Complement and efferocytosis in clearing pyroptotic cells

摘要 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。最直接的方式 对抗这种毒力策略的方法是通过程序性细胞死亡来杀死受感染的细胞。焦亡是一种 由半胱天冬酶-1或-11驱动的gasdermin孔的打开引发的程序性细胞死亡。尽管主机 细胞被杀死，我们已经表明在体内和体外细胞内的细菌存活的过程中的pyroptosis。 然而，我们发现，在pyroptotic细胞内的细菌，而不是分散到细胞内空间， 被困在撕裂但基本完整的质膜中。因为这种诱捕在免疫学上 可用于防止传播，并且由于自燃细胞的结构不同于术语碎片， 我们选择将这种结构命名为“孔诱导的细胞内陷阱”或PIT。 PIT充当补体沉积的病灶，其吸引中性粒细胞至PIT。的 然后嗜中性粒细胞吞噬（死亡细胞的吞噬作用）PIT和被困在其中的细菌。最终它 是嗜中性粒细胞，因此，杀死细胞内的细菌。 鼠伤寒沙门氏菌具有肠毒力因子和细胞内毒力因子。在肠道中， 它表达鞭毛蛋白并利用SPI 1 T3 SS侵入肠上皮细胞。鞭毛蛋白和SPI 1很容易 通过激活半胱天冬酶-1的NLRC 4炎性体检测。然而，在细胞内复制过程中， 在控制全身性疾病的巨噬细胞中，细菌抑制鞭毛蛋白并表达NLRC 4回避蛋白。 SPI2 T3SS。为了在体内研究焦亡，我们对细菌进行了工程改造，使其按需表达鞭毛蛋白。 在目的1中，我们继续使用这种鞭毛蛋白工程化的S。鼠伤寒沙门氏菌研究PIT清除机制 在体外和体内。补体是清除体内PIT及其捕获的细菌所必需的。我们 假设补体激活死细胞的原因是预期它们可能保留 捕获的细胞内细菌。我们研究了由PIT触发的补体起始途径， C5 a和C3 a的重要性，以及补体调理作用对驱动红细胞增多的重要性。 在目标2中，我们回到野生型S。鼠伤寒，询问诱捕概念是否适用于胃肠道 NLRC 4检测到这些细菌的感染阶段。我们假设肠上皮细胞 也会形成陷获细菌的凹坑。我们进一步假设 这种在肠腔中的捕获是重要的，因为它允许浸润的中性粒细胞优先靶向 而不是侵入性细菌。我们假设这是完成的，因为 侵入性细菌被困在脱落的肠上皮细胞内。

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.