Establishment of non-transformed, continuously growing, alternatively activated mouse macrophage cell lines

建立非转化、持续生长、交替激活的小鼠巨噬细胞系

• 批准号: NC/L00058X/1
• 负责人: Gyorgy Fejer
• 金额: $ 9.62万
• 依托单位: Plymouth University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FL00058X%2F1
• 关键词: Establishment; non; transformed; continuously; growing

Macrophages are crucial components of the innate immune system and play key roles in defence against invading pathogens. Macrophages can be switched to perform different functions by chemical messengers (cytokines) produced by cells and tissues in response to infection. Some cytokines switch macrophages to a type (M2) that are beneficial in parasitic diseases and wound healing but play negative roles in certain bacterial infections, and in allergic diseases such as asthma. Recent studies indicate that, in contrast to most other macrophage types, lung M2 macrophages can proliferate in vivo. Due to their key roles and to develop more effective therapies for infections, it is important to understand more about M2 macrophages. A significant part of research on M2 macrophages are done in the mouse system where the availability of various mutant strains makes the understanding of specific pathways possible. Here, researchers rely entirely on the use of living animals. M2 macrophages are isolated and studied directly from organs or grown and differentiated from bone-marrow in vitro. The cells obtained in all of these procedures have a limited life-span, thus requiring the repeated use of animals. The aim of this proposal is to establish permanently growing, alternatively activated (M2) macrophage cell lines in order to reduce the number of animals currently used to provide such cells. Recently we established a novel in vitro model of mouse lung macrophages (MPI cells). In contrast to existing systems, these primary, non-transformed cells can be grown indefinitely from various mouse strains and are available in practically unlimited numbers when cultured with growth factors. Thus, their use can make macrophage experiments that have been previously problematic feasible. For example, a very limited number of lung macrophages, (approximately 300, thousand cells) can be obtained from one mouse by the technique of broncho-alveolar lavage. In comparison, we regularly obtain 20-30 million MPI cells from a single 75 cm2 tissue culture flask.Our most recent data show that IL-4 or IL-13 treatment induces an M2 like phenotype in the MPI cells. These findings have relevance to patients with asthma, where M2 macrophages are predominant, and a higher frequency and more serious outcome of pulmonary virus infections are experienced. Therefore, in this pilot project we want to establish MPI cell cultures as a faithful model of M2 lung macrophages for medically important studies. MPI cells treated with the cytokine IL-4 will be characterized and compared to existing models of alternatively activated macrophages, mouse bone marrow derived macrophages and lung alveolar macrophages treated with these cytokines. We will study the infection of alternatively activated MPI cells with relevant bacterial pathogens currently studied in alternatively activated bone marrow derived macrophages. We will also analyze the characteristics of airborne virus infection in this system because experimental data suggest an important role for M2 macrophages with these microbes in allergic airway diseases, such as asthma.Accomplishment of this project can drastically reduce the number of animals used as a source of alternatively activated macrophages. Furthermore, the establishment of the proposed system would reveal novel mechanisms in biomedical science. By providing a new tool for high throughput screening and drug testing it may open new perspectives for the treatment of important diseases as well.

巨噬细胞是先天免疫系统的重要组成部分，在防御入侵病原体方面发挥关键作用。巨噬细胞可以通过细胞和组织响应感染产生的化学信使（细胞因子）来转换以执行不同的功能。一些细胞因子将巨噬细胞转变为一种类型（M2），这种类型对寄生虫病和伤口愈合有益，但在某些细菌感染和过敏性疾病（如哮喘）中起负面作用。最近的研究表明，与大多数其他巨噬细胞类型相反，肺M2巨噬细胞可以在体内增殖。由于它们的关键作用和开发更有效的感染疗法，重要的是要了解更多关于M2巨噬细胞。对M2巨噬细胞的研究的一个重要部分是在小鼠系统中完成的，其中各种突变株的可用性使得对特定途径的理解成为可能。在这里，研究人员完全依赖于使用活体动物。M2巨噬细胞直接从器官中分离和研究，或在体外从骨髓中生长和分化。在所有这些程序中获得的细胞具有有限的寿命，因此需要重复使用动物。本提案的目的是建立永久生长的、交替激活的（M2）巨噬细胞系，以减少目前用于提供此类细胞的动物数量。最近，我们建立了一种新的小鼠肺巨噬细胞（MPI细胞）体外模型。与现有系统相反，这些原代非转化细胞可以从各种小鼠品系无限生长，并且当与生长因子一起培养时，可以以几乎无限的数量获得。因此，它们的使用可以使以前存在问题的巨噬细胞实验变得可行。例如，通过支气管肺泡灌洗技术可以从一只小鼠获得非常有限数量的肺巨噬细胞（大约30万个细胞）。相比之下，我们定期从单个75 cm 2组织培养瓶中获得2000 - 3000万个MPI细胞，我们最近的数据显示IL-4或IL-13处理诱导MPI细胞中的M2样表型。这些发现与哮喘患者相关，其中M2巨噬细胞占主导地位，并且经历了更高频率和更严重的肺部病毒感染结局。因此，在本试验项目中，我们希望建立MPI细胞培养物作为M2肺巨噬细胞的可靠模型，用于医学重要研究。将表征用细胞因子IL-4处理的MPI细胞，并与用这些细胞因子处理的替代活化的巨噬细胞、小鼠骨髓衍生的巨噬细胞和肺泡巨噬细胞的现有模型进行比较。我们将研究交替激活的MPI细胞与目前在交替激活的骨髓源性巨噬细胞中研究的相关细菌病原体的感染。实验数据表明，M2巨噬细胞与这些微生物在哮喘等过敏性气道疾病中发挥着重要作用，因此我们还将分析该系统中空气传播病毒感染的特征。该项目的完成可以大幅减少用作替代活化巨噬细胞来源的动物数量。此外，该系统的建立将揭示生物医学科学中的新机制。通过为高通量筛选和药物测试提供新的工具，它也可能为重要疾病的治疗开辟新的前景。