Defining the roles of RIPK1 and RIPK3 in emergency hematopoiesis

描述（由申请人提供）：细胞死亡有助于清除感染细胞；它可以由许多刺激触发，这些刺激激活许多生化途径，导致凋亡（免疫“沉默”）以及非凋亡（包括焦亡或caspase -1依赖性，以及坏死亡或RIPK3/ mlkl依赖性）形式的死亡(1)（图1）。最近，我们发现在病毒感染和化疗引起的造血应激中，造血干细胞和祖细胞（HSPC）的焦亡在细胞减少、免疫抑制和骨髓衰竭中的新作用(2)。目前，我们在Cell杂志上发表了初步数据，表明ripk3依赖性坏死细胞死亡限制了致命辐照受体小鼠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是限制HSC“自我更新”的关键生物学过程，并对提高HSC在骨髓、g - csf动员的外周血干细胞、脐带血细胞移植环境中的移植潜力以及基因治疗临床试验具有重要的临床意义。这些数据将改变我们对HSC生物学基础之一的看法：HSC的自我更新以及坏死坏死如何影响终身造血和免疫。我们广泛的初步数据也证实了RIPK1是RIPK3/ mlkl依赖性坏死性坏死的负调节因子。为了进入坏死细胞死亡途径，大多数先前的研究使用caspase抑制剂来阻断caspase -8介导的RIPK3抑制。在这里，我们现在可以在不使用caspase抑制剂治疗原代细胞或小鼠的情况下研究坏死性死亡，预示着一种新的方法来理解坏死性死亡以及这种“非凋亡”细胞死亡途径与刽子手caspase-3等caspase-3可能的相互作用。本应用程序寻求使用这些“坏死性Ripk1-/-嵌合小鼠”特异性研究

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.