This research aims to establish a continuously growing cell line of alternatively activated mouse macrophages to replace the use of cells isolated from mouse organs or bone marrow.
Macrophages are phagocytes and antigen-presenting cells which perform key roles in innate and adaptive immunity. Activated macrophages are classified as M1 or M2. M1 macrophages have an acute inflammatory phenotype and are highly important for fighting bacteria. The alternatively activated anti-inflammatory M2 macrophages on the other hand promote cell proliferation and tissue repair, and are thought to have a role in allergic diseases, such as asthma.
There is increasing interest in the role of M2 macrophages in both allergy and infection. Currently, the only way of obtaining these cells for study relies on harvesting directly from animals. These cells have a limited life-span, thus requiring the repeated use of animals – it is estimated that around 300,000 mice are used for this purpose worldwide each year. Establishing a permanently growing M2 macrophage cell line would replace the number of animals currently used, and potentially provide a screening platform for drugs which modify M2 macrophage function to treat diseases.
Research details and methods
Recently a method to obtain continuously growing non-transformed primary macrophages (MPI cells) from mice without chemical or viral mutagenesis or genetic manipulation has been developed.
The research will establish if MPI cells, when treated with the cytokines interleukin 4 or interleukin 13, can be induced to form an M2-like phenotype. The cells will be characterised and compared to parental MPI cells and bone marrow-derived M2 macrophages using a number of approaches, including gene expression patterns and the induction of inflammatory mediating molecules in response to challenge with pathogens and other innate immune system stimulators.
In addition, the ability of MPI-derived M2 macrophages to control mouse cytomegalovirus infection and replication of some bacterial agents will also be compared to parental MPI cells and bone marrow-derived macrophages.
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Macrophages are crucial in defence against pathogens. They have two major types, the pro-inflammatory classically activated/M1 macrophages and alternatively activated/M2 macrophages. Interleukin (IL)-4 and IL-13 drive the formation of M2 macrophages that are considered to be anti-inflammatory and beneficial in tissue remodelling but harmful in certain bacterial infections and in allergic diseases. Recent studies indicate that, in contrast to most other macrophages, lung M2 macrophages proliferate in response to IL-4 in vivo. A significant part of research on M2 macrophages are done in the mouse system. M2 macrophages currently used in vitro have a limited life-span requiring the extensive and repeated use of animals.
The aim of our proposed studies is to set up permanently growing, primary, alternatively activated macrophage cell lines to reduce the need for animals as a source of similar cells and to improve the existing macrophage research repertoire.
Recently we established a novel method producing primary, GM-CSF dependent, continuously growing lung alveolar macrophage (AM)-like cells (MPI cells). Our most recent data show that IL-4 or IL-13 treatment induces an M2 like phenotype in the MPI cells.
Here, we want to establish IL-4 treated MPI cells as a faithful model of alternatively activated AMs. To prove their usefulness in infection research these MPI cells will be compared to existing primary M2 macrophage systems. We will analyse global gene expression patterns in alternatively activated un-induced or stimulated MPI macrophages with RNA sequencing and measure the levels of relevant proteins important in M2 macrophages and innate signalling as well. We will also characterize alternatively activated MPI cell responses to a range of selected important pulmonary pathogens.
Accomplishment of this project can drastically reduce the number of animals in this area and the proposed system would provide a powerful new tool in biomedical science.
Fejer G, Sharma S, Gyory I (2015). Self-renewing macrophages--a new line of enquiries in mononuclear phagocytes. Immunobiology 220(2): 169-74. doi: 10.1016/j.imbio.2014.11.005.
Principal investigatorDr Gyorgy Fejer
InstitutionUniversity of Southampton
Co-InvestigatorProfessor Simon Jackson
Professor Nigel Silman
Dr Lars Dolken