Lung epithelium collaborates with alveolar macrophages in host defense

描述(由申请人提供)：铜绿假单胞菌(PA)经常感染免疫功能低下的人，感染艾滋病毒-1、癌症和囊性纤维化(CF)。由于PA对抗生素的抗药性越来越强，它的感染通常会导致严重的状态或慢性 持续炎症反应的情况。更好地了解寄主-病原体的相互作用可能会提出一种更有效的方法来对抗这种病原体。单核细胞趋化蛋白-1(MCP-1)是肺泡II型上皮细胞(AECII)分泌的主要趋化因子。最近的研究已经阐明了AECII在PA感染中的免疫作用，但其潜在的机制仍不清楚。我们的长期目标是了解宿主免疫的机制，并开发控制呼吸道感染的新策略。这项应用的目的是阐明AECII的免疫功能，特别是通过它们分泌细胞因子和激活AM我们的中心假设是AECII可以分泌细胞因子(MCP-1)，通过脂筏介导的机制增强AM的抗细菌免疫。我们根据最近的发现提出了这一假设，即AECII和AM都参与了对PA的先天免疫。我们进一步发现，膜脂筏可能有助于调节细胞因子的分泌。使用我们的原代细胞模型，我们发现AECII在使用条件AECII培养基来增强AM的免疫力方面具有免疫作用。我们的数据还表明，AECII通过分泌MCP-1和募集经典激活的巨噬细胞(CAM)在PA感染中发挥关键作用。其基本原理是，阐明AECII如何增强AM免疫将表明一种潜在的策略，以增强对PA的免疫。我们的实验室非常适合这项研究，在AECII a分离和培养以及肺部感染模型方面拥有相关专业知识。我们提出了以下三个特定目标：特定目标1：明确AECII细胞在PA感染过程中分泌细胞因子的免疫作用。我们将通过与AECII标记SPC的原位杂交来鉴定MCP-1的来源。我们还将使用AECII原代培养来显示MCP-1是主要的细胞因子。此外，将检测来自MCP-1-/-小鼠的AM和AECII对PA感染的免疫功能降低。我们将确定AECII招募经典激活的巨噬细胞(CAM)的能力。具体目标2：评估脂筏如何调节AECII中MCP-1的分泌。我们将研究MCP-1分泌的潜在机制，并有望确定富含神经酰胺的膜微域的参与。酸性髓鞘磷脂酶将被siRNA和化学抑制剂阻断，用于分析PA感染早期的鞘脂水解酶。特定目标3：评估超AECII高表达MCP-1在增强人AM抗PA能力方面的潜力我们将使用逆转录病毒载体创建超级AECII，以分泌高水平的MCP-1，并测试其在PA感染中的宿主防御能力。我们还将证明人类AM可以被AECII激活，这项翻译研究可能暗示免疫AECII的临床价值。这项研究将由研究生和本科生进行。我们的努力有望极大地促进对AECII激活AM这一先前未知的免疫功能的了解，并可能为细胞因子分泌机制提供见解，并为治疗这种感染的新疗法的开发提供指征。 公共卫生相关性：铜绿假单胞菌(PA)是一种引起严重感染的细菌，尤其是在患有结核病、癌症、艾滋病、严重烧伤和囊性纤维化的免疫缺陷患者中。由于PA对抗生素的抗药性越来越强，其感染通常会导致持续炎症反应的慢性状态。我们已经有了令人惊讶的发现，MCP-1，一种来自肺泡上皮细胞的多功能细胞因子，调节 PA感染中的宿主防御和炎症反应。我们还注意到，脂筏对于调节细胞因子的产生可能是重要的。通过分泌MCP-1，肺泡上皮细胞可以招募特定的巨噬细胞亚群(即经典激活的巨噬细胞)来迅速对感染做出反应。研究肺泡上皮细胞的免疫作用可能为开发治疗PA感染的新方法提供新的见解。

DESCRIPTION (provided by applicant): P. aeruginosa (PA) frequently infects immunocompromised individuals with HIV-1, cancer, and cystic fibrosis (CF). Since PA is increasingly resistant to antibiotics, its infection often leads to either severe states or chronic situations with a persistent inflammatory response. Better understanding of host-pathogen interaction may suggest a more effective approach to combating this pathogen. MCP-1 is a major chemokine secreted by alveolar epithelial cells type II (AECII). Recent research has illustrated an immune role of AECII in PA infection, but the underlying mechanism remains unidentified. Our long-term goal is to understand the mechanism of host immunity and develop new strategies for controlling respiratory infections. The objective of this application is to elucidate the immune function of AECII, in particular through their secretion of cytokines and activation of AM. Our central hypothesis is that AECII can secrete cytokines (MCP-1) to enhance AM's anti-bacterial immunity through a lipid raft- mediated mechanism. We have formulated this hypothesis based on our recent findings that both AECII and AM participate in innate immunity against PA. We further found that membrane lipid rafts may be instrumental for regulating cytokine secretion. Using our primary cell model, we have discovered an immune role of AECII in enhancing AM's immunity using a conditioned AECII medium. Our data also suggest that AECII play a critical role in PA infection by secreting MCP-1 and recruiting the classically activated macrophages (CAM). The rationale is that elucidating how AECII enhance AM immunity will indicate a potential strategy to bolster immunity against PA. Our laboratory is ideally suited for this research, having the relevant expertise in isolation and culture of AECII a well as in lung infection models. We propose the following three specific aims: Specific Aim 1: Define the immune role of AECII cells in secreting cytokines during PA infection. We will identify the source of MCP-1 using in situ hybridization with AECII marker SPC. We will also use primary AECII culture to show MCP-1 as a dominant cytokine. Furthermore, AM and AECII from MCP-1-/- mice will be examined for their decreased immune function against PA infection. We will determine the ability of AECII in recruiting the classically activated macrophages (CAM). Specific Aim 2: Evaluate how lipid rafts regulate MCP-1 secretion in AECII. We will study the underlying mechanism for MCP-1 secretion and hopefully identify the involvement of ceramide-rich membrane microdomains. Acid shingomyelinase will be blocked by siRNA and chemical inhibitors for analyzing sphingolipid hydrolysis during PA early infection. Specific Aim 3: Assess the potential of super-AECII over-expressing MCP-1 in enhancing anti-PA capacity of human AM. We will create super-AECII using retroviral vectors to secrete high levels of MCP-1 and test their host defense in PA infection. We will also demonstrate that human AM can be activated by AECII and that this translational research may imply the clinical value of the immune AECII. This research will be performed by graduate and undergraduate students. Our efforts are expected to substantially advance understanding of this previously unrecognized immune function of AECII in activating AM, and may provide insights into mechanisms of cytokine secretion, with indications in development of novel therapeutics for treating this infection. PUBLIC HEALTH RELEVANCE: P. aeruginosa (PA) is a bacterium that causes severe infections, particularly in immunodeficient individuals who are suffering tuberculosis, cancer, AIDS, severe burns, and cystic fibrosis. Because PA is increasingly resistant to antibiotics, its infection usually leads to a chronic state of persistent inflammatory response. We have made the surprising discovery that MCP-1, a versatile cytokine from alveolar epithelial cells, regulates host defense and inflammatory response in PA infection. We have also noted that lipid rafts may be important for regulating cytokine production. Through secretion of MCP-1, the alveolar epithelial cells may recruit a particular subset of macrophages (i.e., classically activated macrophages) to promptly respond to infection. Studying the immune role of alveolar epithelial cells may provide new insights into the development of novel treatment for PA infection.