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着关键作用。巨噬细胞可以通过细胞和组织响应感染而产生的化学信使（细胞因子）来切换以执行不同的功能。一些细胞因子将巨噬细胞转变为 M2 型巨噬细胞，这种巨噬细胞对寄生虫病和伤口愈合有益，但在某些细菌感染和哮喘等过敏性疾病中发挥负面作用。最近的研究表明，与大多数其他巨噬细胞类型相比，肺 M2 巨噬细胞可以在体内增殖。由于它们的关键作用以及开发更有效的感染疗法，更多地了解 M2 巨噬细胞非常重要。 M2 巨噬细胞研究的很大一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了解特定途径成为可能。在这里，研究人员完全依赖活体动物的使用。 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。所有这些过程中获得的细胞的寿命有限，因此需要重复使用动物。该提案的目的是建立永久生长的交替激活（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，我们建立了一种新型的小鼠肺巨噬细胞（MPI 细胞）体外模型。与现有系统相比，这些原代非转化细胞可以从各种小鼠品系中无限期地生长，并且在与生长因子一起培养时几乎可以无限数量地获得。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中获得数量非常有限的肺巨噬细胞（大约 300, 1000 个细胞）。相比之下，我们定期从单个 75 cm2 组织培养瓶中获得 20-3000 万个 MPI 细胞。我们最新的数据表明，IL-4 或 IL-13 处理可在 MPI 细胞中诱导 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，肺部病毒感染的频率更高、后果更严重。因此，在这个试点项目中，我们希望建立 MPI 细胞培养物作为 M2 肺巨噬细胞的忠实模型，用于重要的医学研究。用细胞因子 IL-4 处理的 MPI 细胞将被表征，并与用这些细胞因子处理的替代激活巨噬细胞、小鼠骨髓来源的巨噬细胞和肺泡巨噬细胞的现有模型进行比较。我们将研究替代激活的 MPI 细胞与目前在替代激活的骨髓来源的巨噬细胞中研究的相关细菌病原体的感染。我们还将分析该系统中空气传播病毒感染的特征，因为实验数据表明M2巨噬细胞和这些微生物在哮喘等过敏性气道疾病中发挥着重要作用。该项目的完成可以大大减少用作来源的动物数量替代激活的巨噬细胞。此外，所提出的系统的建立将揭示生物医学的新机制。通过提供高通量筛选和药物测试的新工具，它也可能为重要疾病的治疗开辟新的视角。