Role of TLR-4 in Lung Reperfusion Injury

描述（由申请人提供）：15-25%的肺移植受者发生肺缺血再灌注损伤（LIRI），导致MHC II类表达增强、急性移植物功能障碍、闭塞性细支气管炎早期发病和受者死亡率增加。先天免疫在LIRI中的作用尚不清楚，尽管存在于肺泡间隙的细菌产物可能会激活它。供体肺经常定植或有时轻度感染，这是否应促进或阻碍其用于移植仍是一个问题。LIRI的早期阶段与肺泡巨噬细胞（AM）中TNF-a和IL-1定位的增加相关，这可能允许AM激活其他细胞，包括2型肺细胞（T2P）和肺动脉内皮细胞（PAEC）。考虑到这种反应的快速性，这可能是一个激活炎症介质产生导致LIRI的信号事件。IRI的脑血管模型表明，转录因子的激活受丝裂原活化蛋白激酶（MAPK）的调控，包括ERK 1/2和两种应激活化蛋白激酶（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是如何被激活的仍然未知，尽管toll样受体（TLR）是一种可能的候选受体，它是先天免疫系统中涉及的模式识别受体。TLR-4响应许多“警报”信号，包括LPS，而AM需要TLR-2和TLR-4来响应许多刺激。TLR-4具有快速的myd88依赖性信号通路，包括TIRAP和TRAF-6导致SAPK激活，以及较慢的myd88依赖性信号通路，包括TRIF/TRAM和TRAF-3，导致1型干扰素应答和IL-10的产生，这已被证明对LIRI具有保护作用。尽管高剂量气管内脂多糖会导致急性肺损伤，但在许多IRI模型中，低剂量脂多糖具有保护作用，这种保护可能是通过TLR-4接头蛋白的不同募集而发生的，导致TRAF-3相对于TRAF-6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激活转导的，这反过来促进SAPK磷酸化，并导致非AM细胞类型的促炎信号放大。此外，我们假设用LPS预激活TLR-4可以提供缺血耐受性，并通过受体蛋白的差异募集有效降低LIRI的严重程度。在我们的第一个目标中，我们将确定TLR-4在大鼠LIRI模型中的抑制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靶向敲低TLR-4及其接头蛋白TIRAP和TRIF，以确定它们在IRI中的作用。肺损伤以血管通透性、炎症细胞浸润、组织学、SAPK活化、NF？B易位和炎症介质的产生。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，并通过体外培养基转移实验证明AM产品增强这种反应的能力。此外，我们将评估通过抑制TLR-4和myd88依赖的信号传导来预防AM活化是否能有效地消除次级细胞反应放大。将评估T2P和PAEC中ERK 1/2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产品在信号级联中发挥其影响的位置。我们的最终目标将集中在LPS预处理在调节LIRI中的作用，我们认为这是由myd88独立的TRAF-3信号相对于myd88依赖的TRAF-6信号的相对增加来解释的。我们将在缺血和再灌注前对大鼠进行气管内LPS预处理，并评估肺损伤、TLR-4接头蛋白募集和MAPK激活。我们还将使用靶向敲低TIRAP和TRIF的分子来确定lps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。从拟议的研究中获得的信息将有助于描述TLR-4激活和AM调节细胞间信号的作用，以及lps诱导的缺血耐受的机制。这解决了一个重要的临床问题，并将提供有关供体纳入标准和TLR-4信号调节的有用且易于翻译的信息。公共卫生相关性：高达25%的患者在血流重建后，由于移植肺组织损伤的发展，肺移植变得复杂，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，这可能最终允许使用药物进行调节。此外，了解被考虑捐献的肺中细菌存在的重要性可能对供体器官的利用有严重的影响。肺化学再灌注损伤仍然是移植后的一个重要问题，导致移植后发病率和死亡率增加，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。对预处理的研究将为安全扩展可接受供体器官的标准提供见解。

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.