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应答的新信号机制： Gasdermins D和E（GSDMD/E）在粒细胞白细胞中，包括中性粒细胞、肥大细胞和 嗜酸性粒细胞GSDMD在炎性小体细胞凋亡和IL-1β释放中的生理作用 在巨噬细胞和其它单核骨髓白细胞中已经广泛地表征了信号传导。 这涉及通过半胱天冬酶-1切割GSDMD以产生N-GSDMD片段，其在半胱天冬酶-1中寡聚化。 质膜（PM）形成介导IL-1β流出和细胞凋亡的孔。值得注意的是， 髓系白细胞也是多种先天免疫应答中IL-1β的重要来源，但很少有 研究已经检查了GSDMD或其它GSDM家族蛋白在这些细胞中的作用。我们的初步数据 表明中性粒细胞中caspase-1产生的N-GSDMD不定位于PM以形成孔或驱动 但是IL-1β分泌所必需的。这种PM孔的缺乏反映了替代的亚细胞 N-GSDMD与嗜天青分泌颗粒（AG）和LC 3+自噬体相关联。 使用ATG 7缺陷型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的高表达，将塑造粒细胞在 先天免疫我们推测，中性粒细胞、肥大细胞和巨噬细胞分泌颗粒丰富， 细胞和嗜酸性粒细胞是粒细胞特异性机制的基础， 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， 亚细胞运输在目标1中，我们将定义基于炎症激活的信号传导层次， 半胱天冬酶、颗粒来源的丝氨酸蛋白酶、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向分泌颗粒和质膜的运输，以及GSDMD 作为自噬蛋白辅助的proIL-1β分泌的伴侣，proIL-1β可被细胞外切割， 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、凋亡和进展的触发物诱导的途径 到粒细胞衰老。该项目将借鉴其他三个项目的优势， 功能研究。这些结果将为GSDMD和GSDME如何介导 粒细胞特异性信号应答在急性感染期间发挥生理作用，但有助于 组织功能障碍的人类常见的自身免疫性，过敏性或慢性炎症性疾病。

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.