Monocyte-to-Macrophage Differentiation with 1,25-Dihydroxyvitamin D3 Potentially Enhances Macrophage Polarisation and Function in THP-1 Cells

Abstract

Macrophages are innate immune cells, leading the early defence against invading pathogens. The highly versatile and adaptable nature of macrophages is critical in maintaining immune homeostasis. The dysregulation of macrophage function has been linked to various pathologies. Hence, understanding macrophage biology is essential. THP-1 cells are the most widely used in vitro model for studying macrophage biology. Despite its broad adoption, the experimental protocol for obtaining mature macrophages from THP-1 cells has not been standardised. This is particularly evident when it comes to the concentration of phorbol 12-myristate 13-acetate (PMA), a widely used differentiation agent, and its associated impact on the inflammatory state of the derived macrophages. 1,25-Dihydroxyvitamin D3 (1,25D3), a well-known immunomodulator with anti-inflammatory properties, has the potential to augment the limitations of PMA-based differentiation protocols. This study, therefore, hypothesised that a low-dose PMA-based differentiation protocol in the presence of 1,25D3 would yield functional, naive macrophages (M0) that can be polarised into either the pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. To test this premise, THP-1 cells were differentiated with 5 nM PMA, in the presence and absence of 10 nM 1,25D3. To evaluate the functional implication of the differentiation protocol on their polarisation potential, M0 macrophages were stimulated for 24 h with 100 ng/ml lipopolysaccharide (LPS) and 20 ng/ml of interferon-Gamma (IFN-γ) to obtain M1 macrophages or 20 ng/ml interleukin 4 (IL-4) and 20 ng/ml IL-13 for 48 h to obtain M2 macrophages. The surface expression of M1-marker, CD80, and M2-marker, CD206, were then quantified by flow cytometry. As further confirmation of the inflammatory state of the cells, the expression levels of key inflammatory markers (RIG-I, IL-1β, CXCL-10, CCL-2 and IL-10) were quantified using RT-qPCR. To test the functional potential of the macrophages, they were transfected with the retinoic acid-inducible gene I (RIG-I) agonist, 5'ppp-dsRNA, for 2 h. The results revealed that the presence of 1,25D3 caused differentiated cells to adopt a morphology comparable to primary macrophages. Differentiation in the presence of 1,25D3 also supported macrophage polarisation into distinct M1 and M2 phenotypes based on CD80 and CD206 expression in response to IFN-γ + LPS and IL-4 + IL-13, respectively. Moreover, M1 macrophages differentiated from M0 macrophages obtained in the presence 1,25D3 showed significantly higher expression of inflammatory markers RIG-I and CXCL-10, compared to M1 macrophages differentiated from M0 macrophages in the absence of 1,25D3. Regardless of 1,25D3 presence during differentiation, neither M0 nor M1 macrophages launched a notable response to 5'ppp-dsRNA 2 hours post-transfection. However, M2 macrophages differentiated from M0 macrophages in the presence of 1,25D3 responded with a significant increase in inflammatory markers CXCL-10 (p < 0.001) and CCL-2 (p < 0.050) relative to the control. Thus, the presence of 1,25D3 during differentiation appears to support a macrophage phenotype that can respond to both inflammatory and anti-inflammatory signals. Taken together, the results support the premise that 1,25D3 augments low-dose PMA-based differentiation of THP-1 cells into functional macrophages