Ceramide membrane microdomains regulate cytokine secretion

描述(申请人提供)：虽然肺泡上皮II型细胞(AECII)形成了肺泡腔的屏障，并产生表面活性物质来维持肺的完整性，但肺中独特的AECII群体也可能通过分泌细胞因子，如抗铜绿假单胞菌(PA)感染的单核细胞化学吸引蛋白(MCP-1)，在抗微生物免疫中发挥关键作用。然而，细胞因子的分泌机制在很大程度上还不清楚。我们的长期目标是了解肺宿主防御机制，从而确定治疗细菌感染的新策略。我们这项申请的目的是为了刻画 AECII分泌细胞因子。我们假设AECII通过分泌细胞因子发挥免疫作用，而细胞因子是通过膜微域重组机制来调节的。基于我们最近的研究和两条不同的证据，我们提出了这一假设。首先，在高纯度的细胞群中，我们发现来自PA感染的原代AECII的条件培养液增强了AM免疫。另一方面，脂筏(膜微域)对于先天免疫的重要性已被证明，尤其是处于PA感染风险的CF患者，由于神经酰胺缺乏和脂肪酸失衡，经常遭受脂筏功能异常的影响。与此一致，我们发现脂筏可能参与细胞因子MCP-1的分泌，这种细胞因子定位于富含神经酰胺的筏子。我们的理论基础是，阐明富含神经酰胺的微区中的相关机制将定义细胞因子分泌的一般机制。我们的实验室非常适合这项研究，因为我们在AECII分离、PA感染和先进的生化技术方面拥有必要的专业知识。为了验证我们的假设，我们提出了以下两个特定目标：特定目标1：利用成像工具表征AECII和小鼠体内调节细胞因子分泌的膜微域的动态重组。我们的工作假设是，PA感染启动了膜微域的动态变化，从而影响细胞因子的产生。具体目的2：明确膜微区调节PA防御所需的关键细胞因子的机制。我们的假设是神经酰胺是通过水解产生的，并被转移到特定的区域，在那里它启动了产生MCP-1的信号转导。由于细胞因子在各种生理和病理条件下的重要作用，这些新的机制将提高对其他类型细胞、病原体和炎症情况下细胞因子分泌的了解，从而确定新的治疗靶点。 公共卫生相关性：虽然肺泡上皮II型细胞(AECII)是肺中的结构细胞和前体细胞，但它们也通过分泌细胞因子来提供免疫防御。这种独特的免疫力可能极大地增强宿主对铜绿假单胞菌(PA)的抵抗力，PA是一种在免疫缺陷个体中导致严重感染的细菌，如艾滋病、结核病和囊性纤维化。我们假设AECII通过细胞膜微区的变化来分泌细胞因子，这是一种可能影响许多疾病过程的新机制。因此，我们的研究将为开发新的治疗这种反复感染的方法提供新的见解。

DESCRIPTION (provided by applicant): Although alveolar epithelial type II cells (AECII) form the barrier of alveolar spaces and produce surfactants to maintain lung integrity, the unique AECII population in the lung may also play a critical role in anti-microbial immunity by secreting cytokines, such as monocyte chemoattractant protein (MCP-1) against P. aeruginosa (PA) infection. However, the mechanism of cytokine secretion is largely unknown. Our long-term goal is to understand mechanisms of lung host defense, thereby identifying new strategies for treating bacterial infection. Our objective of this application is to characterize the mechanism of cytokine secretion by AECII. We hypothesize that AECII play immune roles by secreting cytokines, which is regulated through a mechanism of membrane microdomain reorganization. We have formulated this hypothesis based on our recent studies and two separate lines of evidence. First, in a high purity cell population, we found that a conditioned medium from PA-infected primary AECII enhanced AM immunity. On the other hand, the importance of lipid rafts (membrane microdomains) for innate immunity has been indicated by the fact that CF patients, who are particularly at risk for PA infection, may often suffer from abnormal lipid raft function due to ceramide deficiency and fatty-acid imbalance. Consistent with this, we showed that lipid rafts may be involved in the secretion of the cytokine MCP-1, which is localized in ceramide-rich rafts. Our rationale is that elucidating the relevant mechanism in ceramide-rich microdomains will define a general mechanism for cytokine secretion. Our laboratory is ideally suited for this research since we have the requisite expertise in AECII isolation, PA infection, and advanced biochemical techniques. To test our hypothesis, we propose the following two specific aims: Specific Aim 1: To characterize dynamic reorganization of membrane microdomains in regulating cytokine secretion in AECII and in mice using imaging tools. Our working hypothesis is that PA infection initiates dynamic changes in membrane microdomains that impact cytokine production. Specific Aim 2: To define the mechanism by which membrane microdomains regulate key cytokines required for PA defense. Our hypothesis is that ceramide is generated by hydrolysis and translocated to specialized domains, where it initiates signaling for production of MCP-1. Due to the important role of cytokines in various physiological and pathological conditions, these novel mechanisms will improve understanding of cytokine secretion for other types of cells, pathogens, and inflammatory situations, and thereby identifying new therapeutic targets. PUBLIC HEALTH RELEVANCE: Although alveolar epithelial type II cells (AECII) are structural and progenitor cells in the lung, they also provide immune defense by secreting cytokines. This unique immunity may critically increase host defense against P. aeruginosa (PA), a bacterium that causes severe infections in immunodeficient individuals, such as AIDS, tuberculosis and cystic fibrosis. We hypothesize that AECII secrete cytokines through changes in cell membrane microdomains, a novel mechanism that may impact many disease processes. Thus, our research will provide new insights into the development of novel treatment for this recurrent infection.