Mechanisms of MCP-1 in lung contusion with and without gastric aspiration

描述(申请人提供)：肺挫伤是机动车事故和爆炸伤受害者的常见损伤。也是ALI、ARDS和呼吸机相关性肺炎发生的独立危险因素。根据我们在动物身上的初步研究，它在临床上经常与创伤时的胃吸入有关，这会进一步增加肺损伤的严重程度和ALI/ARDS的风险。目前的方案研究了孤立性肺挫伤和肺挫伤伴胃抽吸的小鼠模型。重点是从机制上了解MCP-1和MCP-5在这些损伤的单独和联合发病机制中的保护作用，包括这种趋化因子在促进肺泡巨噬细胞的募集和激活以及随后对凋亡的中性粒细胞的吞噬中的特定作用。这笔赠款有三个具体目标。目的1研究单核细胞趋化蛋白-1(MCP-1)、单核细胞趋化蛋白-5(MCP-5)或受体蛋白CCR2缺失的基因敲除小鼠肺挫伤后的肺损伤和先天肺部炎症，以及给予这些分子抗体的野生型小鼠肺挫伤的肺损伤和先天肺部炎症反应。这些体内研究的补充是额外的实验，其中分离的肺泡巨噬细胞在体外检查表型参数、吞噬能力和细胞特异性炎症介质特征。此外，通过使用骨髓来源的嵌合体，对肺挫伤中主要负责产生MCP-1的细胞的来源进行了调查。此外，还研究了TLR9与细胞内接头蛋白myd 88作为中介在识别损伤以及随后激活和形成肺泡巨噬细胞MCP-1过程中的重要性。目的2采用与目的1类似的研究，明确单核细胞趋化蛋白-1和单核细胞趋化蛋白-5在肺挫伤胃联合损伤模型中的具体活性和作用机制。趋化因子活性与肺泡巨噬细胞活性的确切机制以及TLR9在产生MCP-1或MCP-5中的作用与目标1相似。目的3检测通过直接滴注和通过电穿孔促进含有MCP-1的DNA质粒到MCP-1(-/-)肺挫伤和胃吸入肺挫伤后野生型小鼠肺内的作用。后两项研究扩展了目标1和目标2，以进一步证明单核细胞趋化蛋白-1在有和没有胃抽吸的肺挫伤中的功能意义，同时调查基于这种趋化因子或相关药物的肺部递送的潜在未来治疗的有效性。 公共卫生相关性：这笔赠款旨在提高对肺挫伤和胃吸入的机制的了解，这两种重要的人类肺部疾病(肺损伤)经常与钝性胸部创伤一起发生。该提案中的研究研究这些疾病的肺部炎症反应中特定物质的行为，特别是影响巨噬细胞等重要炎症细胞的活动和募集的趋化因子MCP-1和MCP-5。这笔拨款不仅研究了早期增加MCP-1和MCP-5可能有助于及时解决有或不有胃吸入性肺挫伤所致急性炎症损伤的机制，还调查了相关药理学和基因输送方法的效果，为未来可能对这些重要肺部疾病进行基于趋化因子的药物或基因治疗奠定了基础。

DESCRIPTION (provided by applicant): Lung contusion is a common injury sustained by victims of motor vehicular accidents and blast trauma. It is also an independent risk factor for the development of ALI, ARDS and ventilator associated pneumonia. It is frequently associated clinically with gastric aspiration at the time of trauma that can further increase the severity of lung injury and the risk of ALI/ARDS based on our preliminary studies in animals. The current proposal studies murine models of both isolated lung contusion and lung contusion with gastric aspiration. Emphasis is on mechanistic understanding of the protective roles of MCP-1and MCP-5 in the pathogenesis of these injuries alone and in combination, including specific effects of this chemokine in facilitating the recruitment and activation of alveolar macrophages and the subsequent phagocytosis of apoptotic neutrophils. The grant has three Specific Aims. Aim 1 investigates lung injury and innate pulmonary inflammation in lung contusion in knockout mice lacking MCP-1, MCP-5 or the receptor protein CCR2, as well as in wild type mice given antibodies against these molecules. These in vivo studies are complemented by added experiments where isolated alveolar macrophages are examined ex vivo for phenotypic parameters, phagocytic potential, and cell-specific inflammatory mediator profile. Additionally, an investigation of origin of cells primarily responsible for the production of MCP-1 in lung contusion through the use of bone marrow-derived chimeras is proposed. In addition, the importance of TLR9 with the intracellular adaptor protein MyD 88 as an intermediate in the recognition of injury and the subsequent activation and elaboration of MCP-1 from alveolar macrophages is investigated. Aim 2 involves analogous studies to those in Aim 1 to define the detailed activities and mechanisms of action of MCP-1 and MCP-5 in the combined injury model of lung contusion with gastric aspiration. The precise mechanisms of activity of chemokines with alveolar macrophage activity and role of TLR9 in the production of MCP-1 or MCP-5 are similar to aim 1. Aim 3 examines the effects of delivering exogenous MCP-1 to the lungs via direct instillation and by electroporation-facilitated gene delivery of DNA plasmids containing MCP-1 to MCP-1 (-/-) and wild type mice following lung contusion and lung contusion with gastric aspiration. These latter studies extend Aim 1 and 2 to further document the functional significance of MCP-1 in lung contusion with and without gastric aspiration, while at the same time investigating the efficacy of potential future therapies based on the pulmonary delivery of this chemokine or related agents. PUBLIC HEALTH RELEVANCE: This grant seeks to improve understanding about mechanisms contributing to lung contusion and gastric aspiration, two important human pulmonary diseases (lung injuries) that frequently occur in conjunction with blunt chest trauma. Research in the proposal studies the behavior of specific substances in the pulmonary inflammatory response in these diseases, particularly the chemokines MCP-1 and MCP-5 that affect the activity and recruitment of important inflammatory cells such as macrophages. The grant not only studies mechanisms by which early increases in MCP-1 and MCP-5 may aid the timely resolution of acute inflammatory injury during lung contusion with or without gastric aspiration, but also investigates the effects of related pharmacologic and gene delivery approaches to lay the groundwork for possible future chemokine- based drug or gene therapies for these important lung diseases.