Effect of Bacterial Tolerance on TLR4 Signal Transduction

描述（由申请人提供）：尽管现代医学取得了进步，但细菌性脓毒症仍然是全球人类健康的主要威胁之一。仅在美国，它每年影响大约750，000名美国人，相关死亡率> 28%。脂多糖（LPS）是革兰氏阴性菌的外膜组分，革兰氏阳性菌和结核分枝杆菌的各种结构代表了先天免疫应答激活中的起始刺激。单核细胞和巨噬细胞上表达的Toll样受体（TLR）对这些细菌结构的有效识别对于建立强大的第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的显著抑制，包括多种细胞因子的产生受到抑制。这种状态非常令人联想到细菌耐受性，细菌耐受性定义为先前暴露于细菌组分后细胞不应性的短暂状态。因此，细菌耐受性的诱导可以用作描述脓毒症患者单核细胞反应性降低的机制的模型。尽管进行了大量的研究，但诱导细菌耐受性的分子机制仍然在很大程度上未知。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细菌耐受性中的作用。中心假设是，在人单核细胞中诱导细菌耐受性显著改变TLR途径的关键组分之间的信号诱导的复合物形成，导致抑制抗菌反应性的状态的发展。提出以下具体目标：1.检查与TLR 4和TLR 2激动剂耐受性相关的TLR 4复合物关键分子的翻译后修饰和相互作用。2.分析细菌耐受性对关键衔接蛋白的表达、募集、翻译后修饰和IRAK-4活化的影响。3.表征耐受化细胞中TLR介导的信号传导抑制剂，其干扰TLR、衔接蛋白和IRAK激酶之间激动剂诱导的相互作用。预计在完成该资助时，我们将确定负责细菌耐受性发展的关键分子机制，并提供开发新的治疗方法的策略，用于治疗细菌性脓毒症患者，以靶向耐受性和脓毒症中受损的TLR信号通路组分。

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.