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Transcriptomic Analysis of Macrophage Polarization Protocols: Vitamin D3 or IL-4 and IL-13 Do Not Polarize THP-1
Maria Rynikova1, Petra Adamkova1, Petra Hradicka1,2
1Department of Animal Physiology, Faculty of Science, Pavol Jozef Safarik University in Kosice, 041 54 Kosice, Slovakia.
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.
Insights
Comparing macrophage polarization protocols, researchers found that M1 macrophages can be reliably generated using PMA, IFN-γ, and LPS. However, current methods failed to produce consistent M2 macrophages, indicating a need for improved anti-inflammatory macrophage induction protocols.
Area of Science:
- Immunology
- Cell Biology
Background:
- Macrophages are key immune cells with inflammatory (M1) and anti-inflammatory (M2) phenotypes.
- In vitro polarization of monocytic cell lines, like THP-1, is used to generate M1 and M2 macrophages.
- A standardized protocol for reliable M1 and M2 macrophage acquisition from THP-1 cells is lacking.
Purpose of the Study:
- To compare three distinct in vitro polarization protocols for generating M1 and M2 macrophages from the THP-1 cell line.
- To evaluate the transcriptomic profiles of macrophages obtained through different polarization methods.
- To identify the most effective protocol for reliable M1 and M2 macrophage induction.
Main Methods:
- THP-1 cells were subjected to three different polarization protocols.
- Whole transcriptomes were analyzed using RNA-sequencing (RNA-seq).
- Differential gene expression and Gene Ontology (GO) enrichment analysis were performed.
Main Results:
- The protocol using PMA (61.3 ng/mL), IFN-γ, and LPS effectively induced M1 macrophages, showing high expression of M1-associated genes, cytokines, and chemokines.
- GO enrichment analysis confirmed differential expression of genes related to inflammatory and defensive processes in M1 macrophages.
- Protocols utilizing Vitamin D3, IL-13/IL-4, or IL-4 failed to consistently induce a reliable M2 macrophage phenotype.
Conclusions:
- The combination of PMA, IFN-γ, and LPS is a reliable method for M1 macrophage polarization from THP-1 cells.
- Existing protocols using Vitamin D3, IL-13/IL-4, and IL-4 are insufficient for generating reliable M2 macrophages.
- Further research and optimization are required to develop effective protocols for inducing anti-inflammatory M2 macrophages.
