Effect of Bacterial Tolerance on TLR4 Signal Transduction

描述(由申请人提供)：败血症是美国人类健康的主要威胁，每年影响约75万美国人，相关死亡率为28%。许多败血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。这让人联想到内毒素耐受性，这是一种在先前暴露于内毒素后TLR4反应的重新编程状态。内毒素耐受性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，我们发现了内毒素耐受的新特征。这些缺陷包括：(I)TLR4和适配蛋白MAL的酪氨酸磷酸化缺陷；(Ii)抑制内毒素诱导的TLR4、适配蛋白TRIF和激酶TBK1之间的信号体组装；(Iii)TBK1和转录因子IRF-3的缺陷激活；以及(Iv)负调控因子Tollip、IRAK-M、SHIP-1、SOCS-1、SARM和SIKE的表达增加。我们在THP1细胞和人单核细胞中的初步数据显示，脂多糖诱导的c-Src和Lyn磷酸化以及Lyn-TLR4相互作用减少；抑制了K63连接的IRAK1和TRAF-6的激活；抑制了IRAK1和TRAF-6的多泛素化；抑制了泛素化的IRAK1与TRAF6和IKK3的相互作用；以及增加了关键的脱泛素化酶A20的表达。此外，我们发现抑制蛋白酪氨酸磷酸酶或通过A20基因敲除降低了内毒素耐受性诱导，这表明改变的酪氨酸磷酸化和K63连锁的多泛素化在耐受性中起着关键作用。基于这些数据，我们假设脂多糖耐受性改变了TLR4、MAL和IRAK激酶的翻译后修饰，并增加了TLR信号负调控因子的水平，通过改变近端信号体的组成导致TLR反应的重新编程。这一假说将在以下特定目标中得到验证：1.识别参与TLR4和MAL磷酸化的激酶和磷酸酶，并确定脂多糖耐受对其表达和活性的影响；2.确定脂多糖耐受改变信号小体组装和激活近端接头-激酶模块的机制；3.阐明与脂多糖耐受相关的负性调控分子干扰TLR4信号转导的分子基础。这些研究将确定导致耐受性的新机制，并确定受影响的关键中间体。它们将为我们未来对脓毒症动物模型的体内研究和脓毒症患者的转译研究奠定基础，目的是促进开发新的治疗策略，以改善脓毒症患者的治疗。这些进展对于改善美国败血症患者的公共健康至关重要。

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.