Molecular Mechanism of Eosinophil Cell Death

描述（由申请人提供）：细胞死亡是许多生理过程所必需的，其失调是许多人类疾病的特征。因此，深入研究细胞死亡及其机制对于开发新的治疗策略具有巨大的意义。这对于嗜酸性粒细胞尤其如此，其延长的存活和活化或坏死细胞死亡伴随毒性颗粒蛋白的释放导致嗜酸性粒细胞相关疾病中的组织炎症。经典地，细胞死亡被二分为凋亡和坏死;然而，最近的研究表明存在细胞死亡表型的“连续体”，以及新颖的、不同的细胞死亡过程，如调节性坏死和在某些情况下的自噬。重要的是，这些亚型由特定的生化级联差异调节;因此，细胞死亡表型的正确鉴定可能具有重要的治疗意义，因为细胞可以通过诱导或抑制特定细胞死亡模式的方案靶向。 调节性坏死通常发生在细胞接收细胞死亡信号但细胞凋亡被抑制的情况下（例如Fas连接与半胱天冬酶抑制同时发生）。类似地，我们观察到同时用存活因子（例如IL-5、酸度）和细胞死亡诱导剂（抗Fas、抗Siglec-8）处理的嗜酸性粒细胞中细胞死亡的矛盾增强。此外，细胞死亡的“模式”是不同的;抗Siglec-8诱导半胱天冬酶依赖性细胞凋亡，而IL-5处理的嗜酸性粒细胞中的抗Siglec-8引起半胱天冬酶非依赖性细胞死亡。从概念上讲，这些发现与以下观点一致：在组织微环境中，嗜酸性粒细胞同时暴露于多种信号，包括促存活和促细胞死亡信号。事实上，在从嗜酸性粒细胞炎性疾病患者收集的组织样本中，大部分嗜酸性粒细胞显示细胞溶解或坏死的超微结构特征。然而，在嗜酸性粒细胞中诱导的细胞死亡表型谱和导致这些细胞死亡模式的生化机制尚不清楚。本资助申请中提出的研究旨在解决这一知识差距，并作为将这一知识最终转化为临床环境的平台。我们的中心假设是，嗜酸性粒细胞进行调节性坏死，一个有针对性的过程，这在嗜酸性粒细胞相关疾病中具有重要的病理生理意义。我们提出了两个具体的目的来验证这一假设：1）确定人类嗜酸性粒细胞死亡表型的谱，2）确定嗜酸性粒细胞调节性坏死的病理生理学后果。我们将在原发性人类嗜酸性粒细胞，嗜酸性粒细胞疾病患者的活检和动物模型中使用创新方法。我们的研究将提供概念验证，即调节性坏死发生在嗜酸性粒细胞中（目的1），并且在疾病中具有重要意义（目的2），这将为机制R 01级拨款申请提供关键的初步数据。

DESCRIPTION (provided by applicant): Cell death is essential for many physiological processes, and its deregulation characterizes numerous human diseases. Thus, in-depth investigation of cell death and its mechanism has tremendous implications for the development of novel therapeutic strategies. This is especially true for eosinophils, whose extended survival and activation or necrotic cell death with release of toxic granule proteins lead to tissue inflammation in eosinophil-associated diseases. Classically, cell death was divided dichotomously into apoptotic and necrotic; however, recent studies have suggested the existence of a "continuum" of cell death phenotypes, as well as novel, distinct cell death processes such as regulated necrosis and, in certain situations, autophagy. Importantly, these subtypes are differentially regulated by specific biochemical cascades; thus, the correct identification of cell death phenotype may have important therapeutic implications, as cells may be targetable by regimens that induce or inhibit a specific mode of cell death. Regulated necrosis commonly occurs in situations in which cells receive a cell death signal but apoptosis is inhibited (e.g. Fas ligation concurrent with caspase inhibition). Similarly, we observed the paradoxical enhancement of cell death in eosinophils simultaneously treated with survival factors (e.g. IL-5, acidity) and cell death-inducing agents (anti-Fas, anti-Siglec-8). Moreover, th "mode" of cell death was distinct; anti-Siglec-8 induced caspase-dependent apoptosis whereas anti-Siglec-8 in IL-5-treated eosinophils caused caspase- independent cell death. Conceptually, these findings are consistent with the notion that in the tissue microenvironment, eosinophils are exposed to multiple signals simultaneously, including pro-survival and pro- cell death signals. Indeed, in tissue samples collected from patients with eosinophilic inflammatory disease, a large portion of eosinophils display ultrastructural characteristics of cytolysis or necrosis. However, the spectrum of cell death phenotypes induced in eosinophils and the biochemical mechanisms leading to these modes of cell death are not known. The studies proposed in this grant application aim to address this gap in knowledge and to serve as a platform for the eventual translation of this knowledge to clinical settings. Our central hypothesis is that eosinophils undergo regulated necrosis, a targetable process, which has important pathophysiological implications in eosinophil-associated disease. We propose two specific aims to test this hypothesis: 1) to define the spectrum of human eosinophil cell death phenotypes, and 2) to determine the pathophysiological consequences of regulated necrosis of eosinophils. We will use innovative approaches in primary human eosinophils, biopsies from patients with eosinophilic disease, and animal models. Our studies will provide proof-of-concept that regulated necrosis occurs in eosinophils (aim 1) and that it is significant in disease (aim 2), which will provide critical preliminary data for a mechanistic R01-level grant application.