Molecular Mechanism of Eosinophil Cell Death

描述（由申请人提供）：细胞死亡在许多生理过程中是必不可少的，它的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开发新的治疗策略具有重要意义。对于嗜酸性粒细胞尤其如此，其延长的生存和激活或坏死细胞死亡与释放有毒颗粒蛋白导致嗜酸性粒细胞相关疾病的组织炎症。传统上将细胞死亡分为凋亡和坏死；然而，最近的研究表明存在细胞死亡表型的“连续体”，以及新的、独特的细胞死亡过程，如受调节的坏死和在某些情况下的自噬。重要的是，这些亚型受特定生化级联的差异调节；因此，正确识别细胞死亡表型可能具有重要的治疗意义，因为细胞可能是诱导或抑制特定细胞死亡模式的治疗方案的目标。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结扎同时伴有caspase抑制）。同样，我们观察到嗜酸性粒细胞同时接受生存因子（如IL-5、酸度）和细胞死亡诱导剂（抗fas、抗siglece -8）处理时，细胞死亡的矛盾增强。细胞死亡的“方式”明显；抗siglece -8诱导caspase依赖性细胞凋亡，而il -5处理的嗜酸性粒细胞的抗siglece -8诱导caspase非依赖性细胞死亡。从概念上讲，这些发现与在组织微环境中，嗜酸性粒细胞同时暴露于多种信号，包括促生存和促细胞死亡信号的观点是一致的。事实上，在嗜酸性炎症性疾病患者的组织样本中，大部分嗜酸性粒细胞表现出细胞溶解或坏死的超微结构特征。然而，嗜酸性粒细胞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，并作为最终将这些知识转化为临床环境的平台。我们的中心假设是嗜酸性粒细胞经历了受调节的坏死，这是一个可靶向的过程，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标来验证这一假设：1)定义人类嗜酸性细胞死亡表型的谱，以及2)确定嗜酸性细胞调节坏死的病理生理后果。我们将在原发性人类嗜酸性粒细胞、嗜酸性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证明，即调节性坏死发生在嗜酸性粒细胞中（目标1），并且在疾病中具有重要意义（目标2），这将为机械性r01级资助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.