PMA

The choice of phorbol 12-myristate 13-acetate differentiation protocol inﬂuences the response of THP-1 macrophages to a pro-inﬂammatory stimulus

Maria E. Lund ⁎, Joyce To, Bronwyn A. O’Brien, Sheila Donnelly
School of Life Sciences, University of Technology Sydney, New South Wales, Australia

a r t i c l e i n f o

Article history:
Received 9 July 2015
Received in revised form 4 January 2016
Accepted 26 January 2016 Available online xxxx

Keywords: THP-1 cells Macrophages PMA
LPS TNF
Pro-inﬂammatory

a b s t r a c t

The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/ macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inﬂammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated signiﬁcant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differ- entiation of THP-1 macrophages. Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, speciﬁcally in response to the pro- inﬂammatory stimulus, lipopolysaccharide (LPS). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied signiﬁcantly, and was dependent upon the concentration of PMA used to stimulate the differ- entiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24 h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells.
Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.

1.Introduction

The human leukaemia-derived monocytic cell line, THP-1, is the most widely used cell line for in vitro studies investigating primary human macrophage function. This is because, after their differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire phe- notypic and functional characteristics which closely resemble those of primary human macrophages. After exposure of differentiated THP-1 cells to pro-inﬂammatory stimuli, notably lipopolysaccharide (LPS), this maturation phenotype is characterised by increased cell adherence, expression of classical macrophage markers, enhanced phagocytic abil- ity, and secretion of pro-inﬂammatory mediators, such as superoxide and tumour necrosis factor (TNF) (Auwerx, 1991; Schwende et al., 1996a, 1996b).

Abbreviations: FCS, foetal calf serum; HI, heat inactivated; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; TNF, tumour necrosis factor; SSC-H, side scatter height; SEM, standard error of the mean.
⁎ Corresponding author at: School of Life Sciences, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007, Australia.
E-mail address: [email protected] (M.E. Lund).

The elucidation of biological phenomena using cell lines as surro- gates for their primary counterparts is frequently criticised. However, it is often not practical to use primary cells ex vivo. This is especially the case for human macrophages, due to the inability to expand macro- phage populations ex vivo, and their limited life span in culture. Accord- ingly, THP-1 cells have been used as a model system to overcome the experimental limitations of using primary macrophages, and they have become the most widely used cell line for investigations to deter- mine the response of human macrophages to pro-inﬂammatory stimuli (Chanput et al., 2012; Feng et al., 2004; Hjort et al., 2003; Kim et al., 2010; Kim et al., 2008; Ueki et al., 2002; Zhao et al., 2015).
Despite the widespread use of THP-1 cells as representatives of in vivo human macrophages, there has been no standardised protocol established for the differentiation of THP-1 monocytes into bonaﬁde macrophages, using PMA. Typically, THP-1 monocytes are initially acti- vated with PMA, followed by a period of culture in the absence of PMA (to allow a degree of cellular recovery), prior to their use in in vitro stud- ies. However, the concentration of PMA used differs greatly between studies, ranging from 6 to 500 nM, which represents over an 80-fold difference in concentration (Feng et al., 2004; Park et al., 2007; Spano et al., 2007; Tulk et al., 2015). In addition, the length of time that cells

http://dx.doi.org/10.1016/j.jim.2016.01.012

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are exposed to PMA ranges from 3 to 72 h, which represents a 24-fold difference in the length of the stimulation period (Daigneault et al., 2010; Feng et al., 2004). Furthermore, there are signiﬁcant differ- ences in the recovery periods implemented post differentiation, with some studies eliminating the rest period (Chanput et al., 2010; Tulk et al., 2015), and others allowing stimulated THP-1 cells to recover for as long as 5 (Daigneault et al., 2010) or 10 (Solberg et al., 2015) days.
Monocytes and macrophages exhibit considerable plasticity, and gain unique phenotypic and functional characteristics, which are dependent upon the stimulatory environment (Mosser and Edwards, 2008). For example, a pro-inﬂammatory environment drives monocytes/macrophages toward an M1 phenotype, while the presence of speciﬁc stimuli and/or regulatory/anti-inﬂammatory cyto- kines drives the differentiation of cell populations that are collectively termed M2 macrophages (but which consist of subsets, dependent upon the speciﬁc stimulant and cytokines present) (Roszer, 2015). Ac- cordingly, the differentiation conditions (PMA concentration, stimulus duration, and length of rest period) used to generate terminally differ- entiated macrophages, invariably affects the phenotypic and functional characteristics of the population. Furthermore, many studies using THP- 1 macrophages have not rigorously established the phenotypic and functional characteristics of the differentiated cell population(s). As the use of a variety of PMA differentiation protocols likely generates a range of functionally distinct macrophage subsets, clinical applicability and direct comparisons of in vitro studies are compromised. For exam- ple, maturation of THP-1 cells using higher concentrations of PMA, for instance 100 ng/ml (~ 160 nM), increased the expression levels of genes associated with inﬂammation, and enhanced the secretion of pro-inﬂammatory cytokines, including TNF and IL-8, as compared to a concentration of 5 ng/ml (~8 nM) PMA (Park et al., 2007). This suggests that THP-1 cells differentiated using higher concentrations of PMA may acquire a more activated phenotype, as compared to those differentiat- ed using lower PMA concentrations.
Variations in the duration of the rest period also signiﬁcantly im- pact upon the phenotype of macrophage produced. Extended rest periods in the absence of PMA led to the de-differentiation of THP- 1 cells, which was associated with regained proliferative ability, sug- gesting reversion of macrophage populations to a monocytic pheno- type (Spano et al., 2013; Spano et al., 2007). Cellular adhesion and morphological features (such as cell spreading), are generally the accepted criteria for the successful differentiation of THP-1 cells into macrophages. However, these readouts provide limited infor- mation regarding the phenotype, and, consequently, the biological functions of the resultant differentiated macrophage and, therefore, the authenticity of data reported.
THP-1 cells are commonly used to assess compounds/agents for their ability to modulate macrophage activity (Budai et al., 2013; Chanput et al., 2012; Feng et al., 2004; Kim et al., 2010; Kim et al., 2008; Smiderle et al., 2011; Solberg et al., 2015; Ueki et al., 2002). Secre- tion of TNF by differentiated THP-1 cells in response to stimulation with LPS is accepted as a measurable pro-inﬂammatory response (Ueki et al., 2002). Given the widespread use of THP-1 cells for studies of the exac- erbation or inhibition of inﬂammatory responses, it is critically impor- tant to establish and use a standardised, reproducible and reliable protocol for THP-1 cell differentiation. This would ensure that the baseline phenotypic and functional characteristics of the resultant macrophage population are consistent between studies.
Here, we compared the most commonly published PMA differentia-
tion protocols, with a focus on the pro-inﬂammatory response of THP-1 cells to LPS stimulation. We have demonstrated that the choice of proto- col signiﬁcantly inﬂuences the functional activities of macrophages, highlighting the need to establish and use a uniform PMA differentiation protocol. Adoption of such a standard will increase the in vivo relevance of in vitro experimental data, and will ensure consistency between in vitro studies using THP-1 cells.
2.
Materials and methods

2.1.Culture and differentiation of THP-1 cells

THP-1 cells (ATCC) were maintained in RPMI 1640 supplemented with heat inactivated foetal calf serum (FCS; 10% v/v), Hepes (10 mM), sodium pyruvate (1 mM; all from Invitrogen) and β-mercaptoethanol (0.05 mM) (referred to hereafter as media), in a humidiﬁed incubator (37 °C/5% CO2). Cells were grown to a density of 1–8 × 105 cells/ml and used for experiments between passage numbers 5 and 10.
For differentiation to a macrophage phenotype, THP-1 cells (at a concentration of 2 × 105 cells/ml) were incubated with varying concen- trations of PMA (8–200 nM; Sigma Aldrich) diluted in media, in T 75 cm2 tissue culture ﬂasks (BD) or 6 well tissue culture plates (Nalge Nunc), for 48 h. This length of exposure to PMA is commonly used in the literature (Chanput et al., 2010; Chanput et al., 2012; Park et al., 2007; Smiderle et al., 2011; Ueki et al., 2002). TNF secretion in response to LPS peaks following 48 h exposure to PMA (Schwende et al., 1996a, 1996b). Thus, to optimise the measurable TNF response, and to mini- mise PMA exposure, 48 h incubation was chosen. Following differentia- tion, PMA-containing media was replaced with fresh media, and cells were used immediately, or rested in culture for 24 h, 48 h, or 5 days, with media changed daily. To detach adherent cells, the ﬂask or well was rinsed twice with sterile saline (Baxter Healthcare), and then incubated with TrypLE Express (Invitrogen) for 30 min, in a humidiﬁed incubator (37 °C/5% CO2). Cells were then fully detached by gentle scraping, collected and rinsed twice with RPMI 1640. Following collec- tion, cell viability was always N 93%, as assessed microscopically by Trypan Blue exclusion or by ﬂow cytometry using the viability stain Sytox Blue (Invitrogen).

2.2.Assessment of THP-1 cell differentiation

To assess the acquisition of a macrophage-like phenotype (cell adhesion, spreading, and increased cytoplasmic volume), cells were visualised by phase-contrast microscopy at a magniﬁcation of 400 ×, using the Nikon Inverted Microscope DS FI2 (Nikon).
The extent of differentiation was also measured by ﬂow cytometry, using forward and side scatter properties. For this, THP-1 cells differen- tiated with varying PMA concentrations, were gently removed from wells by scraping, and analysed using the BD LSR II (BD Biosciences). Analysis was restricted to live cells using Sytox Blue viability staining.
To determine the percentage adherence, cells were cultured in 6 well plates for 48 h, in triplicate, with varying concentrations of PMA, at a density of 2 × 105 cells/ml. The non-adherent cells were collected, and cell counts were performed. The supernatant was then replaced with fresh media. Cells were incubated for a further 24 h, before super- natants were once again collected, and numbers of non-adherent cells were quantiﬁed. This process was continued until cells had been rested for 5 days. To calculate the percentage adherence for each sample, the total number of non-adherent cells was subtracted from the starting cell number (4 × 105 cells), then divided by the starting cell number, and multiplied by 100. The total number of non-adherent cells for each time point was calculated as the sum of the non-adherent cells recorded up to and including that time point. For example, the number of non-adherent cells following 48 h of rest was calculated as the sum of non-adherent cells recorded at 0 h, 24 h and 48 h.
2.3.Assessment of the pro-inﬂammatory response of differentiated THP-1 cells to LPS stimulation

To measure the response of matured THP-1 macrophages to LPS (Sigma Aldrich; from Escherichia coli 0111.B4), cells were detached using a combination of TrypLE and gentle scraping (as described above), following incubation with varying concentrations of PMA, and various periods of rest in culture. Cells were prepared in media, at a

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density of 1 × 106 cells/ml, and seeded, in triplicate, into 96 well tissue culture plates (Nalge Nunc; 200 μl/well). The cells were allowed to recover for 2 h (37 °C/5% CO2), and then stimulated with LPS for 2 h (for gene expression analysis) or 4 h (for analysis of cytokine levels in supernatants by ELISA). Following centrifugation of the plate (1200 rpm, 5 min), supernatants were collected and frozen at −20 °C, until analysis. To quantify levels of TNF in cell supernatants, a human TNF II ELISA kit (BD) was used, according to the manufacturer’s instructions.
For gene expression analysis, total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Total RNA (500 ng) was reverse transcribed using SuperScript III RT (Thermoﬁsher Scientiﬁc) according to the manufacturer’s instructions. Triplicate, single plex reactions were performed using Taqman Gene Expression Master Mix (2X) (Thermoﬁsher Scientiﬁc), Taqman Gene Expression Assays (20X) (Gapdh: HS03929097_g1; TNF: HS01113624_g1; both FAM;
Thermoﬁsher Scientiﬁc), cDNA representing 25 ng RNA and water to volume. The QuantStudio 12K Flex Real Time PCR System (Thermoﬁsher Scientiﬁc) was used for qRT-PCR with the following steps: 50 °C 2 min, 95 °C 10 min and then 40 cycles of: 95 °C 15 s and 60 °C 1 min. No RT controls (all components of reverse transcription reaction, without RT) were run for each sample to conﬁrm the absence of genomic DNA. The ΔCt value for each sample was calculated from triplicate reactions as (mean Ct target gene (TNF) − mean Ct reference gene (Gapdh)). This ΔCt value was then transformed using the equation 2−ΔCt for individual samples (Schmittgen and Livak, 2008).
2.4.Statistical analysis

Data are reported as the mean ± standard error of the mean (SEM) or the mean ± standard deviation (SD), as speciﬁed in the ﬁgure legends. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc.), using a two-tailed unpaired t-test or a one-way ANOVA, as indicated. Statistical signiﬁcance was deﬁned as p b 0.05, and speciﬁc signiﬁcance values are stated in the ﬁgure legends.

3.Results

3.1.The concentration of PMA used to differentiate THP-1 monocytes does not impact upon cell morphology

The differentiation of THP-1 monocytes with concentrations of PMA ranging from 8 to 200 nM resulted in an increase in cytoplasmic volume (as compared to undifferentiated cells), as measured by an increase in forward scatter by ﬂow cytometry, and visually by phase-contrast microscopy (Fig. 1A). The observed increases in cell volumes were uni- form across the entire range of PMA concentrations tested.
Differentiated macrophages also exhibited an increase in granularity, which was determined by ﬂow cytometry as an increase in side scatter. THP-1 cells differentiated with PMA exhibited higher side scatter (which was uniformly observed, independently of PMA concentration), as compared to undifferentiated THP-1 monocytes (Fig. 1A–B). Collec- tively, these data suggest that the generation of a macrophage phenotype could be obtained using all concentrations of PMA commonly used to differentiate THP-1 cells.

3.2.The adherent properties of THP-1 cells are modulated by the PMA differentiation protocol used

Cell adhesion and spreading are widely accepted hallmarks of the differentiation of monocytes, both primary and THP-1 cells, into macrophages. To determine the effect of PMA concentration, and the subsequent period of cellular rest in the absence of PMA, on the adher- ence of THP-1 cells, the proportion of adherent cells was calculated after differentiation with varying concentrations of PMA (8–200 nM), follow- ed by a rest period of 0 h, 24 h, 48 h or 5 days.

It has been reported that increasing concentrations (6–60 nM) of PMA induced a spectrum of maturation proﬁles in THP-1 macrophages, as indicated by reduced levels of adherence in cell populations that were differentiated with lower concentrations of PMA, following 72 h of PMA withdrawal (Spano et al., 2013). The current study corroborates these ﬁndings, as we observed that the concentration of PMA signiﬁ- cantly affected cellular adherence, but only following a period of PMA withdrawal. Immediately post differentiation (0 h), there was no signif- icant difference in adherence between cells stimulated with varying PMA concentrations (p = 0.02; Fig. 2).
Following 24 h of rest, THP-1 cells that were differentiated with the two highest PMA concentrations (100 and 200 nM) exhibited signiﬁ- cantly more adherence, as compared to those matured with a low con- centration of PMA (8 nM; p = 0.0017 and 0.0192, respectively; Fig. 2). There was a higher proportion of adherent THP-1 cells in cultures matured with 50 nM PMA, as compared to cells exposed to 8 nM PMA, at this time point, although this increase did not reach signiﬁcance (p = 0.0822). Similarly, at later time points (48 h and 5 days), the per- centage of adherent cells was higher in THP-1 cell populations matured in the presence of higher concentrations of PMA, than those differenti- ated using 8 nM PMA.

3.3.The pro-inﬂammatory response of differentiated THP-1 macrophages varies according to the PMA differentiation protocol used

Mature THP-1 macrophages secrete TNF in response to pro- inﬂammatory stimuli, such as LPS (Park et al., 2007). Therefore, the effect of both PMA concentration (8–200 nM) and rest period (0–5 days) on the pro-inﬂammatory response (as determined by TNF secretion) of THP-1 cells was assessed after LPS stimulation. Two concentrations of LPS (10 and 100 ng/ml) were used to compare the effect of different PMA differentiation protocols on the response to weak (10 ng/ml LPS) and stronger (100 ng/ml LPS) pro-inﬂammatory signals. Levels of TNF in the culture supernatant were assessed 4 h post stimulation because at this time point TNF secretion levels in response to LPS were maximal (data not shown). Negligible to no TNF was secreted by differentiated THP-1 cells in the absence of LPS stimulation (data not shown).

3.3.1.PMA concentration modulates the pro-inﬂammatory response of THP-1 macrophages
THP-1 cells differentiated with varying concentrations (8–200 nM) of PMA secreted TNF in response to LPS stimulation. However, the mag- nitude of this pro-inﬂammatory response was dependent upon the PMA concentration, likely indicative of differences in the extent of cellular maturation induced.
THP-1 cells that were differentiated with higher concentrations (50– 200 nM) of PMA secreted signiﬁcantly higher levels of TNF, as compared to those matured in the presence of a low concentration (8 nM) of PMA, in response to both weak and strong LPS stimuli (Fig. 3A–B). For exam- ple, cells differentiated with 100 nM PMA, secreted 352–422 pg/ml TNF, as compared to those matured with 8 nM PMA, which secreted only 57– 88 pg/ml TNF, in response to a weak (10 ng/ml) LPS stimulation (Fig. 3A). Similarly, when stimulated with the higher concentration (100 ng/ml) of LPS, THP-1 cells matured in the presence of 100 nM PMA secreted 264–294 pg/ml TNF, as compared to only 132–187 pg/ml TNF that was secreted by cells exposed to 8 nM PMA (Fig. 3D).
The increased secretion of TNF following LPS stimulation of cells differentiated with higher concentrations of PMA, was reﬂected at the level of gene expression. Cells differentiated with 100 nM PMA, expressed signiﬁcantly higher levels of TNF, as compared to those cells differentiated with 8 nM PMA, in response to both low and high concen- trations of LPS, as assessed by qRT-PCR (Fig. 3B, E; p b 0.0001). It should be noted, however, that the levels of TNF expressed by cells differentiat- ed with 100 nM PMA (in the absence of LPS stimulation) was signiﬁ- cantly higher than those expressed by cells differentiated with 8 nM PMA, under the same conditions. This corroborates the ﬁndings of

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Fig. 1. Morphology of THP-1 macrophages differentiated with varying concentrations of PMA. THP-1 cells were differentiated, in triplicate, with 8, 25, 50, 100 or 200 nM PMA, for 48 h. Cell morphology was then assessed by phase-contrast microscopy, and cell size and granularity were measured by ﬂow cytometry (forward and side scatter properties). A. Forward and side scatter plots (left panels) and phase-contrast images (right panels) of cells differentiated with 0–200 nM PMA. Scale bars represent 30 μm. B. Geometric means obtained from side scatter height (SSC-H) measurements of cells differentiated with 0–200 nM PMA. For statistical analyses, SSC-H measurements for each PMA concentration were compared to SSC-H measurements for undifferentiated (0 nM PMA) THP-1 cells, using an unpaired two-tailed t-test, where ***p b 0.0001. These data are representative of two independent experiments.

Park et al. (2007), who demonstrated that incubation with increasing concentrations of PMA, in the absence of further stimulation, was asso- ciated with increased gene expression of TNF.

3.3.2.The duration of PMA-withdrawal modulates the pro-inﬂammatory response of THP-1 macrophages differentiated with higher concentrations of PMA
The pro-inﬂammatory response of THP-1 cells that were matured with a lower concentration (8–25 nM) of PMA, to a weak LPS stimulus,

was unchanged after any length (1–5 days) of rest in culture following PMA stimulation (Fig. 3C). Similarly, in response to a stronger LPS signal, cells differentiated with 8–25 nM PMA responded in a similar manner, regardless of the length of the subsequent rest period (Fig. 3D).
Conversely, THP-1 cells differentiated with higher concentrations (50–100 nM) of PMA, and then exposed to a weak LPS stimulus, secret- ed different levels of TNF, which were dependent upon the duration of PMA withdrawal (Fig. 3C). Following 24 h of rest in culture, THP-1 cells that were differentiated with 50 nM PMA secreted less TNF in

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Fig. 2. The adherent properties of THP-1 cells are dependent upon the PMA concentration and the duration of PMA withdrawal. THP-1 cells were differentiated with varying concentrations (8–200 nM) of PMA over 48 h, before being cultured in PMA-free media for 0 h, 24 h, 48 h, or 5 days. The proportion of adherent cells was calculated at each time point. The data shown are the mean % adherence (±SEM) of triplicate samples and are representative of three independent experiments. A two-tailed, unpaired t-test was used to determine signiﬁcance for each PMA concentration, as compared to 8 nM PMA, at the same time point, where *p b 0.05, **p b 0.001 and ***p b 0.0001. One-way ANOVAs were performed to compare adherence between THP-1 cells generated after exposure to the 5 concentrations of PMA used, as a group (indicated by the horizontal bars and respective p-values), at a given time point.

response to weak LPS stimulation (10 ng/ml), as compared to immedi- ately (0 h) following PMA activation, albeit that this decrease did not reach signiﬁcance. Following 48 h or 5 days of rest, the same cells secreted signiﬁcantly lower amounts of TNF, as compared to those levels released at 0 h. Similarly, following 24 h, 48 h and 5 days of rest in culture, THP-1 cells matured with 100 nM PMA secreted signiﬁcantly less TNF in response to the LPS pro-inﬂammatory stimulus, as compared to secretion levels immediately following (0 h) PMA treatment. A simi- lar, albeit statistically insigniﬁcant, trend was observed for THP-1 cells treated with 200 nM PMA. For example, after 48 h rest in culture, THP-1 cells that were differentiated with 200 nM PMA secreted less TNF in response to those stimulated immediately after PMA treatment, although this trend failed to reach statistical signiﬁcance (p = 0.1318). Interestingly, differences in the magnitude of the TNF response by THP-1 cells associated with a period of rest in culture were not evident when cells were stimulated with a higher concentration (100 ng/ml) of
LPS (Fig. 3D).

4.Discussion

The initial steps in drug discovery often involve screening com- pounds using cell-based in vitro assays to predict potential in vivo efﬁcacy. Accordingly, selection of a biologically representative cell line (in situations where it is not feasible to use primary cells) and appropri- ate assay conditions are important for generating authentic, reliable results. Irrespective of the model system selected, the most important experimental component is to ensure the use of a consistent and repro- ducible protocol that modulates cellular activity independently of the experimental parameter (such as drug-induced modulation of pheno- type and function) to be assessed. The human monocytic leukaemia cell line, THP-1, is commonly used as an in vitro representative of primary human macrophages. Differentiated THP-1 macrophages, like primary human macrophages, respond to stimulation with pro-inﬂammatory signals (such as LPS) by secreting a range of pro-inﬂammatory media- tors, most notably TNF (Feng et al., 2004; Schwende et al., 1996a, 1996b). This analogous behaviour of THP-1 cells to their primary counterparts has led to the widespread use of this cell line in studies of macrophage activation and differentiation, macrophage responses to inﬂammatory situations, and mechanisms of inﬂammatory disease, with over 100 published reports using THP-1 cells for such applications

to date. THP-1 cells have also been used as a model system to elucidate the mechanisms of action of novel therapeutics as an intermediary step on the path to clinical trials (Bressler et al., 2015).
However, despite the wide acceptance of the THP-1 cell line as a suitable model to evaluate macrophage functions and responses to external stimuli in vitro, there is currently no standardised protocol for the differentiation of THP-1 monocytes to macrophages using PMA. The lack of a uniform protocol impacts greatly upon the interpretation of results, and the ability to compare studies. This is because the exper- imental design does not account for possible phenotypic and/or func- tional differences in macrophage populations that are attributable to the use of different maturation protocols. The differentiation protocol used would represent a signiﬁcant confounding factor, independent of any experimental effects (i.e. the effects of drug co-incubation) being studied. In vivo, macrophages are a dynamic cell population, whose phe- notypic and functional characteristics differ signiﬁcantly, depending upon the composition of the environment in which they mature, and the nature of stimuli added (Mosser and Edwards, 2008; Roszer, 2015). This is also true for THP-1 monocytes, which can be skewed toward different macrophage phenotypes by incubation with speciﬁc inﬂammatory stimuli. For example, THP-1 cells can be driven toward an M1 phenotype using a combination of LPS and IFN-γ (Chanput et al., 2013). Alternatively, an M2a phenotype is generated using IL-4 alone (Chanput et al., 2013) or in combination with IL-13 (Caras et al., 2011). Given that PMA is, in essence, an activating stimulus, it is con- ceivable that different PMA differentiation protocols will generate THP-1 macrophages, which possess characteristics representative of various points on the macrophage activation spectrum.
Indeed, several studies have demonstrated both functional and phe- notypic differences in THP-1 cells matured with different PMA concen- trations (Maeβ et al., 2014; Park et al., 2007; Spano et al., 2013; Spano et al., 2007), or exposed to different periods of rest in the absence of PMA following differentiation (Daigneault et al., 2010). Beyond the use of THP-1 macrophages for studies of the inﬂammatory response in “naïve” macrophages, THP-1 macrophages can be polarised for the study of M1/M2 macrophage phenotypes (Chanput et al., 2013). It is important to note that increasing concentrations of PMA have been associated with the expression of markers of an M1 phenotype and that the concentration of PMA may affect the potential for M1/M2 polarisation (Maeβ et al., 2014). The data presented herein focuses on the effect of PMA on the inﬂammatory response, and recommends a dif- ferentiation protocol based on this speciﬁc application. However, for the use of THP-1 macrophages for polarisation, the optimal differentiation protocol should be determined and the polarising potential of PMA carefully considered.
The current study compared the effect of the most commonly used PMA differentiation protocols on the phenotypic and functional charac- teristics of the resultant population of THP-1 ‘macrophages’ generated. Changes in morphological characteristics, adherence levels and the pro-inﬂammatory response to LPS stimulation, were assessed in THP- 1 cells following incubation with PMA.

5.Conclusions

Collectively, the data of the current study demonstrate that the dif- ferentiation of THP-1 cells, using different PMA differentiation proto- cols, generates macrophages at various stages of maturation. These macrophage populations are functionally different, as they respond variably to the common, and biologically relevant, pro-inﬂammatory stimulus of LPS. Thus, the use of different PMA differentiation protocols introduces the likelihood of naively and incorrectly comparing the response of presumed uniform, yet functionally different, macrophage subsets, between studies, based on the presumption that all investiga- tions have used terminally differentiated macrophages.
This study proposes that a single PMA differentiation protocol should be used for investigations of THP-1 macrophage function, to

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Fig. 3. The pro-inﬂammatory response of THP-1 macrophages is dependent upon the concentration of PMA used to stimulate monocyte differentiation and the period of rest following PMA exposure. THP-1 cells were differentiated with 8–200 nM PMA for 48 h, and then collected immediately (A, D) or immediately, after 24 h, 48 h or 5 days of culture in the absence of PMA (F, G). Cells were collected and co-incubated with LPS at a concentration of 10 ng/ml (A, F) or 100 ng/ml (D, G), for 4 h in culture. Data shows the concentration of TNF measured in supernatants, and bars represent the mean ± SEM of triplicate experimental values. Statistical analyses were performed to compare the levels of TNF secretion by cells differentiated with a given concentration of PMA, as compared to those matured with 8 nM PMA, for each LPS concentration (A, D). Statistical differences were determined using a two-tailed, unpaired t-test, comparing TNF secretion at a given time point to TNF secretion at 0 h, for a given concentration of PMA (F, G). THP-1 cells were differentiated with 8 nM or 100 nM PMA for 48 h, and then stimulated with LPS at a concentration of 10 ng/ml (B), 100 ng/ml (E) or media alone (C) for 2 h. Gene expression of TNF was measured by qRT-PCR, and then compared between samples, following normalisation to the reference gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Data have been expressed as 2−ΔCt values, multiplied by 1000, for individual replicates (n = 4), and bars represent the mean ± SD (B, C and E). Statistical differences between groups were determined using a two-tailed, unpaired t-test. *p b 0.05, **p b 0.001, ***p b 0.0001.

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ensure consistency, so that valid comparisons can be made between studies, speciﬁcally with respect to the response of THP-1 macrophages to a pro-inﬂammatory stimulus.
Following a rest period (≥ 24 h) in the absence of PMA, THP-1 cells
that were differentiated with lower concentrations (8–50 nM) of PMA, responded comparably to a subsequent weak LPS stimulus (10 ng/ml), when rested for different periods of time. However, given that THP-1 macrophage yield was optimal following 24 h of rest in culture, a rest period of 24 h is recommended.
The use of higher concentrations of PMA generated THP-1 cells that responded more strongly to LPS stimulation, as measured by TNF secre- tion. However, here we show that exposure to higher concentrations of PMA, is associated with the up-regulation of inﬂammatory gene expres- sion, as compared to lower concentrations of PMA, in the absence of LPS stimulation. Depending on the strength of the stimulus, this increase in baseline gene expression may mask changes in gene expression attrib- uted to subsequent stimulation. Thus, it is crucial to assess the impact of PMA alone on the gene of interest. Contrary to gene expression, we did not detect an increase in TNF secretion by ELISA, following exposure to higher concentrations of PMA, as compared to lower levels of PMA, in the absence of subsequent stimulation with LPS. Thus, caution should be exercised when comparing different read outs (expression at the level of gene and protein) of inﬂammation, and these results suggest the use of more than one measurement assessing the inﬂammatory re- sponse may be appropriate. Finally, the current study demonstrates that the use of higher PMA concentrations, when investigating the mac- rophage inﬂammatory response, may not be ideal, particularly when using inﬂammatory gene expression as an experimental readout.
Following 24 h rest, cells differentiated with 8 nM PMA secreted very
low levels of TNF in response to LPS stimulation. Thus, to balance the objective of inducing a potent and measurable response, while limiting the inﬂammatory response caused by PMA exposure per se, the present study indicates that a concentration of 25 nM PMA (for 48 h, followed by 24 h rest in the absence of PMA) should be used to induce a consis- tent phenotype of THP-1 macrophage, for the study of inﬂammatory responses under various conditions.

Acknowledgements

This work was funded in part by a Diabetes Australia Research Trust (DART) project grant, and in part by a National Health and Medical Research Council (NHMRC) project grant 1087341.

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Pgc1 Signal

PGC-1α is also the primary regulator of liver gluconeogenesis, inducing increased gene expression for gluconeogenesis.

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