Role of TLR-4 in Lung Reperfusion Injury

TLR-4 在肺再灌注损伤中的作用

• 批准号: 7729936
• 负责人: Michael Scott Mulligan
• 金额: $ 39万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-12 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729936
• 关键词: Accounting; Acute; Acute Lung Injury; Adaptor Signaling Protein; Address; Affect; Alveolar; Alveolar Macrophages; Animal Model; Bacteria; Blood Vessels; Blood flow; Brain Hypoxia-Ischemia; Bronchiolitis Obliterans; Cells; Cerebrum; Chemotactic Factors; Clinical; Development; Dose; Event; Excision; Functional disorder; Generations; Histology; Hypoxia; Immune system; In Situ; In Vitro; Individual; Infiltration; Inflammation Mediators; Inflammatory; Inflammatory Response; Injury; Interferons; Interleukin-1 beta; Interleukin-10; Ischemia; Knockout Mice; Lipopolysaccharides; Lung; Lung Transplantation; MAPK14 gene; MAPK8 gene; MHC Class II Genes; Macrophage Activation; Maps; Mediating; Mitogen-Activated Protein Kinases; Modeling; Molecular Target; Morbidity - disease rate; NF-kappa B; Natural Immunity; Organ Donor; Oxidants; Oxidative Stress; Pathway interactions; Patients; Pattern; Pattern recognition receptor; Phase; Phosphorylation; Prevention; Production; Rattus; Receptor Activation; Receptor Signaling; Regulation; Relative (related person); Reperfusion Injury; Reperfusion Therapy; Research; Role; Severities; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Specificity; Stimulus; TLR4 gene; TNF receptor-associated factor 3; Time; Tissues; Toll-Like Receptor 2; Toll-like receptors; Transplant Recipients; Transplantation; Tumor Necrosis Factor-alpha; Vascular Permeabilities; activation product; cell type; chemokine; cytokine; early onset; in vivo; inclusion criteria; insight; lung injury; lung ischemia; macrophage inflammatory protein 2; macrophage product; mortality; neutrophil; novel; pneumocyte; preconditioning; public health relevance; pulmonary artery endothelial cell; reconstitution; research study; response; stress activated protein kinase; toll-like receptor 4

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.

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II类MHC表达增强、急性移植物功能障碍、闭塞性细支气管炎早期发作和受者死亡率增加。先天免疫在LIRI中的作用尚不清楚，尽管肺泡腔内存在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，这是否应该促进或阻止其用于移植仍然是一个问题。LIRI的早期阶段与仅在肺泡巨噬细胞（AM）中增加的TNF-α和IL-1 β定位相关，这可能允许包括2型肺细胞（T2 P）和肺动脉内皮细胞（PAEC）的其它细胞的AM活化。考虑到这种反应的快速性，它可能是激活炎症介质产生的信号传导事件，导致LIRI。IRI的脑和血管模型已经证明，转录因子激活受丝裂原活化蛋白激酶（MAPK）调节，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，并且原位模型证明了p38和JNK抑制的保护作用。MAPK如何被激活仍然是未知的，尽管参与先天免疫系统的模式识别受体Toll样受体（TLR）是一个可能的候选者。TLR-4对包括LPS在内的许多“警报”信号做出响应，AM需要TLR-2和TLR-4对许多刺激做出响应。TLR-4具有快速的MyD 88依赖性信号传导途径，涉及TIRAP和TRAF-6，导致SAPK活化，以及较慢的MyD 88非依赖性途径，涉及TRIF/TRAM和TRAF-3，导致1型干扰素应答和IL-10产生，其已显示在LIRI中具有保护性。虽然高剂量的气管内LPS导致急性肺损伤，但低剂量的LPS在许多IRI模型中具有保护作用，并且这种保护可能通过差异TLR-4衔接蛋白募集而发生，导致TRAF-3与TRAF-6相比相对增加。我们的总体假设是，在LIRI中，氧化应激最初通过AM中的TLR-4活化转导，这反过来促进SAPK磷酸化并导致非AM细胞类型中促炎信号的放大。此外，我们假设TLR-4与LPS的预激活提供缺血耐受，并通过接头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，我们将确定在LIRI大鼠模型中抑制TLR-4是否具有保护作用，以及与这种损伤相关的下游信号传导事件。我们将利用siRNA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。肺损伤的特征是血管通透性、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和T2 P对氧化应激的促炎反应是否是TLR-2、TLR-4或MyD 88依赖性的，并使用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，我们将评估是否预防AM激活，与TLR-4和MyD 88依赖性信号的敲低，有效地消除二次细胞反应放大。将评估T2 P和PAEC中ERK 1/2活化、潜在TLR活化和衔接蛋白募集的变化，以精确确定这些AM产物在信号级联中的何处发挥其影响。我们的最终目标将集中在LPS预处理在调节LIRI中的作用，我们认为这是通过与MyD 88依赖性TRAF-6信号相比，MyD 88依赖性TRAF-3信号相对增加来解释的。在缺血和再灌注之前，将用气管内LPS预处理大鼠，并且我们将评估肺损伤、TLR-4衔接蛋白募集和MAPK活化。我们还将使用TIRAP和TRIF的靶向分子敲低来确定LPS诱导的缺血耐受被赋予的信号传导途径。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绘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，并将提供有用的和容易翻译的信息，供体纳入标准和TLR-4信号转导的调制。公共卫生相关性：在高达25%的患者中，移植肺在血流重建后发生组织损伤，导致排斥反应和死亡率增加，从而使肺移植变得复杂。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理学药物进行调节。此外，了解考虑捐赠的肺部中存在细菌的重要性可能对供体器官的利用产生严重影响。肺缺血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如何发展，并确定新的治疗靶点。此外，可接受的供体肺的短缺仍然是肺移植的重要限制因素。预处理的研究将为安全地扩大可接受供体器官的标准提供见解。