Establishment of non-transformed, continuously growing, alternatively activated mouse macrophage cell lines
Lead Research Organisation:
Plymouth University
Department Name: Centre for Biomedical Research
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
The proposal contributes to the following 3R areas
Reduction in animal experimentation
- M2 macrophages are important in infectious disease pathogenesis particularly with intracellular pathogens. Such infections and effectiveness of prospective therapies are currently studied using animal models (mice, rats, ferrets) (e. g. inhalation studies with allergens, viral, bacterial and worm infections). The use of our proposed polarized lung alveolar macrophage model cells could significantly reduce these animal numbers by providing a faithful in vitro model system. Such a cell system could be used for preliminary studies and screening for eg effective drug therapies/doses before final trials in existing animal models, thus significantly reducing the numbers of animals used for such studies.
- Replacing live animals in experimental methods
This project can provide polarized macrophage lines able to grow indefinitely and in unlimited numbers and could, therefore, replace animals used as a source of macrophages. A vast number of experimental animals are used currently in macrophage biology mostly in innate immune research. A search for the terms "mouse bone marrow derived macrophage" plus "alternative activation" in the Google Scholar database since 2011 gave 11900 results. Assuming that in each of these outputs 27 animals were used (3 mice/figure, 3 figures with animal data, 3 independent experiments) a total of 321300 mice were used for these studies alone. A significant part of these studies were done to obtain M2 polarized macrophages that we propose to replace here.
In particular, the obtained cells could be very useful for the study of polarized lung alveolar macrophages that play important roles in infectious and allergic lung disease. A very limited number of alveolar macrophages, approximately 3 X 10 (5) (three hundred thousand) cells can be obtained from one mouse by conventional methods. In comparison, we could obtain 2-3 X 10 (7) (twenty-thirty million) alveolar macrophage model cells from a single 75 cm2 tissue culture flask.
Impact on legislation and permits for animal experimentation
In the case of our macrophage lines, we could imagine that the legislator could request experiments with macrophage lines before allowing certain animal experiments in the field. This would be in line with increasing requests of experiments with cell lines before animal experiments are granted by legislators. The herein developed macrophage cell lines will give the community an additional powerful tool to reduce the number of animal experiments in basic research and potentially also in commercial research, particularly drug development, infectious diseases and airway diseases, including allergy research.
Reduction in animal experimentation
- M2 macrophages are important in infectious disease pathogenesis particularly with intracellular pathogens. Such infections and effectiveness of prospective therapies are currently studied using animal models (mice, rats, ferrets) (e. g. inhalation studies with allergens, viral, bacterial and worm infections). The use of our proposed polarized lung alveolar macrophage model cells could significantly reduce these animal numbers by providing a faithful in vitro model system. Such a cell system could be used for preliminary studies and screening for eg effective drug therapies/doses before final trials in existing animal models, thus significantly reducing the numbers of animals used for such studies.
- Replacing live animals in experimental methods
This project can provide polarized macrophage lines able to grow indefinitely and in unlimited numbers and could, therefore, replace animals used as a source of macrophages. A vast number of experimental animals are used currently in macrophage biology mostly in innate immune research. A search for the terms "mouse bone marrow derived macrophage" plus "alternative activation" in the Google Scholar database since 2011 gave 11900 results. Assuming that in each of these outputs 27 animals were used (3 mice/figure, 3 figures with animal data, 3 independent experiments) a total of 321300 mice were used for these studies alone. A significant part of these studies were done to obtain M2 polarized macrophages that we propose to replace here.
In particular, the obtained cells could be very useful for the study of polarized lung alveolar macrophages that play important roles in infectious and allergic lung disease. A very limited number of alveolar macrophages, approximately 3 X 10 (5) (three hundred thousand) cells can be obtained from one mouse by conventional methods. In comparison, we could obtain 2-3 X 10 (7) (twenty-thirty million) alveolar macrophage model cells from a single 75 cm2 tissue culture flask.
Impact on legislation and permits for animal experimentation
In the case of our macrophage lines, we could imagine that the legislator could request experiments with macrophage lines before allowing certain animal experiments in the field. This would be in line with increasing requests of experiments with cell lines before animal experiments are granted by legislators. The herein developed macrophage cell lines will give the community an additional powerful tool to reduce the number of animal experiments in basic research and potentially also in commercial research, particularly drug development, infectious diseases and airway diseases, including allergy research.
