Role of TLR-4 in Lung Reperfusion Injury

赞助商摘要 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，闭塞性毛细支气管炎和 接受者死亡率增加。先天免疫在Liri中的作用尚不清楚，尽管细菌产物 出现在肺泡腔中可能会激活它。供体肺经常被定植或有时很轻微。 感染以及这是否应该促进或阻止它们用于移植仍然是一个问题。 LIRI的早期阶段与肿瘤坏死因子-β和白介素1-β的定位增加有关 肺泡巨噬细胞(AM)，这可能允许AM激活包括2型肺细胞在内的其他细胞 (T2P)和肺动脉内皮细胞(PAEC)。鉴于这一反应的速度，这很可能是一个信号 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是由丝裂原激活的蛋白激酶调节的。 (MAPK)，包括ERK1/2和两个应激激活蛋白激酶(SAPK)：JNK和p38。这些MAPK是 在LIRI中增加，原位模型显示p38和JNK抑制具有保护作用。MAPK是怎样的 激活仍然未知，尽管Toll样受体(TLR)是模式识别受体，参与了 先天免疫系统是一个可能的候选者。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来对多种刺激做出反应。TLR-4既有快速的 MyD88依赖的信号通路，涉及TIRAP和TRAF-6，导致SAPK激活和缓慢 涉及TRIF/TRAM和TRAF-3的MyD88非依赖通路，导致1型干扰素反应和 IL-10的产生，已被证明在Liri具有保护作用。尽管大剂量气管内注射内毒素会导致 急性肺损伤，小剂量内毒素在许多IRI模型中具有保护作用，这种保护可能通过 不同的TLR-4接头蛋白募集导致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TLR-4的激活来转导的， 进而促进SAPK的磷酸化，并导致非炎症信号的放大 AM细胞类型。此外，我们假设，内毒素预激活TLR-4可提供缺血耐受性 并通过不同的接头蛋白招募有效地降低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 与这一损伤相关的下游信号事件。我们将利用siRNA进行靶向的分子敲除 TLR-4及其接头蛋白TIRAP和TRIF，以确定它们在IRI中的作用。肺损伤的特点是 血管通透性、炎性细胞浸润、组织学、SAPK激活、核因子B移位和 炎症介质的产生。我们的第二个目标将确定PAEC的促炎反应 T2P对氧化应激具有TLR-2、TLR-4或MyD88依赖关系，说明AM产物的氧化应激能力 通过体外介质转移实验来增强这种反应。此外，我们将评估是否 通过敲除TLR-4和MyD88依赖的信号，防止AM激活，有效地消除 次级细胞反应扩增。ERK 1/2激活、潜在TLR激活和接头的变化 将评估T2P和PAEC中的蛋白质募集，以准确确定信号级联中的什么位置 这些AM产品正在发挥他们的影响力。我们的最终目标将集中在内毒素预适应在 我们认为，通过MyD88非依赖性TRAF-3信号的相对增加来解释LIRI的调制 与依赖MyD88的TRAF-6信号相比。大鼠将在气管内注射脂多糖进行预处理 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信号转导 内毒素诱导的缺血耐受途径被授予。这将包括对生产的评估 炎症介质以及1型干扰素反应和IL-10的产生。 从拟议研究中获得的信息将有助于描述TLR-4的作用 细胞间信号的激活和AM调控及内毒素诱导脑缺血的机制 宽容。这解决了一个重要的临床问题，并将提供有用的和易于翻译的 关于供体纳入标准和TLR-4信号调制的信息。 与公共卫生相关的声明： 肺移植术后并发移植肺组织损伤 高达25%的患者血流重建，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，最终可能会 使用药理制剂进行调节。此外，了解存在的意义 被考虑捐献的肺部细菌可能会对捐赠者的器官利用产生严重影响。

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.