GSDMD-dependent IL-1 signaling in intestinal inflammation

肠道炎症中 GSDMD 依赖性 IL-1 信号传导

• 批准号: 10441357
• 负责人: Theresa Torres Pizarro
• 金额: $ 54.17万
• 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/10441357
• 关键词: Ablation; Address; Attenuated; Automobile Driving; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Biopsy; CASP1 gene; CASP8 gene; CDC37 gene; CDC37 homolog protein; Cell physiology; Cells; Chronic; Colitis; Colon; Complement; Complex; Crohn' s disease; Data; Disease; Electron Microscopy; Epithelial Cells; Etiology; Event; Extracellular Space; Family member; Financial compensation; Genetic Polymorphism; Helper-Inducer T-Lymphocyte; IL1R1 gene; Immune; In Vitro; Inflammasome; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interleukin-1; Interleukin-1 beta; Intestinal Secretions; Intestines; Knowledge; Length; Lipid Binding; Literature; Maps; Mediating; Modeling; Molecular; Molecular Chaperones; Mus; Mutation; Myeloid Cells; Nonlytic; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peptide Signal Sequences; Persons; Polyubiquitination; Predisposition; Preventive measure; Process; Production; Proteins; Proteomics; Regulation; Role; Secretory Vesicles; Signal Transduction; Sodium Dextran Sulfate; Source; Stimulus; T-Lymphocyte; TCR Activation; Testing; Tissues; Ubiquitination; Ulcerative Colitis; United States; Vesicle; anakinra; base; cell type; cytokine; design; dextran sulfate sodium induced colitis; effective therapy; extracellular vesicles; gut inflammation; in vivo; insight; intestinal epithelium; mouse model; novel; novel therapeutic intervention; programs; public health relevance; rapid growth; receptor; recruit; response; targeted treatment; therapeutic target; ubiquitin-protein ligase; vesicular release

Abstract: About 1.3 million people suffer from IBD (chronic inflammation of the intestine) in the United States. The etiology of IBD remains elusive and preventive measures or a cure are not available. Inflammasomes and derived cytokines IL-1β are being intensely investigated as the signaling hub that is dys-regulated in inflammatory bowel disease (IBD). IL-1β is synthesized as inactive pro-forms with no secretory signal sequence. Recent studies have found that a lipid binding protein, Gasdermin D (GSDMD), is required for release of IL-1β in response to caspase-1/11 inflammasome activation. This breakthrough led to a rapid growth of literature that focuses on the pore forming and associated pyroptotic activity of GSDMD in myeloid cells. Interestingly, we discovered a novel nonpyrototic role of GSDMD in guiding the release of IL-1β containing vesicles from intestinal epithelia cells and T cells in response to Caspase 8 (Casp8) but not casp1 activation. Through unbiased proteomic analysis, we identified a set of novel GSDMD-interacting proteins in intestinal epithelial cells (IECs), including NEDD4 (an E3 ligase) and the Hsp90 co-chaperone CDC37. Ablation of GSDMD or NEDD4 abolished LPS and ATP-induced IL-1β production from IECs. Strikingly, LPS+ATP stimulation led to the polyubiquitination of pro-IL-1β, which was secreted and processed into mature IL-1β along with a complex containing full-length GSDMD, Hsp90/CDC37, NEDD4, Atg7, Casp8 but not Casp1. In vitro ubiquitination assay demonstrated that NEDD4, known to interact with LC3 and promote cargo loading into secretory vesicles, catalyzed the polyubiquitination of pro-IL-1β. Indeed, while GSDMD was associated with LC3+ vesicles; GSDMD-dependent release of extracellular vesicles (< 200 nm) were detected by electron microscopy, which contains the GSDMD/NEDD4/IL-1β complex. Moreover, inactivating mutation in the Asp276 of GSDMD, which abolishes its pore-forming and pyroptotic activity, did not impact the GSDMD-guided IL-1β secretion from IECs. In TH17 cells, a T helper cell subset highly relevant to intestinal inflammation, this GSDMD-guided secretion of polyubiquitinated pro-IL-1β complex (GSDMD, Hsp90, Casp8 and NEDD4) was also readily detected in response to ATP stimulation and TCR activation. Collectively, the data revealed a novel nonpyroptotic role for GSDMD in IL-1β release from non-myeloid cells. The pathogenic role of the GSDMD-guided IL-1β release was investigated in two mouse models of intestinal inflammation. Firstly, while polyubiquitinated pro-IL-1β, mIL-1β and the GSDMD/NEDD4-secretary complex were induced in a GSDMD-dependent manner in the intestinal explants in response to dextran sodium sulfate (DSS)-induce colitis, GSDMD-deficiency attenuated the intestinal inflammation. Secondly, GSDMD or IL-1β deficiency in naïve T cells reduced their ability to elicit intestinal inflammation. Based on these findings, we hypothesize that the GSDMD mediates distinct pathways (guided secretion and pyroptosis) for IL-1β release in non-myeloid and myeloid cells, which jointly contribute to the pathogenesis of intestinal inflammation in a coordinated manner. We will test this hypothesis through the following aims: (1) Investigate the molecular mechanism for GSDMD-guided secretory pathway for IL-1β release; (2) Investigate the cell-type and pathway-specific role of GSDMD-IL-1β axis in intestinal inflammation, including GSDMD-mediated pyroptotic versus GSDMD-guided IL-1β secretion on the intestinal inflammation.

抽象的： 在美国，大约有 130 万人患有 IBD（慢性肠道炎症）。 IBD 的发病率仍然难以捉摸，并且尚无炎症小体及其衍生的预防措施或治疗方法。 细胞因子 IL-1β 作为炎症性肠中失调的信号中枢正在受到深入研究 IL-1β 被合成为无活性的前体形式，没有分泌信号序列。 已经发现，脂质结合蛋白 Gasdermin D (GSDMD) 是释放 IL-1β 所必需的，以响应 caspase-1/11 炎症小体激活这一突破导致关注 caspase-1/11 炎症小体激活的文献迅速增长。 骨髓细胞中 GSDMD 的孔形成和相关的焦亡活性。 GSDMD 在引导肠上皮细胞释放含有 IL-1β 的囊泡中的非热解作用 T 细胞响应 Caspase 8 (Casp8)，但不响应 casp1 激活。 在肠上皮细胞 (IEC) 中鉴定了一组新型 GSDMD 相互作用蛋白，包括 NEDD4（一种 E3 连接酶）和 Hsp90 共伴侣 CDC37 的消除 GSDMD 或 NEDD4 消除了 LPS 和 ATP 诱导的。 引人注目的是，LPS+ATP 刺激导致 IL-1β 前体的多泛素化。 分泌并加工成成熟的 IL-1β 以及含有全长 GSDMD、Hsp90/CDC37 的复合物， NEDD4、Atg7、Casp8 但不包括 Casp1 体外泛素化测定表明 NEDD4 会相互作用。 与 LC3 结合并促进货物装载到分泌囊泡中，催化 pro-IL-1β 的多泛素化。 而 GSDMD 与 LC3+ 囊泡依赖性细胞外囊泡释放相关（< 200） nm）通过电子显微镜检测，其中含有GSDMD/NEDD4/IL-1β复合物。 GSDMD 的 Asp276 失活突变消除了其成孔和焦亡活性，但并未 影响 GSDMD 引导的 IEC 分泌 IL-1β 在 TH17 细胞中，TH17 细胞与 T 辅助细胞亚群高度相关。 肠道炎症，这种 GSDMD 引导的多泛素化前 IL-1β 复合物（GSDMD、Hsp90、 Casp8 和 NEDD4) 也很容易检测到对 ATP 刺激和 TCR 激活的反应。 数据揭示了 GSDMD 在非骨髓细胞释放 IL-1β 中的新的非焦亡作用。 在两种肠道炎症小鼠模型中研究了 GSDMD 引导的 IL-1β 释放的作用。 而多泛素化​​的 pro-IL-1β、mIL-1β 和 GSDMD/NEDD4 分泌复合物在 肠外植体中 GSDMD 依赖性方式响应葡聚糖硫酸钠 (DSS) 诱导的结肠炎， 其次，幼稚 T 细胞中 GSDMD 或 IL-1β 缺乏会减轻肠道炎症。 降低了它们引发肠道炎症的能力。基于这些发现，我们发现 GSDMD 是有效的。 介导非髓细胞和髓细胞中 IL-1β 释放的不同途径（引导分泌和细胞焦亡）， 它们以协调的方式共同促进肠道炎症的发病机制，我们将对此进行测试。 通过以下目标提出假设：（1）研究GSDMD引导分泌的分子机制 IL-1β 释放途径；(2) 研究 GSDMD-IL-1β 轴在细胞类型和途径中的特异性作用 肠道炎症，包括 GSDMD 介导的焦亡与 GSDMD 引导的 IL-1β 分泌 肠道炎症。