Establishment of novel macrophage cell lines to study the pathogenesis of respiratory bacterial pathogens in lung alveolar macrophages
Lead Research Organisation:
Plymouth University
Department Name: Sch of Biomedical and Healthcare Sci
Abstract
Macrophages are important immune cells in the first line of defence against infectious pathogens.
To understand macrophage-bacteria interactions mice are frequently used. Macrophages can be best investigated using purified cells from organs or by producing them from bone marrow in tissue culture, but the availability of such cells is limited.
Lung alveolar macrophages (AMs) play central roles in defence against respiratory bacterial pathogens. Infection of AMs can result in direct pathogen elimination, however, bacteria may overcome macrophage killing mechanisms allowing pathogen replication or persistence inside the cell. The activation of a plethora of pathogen sensing mechanisms play key roles in these processes and their understanding is key to the efficient treatment of respiratory infections. The therapy of bacterial infections of the airways can be still problematic and macrophages play key roles in these processes as bacteria can be unavailable to drugs when persist or replicate in macrophages.
We described a novel, continuously growing, murine model of lung alveolar macrophages (AMs), providing unrestricted amounts of primary macrophages (MPI cells). Using this system we have demonstrated the very high sensitivity of MPI cells and AMs to respiratory pathogens and have shown the existence of unique immune mechanisms in AMs.
The sensing of respiratory bacteria by AMs involves various immune receptors, however, the understanding of their contribution to pathogenesis mechanisms is hampered by the restricted availability of AMs.
Here we will transfer the MPI cell technology to the Kings College London (KCL) and the Institut Pasteur, Korea (IPK) to study bacterial pathogenesis in lung macrophages. We will establish new MPI cell lines from relevant genetically modified mice lacking various bacterial pathogen recognition sensors. To better understand respiratory macrophage-bacteria interactions we will establish MPI cells from these g mice available at KCL, the Sanger Institute and the IPK. To make the cells accessible to other researchers we will make a repository of the new cell lines and/or distribute them through commercial vendors to the wider community.
We have ongoing in vivo and in vitro studies with the above listed pathogens. We will use the established new cell lines to complement these studies and obtain mechanistic data regarding the activation of innate immune pathways to the sensing and the treatment of respiratory bacterial infections.
To understand macrophage-bacteria interactions mice are frequently used. Macrophages can be best investigated using purified cells from organs or by producing them from bone marrow in tissue culture, but the availability of such cells is limited.
Lung alveolar macrophages (AMs) play central roles in defence against respiratory bacterial pathogens. Infection of AMs can result in direct pathogen elimination, however, bacteria may overcome macrophage killing mechanisms allowing pathogen replication or persistence inside the cell. The activation of a plethora of pathogen sensing mechanisms play key roles in these processes and their understanding is key to the efficient treatment of respiratory infections. The therapy of bacterial infections of the airways can be still problematic and macrophages play key roles in these processes as bacteria can be unavailable to drugs when persist or replicate in macrophages.
We described a novel, continuously growing, murine model of lung alveolar macrophages (AMs), providing unrestricted amounts of primary macrophages (MPI cells). Using this system we have demonstrated the very high sensitivity of MPI cells and AMs to respiratory pathogens and have shown the existence of unique immune mechanisms in AMs.
The sensing of respiratory bacteria by AMs involves various immune receptors, however, the understanding of their contribution to pathogenesis mechanisms is hampered by the restricted availability of AMs.
Here we will transfer the MPI cell technology to the Kings College London (KCL) and the Institut Pasteur, Korea (IPK) to study bacterial pathogenesis in lung macrophages. We will establish new MPI cell lines from relevant genetically modified mice lacking various bacterial pathogen recognition sensors. To better understand respiratory macrophage-bacteria interactions we will establish MPI cells from these g mice available at KCL, the Sanger Institute and the IPK. To make the cells accessible to other researchers we will make a repository of the new cell lines and/or distribute them through commercial vendors to the wider community.
We have ongoing in vivo and in vitro studies with the above listed pathogens. We will use the established new cell lines to complement these studies and obtain mechanistic data regarding the activation of innate immune pathways to the sensing and the treatment of respiratory bacterial infections.
Technical Summary
Macrophages represent the first line of defence against pathogens and play important roles in major infectious diseases.
Because of the availability of inbred strains and gene deficient animals, macrophage-pathogen interactions are studied largely using mice. Macrophages can be best investigated using either organ-derived or ex vivo produced primary macrophages (BMDMs), but the availability of such cells is limited.
lung alveolar macrophages (AMs) play central roles in defence against respiratory bacterial pathogens, however, bacteria may overcome macrophage killing mechanisms. Bacteria unavailable to drugs inside macrophages may represent an important problem in the treatment of bacterial infections of the airways.
We described a novel, self-renewing, non-transformed, primary murine model of AMs, providing unrestricted amounts of primary macrophages. Using this system we have demonstrated the very high sensitivity of MPI cells and AMs to respiratory pathogens and have shown the existence of unique innate pathogen sensing pathways in these cells.
The innate recognition of respiratory bacteria by AMs involves various sensors, but the understanding of their contribution to pathogenesis mechanisms is hampered by the restricted availability of AMs.
Here we will transfer the MPI cell technology to the King's College London and the Institut Pasteur, Korea to study bacterial pathogenesis in lung macrophages. We will establish new MPI cell lines from mice lacking various bacterial pathogen recognition sensors such as scavenger and C-type lectin receptors, Toll-like Receptors and cytoplasmic sensors. To make the cells accessible to other researchers we will make a repository of the new cell lines and/or distribute them through commercial vendors to the wider community.
We have ongoing studies with these bacteria and will use the new cell lines to obtain mechanistic data regarding the activation of innate pathways to the sensing of respiratory bacterial infections.
Because of the availability of inbred strains and gene deficient animals, macrophage-pathogen interactions are studied largely using mice. Macrophages can be best investigated using either organ-derived or ex vivo produced primary macrophages (BMDMs), but the availability of such cells is limited.
lung alveolar macrophages (AMs) play central roles in defence against respiratory bacterial pathogens, however, bacteria may overcome macrophage killing mechanisms. Bacteria unavailable to drugs inside macrophages may represent an important problem in the treatment of bacterial infections of the airways.
We described a novel, self-renewing, non-transformed, primary murine model of AMs, providing unrestricted amounts of primary macrophages. Using this system we have demonstrated the very high sensitivity of MPI cells and AMs to respiratory pathogens and have shown the existence of unique innate pathogen sensing pathways in these cells.
The innate recognition of respiratory bacteria by AMs involves various sensors, but the understanding of their contribution to pathogenesis mechanisms is hampered by the restricted availability of AMs.
Here we will transfer the MPI cell technology to the King's College London and the Institut Pasteur, Korea to study bacterial pathogenesis in lung macrophages. We will establish new MPI cell lines from mice lacking various bacterial pathogen recognition sensors such as scavenger and C-type lectin receptors, Toll-like Receptors and cytoplasmic sensors. To make the cells accessible to other researchers we will make a repository of the new cell lines and/or distribute them through commercial vendors to the wider community.
We have ongoing studies with these bacteria and will use the new cell lines to obtain mechanistic data regarding the activation of innate pathways to the sensing of respiratory bacterial infections.
Planned Impact
Mouse models are used extensively to study respiratory bacterial pathogenesis in macrophages. Here, experiments are frequently done in vivo causing significant suffering to animals. Mice are also used extensively to obtain lavaged alveolar macrophages (AMs) and ex vivo differentiated bone marrow derived macrophages (BMDMs) for in vitro studies. Genetically targeted animals are commonly used in these settings for mechanistic pathogenesis studies.
Our proposed studies can contribute to the reduction and the replacement of experimental animal use. By providing scientifically attractive and cheap relevant AM-like cell lines our model can significantly reduce the number of in vivo experiments with many bacterial species as well as AM studies in vitro. Moreover, BMDM experiments can be replaced almost entirely with lung pathogens as MPI cells represent a more relevant model for them than BMDMs. By using the established MPI lines (wt and MARCO deficient cells) about 100 wild-type mice could be saved in both the King's College London and in the Institut Pasteur Korea in ongoing work annually. We achieved a similar reduction with our MARCO deficient MPI line during our recently published work (Maler et al, Mbio). Thus, just the additional seven MPI macrophage lines (Phagocytosis receptors, ALPK1, IFN signalling, Lpr1) intended to use immediately at KCL, IPK and PU could contribute to the saving of up to 700 more mice in these institutions annually. Nevertheless, the other proposed relevant macrophage lines will contribute to further reduction/replacement and other groups are also interested in the proposed cell lines to study respiratory bacterial infections and to minimize their animal use (see attached letters of support).
Dr Fejer gets requests for the original MPI line frequently and received reports of significant animal use reduction (100-300 mice/year) indicating also a consistent, similar animal reduction with the new cell lines.
By providing valuable tools to study the mechanisms of respiratory bacterial infection in primary, relevant macrophages our newly established cell lines will promote the application of our in vitro model for pathogenesis studies in both wild type and modified genetic backgrounds. A search for the terms "mouse", "mycobacteria" and "macrophage" with Google Scholar in 2019 gave 3930 results. Checking the first 100 entries, actually 33 were relevant (mycobacterial infection of primary macrophages in vivo or in vitro). Each study used minimally 20-30 mice, thus, a total of around 25000 mice could have been used here. Using similar calculations we can estimate the annual use of approximately 9000 and 18000 mice in connection with Klebsiella pneumoniae and Pseudomonas aeruginosa research. However, this is probably an underestimate not considering breeding and unpublished data. We propose that the identification and pre-validation of potential mechanisms in our highly-validated and quality-controlled model could reduce and/or replace many of these animals.
We expect that the established and easily available cell lines will be used further in related fields and will contribute to the reduction of animal experimentation. These fields include Infections with (i) other respiratory bacteria (Legionella, Burkholderia, Chlamydia, Mycoplasma species), (ii) viruses (e g influenza, corona, adeno, rhino, respiratory synctitial viruses), (iii) Fungi (Cryptococcus, Aspergillus, Candida), (iv) Environmental pollution and toxicology (compost, smoke, toxic vapours) and (v) drug development (eg against influenza responses).
Our proposed studies can contribute to the reduction and the replacement of experimental animal use. By providing scientifically attractive and cheap relevant AM-like cell lines our model can significantly reduce the number of in vivo experiments with many bacterial species as well as AM studies in vitro. Moreover, BMDM experiments can be replaced almost entirely with lung pathogens as MPI cells represent a more relevant model for them than BMDMs. By using the established MPI lines (wt and MARCO deficient cells) about 100 wild-type mice could be saved in both the King's College London and in the Institut Pasteur Korea in ongoing work annually. We achieved a similar reduction with our MARCO deficient MPI line during our recently published work (Maler et al, Mbio). Thus, just the additional seven MPI macrophage lines (Phagocytosis receptors, ALPK1, IFN signalling, Lpr1) intended to use immediately at KCL, IPK and PU could contribute to the saving of up to 700 more mice in these institutions annually. Nevertheless, the other proposed relevant macrophage lines will contribute to further reduction/replacement and other groups are also interested in the proposed cell lines to study respiratory bacterial infections and to minimize their animal use (see attached letters of support).
Dr Fejer gets requests for the original MPI line frequently and received reports of significant animal use reduction (100-300 mice/year) indicating also a consistent, similar animal reduction with the new cell lines.
By providing valuable tools to study the mechanisms of respiratory bacterial infection in primary, relevant macrophages our newly established cell lines will promote the application of our in vitro model for pathogenesis studies in both wild type and modified genetic backgrounds. A search for the terms "mouse", "mycobacteria" and "macrophage" with Google Scholar in 2019 gave 3930 results. Checking the first 100 entries, actually 33 were relevant (mycobacterial infection of primary macrophages in vivo or in vitro). Each study used minimally 20-30 mice, thus, a total of around 25000 mice could have been used here. Using similar calculations we can estimate the annual use of approximately 9000 and 18000 mice in connection with Klebsiella pneumoniae and Pseudomonas aeruginosa research. However, this is probably an underestimate not considering breeding and unpublished data. We propose that the identification and pre-validation of potential mechanisms in our highly-validated and quality-controlled model could reduce and/or replace many of these animals.
We expect that the established and easily available cell lines will be used further in related fields and will contribute to the reduction of animal experimentation. These fields include Infections with (i) other respiratory bacteria (Legionella, Burkholderia, Chlamydia, Mycoplasma species), (ii) viruses (e g influenza, corona, adeno, rhino, respiratory synctitial viruses), (iii) Fungi (Cryptococcus, Aspergillus, Candida), (iv) Environmental pollution and toxicology (compost, smoke, toxic vapours) and (v) drug development (eg against influenza responses).
