Role of TLR-4 in Lung Reperfusion Injury

描述(由申请人提供)：肺缺血再灌注损伤(LIRI)发生在15%-25%的肺移植受者中，并导致MHC II类表达增强，急性移植物功能障碍，闭塞性细支气管炎的更早发病，以及受者死亡率的增加。先天免疫在LIRI中的作用尚不清楚，尽管肺泡腔中存在的细菌产物可能会激活它。供体肺经常被定植，有时受到轻微感染，这是否应该促进或阻止它们用于移植仍然是一个问题。LIRI的早期与肺泡巨噬细胞(AM)内的肿瘤坏死因子-α和IL-1β的定位增加有关，这可能允许肺泡巨噬细胞激活其他细胞，包括2型肺泡细胞(T2P)和肺动脉内皮细胞(PAEC)。鉴于这种反应的快速，这很可能是一个信号事件，激活了炎症介质的产生，导致LIRI。脑和血管模型已经证明，转录因子的激活受丝裂原激活的蛋白激酶(MAPK)的调节，包括ERK1/2和两个应激激活的蛋白激酶(SAPK)：JNK和p38。这些MAPK在LIRI中增加，原位模型显示p38和JNK抑制具有保护作用。MAPK是如何被激活的尚不清楚，尽管参与先天性免疫系统的模式识别受体Toll样受体(TLR)可能是一个候选。TLR-4对包括内毒素在内的许多“警报”信号作出反应，AM需要TLR-2和TLR-4来对一些刺激作出反应。TLR-4既有一个快速的MyD88依赖的信号通路，涉及TIRAP和TRAF-6，导致SAPK激活，也有一个较慢的MyD88依赖的信号通路，涉及TRIF/TRAM和TRAF-3，导致1型干扰素反应和IL-10的产生，这已被证明在LIRI中具有保护作用。虽然大剂量气管内注射内毒素可导致急性肺损伤，但小剂量内毒素在许多IRI模型中具有保护作用，这种保护作用可能是通过不同的TLR-4接头蛋白募集而实现的，导致TRAF-3的表达相对高于TRAF-6。我们的总体假设是，在LIRI中，氧化应激最初是通过AM中TLR-4的激活而转导的，这反过来又促进SAPK磷酸化，并导致非AM细胞类型的促炎信号的放大。此外，我们假设TLR-4与脂多糖一起预激活提供了缺血耐受性，并通过适配器蛋白的不同招募有效地降低了LIRI的严重程度。在我们的第一个目标中，我们将确定抑制TLR-4在LIRI大鼠模型中是否具有保护作用，以及与这种损伤相关的下游信号事件。我们将利用siRNA靶向分子敲除TLR-4及其适配蛋白TIRAP和TRIF，以确定它们在IRI中的作用。肺损伤以血管通透性、炎性细胞浸润、组织学、SAPK活化、核因子B移位和炎性介质产生为特征。我们的第二个目标是确定PAEC和T2P对氧化应激的促炎反应是否依赖于TLR-2、TLR-4或MyD88，并通过体外介质转移实验证明AM产品增强这一反应的能力。此外，我们将评估通过抑制TLR-4和MyD88依赖的信号通路来阻止AM激活是否有效地消除二级细胞反应放大。将评估T2P和PAEC中ERK1/2激活、潜在TLR激活和接头蛋白募集的变化，以准确确定这些AM产物在信号级联中发挥影响的位置。我们的最终目标将集中在脂多糖预适应在调节LIRI中的作用，我们认为这是因为与MyD88依赖的TRAF-6信号相比，MyD88依赖的TRAF-3信号相对增加。大鼠在缺血和再灌注前气管内注射脂多糖，我们将评估肺损伤、TLR-4接头蛋白募集和MAPK激活。我们还将使用TIRAP和TRIF的靶向分子敲除来确定内毒素诱导的缺血耐受的信号通路。这将包括评估炎症介质的产生，以及1型干扰素反应和IL-10的产生。从这些研究中获得的信息将有助于阐明TLR-4激活和AM在细胞间信号转导中的作用以及内毒素诱导缺血耐受的机制。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信息，关于供体纳入标准和TLR-4信号的调节。公共卫生相关性：在高达25%的患者中，在血流重建后，肺移植并发移植肺的组织损伤，导致排斥反应和死亡率增加。这项研究将有助于了解与这种损伤相关的信号通路，最终可能允许使用药理学试剂进行调节。此外，了解考虑捐献时肺部细菌存在的重要性可能会对捐献器官的利用产生严重影响。项目简介肺缺血再灌注损伤在移植后仍然是一个重要的问题，占移植后发病率和死亡率增加的原因，这些研究将确定损伤是如何发展的，并确定新的治疗靶点。此外，缺乏可接受的供体肺仍然是肺移植的一个重要限制因素。有关预适应的研究将为安全地扩大可接受供体器官的标准提供洞察力。

DESCRIPTION (provided by applicant): 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-a 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. PUBLIC HEALTH RELEVANCE: 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. Project Narrative Lung is chemia reperfusion injury continues to be a significant problem after transplantation, accounting for increased morbidity and mortality after transplantation, these studies will identify how injury develops and identify novel targets for therapy. Additionally, a shortage of acceptable donor lungs continues to be a significant limiting factor in lung transplantation. The studies on preconditioning will provide insight into safely expanding the criteria for acceptable donor organs.