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.

摘要： 在美国，大约有130万人患有IBD（慢性肠道炎症）。病因 IBD仍然难以捉摸，预防措施或治愈方法不可用。炎性小体及其衍生物 细胞因子IL-1β作为炎症性肠病中调节异常的信号中枢， 疾病（IBD）。IL-1β作为无分泌信号序列的无活性前体合成。最近的研究 已经发现一种脂质结合蛋白Gasdermin D（GSDMD）是响应于 caspase-1/11炎性体激活。这一突破导致了文学的快速增长，重点是 骨髓细胞中GSDMD的孔形成和相关的致热活性。有趣的是，我们发现了一本小说 GSDMD在引导含IL-1β的囊泡从肠上皮细胞释放中的非致热作用， T细胞响应于Caspase 8（Casp 8）而非Casp 1活化。通过无偏的蛋白质组学分析，我们 在肠上皮细胞（IEC）中鉴定了一组新的GSDMD相互作用蛋白，包括NEDD 4（E3 连接酶）和Hsp 90共分子伴侣CDC 37。GSDMD或NEDD 4的消融消除了LPS和ATP诱导的细胞凋亡。 来自IEC的IL-1β产生。引人注目的是，LPS+ATP刺激导致pro-IL-1β的多泛素化， 分泌并加工成成熟的IL-1β沿着含有全长GSDMD，Hsp 90/CDC 37， NEDD 4，Atg 7，Casp 8，但不是Casp 1。体外泛素化分析表明，已知与NEDD 4相互作用的NEDD 4， 与LC 3和促进货物装载到分泌囊泡中，催化pro-IL-1β的多聚泛素化。的确， 而GSDMD与LC 3+囊泡相关; GSDMD依赖的细胞外囊泡（< 200 透射电镜下观察到GSDMD/NEDD 4/IL-1β复合物的存在。此外，委员会认为， GSDMD的Asp 276中的失活突变消除了其孔形成和致热原活性， 影响GSDMD引导的IL-1β从IEC分泌。在TH 17细胞中，一个与T细胞亚群高度相关的T辅助细胞亚群， 肠道炎症，这种GSDMD引导的多聚泛素化前IL-1β复合物（GSDMD，Hsp 90， Casp 8和NEDD 4）也响应于ATP刺激和TCR活化而容易检测到。总的来说， 这些数据揭示了GSDMD在IL-1β从非骨髓细胞释放中的新的非致热作用。致病 在两种小鼠肠道炎症模型中研究了GSDMD引导的IL-1β释放的作用。第一、 而多泛素化的pro-IL-1β、mIL-1β和GSDMD/NEDD 4分泌复合物则被诱导为一种新的细胞因子。 葡聚糖硫酸钠（DSS）诱导的结肠炎在肠外植体中的GSDMD依赖性方式， GSDMD缺乏减轻了肠道炎症。第二，幼稚T细胞中的GSDMD或IL-1β缺乏 降低了它们引发肠道炎症的能力。基于这些发现，我们假设GSDMD 介导非髓细胞和髓细胞中IL-1β释放的不同途径（引导分泌和焦亡）， 它们以协调的方式共同促进肠道炎症的发病。我们将测试这个 本研究通过以下几个方面对GSDMD的分子机制进行了初步探讨：（1）探讨GSDMD介导的细胞分泌的分子机制 （2）研究GSDMD-IL-1β轴在IL-1β释放中的细胞类型和途径特异性作用。 肠道炎症，包括GSDMD介导的焦萎与GSDMD引导的IL-1β分泌， 肠道炎症