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

摘要 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 Gasdermins D和E(GSDMD/E)在粒细胞中的表达，包括中性粒细胞、肥大细胞和 嗜酸性粒细胞。GSDMD在炎症性上睑下垂和IL-1β释放中的生理作用 信号转导在巨噬细胞和其他单核髓系白细胞中广泛存在。 这涉及到caspase-1裂解GSDMD以产生N-GSDMD片段，这些片段在 质膜(PM)形成毛孔，介导IL-1β外流和下垂。值得注意的是，粒细胞 在多种天然免疫反应中，髓系白细胞也是IL-1β的重要来源，但很少 研究已经检测了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产生的N-GSDMD不定位于PM形成毛孔或驱动 下垂，但IL-1β的分泌是必需的。这种PM毛孔的缺失反映了另一种亚细胞 N-GSDMD与亲天青分泌颗粒(AG)和Lc3+自噬小体结合的运输。 利用缺乏ATG7的中性粒细胞进行的分析表明，IL-1β是通过自噬从中性粒细胞分泌出来的 机械辅助机构。这些发现揭示了GSDM生物学上的根本差异，包括 GSDME在粒细胞和巨噬细胞之间的高表达，将塑造粒细胞在 先天免疫力。我们假设，定义中性粒细胞的丰富分泌颗粒，肥大 细胞和嗜酸性粒细胞是粒细胞特异性机制的基础，可非规范地产生 生物活性IL-1β和通过GSDM家族加工的替代途径调节细胞死亡和 亚细胞贩卖。在目标1中，我们将定义基于炎症激活的信号层次 Caspase、颗粒衍生丝氨酸蛋白酶、凋亡caspase、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 GSDMD/E转运至分泌颗粒与质膜及GSDMD结合能力的分析 作为自噬蛋白辅助分泌可被细胞外切割的ProIL-1β的伴侣 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 细胞外DNA TRAP释放/释放、细胞凋亡和进展所诱导的通路 导致粒细胞衰老。该项目将汲取其他三个项目的优势，以促进我们的 功能研究。这一结果将定义GSDMD和GSDME如何进行调解的新机制 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、过敏性或慢性炎症性疾病的人类的组织功能障碍。

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.