Perturbations of host cell signaling by a complex hepatotropic pathogen

摘要 在疟原虫肝脏阶段感染期间，寄生虫通过阻止其 死亡和开发宿主资源以实现增长和发展。这个过程是不完美的，就像一些寄生虫 屈服于宿主防御，并被宿主细胞死亡消除；这反过来又引发免疫反应。这 提案的重点是研究肝细胞中抑制或促进 疟疾寄生虫感染和这些结果的免疫学后果。在通过 蚊子、疟原虫寄生虫由血流携带到肝脏，并侵入单个肝细胞以 形成肝期。在整个肝脏阶段，我们已经证明了细胞凋亡和脂质过氧化都是由 死亡途径可以减少感染。被感染的细胞是存活还是死于宿主细胞 超越个体感染的后果；通过肝细胞清除的寄生虫 死亡可以为随后的免疫反应和免疫的性质提供强有力的刺激。 刺激部分依赖于参与调节的细胞死亡的类型。事实上，我们证明了这种免疫力 细胞亚群，特别是具有与耐受性巨噬细胞一致的标记的免疫细胞，隔离 在肝脏感染期间绕过寄生虫。此外，消除细胞死亡的一种无菌形式的细胞凋亡， 降低对整个寄生虫疫苗接种的免疫力。这项提议旨在检验这样一种假设 多种形式的细胞死亡可以控制疟原虫感染，改变不育和不育之间的平衡 炎症形式的细胞死亡会影响随后对寄生虫感染的免疫力。为了检验这一假设，我们 将广泛使用动物和技术工具。首先，我们将使用具有细胞死亡途径的小鼠 仅在肝细胞中消除，以检查每种类型的细胞死亡在调节野生型 型寄生虫和具有多种生物学缺陷的寄生虫。我们将使用全基因组CRISPR/Cas9 筛选以确定受感染细胞中细胞死亡的特定遗传调节因素，我们将利用蚊子 我们最近设计、制造和测试的解剖机器人，可以执行更大、更公正的分析 比以前可能的要多。我们还将利用最先进的空间剖面图方法 促进原位收集感染疟原虫肝脏的蛋白质组和转录组信息 检查被感染的细胞和周围的免疫环境。除了确定最初的调解人 感染，将使用这些工具来询问改变的肝细胞死亡如何启动CD8 T细胞的反应和通知 对后续感染具有免疫力。实现我们的目标能够改变关键的主机因素，用小的- 可以防止野生型寄生虫进展为有症状的红细胞感染或 以一种能引起对后续挑战的有效免疫反应的方式消除感染。

ABSTRACT During Plasmodium parasite liver stage infection, parasites protect their host hepatocyte by preventing its death and exploiting host resources for growth and development. This process is imperfect, as some parasites succumb to host defenses and are eliminated by host cell death; this, in turn, elicits an immune response. This proposal is focused on the investigation of molecular mechanisms in the hepatocyte that curtail, or promote, malaria parasite infection and the immunological consequences of those outcomes. After transmission by a mosquito, Plasmodium parasites are carried by the blood stream to the liver and invade a single hepatocyte to form a liver stage. Throughout the liver stage we have shown that both apoptosis and lipid peroxide mediated death pathways curtail infection. Whether the infected cell survives or succumbs to host cell death has consequences that expand beyond the individual infection; parasites that are eliminated via hepatocyte cell death can provide potent stimuli for the subsequent immune response and the nature of the immune stimulation is partially dependent on the type of regulated cell death engaged. Indeed, we show that immune cell subsets, in particular immune cells with markers consistent with tolerogenic macrophages, sequester around the parasite during liver infection. Additionally, eliminating apoptosis, a sterile form of cell death, reduces immunity in response to whole parasite vaccination. This proposal aims to test the hypothesis that multiple forms of cell death can control Plasmodium infection and that altering the balance between sterile and inflammatory forms of cell death impacts subsequent immunity to parasite infection. To test this hypothesis, we will make extensive use of animal and technological tools. First, we will use mice with cell death pathways eliminated exclusively in hepatocytes to examine the specific role of each type of cell death in regulating wild type parasites and parasites with multiple biological deficiencies. We will use genome wide CRISPR/Cas9 screens to identify specific genetic regulators of cell death in infected cells and we will make use of a mosquito dissection robot that we have recently designed, built, and tested to perform larger, more unbiased analyses than previously possible. We will also take advantage of state-of-the-art spatial profiling approaches that facilitate the collection of proteomic and transcriptomic information in situ for Plasmodium-infected livers to examine infected cells, and the surrounding immune environment. In addition to identifying mediators of initial infection, will use these tools to ask how altered hepatocyte death primes CD8 T cell responses and informs immunity to subsequent infection. Accomplishing our aims enables altering key host factors with small- molecules that could prevent a wild-type parasite from progressing to symptomatic erythrocytic infection or eliminate infection in a way that elicits a potent immune response against subsequent challenge.