Alternative pathways of gasdermin function and IL-1beta secretion in granulocytic leukocytes

粒细胞白细胞中 Gasdermin 功能和 IL-1β 分泌的替代途径

• 批准号: 10654570
• 负责人: GEORGE R DUBYAK
• 金额: $ 41万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-24 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654570
• 关键词: Allergic; Allergic Disease; Apoptosis; Apoptotic; Autoimmune; Autophagocytosis; Autophagosome; Biology; CASP1 gene; Caspase; Cell Death; Cell membrane; Cells; Cessation of life; Cytoplasmic Granules; Cytosol; DNA; Data; Effector Cell; Exocytosis; Extravasation; Family; Feedback; Functional disorder; Homeostasis; Human; Immune; Immune signaling; Immunologic Stimulation; Inflammasome; Inflammatory; Innate Immune Response; Interleukin-1 beta; Leukocyte Elastase; Leukocytes; Lytic; Macrophage; Mediating; Membrane Lipids; Metalloproteases; Molecular Chaperones; Mononuclear; Myelogenous; Natural Immunity; Nonlytic; Nuclear Envelope; Pathway interactions; Permeability; Physiological; Play; Process; Production; Property; Protease Inhibitor; Protein Family; Proteins; Regulation; Reporting; Role; Secondary to; Secretory Vesicles; Serine Protease; Serpins; Shapes; Signal Transduction; Signaling Protein; Source; Stimulus; Testing; Tissues; acute infection; cell type; chronic inflammatory disease; cytokine; eosinophil; extracellular; granulocyte; mast cell; neutrophil; novel; public health relevance; response; senescence; trafficking

Abstract This project aims to define novel signaling mechanisms for the regulation of innate immune responses by Gasdermins D and E (GSDMD/E) in granulocytic leukocytes that include neutrophils, mast cells, and eosinophils. Physiological roles for GSDMD in both pyroptosis and IL-1β release during inflammasome signaling have been extensively characterized in macrophages and other mononuclear myeloid leukocytes. This involves cleavage of GSDMD by caspase-1 to generate N-GSDMD fragments which oligomerize in the plasma membrane (PM) to form pores that mediate IL-1β efflux and pyroptosis. Notably, the granulocytic myeloid leukocytes are also important sources of IL-1β during multiple innate immune responses, but few studies have examined roles for GSDMD or other GSDM-family proteins in these cells. Our preliminary data shows that caspase-1-generated N-GSDMD in neutrophils does not localize to the PM to form pores or drive pyroptosis but is required for IL-1β secretion. This absence of PM pores reflects alternative subcellular trafficking whereby N-GSDMD associates with azurophilic secretory granules (AG) and LC3+ autophagosomes. Analyses using ATG7-deficient neutrophils indicate that IL-1β is secreted from neutrophils via an autophagy machinery-assisted mechanism. These findings reveal fundamental differences in GSDM biology, including high expression of GSDME, between granulocytes and macrophages that will shape granulocyte roles in innate immunity. We hypothesize that the abundant secretory granules which define neutrophils, mast cells and eosinophils underlie granulocyte-specific mechanisms for non-canonical production of bioactive IL-1β and for regulated cell death via alternative pathways of GSDM family processing and subcellular trafficking. In Aim 1, we will define signaling hierarchies based on activation of inflammatory caspases, granule-derived serine proteases, apoptotic caspases, GSDMD, and GSDME that facilitate alternative paths of bioactive IL-1β production by neutrophils, mast cells, and eosinophils. Studies will include analyses of GSDMD/E trafficking to secretory granules versus the plasma membrane and GSDMD's ability to act as a chaperone for autophagy protein-assisted secretion of proIL-1β that can be extracellularly cleaved by serine proteases. In Aim 2, we will define GSDMD/E signaling networks that regulate granulocyte death pathways which are induced by triggers of extracellular DNA trap release/ETosis, apoptosis, and progression to granulocyte senescence. The project will draw on the strengths of the other three projects to facilitate our functional studies. The results will define novel mechanisms for how GSDMD and GSDME mediate granulocyte-specific signaling responses which play physiological roles during acute infection but contribute to tissue dysfunction in humans with common autoimmune, allergic, or chronic inflammatory diseases.

抽象的 该项目旨在定义新的信号传导机制，以调节先天免疫反应 粒细胞白细胞中的Gasdermins D和E（GSDMD/E），包括中性粒细胞，肥大细胞和 嗜酸性粒细胞。 GSDMD在炎性体过程中的生理作用和IL-1β释放 信号传导在巨噬细胞和其他单核髓粒白细胞中得到了广泛的特征。 这涉及caspase-1对GSDMD的裂解，以产生N-GSDMD片段，这些片段在 质膜（PM）形成介导IL-1β外排和凋亡的孔。值得注意的是，粒细胞 髓样白细胞也是多种先天免疫反应期间IL-1β的重要来源，但很少 研究检查了这些细胞中GSDMD或其他GSDM家庭蛋白的作用。我们的初步数据 表明中性粒细胞中的caspase-1生成的N-GSDMD不会定位于PM以形成毛孔或驱动 凋亡是IL-1β分泌所必需的。不存在PM孔反映了替代的亚细胞 N-GSDMD与Azurophilic分泌颗粒（AG）和LC3+自噬体相关联。 使用ATG7缺乏性嗜中性粒细胞的分析表明，IL-1β通过自噬从中性粒细胞分泌 机械辅助机制。这些发现揭示了GSDM生物学的根本差异，包括 GSDME的高表达，在粒细胞和巨噬细胞之间，将塑造粒细胞在 先天免疫。我们假设定义中性粒细胞的丰富秘密颗粒，桅杆 细胞和嗜酸性粒细胞是粒细胞特异性机制，用于非规范生产 生物活性IL-1β和通过GSDM家族加工和 亚细胞贩运。在AIM 1中，我们将根据炎症激活定义信号层次结构 胱天蛋白酶，颗粒衍生的序列蛋白酶，凋亡胱天蛋白酶，GSDMD和GSDME 嗜中性粒细胞，肥大细胞和嗜酸性粒细胞生物活性IL-1β产生的替代路径。研究将包括 GSDMD/E运输分泌颗粒的分析与质膜和GSDMD的能力 充当proil-1β的自噬蛋白辅助分泌的伴侣，可以细胞外裂解 连续蛋白酶。在AIM 2中，我们将定义调节粒细胞死亡的GSDMD/E信号网络 由细胞外DNA陷阱释放/Etosis，凋亡和进展引起的诱导的途径 至粒细胞感应。该项目将利用其他三个项目的优势来促进我们 功能研究。结果将定义GSDMD和GSDME如何介导的新机制 粒细胞特异性信号反应在急性感染期间起身体作用，但有助于 患有常见自身免疫，过敏或慢性炎症性疾病的人类组织功能障碍。