may open novel therapeutic perspectives for patients with end-stage liver disease.     

Autophagy and phagocytosis converge for better vision

The retinal pigment epithelium (RPE) is a single layer of nonregenerating cells essential to homeostasis in the retina and the preservation of vision. While the RPE perform a number of important functions, 2 essential processes are phagocytosis, which removes the most distal tips of the photoreceptors to support disk renewal, and the visual cycle, which maintains the supply of chromophore for regeneration of photo-bleached visual pigments. We recently reported that these processes are linked by a noncanonical form of autophagy termed LC3-associated phagocytosis (LAP) in which components of the autophagy pathway are co-opted by phagocytosis to recover vitamin A in support of optimal vision. Here we summarize these findings.