Bioluminescence response curves demonstrate variable monocyte to macrophage differentiation using GM-CSF, M-CSF, or PMA

Introduction:: Monocyte differentiation shapes the outcomes of local inflammatory processes. Monocytes circulate through the blood then infiltrate inflamed tissue where they can differentiate into tissue resident macrophages or dendritic cells. Environment signals in inflamed tissue, like chemokines and growth factors, control the recruitment and differentiation of these monocytes. The concentration and duration of exposure to these signals affect the differentiation and polarization outcome of the monocytes. This study uses bioluminescence temporal spectroscopy to quantify monitor changes in the expression of polarization-relevant transcription factors during monocyte to macrophage differentiation.

We developed THP-1 monocyte reporter lines that align with both the M1 and M2 phenotypes: THP-1 NFκB FLuc and THP-1 STAT6 FLuc. NFκB is a key pro-inflammatory transcription factor associated with M1 polarization within macrophages. It can be activated through both its canonical and non-canonical pathways, originating from TLRs and TNFR or CD40 and RANK respectively. STAT6 meanwhile is a transcription factor associated with anti-inflammatory activation and the M2 polarization, whose pathway begins with activation via IL4 and IL13. Furthermore, our THP-1 CMV FLuc line is constitutively active, relating to the overall protein expression of the cell.

Phorbol 12-myristate 13-acetate (PMA), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage colony-stimulating factor (M-CSF) are commonly used to differentiate monocytes into macrophages for in-vitro studies. The creation of temporal polarization profiles allows us to better understand the relationship between environmental stimuli and cell fate during monocyte differentiation.


Materials and Methods:: THP-1 wt, THP-1 CMV FLuc, THP-1 NFκB FLuc, and THP-1 STAT6 FLuc cells, monocytes were cultured in Roswell Memorial Institute (RPMI) 1640 Medium supplemented with 10% Fetal Bovine Serum. Cells were kept in an incubator at 37°C and 5% CO₂. Cells were used before 20 passages. For the bioluminescence temporal spectroscopy (BTS), we seeded 10,000 cells per well in a clear bottom white 96 well plate with either PMA, GM-CSF, M-CSF at a concentration between 5 ng/mL to 200 ng/mL. The cells are allowed to rest overnight before the media is replaced with media containing 1 mM of D-Luciferin. Luminescence is then measured using a plate reader before returning to the incubator. Every three days the media is replaced with new media containing 1 mM of D-Luciferin and the luminescence is measured (Figure 1). The inducible luminescence is normalized with the protein concentration of the media at the time, measured by a BCA protein assay (Thermo Scientific) using the manufacturer’s protocol.


Results, Conclusions, and Discussions:: We investigated the effects of PMA, GM-CSF, and M-CSF on changes in protein expression and polarization-related activation during monocyte to macrophage differentiation. We utilized constitutively active bioluminescent reporters (THP-1 CMV FLuc) to measure changes in protein expression during differentiation. Our results indicate an agent-dependent effect on luminescence over time (Figure 2). Although GM-CSF and M-CSF demonstrate a similar positive trend, PMA-differentiated macrophages exhibit a noticeable decrease in protein expression over the first 9 days, which eventually increases slightly over the next 9 days but remains lower than the initial level.

We investigated the polarization-related transcription factors using inducible reporter lines. (Figure 3) demonstrates changes in NFκB (M1, pro-inflammatory) and STAT6 (M2a, regulatory) activation over 18 days, normalized by media protein content. Results show that each stimulus has a unique, dose-dependent temporal activation profile. PMA-macrophages exhibit a early pro-inflammatory phenotype with strong NFκB activation, which rapidly decreases in the last 9 days. Their STAT6 activation is downregulated, except for a spike at day 6, like what was seen in their NFκB profile. In contrast, GM-CSF-macrophages demonstrate relatively constant NFκB activation and constant STAT6 activation in a dose-dependent manner. M-CSF-macrophages exhibit a relatively neutral profile, with lower NFκB activation than the other two types and similar STAT6 activation to GM-CSF. These results fit into established literature.

The advantage of using BTS method is adding a temporal element to polarization information. Methods like qPCR and flow cytometry are end point assays, giving precise but static polarization data. However, polarization, and differentiation, are inherently dynamic processes, as evidenced by the temporal curves made during this study. By adding temporal measurement, we are able to better track monocyte and macrophage polarization during the differentiation process.

Our results demonstrate that different stimuli lead to different temporal profiles of monocyte to macrophage differentiation in both their overall protein expression and polarization-specific transcription factor activation. GM-CSF- & PMA-macrophages had an inflammatory profile, though the PMA-macrophages had more quickly resolving response compared to the constant elevated activation in GM-CSF. M-CSF differentiated macrophages displayed an anti-inflammatory phenotype. This demonstrated how important picking differentiation protocols are on the resulting macrophage phenotype.


Acknowledgements (Optional): :

References (Optional): : Xia, L. et al. Role of the NFκB-signaling pathway in cancer. OncoTargets Ther. 11, 2063–2073 (2018).

Ushach, I. & Zlotnik, A. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage. J. Leukoc. Biol. 100, 481–489 (2016).

Takashiba, S. et al. Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB. Infect. Immun. 67, 5573–5578 (1999).

Li, H. et al. Transcriptional Regulation of Macrophages Polarization by MicroRNAs. Front. Immunol. 9, (2018).

Liu, T. et al. NF-κB signaling in inflammation. Sig Transduct Target Ther 2, 17023 (2017).