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

描述（由申请人提供）：我们这些研究的总体目标是阐明应激过程中toll样受体（TLR）介导的免疫抑制机制。生理和心理压力可以改变人类和动物的免疫系统。压力也是许多人类疾病的已知风险因素。TLR介导的信号通路在免疫应答的诱导中起关键作用。tlr介导应激免疫反应；然而，其机制仍有待阐明。为了研究应激诱导的免疫反应机制和治疗策略，我们建立了约束应激动物模型来研究免疫抑制。利用该模型，我们揭示了小鼠的约束应激通过细胞凋亡机制调节免疫系统。我们发现TLR4缺陷小鼠对应激诱导的脾细胞减少具有抗性。TLR4通常与高迁移率组盒-1蛋白（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。磷酸肌肽3-激酶（PI3K）及其下游底物Akt可以通过减少炎症和抑制细胞凋亡来调节tlr介导的反应。我们已经证明，TLR4在小鼠中缺乏可通过PI3K介导脾细胞数量的保护。总的来说，我们的数据表明TLR4和PI3K/Akt信号在应激时的免疫反应中发挥作用。目前，我们尚不清楚TLR4介导的信号通路在应激诱导淋巴细胞凋亡中的确切作用，也不清楚TLR4在应激时负调控PI3K/Akt信号通路在免疫抑制中的确切作用。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。我们提出以下目标来检验这一假设。目的1将定义应激后tlr4介导的凋亡信号通路的作用。为了阐明TLR4是否在应激状态下激活凋亡信号通路，我们将首先研究TLR4介导的信号通路对淋巴细胞凋亡的影响，包括CD4+和CD8+ T细胞凋亡。接下来，我们将研究TLR4缺陷是否激活应激诱导淋巴细胞凋亡中的PI3K/Akt信号。目的2将明确TLR4负调控PI3K/Akt信号在应激诱导免疫抑制中的作用。为了研究TLR4缺失是否激活PI3K/Akt信号从而产生免疫保护作用，我们将TLR4敲除小鼠和野生型小鼠分别给予PI3K抑制剂，然后进行不同时间的应激。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，我们将确定HMGB1和多功能适配器2-抑制蛋白2在应激后tlr4介导的PI3K/Akt信号传导中的作用。我们的研究将引导我们了解tlr4介导的信号通路对免疫系统的影响，为开发免疫抑制的新策略和治疗方法提供机会。

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.