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: 10654589
• 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/10654589
• 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; Compensation; Complement; Complex; Crohn' s disease; Data; Disease; Electron Microscopy; Epithelial Cells; Etiology; Event; Extracellular Space; Family member; 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; 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

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(慢性肠炎)。病因学 IBD的发病率仍然难以捉摸，也没有预防措施或治愈方法。炎性小体及其衍生 细胞因子IL-1β作为炎症性肠病中异常调节的信号中枢正被广泛研究 疾病(IBD)。IL-1β是以无分泌信号序列的非活性前体形式合成的。最新研究 发现一种脂质结合蛋白GasderminD(GSDMD)是释放IL-1β所必需的 Caspase-1/11炎症体激活。这一突破导致了以文学为中心的文学的迅速发展 GSDMD在髓系细胞中的孔道形成和相关的嗜热活性。有趣的是，我们发现了一本小说 GSDMD在引导肠上皮细胞释放含有IL-1β的囊泡中的非解热作用 T细胞对Caspase 8(Caspase8)的反应，但对CASP1的激活无反应。通过无偏见的蛋白质组分析，我们 在肠上皮细胞中发现了一组新的GSDMD相互作用蛋白，包括NEDD4(一个E3 连接酶)和Hsp90辅助伴侣CDC37。消融GSDMD或NEDD4可消除内毒素和三磷酸腺苷诱导的 IECS生产IL-1β。值得注意的是，内毒素+三磷酸腺苷刺激导致前-IL-1β的多泛素化，这是 分泌并加工成成熟的IL-1β以及含有全长GSDMD、Hsp90/CDC37、 NEDD4、ATG7、Casp8，但不包括CASP1。体外泛素化实验表明，已知相互作用的NEDD4 与Lc3结合，促进货物装入分泌囊泡，催化前IL-1β的多泛素化。的确， 而GSDMD与Lc3+囊泡有关；依赖GSDMD的胞外囊泡释放(&lt；200 NM)，其中含有GSDMD/NEDD4/IL-1β复合体。此外， GSDMD的Asp276的失活突变，取消了其成孔和焦链活性，但没有 影响GSDMD诱导的IECs分泌IL-1β。在TH17细胞中，高度相关的辅助性T细胞亚群 肠道炎症，这种由GSDMD引导的多泛素化前IL-1β复合体(GSDMD，Hsp90， Casp8和NEDD4)也很容易在ATP刺激和TCR激活时检测到。总而言之， 这些数据揭示了GSDMD在非髓系细胞释放IL-1β中的一种新的非嗜热性作用。致病的 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。首先， 而多泛素化的前IL-1β、mIL-1β和GSDMD/NEDD4-分泌复合体在 葡聚糖硫酸钠(DSS)诱导的结肠炎时肠道组织中GSDMD依赖的方式， GSDMD缺乏可减轻肠道炎症。第二，幼稚T细胞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 介导非髓系细胞和髓系细胞释放IL-1β的不同途径(引导分泌和下垂)， 它们共同作用于肠道炎症的发病机制。我们将对此进行测试 通过以下目的提出假说：(1)探讨GSDMD诱导分泌的分子机制 IL-1β释放途径；(2)探讨GSDMD-IL-1β轴的细胞类型和途径特异性作用。 肠道炎症，包括GSDMD介导的嗜热性与GSDMD引导的IL-1β的分泌 肠道发炎。