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: 10024455
• 负责人: Xiaoxia Li
• 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/10024455
• 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; Cellular Structures; 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; Polyubiquitination; Predisposition; Preventive measure; Process; Production; Proteins; Proteomics; Regulation; Role; Secretory Vesicles; Signal Transduction; Sodium Dextran Sulfate; Source; Stimulus; Structure; 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; in vivo; inflammatory disease of the intestine; insight; intestinal epithelium; mouse model; novel; novel therapeutics; 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（肠道慢性感染）。病因 IBD仍然难以捉摸，预防措施或治疗不可用。炎症并得出 细胞因子IL-1β正在积极研究为炎症肠中调节的信号枢纽 疾病（IBD）。 IL-1β被合成为无秘书信号序列的非活性促成形式。最近的研究 已经发现，释放IL-1β的脂质结合蛋白Gasdermin d（GSDMD）响应于 caspase-1/11炎性体激活。这一突破导致文学的迅速增长，重点是 GSDMD在髓样细胞中的孔形成和相关的凋亡活性。有趣的是，我们发现了一本小说 GSDMD在指导含肠上皮细胞和含有IL-1β的蔬菜的非流动性作用 T细胞响应caspase 8（CASP8）而不是CASP1激活。通过公正的蛋白质组学分析，我们 确定了一组新型的GSDMD相互作用蛋白在肠上皮细胞（IEC）中，包括NEDD4（E3 连接酶）和HSP90副酮CDC37。 GSDMD或NEDD4消除了LPS和ATP诱导的 IEC的IL-1β产生。令人惊讶的是，LPS+ATP刺激导致了Pro-IL-1β的多泛素化，这是 分泌并加工成成熟的IL-1β，以及包含全长GSDMD的复合物，HSP90/CDC37， NEDD4，ATG7，CASP8，但不是CASP1。体外泛素化测定法证明了NEDD4，已知可以相互作用 使用LC3并促进货物加载到秘密蔬菜中，催化了Pro-IL-1β的多泛素化。的确， GSDMD与LC3+蔬菜有关； GSDMD依赖性细胞外蔬菜的释放（<200 通过含有GSDMD/NEDD4/IL-1β复合物的电子显微镜检测到NM）。而且， GSDMD的ASP276中的灭活突变，废除了其孔形成和凋亡活性 影响IEC的GSDMD引导的IL-1β分泌。在Th17细胞中，T辅助细胞子集与 肠炎，这种GSDMD引导的多泛素化pro-IL-1β复合物的分泌（GSDMD，HSP90， 还响应ATP刺激和TCR激活而容易检测到CASP8和NEDD4）。共同 该数据揭示了GSDMD在非乳突细胞中释放IL-1β中的新型非凋亡作用。致病性 在两种小鼠注射小鼠模型中，研究了GSDMD引导的IL-1β释放的作用。首先， 虽然在A中诱导了多泛素化的Pro-IL-1β，MIL-1β和GSDMD/NEDD4秘书络合物 肠外植物中的GSDMD依赖性方式响应硫酸葡萄糖钠（DSS）诱导的结肠炎， GSDMD缺陷减弱了肠道注射。其次，幼稚的T细胞中的GSDMD或IL-1β缺乏症 降低了他们引起肠道炎症的能力。基于这些发现，我们假设GSDMD 介导非肌动物和髓样细胞中的IL-1β释放的不同途径（引导秘密和凋亡）， 这共同以协调的方式共同导致肠道炎症的发病机理。我们将测试这个 通过以下目的假设：（1）研究GSDMD引导分泌的分子机制 IL-1β释放的途径； （2）研究GSDMD-IL-1β轴在细胞类型和途径特异性的作用 肠炎，包括GSDMD介导的凋亡与GSDMD引导的IL-1β分泌 肠炎。