Transcriptomic Analysis of Macrophage Polarization Protocols: Vitamin D₃ or IL-4 and IL-13 Do Not Polarize THP-1 Monocytes into Reliable M2 Macrophages

Abstract

Two main types of macrophages (Mφ) include inflammatory (M1) and anti-inflammatory (M2) macrophages. These cells can be obtained in vitro by polarization of monocytic cell lines using various stimuli. Since there is currently no consensus on the best method for the acquisition of reliable M1 and M2 macrophages from the THP-1 cell line, we decided to compare three different polarization protocols at the transcriptomic level. Whole transcriptomes of Mφ polarized according to the chosen protocols were analyzed using RNA-seq. Differential expression of genes and functional enrichment for gene ontology terms were assessed. Compared with other protocols, M1 macrophages polarized using PMA (61.3 ng/mL) and IFN-γ along with LPS had the highest expression of M1-associated regulatory genes and genes for M1 cytokines and chemokines. According to the GO enrichment analysis, genes involved in defensive and inflammatory processes were differentially expressed in these Mφ. However, all three chosen protocols which use Vit D3, IL-13/IL-4, and IL-4, respectively, failed to promote the polarization of macrophages with a reliable M2 phenotype. Therefore, optimization or development of a new M2 polarization protocol is needed to achieve macrophages with a reliable anti-inflammatory phenotype.

Keywords

• M1 macrophages
• M2 macrophages
• polarization protocol
• transcriptomic analysis
• differential gene expression
• RNA-seq