Role of Toll-Like Receptor 4 Signaling in Stress-Induced Lymphocyte Apoptosis

描述(申请人提供)：我们这些研究的总体目标是阐明Toll样受体在应激状态下介导免疫抑制的机制(S)。生理和心理压力都会改变人和动物的免疫系统。压力也是许多人类疾病的已知风险因素。TLR介导的信号通路在诱导免疫应答中起着至关重要的作用。TLRs介导了对应激的免疫反应；然而，其机制仍有待阐明。为了研究应激诱导免疫反应的机制和设计治疗策略，我们建立了束缚应激动物模型来研究免疫抑制。利用这个模型，我们揭示了小鼠的束缚应激通过一种细胞凋亡机制来调节免疫系统。我们发现，TLR4缺陷小鼠对应激诱导的脾细胞减少具有抵抗力。TLR4通常与高迁移率族蛋白-1(HMGB1)相互作用。我们的初步数据表明，应激通过TLR4提高血浆HMGB1水平。磷脂酰肌醇3-激酶(PI3K)及其下游底物Akt可通过减轻炎症反应和抑制细胞凋亡来调节TLR介导的反应。我们已经证明，TLR4缺乏对小鼠脾细胞数量的保护作用是通过PI3K介导的。总之，我们的数据表明，TLR4和PI3K/Akt信号在应激期间的免疫反应中发挥了作用。目前，我们还不清楚TLR4介导的信号在应激诱导的淋巴细胞凋亡中的确切作用，也不清楚TLR4在应激过程中负调控PI3K/Akt信号通路中的确切作用。我们的假设是，TLR4和PI3K/Akt信号通路的调节决定了淋巴细胞在应激反应中的命运。为了检验这一假说，我们提出了以下建议。目标1将确定应激后TLR4介导的细胞凋亡信号通路的作用。为了阐明TLR4在应激过程中是否激活了凋亡信号通路，我们将首先研究TLR4介导的信号对淋巴细胞凋亡的影响，包括CD4+和CD8+T细胞的凋亡。然后，我们将研究TLR4缺乏是否激活了应激诱导的淋巴细胞凋亡中的PI3K/Akt信号。目的2明确TLR4对PI3K/Akt信号的负调控在应激诱导的免疫抑制中的作用。为了检测TLR4缺乏是否激活PI3K/Akt信号通路，导致免疫保护作用，对TLR4基因敲除小鼠和野生型小鼠给予PI3K抑制剂，然后在不同的时间段应激。我们将明确TLR4介导的信号在应激诱导的免疫抑制中的作用，然后确定TLR4缺乏是否会导致PI3K/Akt信号的激活。此外，我们还将确定HMGB1和多功能适配器2-arrestin 2在TLR4介导的应激后PI3K/Akt信号转导中的作用。我们的研究将有助于我们理解TLR4介导的信号通路对免疫系统的影响，为开发新的免疫抑制策略和治疗方法提供机会。 公共卫生相关性：心理和身体压力会对免疫系统产生显著影响。从历史上看，这种影响归因于压力荷尔蒙的释放，尽管其他因素似乎起到了重要作用。利用物理束缚应激的小鼠模型，我们研究了一些可能解释应激和免疫系统之间联系的分子介体。这项研究将研究应激影响免疫系统的机制，希望能导致特定治疗方法或药物的开发，进而可能改善这种有时有害的相互作用。

DESCRIPTION (provided by applicant): Our overall goal of these studies is to elucidate the mechanism(s) underlying Toll-like receptor (TLR) mediated immune suppression during stress. Physical and psychological stress can alter the immune system in both humans and animals. Stress is also a known risk factor for numerous human diseases. TLR mediated signaling pathways play a critical role in the induction of immune response. TLRs mediate the immune response to stress; however, the mechanisms remain to be elucidated. In order to study the mechanisms of stress-induced immune responses and to design strategies for therapeutics, we established an animal model for restraint stress to study immune suppression. Using this model we have revealed that restraint stress of mice modulates the immune system through a cell apoptotic mechanism. We found that TLR4 deficient mice are resistant to stress-induced splenocyte reduction. TLR4 typically interacts with high mobility group box-1 protein (HMGB1). Our preliminary data indicate that stress enhances plasma HMGB1 levels through TLR4. The phosphoinositide 3-kinases (PI3K) and their downstream substrate Akt can modulate TLR-mediated responses by decreasing inflammation and inhibiting apoptosis. We have shown that TLR4 deficiency in mice induces protection in splenocyte numbers which is mediated through PI3K. Collectively, our data suggest that TLR4 and PI3K/Akt signaling play a role in immune responses during stress. At present, we do not understand the precise role of TLR4-mediated signaling in stress-induced lymphocyte apoptosis, nor do we understand the precise role of TLR4 in negative regulation of the PI3K/Akt signaling in immune suppression during stress. Our hypothesis is that modulation of TLR4 and the PI3K/Akt signaling pathways determines the fate of lymphocytes in response to stress. We propose the following aims to test this hypothesis. Aim 1 will define the role of TLR4-mediated apoptotic signaling pathways following stress. To elucidate whether TLR4 activates apoptotic signaling pathways during stress, we will first investigate the effects of TLR4-mediated signaling on lymphocyte apoptosis, including CD4+ and CD8+ T cell apoptosis. We will then examine whether TLR4 deficiency activates PI3K/Akt signaling in stress-induced lymphocyte apoptosis. Aim 2 will define the role TLR4 negative regulation of PI3K/Akt signaling in stress-induced immune suppression. To examine whether TLR4 deficiency activates PI3K/Akt signaling, resulting in immune protection, TLR4 knockout mice and wild type mice will be administered PI3K inhibitors, and then subjected to stress for different time periods. We will define the role of TLR4 mediated signaling in stress-induced immune suppression, and then determine whether TLR4 deficiency results in activation of PI3K/Akt signaling. In addition, we will determine the role of HMGB1 and a multifunctional adaptor 2-arrestin 2 in TLR4-mediated PI3K/Akt signaling following stress. Our studies should lead to our understanding of the effects of TLR4-mediated signaling pathways on the immune system, providing the opportunity to develop novel strategies and therapeutics for immune suppression. PUBLIC HEALTH RELEVANCE: Psychological and physical stress can have pronounced effects on the immune system. Historically this affect was attributed to the release of stress hormones although other factors appear to play an important role. Using a mouse model of physical restraint stress we have investigated some of the molecular mediators which may account for the connection between stress and the immune system. This research will examine the mechanisms whereby the immune system is influenced by stress with the hope of leading to the development of specific treatments or drugs which in turn may ameliorate this sometimes deleterious interaction.