Effect of Bacterial Tolerance on TLR4 Signal Transduction

细菌耐受性对 TLR4 信号转导的影响

基本信息

批准号：
8505354
负责人：
ANDREI E MEDVEDEV
金额：
$ 24.33万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8505354
关键词：
Address Affect American Animal Model Anti-Inflammatory Agents Anti-inflammatory Area Autoimmune Diseases Biological Assay Biological Markers Cells Cessation of life Chemicals Communicable Diseases Cytokine Inducible SH2-Containing Protein Data Deubiquitinating Enzyme Development Dimerization Disease Docking Endotoxins Event Exposure to Family Dasypodidae Funding Gene Silencing Genes Goals Gram-Negative Bacteria Health Host Defense Human IRAK1 gene IRAK4 gene Immune Immunocompromised Host Immunosuppression Incidence Inflammation Inflammatory Interferons Life Ligands Link Lipopolysaccharides MAP3K7 gene Mediating Modeling Molecular National Institute of Allergy and Infectious Disease Pathogenesis Patients Phase Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Polyubiquitination Post-Translational Protein Processing Production Protein Isoforms Protein Tyrosine Phosphatase Public Health RNA Splicing Receptor Signaling Research Role SRC gene Sepsis Septic Shock Signal Pathway Signal Transduction Signaling Molecule Sterility TANK-binding kinase 1 TBK1 gene TLR4 gene TRAF6 gene Testing Toll-Like Receptor Pathway Toll-like receptors Tumor Necrosis Factor Receptor Tyrosine Phosphorylation Ubiquitination adapter protein antimicrobial base chemokine cytokine design health science research human IRF3 protein human TOLLIP protein improved in vivo inhibitor/antagonist inositol-1,4,5-trisphosphate 5-phosphatase insight interferon regulatory factor-3 macrophage microbial monocyte mortality neutrophil novel therapeutic intervention novel therapeutics pathogen prevent programs receptor response secondary infection septic toll-like receptor 4 transcription factor translational study ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Sepsis is a major threat to human health in the US, affecting ~750,000 Americans per year, with an associated mortality rate >28%. Many septic patients develop profound immunosuppression manifested by decreased production of pro-inflammatory cytokines and chemokines. This is highly reminiscent of endotoxin tolerance, a state of re-programming of TLR4 responses after a prior exposure to LPS. Endotoxin tolerance can be used as a model to delineate mechanisms that underlie altered monocyte responses in patients with sepsis. During the period of funding, we have identified new hallmarks of endotoxin tolerance. These include: (i) deficient tyrosine phosphorylation of TLR4 and adapter protein Mal; (ii) suppressed LPS-induced signalosome assembly amongst TLR4, adapter protein TRIF and kinase TBK1; (iii) deficient activation of TBK1 and transcription factor IRF-3; and (iv) increased expression of negative regulators Tollip, IRAK-M, SHIP-1, SOCS-1, SARM, and SIKE. Our preliminary data in THP1 cells and human monocytes show decreased LPS- induced c-Src and Lyn phosphorylation and Lyn-TLR4 interactions; inhibited activation of kinases IRAK4 and TAK-1; impaired K63-linked polyubiquitination of IRAK1 and TRAF-6; suppressed interactions of ubiquitinated IRAK1 with TRAF6 and IKK3; and increased expression of A20, a key deubiquitinating enzyme. Furthermore, we found reduced LPS tolerance induction upon inhibition of protein tyrosine phosphatases or by A20 gene knockdown, indicating a crucial role for altered tyrosine phosphorylation and K63-linked polyubiquitination in tolerance. Based on these data, we hypothesize that LPS tolerance alters post-translational modifications of TLR4, Mal and IRAK kinases and increases levels of negative regulators of TLR signaling, leading to reprogramming of TLR responses through changes in proximal signalosome compositions. This hypothesis will be tested in the following Specific Aims: 1. Identify kinases and phosphatases involved in phosphorylation of TLR4 and Mal and determine the impact of LPS tolerance on their expression and activities; 2. Define mechanisms by which LPS tolerance alters signalosome assembly and activation of proximal adapter-kinase modules; and 3. Elucidate molecular basis of interference in TLR4 signaling by negative regulatory molecules associated with LPS tolerance. These studies will determine new mechanisms responsible for tolerance and identify key intermediates affected. They will lay the groundwork for our future research in animal models of sepsis in vivo and translational studies in septic patients, with the goal of facilitating development of new therapeutic strategies to improve treatments for septic patients. These advances would be of key importance for improving public health in septic patients in the U.S.

描述（由申请人提供）：脓毒症是美国人类健康的主要威胁，每年影响约 750,000 名美国人，相关死亡率 >28%。许多脓毒症患者出现严重的免疫抑制，表现为促炎细胞因子和趋化因子的产生减少。这很容易让人想起内毒素耐受，这是一种在先前暴露于 LPS 后 TLR4 反应重新编程的状态。内毒素耐受性可作为模型来描述脓毒症患者单核细胞反应改变的机制。在资助期间，我们发现了内毒素耐受的新标志。这些包括： (i) TLR4 和接头蛋白 Mal 的酪氨酸磷酸化缺陷； (ii) 抑制 LPS 诱导的 TLR4、接头蛋白 TRIF 和激酶 TBK1 之间的信号体组装； (iii) TBK1 和转录因子 IRF-3 激活不足； (iv) 负调节因子 Tollip、IRAK-M、SHIP-1、SOCS-1、SARM 和 SIKE 的表达增加。我们在 THP1 细胞和人类单核细胞中的初步数据显示 LPS 诱导的 c-Src 和 Lyn 磷酸化以及 Lyn-TLR4 相互作用减少；抑制激酶 IRAK4 和 TAK-1 的激活； IRAK1 和 TRAF-6 的 K63 连接多聚泛素化受损；抑制泛素化 IRAK1 与 TRAF6 和 IKK3 的相互作用；并增加了 A20（一种关键的去泛素化酶）的表达。此外，我们发现抑制蛋白酪氨酸磷酸酶或通过敲低 A20 基因可降低 LPS 耐受性诱导，表明酪氨酸磷酸化和 K63 连接的多泛素化的改变在耐受性中发挥着至关重要的作用。基于这些数据，我们假设 LPS 耐受改变了 TLR4、Mal 和 IRAK 激酶的翻译后修饰，并增加了 TLR 信号传导负调节因子的水平，从而通过近端信号体组成的变化导致 TLR 反应的重新编程。该假设将在以下具体目标中得到检验： 1. 鉴定参与 TLR4 和 Mal 磷酸化的激酶和磷酸酶，并确定 LPS 耐受对其表达和活性的影响； 2. 定义 LPS 耐受性改变信号体组装和近端接头激酶模块激活的机制； 3. 阐明与 LPS 耐受相关的负调节分子干扰 TLR4 信号传导的分子基础。这些研究将确定负责耐受性的新机制并确定受影响的关键中间体。它们将为我们未来脓毒症动物模型的体内研究和脓毒症患者的转化研究奠定基础，目标是促进新的治疗策略的开发，以改善脓毒症患者的治疗。这些进展对于改善美国脓毒症患者的公共卫生至关重要。