Dose-dependent phorbol 12-myristate-13-acetate-mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP-1 cells

Abstract

1, 2). Monocytes in the blood circulation migrate to the site of infection/inflammation and 48 differentiate into macrophages for effective host defense, tissue remodeling, and repair (3). 49Furthermore, macrophages exhibit a high level of plasticity, depending on their local 50 microenvironment, specialized functions and varied phenotype acquired (1, 4). 51Several models are employed to study the mechanisms of immune-modulation in monocytes 52 and macrophages during inflammatory diseases. The most frequently used include primary 53 peripheral blood mononuclear cells (PBMCs) and monocyte cell lines. However, due to donor-54 to-donor variations and technical disparities involved in handling of PBMCs in vitro, the 55 human leukemia monocytic cell line, THP-1, is widely accepted and used as a 56 monocyte/macrophage model (5, 6). Several studies have indicated that THP-1 cells can be 57 differentiated into macrophage-like cells using phorbol-12-myristate-13-acetate (PMA), 58 which markedly resembles PBMC monocyte-derived macrophages (MDMs) in cytokine 59 production, metabolic and morphological properties, including differential expression of 60 macrophage surface markers such as CD14, CD11b (ITGAM) and scavenger receptors-CD163, 61 MSR1, and SCARB2 (5, 7-10). Nonetheless, depending on the parameters of the differentiation 62 protocol employed, such as the concentration (ranging from 5 to 400 ng/mL), and duration of 63 incubation (1 to 5 days) with PMA; the degree of differentiation and the functional changes 64 may vary significantly (5,(11)(12)(13)(14)(15). 65At the molecular level, multiple proteins including growth factors, antigenic markers, 66 chemokine-receptors, cytokines, and cell adhesion molecules, are known to govern and reflect 67 underlying monocyte-macrophage differentiation processes (16)(17)(18)(19)(20). However, the effect of 68 various differentiation protocols on the cellular proteome and intracellular signaling networks 69 4 during monocyte-to-macrophage differentiation remains poorly understood. Hence, it is crucial 70 to determine the most suitable differentiation conditions when using these cells as model 71 system, as this can significantly impact their response to various innate immune stimuli. 72Quantitative high-resolution mass spectrometry-based proteomic approaches have been widely 73 employed to investigate the proteomes of monocytes and macrophages as well as altered 74 cellular proteomes and complex cellular/biological mechanisms in several biological 75 conditions (21-23). However, to date, no studies have directly compared the proteome changes 76 of the PMA-mediated differentiation process. 77In the present study, we evaluate the effect of three PMA-based differentiation protocols on 78 the changes in the proteome profiles upon THP-1 differentiation using stable isotope dimethyl 79 labeling quantitative proteomics. We demonstrate that various differentiation conditions such 80 as concentration and incubation time, prior to any stimuli, are critical consideration factors for 81 hetero...

SiO2 Fibers of Two Lengths and Their Effect on Cellular Responses of Macrophage-like Cells

“…After differentiation, the THP-1 cells became adherent with a lower proliferation rate. In agreement with the literature [ 41 , 42 ], the increase in the CD14 marker on the cell surface was another sign of completed differentiation ( Figure S1 in the Supplementary Materials ).…”

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

“…Data storage and handling are supported under the NIRD/Notur project NN9036K. This manuscript has been released as a pre-print at bioRxiv ( 61 ).…”

A data-driven computational model enables integrative and mechanistic characterization of dynamic macrophage polarization

“…It has been shown that GM-CSF and M-CSF, two commonly used factors in the experimental differentiation of macrophages from monocytes, can differentially regulate M1-M2 marker expression at baseline and also predispose macrophages to enhanced M1 and M2 responses, respectively, when further challenged by other stimuli ( Hamilton et al., 2014 ; Fleetwood et al., 2007 ). Different concentrations and durations of PMA (phorbol 12-myristate-13-acetate), which is widely used to condition the human monocytic THP-1 cells, also results in wide variations of downstream protein expression and ultimately nontrivial differences in their polarization responses ( Pinto et al., 2020 ). In addition, innate genetic differences between human and mouse macrophage cell lines ( Thomas and Mattila, 2014 ), between cell lines of human origin ( Mendoza-Coronel and Castanon-Arreola, 2016 ; Shiratori et al., 2017 ), and between mouse cell lines derived from different strains ( Santos et al., 2006 ) can all lead to differential macrophage phenotype outcomes (e.g., inconsistent marker expression patterns) when these cells are treated with the same stimulation.…”