Complement and efferocytosis in clearing pyroptotic cells

摘要 许多病原体入侵宿主细胞，并在受保护的细胞内生态位中复制。最直接的方法是 对抗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。上睑下垂是一种 细胞程序性死亡由caspase-1或-11驱动的Gasdermin孔打开而启动。虽然主持人 细胞被杀死，我们已经在体内和体外证明了细胞内的细菌在松垂的过程中存活下来。 然而，我们发现，细菌不是分散到细胞内空间，而是在嗜热细胞内 被困在撕裂但基本完好的质膜中。因为这种诱捕是免疫的 有助于防止扩散，而且由于焦化细胞的结构不同于术语碎片， 我们选择将这种结构命名为“孔洞诱导的细胞内陷阱”或坑。 小窝是补体沉积的核心，它将中性粒细胞吸引到小窝。这个 然后，中性粒细胞吞噬(吞噬死亡细胞)进入坑内，细菌被困在坑内。最终，它 因此，是中性粒细胞杀死了细胞内的细菌。 鼠伤寒沙门氏菌具有肠道毒力因子和细胞内毒力因子。在肠道中， 它表达鞭毛蛋白，并通过SPI1 T3SS侵袭肠上皮细胞。鞭毛蛋白和SPI1很容易 被激活caspase-1的NLRC4炎症体检测到。然而，在细胞内复制期间， 主宰全身疾病的巨噬细胞，细菌抑制鞭毛蛋白并表达NLRC4逃逸 SPI2 T3SS。为了研究活体上睑下垂，我们设计了按需表达鞭毛蛋白的细菌。 在目标1中，我们继续使用这种鞭毛蛋白工程的鼠伤寒沙门氏菌来研究排坑机制。 在体外和体内。补体是清除坑和体内捕获的细菌所必需的。我们 假设补体在死亡细胞上激活的原因是预期它们可能会保留 捕获了细胞内的细菌。我们研究了由PIT触发的补体起始通路， C5a和C3a的重要性，以及补体调理对驱动胞吐的重要性。 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。我们假设肠上皮细胞 对细菌入侵作出反应而脱落的细菌也会形成坑来捕获细菌。我们进一步假设 这种在肠腔中的捕获很重要，因为它允许渗入的中性粒细胞优先靶向 入侵细菌而不是共生的管腔细菌。我们假设这是完成的，因为 侵袭性细菌被困在脱落的肠道上皮细胞中。

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.