Effect of Bacterial Tolerance on TLR4 Signal Transduction

描述(申请人提供)：尽管现代医学取得了进步，但细菌败血症仍然是全球人类健康的主要威胁之一。仅在美国，每年就有大约75万美国人受到影响，与之相关的死亡率为28%。内毒素是革兰氏阴性菌的外膜成分，革兰氏阳性菌和结核分枝杆菌的各种结构代表着天然免疫反应的启动刺激。通过表达在单核细胞和巨噬细胞上的Toll样受体(TLR)有效识别这些细菌结构，对于建立强大的一线防御和预防脓毒症至关重要。许多脓毒症患者出现低炎症状态，表现为单核细胞功能受到明显抑制，包括一些细胞因子的产生受到抑制。这种状态使人高度联想到细菌耐受性，其定义是在先前接触细菌成分后细胞不耐受的瞬时状态。因此，细菌耐受性的诱导可以作为描述脓毒症患者单核细胞反应性降低的机制的模型。尽管有许多研究，但诱导细菌耐药的分子机制在很大程度上仍不清楚。该项目的总体目标是更好地了解宿主对微生物反应的分子机制。我们的目标是确定TLR途径的受体和信号转导分子在诱导和维持单核细胞对细菌的耐受性中的作用。核心假设是，在人类单核细胞中诱导细菌耐受显著改变了TLR途径关键成分之间由信号诱导的复合体的形成，导致了一种受抑制的抗菌反应状态的发展。1.研究与TLR4和TLR2激动剂耐受性相关的TLR4复合体关键分子的翻译后修饰和相互作用。2.分析细菌耐受性对IRAK-4的表达、募集、翻译后修饰和激活的影响。3.鉴定耐受细胞中干扰激动剂诱导的TLR、适配蛋白和IRAK激酶之间相互作用的TLR介导的信号抑制物。预计在完成这项拨款后，我们将确定导致细菌耐受性发展的关键分子机制，并为开发治疗细菌性脓毒症患者的新治疗方法提供策略，以靶向TLR信号通路中在耐受性和败血症方面受到损害的组件。

DESCRIPTION (provided by applicant): Despite advances of modern medicine, bacterial sepsis remains one of the major threats to human health worldwide. In the U.S.A alone, it affects approximately 750,000 Americans per year, with an associated mortality rate >28%. The lipopolysaccharide (LPS), an outer membrane component of Gram negative bacteria, and various structures of Gram positive bacteria and Mycobacterium tuberculosis, represent the initiating stimuli in the activation of the innate immune response. Effective recognition of these bacterial structures by Toll-like receptors (TLR) expressed on monocytes and macrophages is critical for mounting a strong first line defense and prevention of sepsis. Many patients with sepsis develop a hypoinflammatory state that is manifested by marked inhibition of monocyte functions, including suppressed production of a number of cytokines. This state is highly reminiscent of bacterial tolerance defined as a transient state of cell refractoriness following a prior exposure to bacterial components. Therefore, induction of bacterial tolerance can be used as a model to delineate mechanisms that underlie decreased monocyte responsiveness in patients with sepsis. Despite numerous studies, the molecular mechanisms that underlie induction of bacterial tolerance are still largely unknown. The overall goal of this project is to gain a better understanding of the molecular mechanisms of host responses to microbes. Our objective is to define the role of receptors and signal transducing molecules of the TLR pathway in induction and maintenance of bacterial tolerance in monocytes. The central hypothesis is that induction of bacterial tolerance in human monocytes dramatically alters signal-induced complex formation among key components of the TLR pathway, leading to development of a state of suppressed antibacterial responsiveness. The following Specific Aims are proposed to: 1. Examine post-translational modifications and interactions of key molecules of TLR4 complexes associated with tolerance to TLR4 and TLR2 agonists. 2. Analyze the effect of bacterial tolerance on expression, recruitment, post-translational modifications of key adapter proteins and activation of IRAK-4. 3. Characterize suppressors of TLR-mediated signaling in tolerized cells that interfere with agonist-induced interactions among TLRs, adapter proteins, and IRAK kinases. It is expected that at the completion of this grant, we will have identified key molecular mechanisms responsible for development of bacterial tolerance, and have provided strategies for development of new therapeutic approaches for treatment of patients with bacterial sepsis to target components of the TLR signaling pathway that are compromised in tolerance and sepsis.