Effect of Bacterial Tolerance on TLR4 Signal Transduction

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。这高度地使人联想到内毒素耐受性，即在先前暴露于LPS之后TLR 4应答的重编程状态。内毒素耐受可作为描述脓毒症患者单核细胞反应改变的机制的模型。在资助期间，我们已经确定了内毒素耐受性的新标志。其中包括：（i）TLR 4和衔接蛋白Mal的酪氨酸磷酸化缺陷;（ii）抑制LPS诱导的TLR 4、衔接蛋白TRIF和激酶TBK 1之间的信号体组装;（iii）TBK 1和转录因子IRF-3的激活缺陷;和（iv）负调节因子Tollip、IRAK-M、SHIP-1、SOCS-1、SARM和SIKE的表达增加。我们在THP 1细胞和人单核细胞中的初步数据显示，LPS诱导的c-Src和林恩磷酸化以及Lyn-TLR 4相互作用降低;激酶IRAK 4和TAK-1的活化受到抑制; IRAK 1和TRAF-6的K63连接的多聚泛素化受损;泛素化IRAK 1与TRAF-6和IKK 3的相互作用受到抑制; A20（一种关键的去泛素化酶）的表达增加。此外，我们发现减少LPS耐受诱导蛋白酪氨酸磷酸酶抑制或A20基因敲除，表明改变酪氨酸磷酸化和K63连接的多泛素化的耐受性的关键作用。基于这些数据，我们假设LPS耐受性改变了TLR 4、Mal和IRAK激酶的翻译后修饰，并增加了TLR信号传导的负调节因子的水平，从而通过近端信号体组成的变化导致TLR应答的重编程。这一假设将在以下具体目标中得到检验：1.鉴定参与TLR 4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;和3.阐明与LPS耐受相关的负调控分子干扰TLR 4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。他们将为我们未来在体内脓毒症动物模型和脓毒症患者转化研究中的研究奠定基础，目的是促进新治疗策略的开发，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。

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.