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Role of TLR-4 in Lung Reperfusion Injury

TLR-4 在肺再灌注损伤中的作用

基本信息

批准号：
8514044
负责人：
Michael Scott Mulligan
金额：
$ 36.76万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-12 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8514044
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 Histology Hypoxia Immune system In Situ In Vitro Individual Infiltration Inflammation Mediators Inflammatory Injury Interferons Interleukin-1 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 Kinase 3 Mitogen-Activated Protein Kinases Modeling Molecular Target Morbidity - disease rate NF-kappa B Natural Immunity Organ Donor Oxidants Oxidative Stress Pathway interactions Patients Pattern recognition receptor Phase Phosphorylation Prevention Production Public Health 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 gene TNF receptor-associated factor 3 Time Tissues Toll-Like Receptor 2 Toll-like receptors Transplant Recipients Transplantation Tumor Necrosis Factor-alpha Vascular Permeabilities abstracting 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 pulmonary artery endothelial cell reconstitution research study response stress activated protein kinase toll-like receptor 4

项目摘要

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.

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