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: 10223157
• 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/10223157
• 关键词: Allergic; Allergic Disease; Apoptosis; Apoptotic; Autoimmune; Autophagocytosis; Autophagosome; Biology; CASP1 gene; Caspase; Cell Death; Cell membrane; Cells; Cessation of life; Cleaved cell; Cytoplasmic Granules; Cytosol; DNA; Data; Effector Cell; Exocytosis; Extravasation; Family; Feedback; Functional disorder; Homeostasis; Human; Immune; Immune signaling; Immunization; Inflammasome; Inflammatory; Innate Immune Response; Interleukin-1 beta; Leukocyte Elastase; Leukocytes; Lytic; Mediating; Membrane Lipids; Metalloproteases; Molecular Chaperones; Mononuclear; Myelogenous; Natural Immunity; Nonlytic; Nuclear Envelope; Pathway interactions; 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; base; cell type; chronic inflammatory disease; cytokine; eosinophil; extracellular; granulocyte; macrophage; 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 该项目旨在通过以下方式定义调节先天免疫反应的新信号机制 粒细胞白细胞中的 Gasdermin D 和 E (GSDMD/E)，包括中性粒细胞、肥大细胞和 嗜酸性粒细胞。 GSDMD 在炎症小体焦亡和 IL-1β 释放中的生理作用 信号传导已在巨噬细胞和其他单核髓系白细胞中得到广泛表征。 这涉及 caspase-1 裂解 GSDMD 以生成 N-GSDMD 片段，该片段在 质膜 (PM) 形成介导 IL-1β 外流和细胞焦亡的孔。值得注意的是，粒细胞 髓系白细胞也是多种先天免疫反应过程中 IL-1β 的重要来源，但很少有 研究检验了 GSDMD 或其他 GSDM 家族蛋白在这些细胞中的作用。我们的初步数据 显示中性粒细胞中 caspase-1 生成的 N-GSDMD 不会定位于 PM 形成孔或驱动 细胞焦亡，但是 IL-1β 分泌所必需的。 PM 孔的缺失反映了替代的亚细胞 N-GSDMD 与嗜天青分泌颗粒 (AG) 和 LC3+ 自噬体结合的运输。 使用 ATG7 缺陷的中性粒细胞进行的分析表明，IL-1β 通过自噬从中性粒细胞中分泌 机械辅助机构。这些发现揭示了 GSDM 生物学的根本差异，包括 GSDME 在粒细胞和巨噬细胞之间高表达，这将塑造粒细胞在 先天免疫。我们假设定义中性粒细胞、肥大细胞的丰富的分泌颗粒 细胞和嗜酸性粒细胞是粒细胞特异性机制的基础，用于非规范的生产 生物活性 IL-1β 并通过 GSDM 家族加工的替代途径调节细胞死亡 亚细胞贩运。在目标 1 中，我们将根据炎症激活来定义信号传导层次 半胱天冬酶、颗粒源丝氨酸蛋白酶、凋亡半胱天冬酶、GSDMD 和 GSDME 中性粒细胞、肥大细胞和嗜酸性粒细胞产生生物活性 IL-1β 的替代途径。研究将包括 分析 GSDMD/E 向分泌颗粒的运输与质膜的比较以及 GSDMD 的能力 作为自噬蛋白辅助分泌 proIL-1β 的伴侣，proIL-1β 可通过细胞外裂解 丝氨酸蛋白酶。在目标 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、细胞凋亡和进展的触发因素诱导的途径 导致粒细胞衰老。该项目将借鉴其他三个项目的优势，以促进我们的 功能研究。结果将定义 GSDMD 和 GSDME 如何调节的新机制 粒细胞特异性信号反应，在急性感染期间发挥生理作用，但有助于 患有常见自身免疫性疾病、过敏性疾病或慢性炎症性疾病的人类组织功能障碍。