Defining the roles of RIPK1 and RIPK3 in emergency hematopoiesis

描述（由申请人提供）：细胞死亡促进感染细胞的清除;它可以由许多刺激物触发，这些刺激物激活导致细胞凋亡（免疫学“沉默”）以及非细胞凋亡（包括细胞凋亡或Caspase-1依赖性和坏死性细胞凋亡或RIPK 3/MLKL依赖性）形式的死亡（1）的许多生化途径（图1）。最近，我们揭示了在病毒感染和化疗诱导的造血应激过程中，造血干细胞和祖细胞（HSPC）中细胞凋亡的新作用，导致血细胞减少、免疫抑制和骨髓衰竭（2）。我们现在提供了目前在Cell出版的初步数据，即RIPK 3依赖性坏死性细胞死亡限制了致死性辐射受体小鼠中LT-HSC的自我更新能力（图2）。这一结果揭示了非凋亡性细胞死亡作为限制HSC“自我更新”的关键生物学过程，并且对于使用骨髓、G-CSF动员的外周血干细胞、脐带血单位在移植环境中改善HSC的植入潜力和用于基因治疗临床试验具有重大临床意义。这些数据将改变我们对HSC生物学基石之一的看法：HSC的自我更新以及坏死性凋亡如何影响终身造血和免疫。我们广泛的初步数据也确立了RIPK 1作为RIPK 3/MLKL依赖性坏死性凋亡的负调节因子。为了获得坏死性细胞死亡途径，大多数先前的研究利用半胱天冬酶抑制剂来阻断半胱天冬酶-8介导的RIPK 3抑制。在这里，我们现在可以研究坏死性凋亡，而不需要用半胱天冬酶抑制剂处理原代细胞或小鼠，这预示着一种新的方法来理解坏死性凋亡以及这种“非凋亡”细胞死亡途径与执行者半胱天冬酶（如半胱天冬酶-3）的可能相互作用。本申请寻求使用这些“坏死性凋亡Ripk 1-/-嵌合小鼠”来具体研究Ripk 1-/-嵌合小鼠的免疫功能。 坏死性凋亡的生理后果及其在造血中通过RIPK 1的负调节。我们将研究以下具体目标，以了解坏死性凋亡如何影响HSPC：目标1。确定RIPK 1和MLKL在HSC自我更新和化疗恢复中的作用。目标2.离体坏死性凋亡的生物化学、形态学和动力学分析。目标3.研究RIPK 1和MLKL在病毒感染HSPC中的作用。

DESCRIPTION (provided by applicant): Cell death facilitates the removal of infected cells; it can be triggered by many stimuli, which activate numerous biochemical pathways that lead to apoptotic (immunologically "silent"), as well as non-apoptotic (including pyroptosis or Caspase-1-dependent, and necroptosis or RIPK3/MLKL-dependent) forms of death (1) (Figure 1). Recently we revealed a new role for pyroptosis in hematopoietic stem and progenitor cells (HSPC) during hematopoietic stress induced by viral infection and chemotherapy causing cytopenia, immunosuppression and bone marrow failure (2). We now present preliminary data, currently in press at Cell, that RIPK3-dependent necroptotic cell death limits the self-renewal capacity of LT-HSC in lethally-irradiated recipient mice (Figure 2). This result unveils non-apoptotic cell death as a key biological process restricting HSC "self-renewal", and has major clinical implications for improving the engraftment potential of HSC in transplantation settings using bone marrow, G-CSF-mobilized peripheral blood stem cells, cord blood units, and for gene therapy clinical trials. These data will change our view of one of the cornerstones of HSC biology: the self-renewal of HSC and how necroptosis impacts lifelong hematopoiesis and immunity. Our extensive preliminary data also establishes RIPK1 as a negative regulator of RIPK3/MLKL-dependent necroptosis. To access necroptotic cell death pathways, most previous studies utilize caspase inhibitors to block Caspase-8-mediated inhibition of RIPK3. Here we can now investigate necroptosis without treating primary cells or mice with caspase inhibitors, heralding a new approach to understanding necroptosis and the possible interaction of this "non-apoptotic" cell death pathway with executioner caspases such as caspase-3. This application seeks to use these "necroptotic Ripk1-/- chimeric mice" to specifically investigate the physiological consequences of necroptosis and its negative regulation by RIPK1 in hematopoieisis. We will investigate the following specific aims to understand how necroptosis affects HSPC: Aim 1. Define the role of RIPK1 and MLKL in the self-renewal of HSC and recovery from chemotherapy. Aim 2. Biochemical, morphological, and kinetic analysis of necroptosis ex vivo. Aim 3. Study the role of RIPK1 and MLKL in viral-infected HSPC.