Role of TLR-4 in Lung Reperfusion Injury

申办方摘要 肺缺血再灌注损伤（LIRI）在15-25%的肺移植受者中发生，并导致 增强的MHC II类表达，急性移植物功能障碍，闭塞性细支气管炎的早期发作， 增加了患者死亡率。先天免疫在LIRI中的作用尚不清楚，尽管细菌产物 肺泡腔中存在的细菌可能会激活它。供体肺经常定植或有时轻度定植 感染，这是否应该促进或阻止他们用于移植仍然是一个问题。 LIRI的早期阶段与TNF-α和IL-1的定位增加相关，仅在 肺泡巨噬细胞（AM），其可允许AM活化其它细胞，包括2型肺细胞 (T2P)和肺动脉内皮细胞（PAEC）。鉴于这种反应的迅速性，这可能是一个信号， 激活炎症介质产生导致LIRI的事件。IRI的脑和血管模型 已经证明转录因子的激活受丝裂原活化蛋白激酶的调节 （MAPK），包括ERK 1/2和两种应激活化蛋白激酶（SAPK），JNK和p38。这些MAPK是 LIRI增加，原位模型显示p38和JNK抑制的保护作用。MAPK如何 激活仍然未知，尽管Toll样受体（TLR）是参与 先天免疫系统是一个可能的候选者。TLR-4会响应许多“警报”信号，包括LPS， TLR-2和TLR-4都是AM对多种刺激作出反应所必需的。TLR-4具有快速的 MyD 88依赖性信号通路，涉及TIRAP和TRAF-6，导致SAPK激活和更慢的 涉及TRIF/TRAM和TRAF-3的MyD 88非依赖性途径，导致1型干扰素应答， IL-10的产生，这已被证明是保护LIRI。虽然大剂量的内毒素会导致 急性肺损伤，低剂量LPS在许多IRI模型中具有保护作用，这种保护作用可能通过以下途径发生： 差异TLR-4衔接蛋白募集导致TRAF-3与TRAF-6相比相对增加。 我们的总体假设是，在LIRI中，氧化应激最初是通过AM中的TLR-4活化转导的， 这反过来又促进SAPK磷酸化，并导致非炎症细胞中促炎信号的放大。 AM细胞类型。此外，我们假设LPS预激活TLR-4可提供缺血耐受， 并通过接头蛋白的差异募集有效降低LIRI严重性。 在我们的第一个目标中，我们将确定在LIRI的大鼠模型中TLR-4的抑制是否具有保护作用， 与这种损伤相关的下游信号传导事件。我们将利用siRNA进行靶向分子敲除， TLR-4及其衔接蛋白TIRAP和TRIF，以确定它们在IRI中的作用。肺损伤的特征为： 血管通透性、炎性细胞浸润、组织学、SAPK活化、NF B易位和 炎症介质产生。我们的第二个目标是确定PAEC的促炎反应是否 而T2 P对氧化应激的反应是TLR-2、TLR-4或MyD 88依赖性的，并证明了AM产物的能力 使用体外培养基转移实验来增强这种反应。此外，我们还将评估 通过敲低TLR-4和MyD 88依赖性信号传导来预防AM活化， 二次细胞反应放大ERK 1/2激活、潜在TLR激活和衔接子的变化 将评估T2 P和PAEC中的蛋白质募集，以准确确定信号级联中的位置 这些增材制造产品正在发挥其影响力。我们的最终目标将集中在LPS预处理在 调节LIRI，我们认为这是通过MyD 88-独立TRAF-3信号转导的相对增加来解释的。 与MyD 88依赖的TRAF-6信号转导相比。大鼠将在注射前用肠内LPS预处理。 我们将评估肺损伤、TLR-4衔接蛋白募集和MAPK activation.我们还将使用TIRAP和TRIF的靶向分子敲低来确定信号传导， LPS诱导的缺血耐受性的途径。这将包括评估生产 炎症介质，以及1型干扰素反应和IL-10的产生。 从拟议的研究中获得的信息将有助于界定TLR-4的作用 细胞间信号的激活和AM调节以及LPS诱导缺血性脑损伤的机制 宽容这解决了一个重要的临床问题，并将提供有用的和容易翻译的 关于供体纳入标准和TLR-4信号传导调节的信息。 关于公共卫生相关性的声明： 肺移植是复杂的发展组织损伤移植肺后， 高达25%的患者出现血流重建，导致排斥反应和死亡率增加。本研究 将有助于了解与这种损伤相关的信号通路， 用于使用药理学试剂进行调节。此外，了解存在的意义， 考虑捐赠的肺部中的细菌可能对捐赠器官的利用有严重的影响。

Sponsor Abstract Lung ischemia reperfusion injury (LIRI) develops in 15-25% of lung transplant recipients and leads to enhanced MHC class II expression, acute graft dysfunction, earlier onset of bronchiolitis obliterans and increased recipient mortality. The role of innate immunity in LIRI is not yet known, though bacterial products present in the alveolar space would likely activate it. Donor lungs are frequently colonized or at times mildly infected and whether this should promote or discourage their use for transplantation remains a question. The early phase of LIRI correlates with increased TNF-¿ and IL-1¿ localization exclusively in the alveolar macrophage (AM) which may allow for AM activation of other cells including type 2 pneumocytes (T2P) and pulmonary artery endothelial cells (PAEC). Given the rapidity of this response, it is likely a signaling event that activates inflammatory mediator production leading to LIRI. Cerebral and vascular models of IRI have demonstrated that transcriptional factor activation is regulated by mitogen-activated protein kinases (MAPK), including ERK 1/2 and two stress-activated protein kinases (SAPK), JNK and p38. These MAPK are increased in LIRI, and in situ models demonstrate protection with p38 and JNK inhibition. How MAPK are activated remains unknown, though toll-like receptors (TLR) which are pattern recognition receptors involved in the innate immune system are a likely candidate. TLR-4 responds to numerous "alarm" signals, including LPS, and both TLR-2 and TLR-4 are required by AM to respond to a number of stimuli. TLR-4 has both a rapid MyD88-dependent signaling pathway, involving TIRAP and TRAF-6 leading to SAPK activation and a slower MyD88-independent pathway involving TRIF/TRAM and TRAF-3, leading to type 1 interferon responses and IL-10 production, which have been shown to be protective in LIRI. Though high-dose intratracheal LPS causes acute lung injury, low-dose LPS is protective in many IRI models and this protection likely occurs through differential TLR-4 adaptor protein recruitment resulting in a relative increase in TRAF-3 compared to TRAF-6. Our overall hypothesis is that in LIRI, oxidative stress is initially transduced via TLR-4 activation in the AM, which in turn promotes SAPK phosphorylation and leads to amplification of proinflammatory signaling in non- AM cell types. Additionally, we hypothesize that preactivation of TLR-4 with LPS provides ischemic tolerance and effectively reduces LIRI severity through differential recruitment of adaptor proteins. In our first aim, we will determine if inhibition of TLR-4 in a rat model of LIRI is protective and the downstream signaling events related to this injury. We will utilize siRNA for targeted molecular knockdown of TLR-4 and its adaptor proteins, TIRAP and TRIF, to define their role in IRI. Lung injury will be characterized by vascular permeability, inflammatory cell infiltration, histology, SAPK activation, NF¿B translocation and inflammatory mediator production. Our second aim will determine if the proinflammatory response of PAEC and T2P to oxidative stress is TLR-2, TLR-4, or MyD88 dependent and demonstrate the ability of AM products to augment this response using in vitro media transfer experiments. In addition, we will evaluate whether prevention of AM activation, with knockdown of TLR-4 and MyD88-dependent signaling, effectively eliminates secondary cell response amplification. Changes in ERK 1/2 activation, potential TLR activation and adaptor protein recruitment in T2P and PAEC will be assessed to determine precisely where in the signaling cascade these AM products are exerting their influence. Our final aim will focus on the role of LPS preconditioning in modulating LIRI which we believe is explained by a relative increase in MyD88-independent TRAF-3 signaling compared to MyD88-dependent TRAF-6 signaling. Rats will be pretreated with intratracheal LPS prior to ischemia and reperfusion and we will assess lung injury, TLR-4 adaptor protein recruitment and MAPK activation. We will also use targeted molecular knockdown of TIRAP and TRIF to determine the signaling pathway whereby LPS-induced ischemic tolerance is conferred. This will include assessment of the production of inflammatory mediators, as well as type 1 interferon responses and IL-10 production. The information garnered from the proposed studies will assist in delineating the role of TLR-4 activation and AM modulation of intercellular signaling as well as the mechanism of LPS-induced ischemic tolerance. This addresses an important clinical problem, and will provide useful and readily translatable information regarding donor inclusion criteria and modulation of TLR-4 signaling. Statement Regarding Relevance to Public Health: Lung transplantation is complicated by the development of tissue injury in the transplanted lung after reconstitution of blood flow in up to 25% of patients, leading to increased rejection and mortality. This research will help gain an understanding of the signaling pathways associated with this injury which may ultimately allow for modulation using pharmacologic agents. In addition, understanding the significance of the presence of bacteria in lungs being considered for donation may have serious implications on donor organ utilization.