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.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes

“…These genes include AREG , EREG , BCL6 , IL10, and IL18 which have been described as anti-inflammatory tissue repair genes <Fig 7G> (58,59). Thus, these cells were designated as anti-inflammatory, although they do not follow the traditional M2 polarization phenotype described in human monocytes (60). Our data suggests a novel classical monocyte subtype with an anti-inflammatory phenotype.…”

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody

“…Therefore, we aim to polarize CD147 KO THP-1 cells into an M1-like macrophage phenotype. This polarization was achieved using LPS or IFN-γ independently or in combination as described previously [ 39 , 44 ]. The phagocytic function of CD147 KO THP-1-derived M-LPS was enhanced by Takatamab to promote ADCP responses to Jurkat T cells.…”

“…A real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was utilized to analyze TNF-α and IL-10 gene expression. WT THP-1 or CD147 KO THP-1 cells were differentiated into M0-like macrophages via incubation with 61.3 ng/mL of PMA for 6 h [ 44 ]. After incubation, the PMA-containing medium was removed and replaced by 20 ng/mL of LPS for 48 h [ 39 ] to produce M-LPS macrophages or 20 ng/mL IL-4 for 48 h to obtain M-IL-4 macrophages [ 44 ].…”

“…WT THP-1 or CD147 KO THP-1 cells were differentiated into M0-like macrophages via incubation with 61.3 ng/mL of PMA for 6 h [ 44 ]. After incubation, the PMA-containing medium was removed and replaced by 20 ng/mL of LPS for 48 h [ 39 ] to produce M-LPS macrophages or 20 ng/mL IL-4 for 48 h to obtain M-IL-4 macrophages [ 44 ]. The total RNA was extracted from the cells using RNeasy Mini Kit, and subsequently converted to cDNA using the SuperScript TM III First-Strand Synthetic System (Thermo Fisher Scientific, Carlsbad, CA, USA).…”

Delineation of signaling routes that underlie differences in macrophage phenotypic states

“…In vitro assays of human macrophages are largely performed either on the differentiated THP-1 cell line or on primary blood-derived macrophages. THP-1 is a human leukemia monocytic cell line, which does not accurately represent relevant immune processes or reflect inter-individual variability in macrophage responses to stimuli 19 .…”