Comparative Proteomic Analysis Reveals Varying Impact on Immune Responses in Phorbol 12-Myristate-13-Acetate-Mediated THP-1 Monocyte-to-Macrophage Differentiation

Abstract

Macrophages are sentinels of the innate immune system, and the human monocytic cell line THP-1 is one of the widely used in vitro models to study inflammatory processes and immune responses. Several monocyte-to-macrophage differentiation protocols exist, with phorbol 12-myristate-13-acetate (PMA) being the most commonly used and accepted method. However, the concentrations and duration of PMA treatment vary widely in the published literature and could affect the probed phenotype, however their effect on protein expression is not fully deciphered. In this study, we employed a dimethyl labeling-based quantitative proteomics approach to determine the changes in the protein repertoire of macrophage-like cells differentiated from THP-1 monocytes by three commonly used PMA-based differentiation protocols. Employing an integrated network analysis, we show that variations in PMA concentration and duration of rest post-stimulation result in downstream differences in the protein expression and cellular signaling processes. We demonstrate that these differences result in altered inflammatory responses, including variation in the expression of cytokines upon stimulation with various Toll-like receptor (TLR) agonists. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics with one of the most common in vitro models for macrophages, which in turn has a profound impact on the immune as well as inflammatory responses being studied.

Macrophage uptake of oxidized and acetylated low-density lipoproteins and generation of reactive oxygen species are regulated by linear stiffness of the growth surface

“…THP1 cells require PMA stimulation to induce the adherent macrophage phenotype, and human monocytes much be differentiated for seven days in GM-CSF, in order to study the effects of extracellular stiffness in our model [ 106 ]. PMA treatment would make interpretation of these studies challenging since it changes in expression of CD36, a receptor we showed in this paper is also regulated by surface stiffness [ 107 ]. Furthermore, compared to primary monocytes, THP1 cells are deficient in CD14, and are thus hyporesponsive to LPS and may result in decreased uptake of minimally oxidized LDL [ 101 , 108 , 109 ].…”

UMP-CMP kinase 2 gene expression in macrophages is dependent on the IRF3-IFNAR signaling axis

“…Human THP-1 monocytic cells (ATCC) were grown in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-activated fetal calf serum (FCS), 2 mM L-glutamine, 100 nM penicillin/streptomycin (Thermo Fisher Scientific), and 50 μM β -mercaptoethanol (Sigma-Aldrich) and cultured at 37°C in a 5% CO 2 atmosphere. THP-1 cells were differentiated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, Darmstadt, Germany) overnight (16 hours) and rested in a PMA-free medium for 48 hours before further experiments [ 30 ].…”

Temporal Quantitative Phosphoproteomics Profiling of Interleukin-33 Signaling Network Reveals Unique Modulators of Monocyte Activation

“…Transcription factors are well-known regulators of gene expression. Several of these play important roles in monocyte biology regulating development [ 58 , 59 ], regulating monocyte-to-macrophage differentiation [ 60 , 61 ] and causing the overexpression of cell-specific cytokines and chemokines [ 62 ]. In our study, IL-33 was found to regulate the phosphorylation dynamics of 178 transcription factors, including several members of the forkhead-box (FOX) family of transcription factors, which play a critical role in immunoregulation in several immune cell lineages.…”