Modulation of macrophage differentiation and activation by paracrine signals from cardiac progenitor cells
File(s)
Author(s)
Samari, Sara
Type
Thesis
Abstract
Myocardial infarction (MI) triggers an inflammatory response that contributes to cardiac remodelling in adult mice. In contrast, the heart of the post-MI neonatal mouse regenerates without scarring due to an anti-inflammatory macrophage-dependent
process. Previously, Noseda and colleagues demonstrated that intramyocardial injection of Lin-Sca1+CD31-PDGFRalpha+SP+ cardiac progenitor/stem cells (CSCs) improved cardiac function. However, despite the lack of long-term engraftment, the
cardiac improvement could be explained by paracrine factors released by CSCs. The paracrine effects of CSCs could affect several cell types in the heart, including macrophages. Therefore, this PhD thesis hypothesises that CSCs release paracrine
factors that promote a subtype of anti-inflammatory macrophage phenotype to support cardiac regeneration.
After flow sorting, single F4/80+CD11b+ macrophages were analysed by single qRT PCR. GM-CSF+LPS+IFNγ-driven M, identified as CX3CR1+CD11b+F4/80loCD206-, co-expressed pro-inflammatory genes, Nos2, Cxcl9, Cxcl10 and Il-6. On the other hand, M-CSF+IL-4+IL-13-driven Mf identified as CX3CR1+CD11b+F4/80+CD206+, upregulated anti-inflammatory genes, Arg1, Angpt2, and Igf1. Notably, CSCs conditioned media (CSC CondM)-driven macrophages were indistinguishable from M-CSF+IL-4+IL-13-Mf. Lastly, CSC CondM+IL4+IL13-driven M group was identified as CX3CR1+CD11b+F4/80+CD206+, expressing Arg1, Angptl2, Igf1, Il1rl1, and Mrc1, and showing a distinct subtype of anti-inflammatory macrophage phenotype compared to all prior reports of anti-inflammatory/M2-like macrophages.
After confirming that CSCs secrete M-CSF (20pg/mL) as the potential protective factor, the M-CSF/CSF1R pathway was inhibited by using first the pharmacological inhibitor BLZ945 and then a monoclonal antibody against CSF1R. These inhibitory experiments demonstrated that the inhibition of M-CSF activity reduced cell viability, the percentage of F4/80+CD11b+CX3CR1+CD206+ macrophages, expression of anti-inflammatory and pro-fibrotic genes (Arg1, Angpt2, Igf1, Il1rl1, Mrc1 and Gdf15), and phagocytosis activity. Multiplex bead-based flow immunoassays also showed that CSC CondM+LPS+IFN-Mf secrete IL-10 depending upon the M-CSF/CSF1R pathway. In conclusion, CSC-secreted M-CSF is the indispensable paracrine factor that induces macrophages with an anti-inflammatory phenotype. In conclusion, CSC-secreted M-CSF is the indispensable paracrine factor that induces macrophages with an anti-inflammatory phenotype.
process. Previously, Noseda and colleagues demonstrated that intramyocardial injection of Lin-Sca1+CD31-PDGFRalpha+SP+ cardiac progenitor/stem cells (CSCs) improved cardiac function. However, despite the lack of long-term engraftment, the
cardiac improvement could be explained by paracrine factors released by CSCs. The paracrine effects of CSCs could affect several cell types in the heart, including macrophages. Therefore, this PhD thesis hypothesises that CSCs release paracrine
factors that promote a subtype of anti-inflammatory macrophage phenotype to support cardiac regeneration.
After flow sorting, single F4/80+CD11b+ macrophages were analysed by single qRT PCR. GM-CSF+LPS+IFNγ-driven M, identified as CX3CR1+CD11b+F4/80loCD206-, co-expressed pro-inflammatory genes, Nos2, Cxcl9, Cxcl10 and Il-6. On the other hand, M-CSF+IL-4+IL-13-driven Mf identified as CX3CR1+CD11b+F4/80+CD206+, upregulated anti-inflammatory genes, Arg1, Angpt2, and Igf1. Notably, CSCs conditioned media (CSC CondM)-driven macrophages were indistinguishable from M-CSF+IL-4+IL-13-Mf. Lastly, CSC CondM+IL4+IL13-driven M group was identified as CX3CR1+CD11b+F4/80+CD206+, expressing Arg1, Angptl2, Igf1, Il1rl1, and Mrc1, and showing a distinct subtype of anti-inflammatory macrophage phenotype compared to all prior reports of anti-inflammatory/M2-like macrophages.
After confirming that CSCs secrete M-CSF (20pg/mL) as the potential protective factor, the M-CSF/CSF1R pathway was inhibited by using first the pharmacological inhibitor BLZ945 and then a monoclonal antibody against CSF1R. These inhibitory experiments demonstrated that the inhibition of M-CSF activity reduced cell viability, the percentage of F4/80+CD11b+CX3CR1+CD206+ macrophages, expression of anti-inflammatory and pro-fibrotic genes (Arg1, Angpt2, Igf1, Il1rl1, Mrc1 and Gdf15), and phagocytosis activity. Multiplex bead-based flow immunoassays also showed that CSC CondM+LPS+IFN-Mf secrete IL-10 depending upon the M-CSF/CSF1R pathway. In conclusion, CSC-secreted M-CSF is the indispensable paracrine factor that induces macrophages with an anti-inflammatory phenotype. In conclusion, CSC-secreted M-CSF is the indispensable paracrine factor that induces macrophages with an anti-inflammatory phenotype.
Version
Open Access
Date Issued
2022-07
Date Awarded
2022-12
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Schneider, Michael
Haskard, Dorian
Noseda, Michela
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)