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 许多病原体侵入宿主细胞并在受保护的细胞内生态位中复制。最直接的方法是 抵消这种毒力的策略是通过程序性细胞死亡来杀死受影响的细胞。焦亡是一种形式 由 caspase-1 或 -11 驱动的 Gasdermin 孔打开引发的程序性细胞死亡。虽然主持人 细胞被杀死后，我们在体内和体外都证明了细胞内细菌在焦亡过程中存活下来。 然而，我们发现焦亡细胞内的细菌并没有分散到细胞内空间，而是 被困在撕裂但大部分完整的质膜中。因为这种捕获是免疫学上的 对于防止传播很有用，并且因为焦亡细胞的结构与术语碎片不同， 我们选择将这种结构命名为“孔诱导的细胞内陷阱”或 PIT。 PIT 作为补体沉积的巢穴，吸引中性粒细胞到 PIT。这 然后中性粒细胞会吞噬（吞噬死细胞）PIT 和其中捕获的细菌。最终它 因此，杀死细胞内细菌的是中性粒细胞。 鼠伤寒沙门氏菌具有肠道毒力因子和细胞内毒力因子。在肠道内， 它表达鞭毛蛋白并使用 SPI1 T3SS 侵入肠上皮细胞。鞭毛蛋白和 SPI1 很容易 由激活 caspase-1 的 NLRC4 炎症小体检测到。然而，在细胞内复制过程中 巨噬细胞主导全身性疾病，细菌抑制鞭毛蛋白并表达 NLRC4 逃避性 SPI2 T3SS。为了研究体内焦亡，我们对细菌进行了改造，使其能够按需表达鞭毛蛋白。 在目标 1 中，我们继续使用这种鞭毛蛋白改造的鼠伤寒沙门氏菌来研究 PIT 清除机制 体外和体内。体内清除 PIT 及其捕获的细菌需要补体。我们 假设补体在死亡细胞上激活的原因是预期它们可能保留 捕获细胞内细菌。我们研究由 PIT 触发的补体启动途径， C5a 和 C3a 的重要性，以及补体调理作用对驱动胞吞作用的重要性。 在目标 2 中，我们回到野生型鼠伤寒沙门氏菌，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。我们推测肠上皮细胞 因细菌入侵而剥落的细胞也会形成 PIT 来捕获细菌。我们进一步假设 这种在肠腔中的捕获很重要，因为它允许浸润的中性粒细胞优先瞄准 入侵细菌而不是共生腔细菌。我们假设这是因为 侵入性细菌被困在脱落的肠上皮细胞内。