Conference 2021 Poster Presentation
Project title
Granulocyte Macrophage Colony Stimulating Factor exerts dominant effects over Macrophage Colony Stimulating Factor during Macrophage differentiation.
Authors and Affiliations
Maria Petrina1, Torki Alothaimeen1, Evan Trus1, Andra Banete1, Katrina Gee1, Sameh Basta1
1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Canada
Abstract
Background
Macrophages (Mϕ) are able to modify their phenotype and effector functions depending on the cytokines present in their microenvironment. This phenotypic plasticity has led to a broad classification system that includes pro-inflammatory M1 Mϕ and anti-inflammatory M2 Mϕ. Recent discoveries suggest that the cytokines Macrophage Colony Stimulating Factor (M-CSF) and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) can influence Mϕ differentiation in opposing manners. These CSFs have various roles in the immune system because they can influence the proliferation, differentiation, and polarization of Mϕ. Mϕ exposed to M-CSF acquire a neutral/unstimulated M0 Mϕ phenotype, where stimulation with GM-CSF results in an “M1-like” Mϕ phenotype. The CSFs can be used to differentiate two different phenotypes of murine Mϕ in vitro; however, the relationship between GM-CSF and M-CSF when administered to Mϕs simultaneously has not been well defined. This study aims to define the competition between GM-CSF and M-CSF antagonism, with respect to Mϕ differentiation and polarization. We hypothesize that GM-CSF will exert dominance over M-CSF with respect to Mϕ phenotype, immune functions, and cytokine expression profiles.
Methods
Bone marrow derived Mϕs (BMDM) sourced from six- to eight-week-old C57BL/6 mice were differentiated with varying ratios of recombinant GM-CSF and M-CSF in RPMI media + 10% fetal calf serum at 37oC with 5% CO2 for 7 days. The active biological units of both cytokines were altered in culture using different ratios while keeping the total number of active units per ml constant in all conditions. On day 7, cells were stimulated with 1μg/mL of lipopolysaccharide for 24 hours. Cells were imaged for cell morphology analysis via Brightfield microscopy. Supernatants were collected for cytokine analysis (IL-6, IL-12p40, IL-10, TNF- α) via ELISA and cell pellets were collected and stained for surface marker expression analysis (CD86, MHC-I, MHC-II, PD-L1) via flow cytometry.
Results
We found that GM-CSF and M-CSF exert competing actions on Mϕ differentiation and polarization. GM-CSF is dominant over M-CSF, in terms of cytokine production, cell morphology, and surface marker expression.
Conclusions
GM-CSF is dominant over M-CSF. These results offer insight into the effects of CSFs on Mϕ activity simultaneously and could theoretically lead to imperative clinical applications in diseases or pathologies where Mϕ play a significant role.
