Complement and efferocytosis in clearing pyroptotic cells

清除焦亡细胞中的补体和胞吞作用

• 批准号: 10308488
• 负责人: Edward A Miao
• 金额: $ 45.56万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308488
• 关键词: Anaphylatoxins; Apoptosis; Apoptotic; Area; Bacteria; Bacterial Infections; Biological Response Modifiers; CASP1 gene; Cell membrane; Cells; Chemotactic Factors; Chemotaxis; Complement; Complement 3a; Complement 3b; Complement 5a; Consumption; Deposition; Eating; Eicosanoids; Engineering; Epithelial Cells; Flagellin; Flow Cytometry; Grant; Health; Image; Immune; Immunologics; In Vitro; Infection; Infectious Agent; Inflammasome; Innate Immune System; Interleukin-18; Intestines; Intracellular Space; Invaded; Knowledge; Lead; Mediator of activation protein; Membrane; Names; Necrosis; Neutrophilic Infiltrate; Organelles; Pathway interactions; Persons; Phagocytosis; Phase; Physiological; Process; Rupture; SPI1 gene; Salmonella; Salmonella infections; Salmonella typhimurium; Specificity; Structure; Swelling; Systemic disease; Systemic infection; Type III Secretion System Pathway; Virulence; Virulence Factors; cell killing; enteric infection; extracellular; gastrointestinal; gastrointestinal infection; gut bacteria; hazard; in vivo; intestinal epithelium; macrophage; neutrophil; novel; pathogen; prevent; response; sensor; tool

ABSTRACT Many pathogens invade host cells and replicate in the protected intracellular niche. The most direct way to counteract this virulence strategy is to kill the afflicted cell by programmed cell death. Pyroptosis is a form of programmed cell death initiated by caspase-1- or -11-driven opening of the gasdermin pore. Although the host cell is killed, we have shown both in vivo and in vitro the intracellular bacteria survive the process of pyroptosis. However, we found that instead of being dispersed into the intracellular space, bacteria within pyroptotic cells become trapped in the torn but mostly intact plasma membrane. Because this trapping is immunologically useful to prevent dissemination, and because the structure of a pyroptotic cell is different than the term debris, we chose to name this structure as a “pore-induced intracellular trap” or PIT. The PIT serves as a nidus for complement deposition, which attracts neutrophils to the PIT. The neutrophils then efferocytose (phagocytosis of a dead cell) the PIT and the bacteria trapped within. Ultimately it is the neutrophil, therefore, that kills the intracellular bacteria. Salmonella Typhimurium has intestinal virulence factors and intracellular virulence factors. In the intestine, it expresses flagellin and invades intestinal epithelial cells using the SPI1 T3SS. Flagellin and SPI1 are readily detected by the NLRC4 inflammasome that activates caspase-1. However, during intracellular replication in macrophages that dominates systemic disease, the bacteria repress flagellin and express the NLRC4-evasive SPI2 T3SS. In order to study pyroptosis in vivo, we engineered the bacteria to express flagellin on demand. In Aim 1 we continue to use this flagellin engineered S. Typhimurium to study PIT clearance mechanisms in vitro and in vivo. Complement is required for clearance of the PIT and its trapped bacteria in vivo. We hypothesize that the reason that complement activates on dead cells is in anticipation that they may retain trapped intracellular bacteria. We investigate the complement initiation pathways that are triggered by the PIT, the importance of C5a and C3a, and the importance of complement opsonization to drive efferocytosis. In Aim 2 we return to wild type S. Typhimurium, asking if the trapping concepts apply to the gastrointestinal phase of infection where these bacteria are detected by NLRC4. We hypothesize that intestinal epithelial cells that exfoliate in response to bacterial invasion also form PITs that trap the bacteria. We further hypothesize that this trapping in the gut lumen is important because it allows infiltrating neutrophils to preferentially target invasive bacteria instead of commensal luminal bacteria. We hypothesize that this is accomplished because invasive bacteria are trapped within the exfoliated intestinal epithelial cells.

抽象的 许多病原体会侵入宿主细胞并在受保护的细胞内小裂中复制。最直接的方法 抵消这种病毒策略是通过编程细胞死亡杀死受伤的细胞。凋亡是 由caspase-1-或-11驱动的Gasdermin孔启动的程序性细胞死亡。虽然是主人 细胞被杀死，我们在体内和体外表现出细胞内细菌的生存过程。 但是，我们发现，不是被分散到细胞内空间，而是在凋亡细胞内 被困在撕裂但主要是完整的质膜中。因为这种诱捕是免疫学的 对于防止传播有用，并且因为凋亡细胞的结构与术语碎屑不同，所以 我们选择将此结构命名为“孔引起的细胞内陷阱”或坑。 该坑用作完成沉积的Nidus，吸引了中性粒细胞到达坑中。 然后嗜中性粒细胞for吞（死细胞的吞噬作用）和被困在其中的细菌。最终 因此，是杀死细胞内细菌的中性粒细胞。 鼠伤寒沙门氏菌具有肠道病毒因子和细胞内病毒因子。在肠中， 它表达鞭毛蛋白并使用SPI1 T3S侵入肠上皮细胞。鞭毛和SPI1很容易 由激活caspase-1的NLRC4炎性小体检测到。但是，在细胞内复制中 巨噬细胞主导全身性疾病，细菌反映鞭毛蛋白并表达NLRC4-渗透 SPI2 T3SS。为了在体内研究凋亡，我们设计了细菌以按需表达鞭毛蛋白。 在AIM 1中，我们继续使用此鞭毛蛋白工程链球菌来研究坑间隙机制 体外和体内。清除凹坑及其在体内被捕获的细菌需要补体。我们 假设补体在死细胞上激活的原因是预期它们可能会保留 捕获的细胞内细菌。我们研究了由坑触发的完成倡议途径， C5A和C3A的重要性以及完成调整对推动效率的重要性。 在AIM 2中，我们返回野生型S.鼠伤寒S，询问诱捕概念是否适用于胃肠道 NLRC4检测到这些细菌的感染阶段。我们假设肠上皮细胞 响应细菌侵袭的角质也形成了捕获细菌的凹坑。我们进一步假设 肠道中的捕获很重要，因为它允许浸润的中性粒细胞优先靶向 侵入性细菌而不是共生腔细菌。我们假设这是因为 侵入性细菌被困在去角质肠上皮细胞中。