Synergism between phorbol-12-myristate-13-acetate and vitamin D3 in the differentiation of U937 cells to monocytes and macrophages
Synergie entre le phorbol-12-myristate-13-acétate et la vitamine D3 pour la différenciation des cellules U937 en monocytes et macrophages
J.F. Valdés López, S. Urcuqui-Inchima ∗
Grupo Inmunovirología, Facultad de Medicina, Universidad de Antioquia (UdeA), Calle 70 No. 52-21, Medellín, Colombia
Summary
Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines, but the extent of differentiation in comparison to primary tissue macrophages is unclear. Here, we examine the morphological/phenotypic markers associated with differentiation of U937 cells into monocytes/macrophages, in response to PMA or VD3 treatment. PMA stimulus but not with VD3, induced changes in cell morphology indicative of differentiation, but did not show differ- entiation comparable to monocyte-derive macrophage (MDM). The cells treated with PMA + VD3 for 2 days (d) acquired morphological/phenotypic features similar to those acquired by mono- cytes. In contrast, U937 cells treated for 2d with PMA and VD3 followed by 6d of resting in culture without PMA but in the presence of VD3 acquired morphological and phenotypic mark- ers similar to those of MDM; i.e. reduced nucleus/cytoplasmic ratio, high auto-fluorescence and cytoplasmic complexity. Furthermore, low expression of CD14/TLR2 and high expression of CD68/CD86 were observed. In conclusion, our results indicate a synergistic effect between PMA and VD3 in U937 cells differentiation into both monocytes or macrophages and we propose a modified PMA differentiation protocol to enhance monocyte/macrophage differentiation of U937 cells.
Introduction
Monocytes are the principal precursors of macrophages [1,2]. They are recruited from the blood to the tissues where their phenotype and their differentiation process are modulated by environmental stimuli including micro- bial products, cytokines/chemokines and growth factors [3,4]. Macrophage differentiation from monocytes occurs in conjunction with the acquisition of a functional phe- notype that depends on micro-environmental signals. This process is characterized by a change in cell morphology, enhanced cytoplasm complexity, increase in cellular auto- fluorescence; and down-regulation of membrane proteins such as the lipopolysaccharide co-receptor (CD14) and the Toll-like receptors 2 (TLR2) and TLR4 [5—9]. Conversely, the expression of α-integrin M (CD11b), α-integrin X (CD11c), the oxidized low density lipoprotein receptor (CD68) and the glycoprotein B7-2 (CD86) are up-regulated in macrophage differentiation [2,10—15]. In addition, there are impor- tant differences between the cytokine expression profiles of monocytes and macrophages in response to TLR lig- ands; e.g. IL-1β is strongly induced in response to TLR2 or TLR4 activation on monocytes while this response is down- regulated in alveolar macrophages [6,15]. However, primary macrophages are highly heterogeneous in their phenotype [2,16] and once differentiated, they become long-living cells and develop specialized functions, including maintaining tissue homeostasis and defense against invading microorgan- isms [16]. Likewise, macrophages are involved in mediating innate immune responses and participate in the adaptive immune response [17,18].
Primary tissue macrophages and monocytes cannot be readily expanded ex vivo, their isolation requires blood donation or their collection from a specific tissue by invasive procedures including biopsy and only a limited number of cells are retrieved, constituting a barrier to the use of these primary cells [19]. Consequently, monocytic cell lines of varying degrees of differentiation are commonly used to study monocyte and macrophage functions, including affinity of macrophages for Beta- tricalcium phosphate, which have been used in vitro to study the behavior of macrophages [20]. Indeed, monocytic cell lines are more suitable than primary macrophages because they are easy to obtain and cultivate; yet, they do not always reflect the behavior or function of primary mono- cytes and/or macrophages. Accordingly, attempts using diverse cellular differentiation protocols have been per- formed to obtain macrophages from monocytic cell lines, including U937 cells. This monocytic cell line derived from a histiocytic lymphoma with characteristics of monoblasts [21—23] and with diverse soluble stimuli including phorbol- 12-myristate-13-acetate (PMA), 1α, 25-dihydroxyvitamin D3 (VD3), interferon and retinoic acid triggers highly vari- able macrophage phenotypes, depending on the stimulus and duration of differentiation [19,24—28]. Both, PMA and VD3 have been widely used in macrophage differentiation from U937 cells [25,29,30]. However, despite the fact that different studies with this cellular model have been per- formed with various differentiation protocols, it is unclear whether macrophages obtained from U937 cells treated with PMA or VD3 have the morphological and phenotypical characteristics of tissue macrophages or monocyte-derived macrophages (MDM). Herein, we examined the morphologi- cal and phenotypic markers associated with differentiation of U937 cells into monocytes (Mo) and macrophages, in response to PMA and/or VD3 treatment as compared to primary Mo and MDM.
Materials and methods
Cell culture and differentiation
The U937 cell line was obtained from ATCC and main- tained at 1 × 105—1 × 106 cells/mL in RPMI 1640 medium (Sigma-Aldrich, St. Louis, USA) supplemented with 5.0% heat-treated fetal bovine serum (FBS; Sigma-Aldrich), 4 mM L-glutamine (Sigma-Aldrich) and 0.3% NaCO3 (Sigma-Aldrich) in cell culture flasks. For cell differentiation, U937 cells (5 × 105 cells/well) were seeded onto 12-well plates and treated with 25 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) or 20 nM vitamin D3 (VD3; Sigma-Aldrich), or co-stimulated with 25 nM PMA + 20 nM VD3 in RPMI 1640 medium supplemented with 0.5% FBS, 4 mM L-glutamine and 0.3% NaCO3 and were incubated overnight at 37 ◦C with 5% CO2 and then the cell cultures were supplemented with 5% FBS and incubated for 24 h to obtain PMA- or VD3- or PMA + VD3-treated cells, respectively. Differentiation of PMA-, VD3- and PMA + VD3-treated cells was enhanced after the initial 2d stimulus by removing the PMA containing media then incubating the cells in fresh RPMI 1640 supplemented with 5% FBS, 4 mM L-glutamine and 0.3% NaCO3, in the absence of PMA but in the presence of 20 nM VD3 for a fur- ther 6d to obtain PMAr- or VD3r- or PMAr + VD3-treated cells. Since DMSO is the thinner of PMA and VD3 (vehicle), we used cells treated with 21.68 µM DMSO (Carlo Erba Reagents) for 2 or 8d (DMSO-treated cells) as negative controls.
Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll histopaque-1077 (Sigma-Aldrich) density centrifugation from whole blood donated by healthy volun- teers. The percentage of CD14 positive cells was determined by flow cytometry. Then, 5.0 × 105 CD14 positive cells/well were plated in RPMI 1640 medium supplemented with 0.5% autologous serum (AS), 4 mM L-glutamine and 0.3% NaCO3 onto 24-well plates. Mo were enriched by adherence to the plate for 2 h after which non-adhering cells were removed, and cells were cultured in RPMI 1640 medium supplemented with 10% AS, 4 mM L-glutamine and 0.3% NaCO3, and incubated at 37 ◦C and 5% CO2 for 1d to obtain Mo or 6d for days to obtain MDM [11,31,32].
Microscopy
Mo, MDM and PMA-, VD3-, PMA + VD3-, PMAr-, VD3r- and PMArVD3-, DMSO- and DMSOr-treated cells (treated cells) were visualized and photographed in an Axio Vert.A1 micro- scope (ZEISS, Oberkochen, Germany) equipped with a Ds-fi1 camera (Nikon, Tokyo, Japan) and adapted to microscopy of transmitted light in phase-contrast and epi-fluorescence microscopy. Pictures were processed and analyzed using the software IrfanView [33], ImageJ [34] and GIMP [35]. To ana- lyze the nucleocytoplasmic ratio, primary Mo, MDM and the different treated cells were stained with SYBR-Green 10000X (1:1667 dilution, Invitrogen) [36] for 30 min at 37 ◦C, and the cells were visualized and photographed in transmitted light in phase-contrast to observe the cellular morphology and in epi-fluorescence microscopy to observe the nuclear morphology, and then the image merge between the two pic- tures was performed using the software ImageJ. The cells were not fixed before staining with SYBR-Green or visual- ization by microscopy. For the generation of scale bars, the IrfanView software was calibrated using photomicrographs taken with the 40X objective to a stage micrometer [37].
Flow cytometry
Flow cytometry measurements were performed using a three color FACSCanto II (Becton Dickinson, NJ, USA). We performed an exclusion of debris after which forward and side scattered light was used to identify cell populations (Mo, MDM, and treated cells) and measure the size and gran- ularity of the cells. Cell auto-fluorescence was recorded by analyzing unstained cells in the FL1 channel (blue laser-488 nm and filter 530/30) as was previously described [6]. To detect cell surface markers, treated cells were detached mechanically from the culture plates and incubated for 40 min at 4 ◦C in the presence of monoclonal mouse anti-human antibodies CD14-Fluorescein isothiocyanate (FITC; 2 ng/µL; clone: 61D3), TLR2-Phycoerythrin (PE; 2 ng/µL; clone: TLR2.1), CD11b/Mac1-PE/Cyanine5 (PE/Cy5; 2 ng/µL; clone: ICRF44), CD86-PE (2 ng/µL; clone: FUN-1), CD11c-PE/Cy5 (2 ng/µL; clone: B-ly6) or the rele- vant isotypes. To detect intracellular markers, treated cells were fixed and permeabilized using the FOXP3 permeabi- lization kit (eBioscience, Massachusetts, USA) according to the manufacturer’s recommendations and incubated 40 min at 4 ◦C in the presence of mouse anti-human CD68-PE (0.001 µg/µL, clone: eBioy1/82A). The samples were then washed and resuspended in PBS 1X and 10,000 events were recorded. Positive labeling cells were defined based on isotype-controls and a compensation matrix was performed to compensate the spectral overlap. To quantify the percentage of CD14 positive cells from PBMCs, 5 × 105 PBMCs were incubated 20 min at 4 ◦C in the presence of monoclonal mouse anti-human CD14-FITC antibody (2 ng/µL, clone: 61D3). Following incubation, the PBMCs were washed and resuspended in PBS 1X, and 10,000 events were recorded. For each experiment, unstained cells and conjugated isotype antibodies were included as controls. Expression of differ- ent markers was expressed as the mean fluorescent intensity (MFI) and percentage of positive cells of the overall treated cells after subtraction of the isotype-control.
Cytokine production
This issue was only evaluated in PMAr- and PMAr + VD3- treated cells since we were interested in determining if these treated cells responded to TLR activation. Thus, PMAr and PMAr + VD3 cultures were unstimulated (control) or stimulated with 20 ng/mL Pam2CSK4 (Invitrogen, California, USA), 10 µg/mL Poly (I:C) (Invitrogen), 20 ng/mL ultrapure LPS (Invitrogen) or 5 µg/mL R848 (Invitrogen) as ligands for TLR 2/6, 3, 4 and 7/8, respectively [38]. The supernatants were harvested after 24 h and stored at −80 ◦C until analy- sis. IL-1β and IL-6 levels were measured by ELISA (BD OptEIA, NJ, USA). The detection limits were 1 pg/mL.
Statistical analysis
All data were recorded as the mean ± standard deviation (SD). A Friedman test with Dunn’s multiple comparison test was performed. All data were plotted and analized using GraphPad Prism7 (GraphPad Software Inc. San Diego, USA). Significant results were defined as P < 0.05 (*), P < 0.01 (**) and highly significant results as P < 0.001 (***). The experi- ments were done between 3 and 5 times independently and was indicated in the legend of figures. Results Morphological features of U937 cells following differentiation Primary Mo are adherent cells with spherical morphology, cytoplasmic projections and a high nucleocytoplasmic ratio (Fig. 1a and b). In contrast, MDMs acquired either an oval cell morphology or a rod cell morphology, increased their cyto- plasmic complexity and increased their cytoplasmic volume with a reduction in their nucleocytoplasmic ratio, as com- pared to monocytes (Fig. 1a and b). These changes were not observed in U937-treated cells either with VD3 or the control (DMSO) that in addition were non-adherent cells and presented a spherical morphology without cytoplasmic projections (Fig. 1a). Treatment of U937 cells with PMA or PMA + VD3 for 2 days induced changes in cell morphol- ogy, increased cellular adherence and cytoplasmic volume (Fig. 1a), but in a very small number of cells. In contrast, when U937 cells were treated with PMA for 2 days and then followed by 6d of resting in culture without PMA (PMAr) or without PMA but in the presence of VD3 (PMAr + VD3), they acquired an amoeboid morphology as compared with the typical morphology of MDMs (Fig. 1a; compare MDM with PMAr or with PMAr + VD3). In addition, PMAr and PMAr + VD3 cells increased their cytoplasmic complexity and cytoplas- mic volume with a reduction in their nucleocytoplasmic ratio (Fig. 1a and b). The treatment with VD3 for 2d (VD3) or for 8d (VD3r) did not induce morphological changes (Fig. 1). How- ever, the cells were slightly adherent and tended to form aggregates. As observed in Fig. 2a, MDM increased both SSC (granularity) and auto-fluorescence on flow cytometry com- pared with Mo. The treatment of U937 cells with PMA, or PMA + VD3 for 2 days increased their granularity but not their auto-fluorescence comparable to DMSO- or VD3-treated cells (Fig. 2b). Additionally, PMA and PMA + VD3 cells increased their granularity and auto-fluorescence to a level compara- ble to that of Mo (Fig. 2a and b). PMAr and PMAr + VD3 cells increased their granularity and auto-fluorescence to a level comparable to that of MDM (Fig. 2a and 2b). The expression of surface and intracellular markers is altered in PMAr + VD3 cells Macrophage heterogeneity that is influenced by the state of differentiation with marked differences between monocytes and macrophages has been extensively studied [39,40]. Consequently, in the present study we only focused on identifying a number of monocyte and macrophage charac- teristics associated with differentiation in U937 cells treated according to different protocols. We observed that following treatment of U937 cells with PMA or VD3 for 2 days, the per- centage of positive CD14 cells was 22.55% (MFI = 206.5) and 10.22% (MFI = 75.75), respectively (Fig. 3a and c; Table 1). However, the percentage of positive CD14 cells was signifi- cantly higher (85.97%; MFI = 2336.75) in U937 cells treated with PMA + VD3 (Fig. 3a and c), indicating a synergic effect between PMA and VD3 on the expression of CD14 dur- ing U937 differentiation. Interestingly, the percentage of positive CD14 cells and the MFI decreased significantly in PMAr + VD3 (34.78% and 612, respectively). Furthermore, the percentage of positive CD14 cells was 29.87% in VD3r cells with a significant increase in the MFI (537.0), similar to that observed in PMAr + VD3 cells (Fig. 3c), indicating a stronger CD14 surface expression in response to treatment only with VD3, in comparison to PMAr cells. U937 differentiation protocol did not induce significant changes in the expression level of TLR2 when comparing PMA-, VD3- or PMA + VD3-treated cells versus negative con- trols (DMSO), neither at the percentage of positive cells nor the MFI (Fig. 3b and d; Table 1). However, the MFI of TLR2 increased slightly in a time-dependent manner in VD3r- versus VD3-treated cells. However, the TLR2 positive cells increased significantly in VD3-treated cells compared to PMA + VD3-treated cells (Fig. 3d). There was a decrease in the percentage of TLR2 positive cells as well as in the MFI in both PMAr- and PMAr + VD3-treated cells. Furthermore, as we can observe in Fig. 4c, PMA + VD3- and PMAr + VD3-treated cells have greater CD11b surface expression vs. control (DMSO). Likewise, we also observed an increased expression of CD11c marker in PMA-, PMAr- , PMA + VD- and PMAr + VD3-treated cells, but not in VD3- or VD3r-treated cells (Fig. 4b and d). PMAr + VD3-treated cells decreased the percentage of cells expressing CD68 in comparison to DMSO, but not compared with VD3r- or PMAr- treated cells (Fig. 5a and b). However, the MFI was higher in PMAr than in DMSO (Fig. 5c). In addition, we found that PMA- treated cells significantly induced the expression of CD86 cells in comparison to DMSO (Fig. 6a and b; Table 1). Similarly, we observed a significant increase in the MFI in PMAr vs. DMSO (Fig. 6c). Cytokine profiles in PMAr- and in PMAr + VD3-treated cells stimulated with TLR agonists Here, we found that PMAr-treated cells produced higher lev- els of IL-1β in comparison to PMAr + VD3-stimulated cells (Fig. 7a). Conversely, PMAr-treated cells secreted lower lev- els of IL-6 than PMAr + VD3-treated cells (Fig. 7a). Since the cytokines are markedly induced following TLRs activation, we quantified the cytokine profiles in PMAr- and PMAr + VD3- treated cells stimulated with the TLR2/6, TLR3, TLR4 or TLR7/8 agonists. The LPS stimulus significantly increased of IL-1β and IL-6 production in PMAr + VD3-treated cells vs. unstimulated with TLRs agonists (control) or other stimulus treated cells (Fig. 7b and c, respectively). Despite variable constitutive levels none of the other treated cell types had significant IL-1β and Il-6 production in response to poly (I:C), R848 and Pam2CSK4 stimulus. Discussion The use of monocytic cell lines as macrophage models and of diverse protocols to reach macrophage differenti- ation revealed some aspects of effector mechanisms and macrophage biology [41,42]; however, the protocol rec- ommended to produce a highly differentiated macrophage phenotype is not well established. We show that PMA- and PMA + VD3-treated cells followed by 6d of resting in culture without PMA (PMAr) or without PMA but in the pres- ence of VD3 (PMAr + VD3) induce differentiation towards a more highly differentiated macrophage phenotype; i.e. more comparable to that of MDM, than PMA- or PMA + VD3- treated cells. Furthermore, PMAr- and PMAr + VD3-treated cells expressed a lower level of CD14 and TLR2 than PMA- and PMA + VD3-treated cells, respectively, and increased expres- sion of surface markers such as, CD11b, CD11c and CD86, a phenotype that is closer to that of primary macrophages. The results can suggest that U937 cell differentiation to macrophage is related with the protocol used. These changes more closely resembled MDM in PMAr + VD3-treated cells than in PMAr- or VD3r-treated cells. Furthermore, the PMAr + VD3-treated cells have a better response to stimula- tion with LPS. In accordance with our results, Sokol et al.[43] also reported that changes normally occur during maturation of human Mo into macrophages; they observed that the entire cell and its cytoplasmic volume increased while the nucleocytoplasmic ratio was reduced during culture and in addition, that cells contain a wealth of organelles. Fur- thermore, macrophage differentiation was associated with enhanced granularity and auto-fluorescence and it was low in Mo and myeloid dendritic cells [40,44], as we observed. More recently and in line with our results, changes in cell morphology and nucleocytoplasmic ratio were reported in VD3- and PMA-treated THP-1 cells, but the differentiation was not comparable to MDM [6]. Based on our results, we postulated that in U937 cells a synergistic effect between PMA and VD3 contributed to U937 cell differentiation to macrophages and that their morphological and phenotypic features are closer to MDM than differentiation with PMA or VD3 at doses commonly used in the scientific litera- ture [42]. Since MDM represents a good surrogate of tissue macrophages [45], we show that PMAr + VD3 treatment might represent a better protocol to obtain macrophages and study their function. In contrast, based on our results the treatment with PMA or PMA + VD3 for 2 days, results in cells that are more closely related to the monocyte morpho- type/phenotype and might represent a useful model for the study of monocytes as has been proposed [6]. Figure 1 Morphological changes in macrophages during differentiation. Representative photomicrographs were taken in phase- contrast from each culture (a). Merge images were performed from pictures of cell cultures of Mo, MDM or U937-treated cells (DMSO, VD3, PMA, PMA + VD3, DMSOr, VD3r, PMAr and PMAr + VD3) (b). The cells were stained with SYBR-Green and photomicrographs were taken by phase-contrast and epi-fluorescence. The data are representatives of at least three independent experiments. Scale bar equals 100 µm. Figure 2 Changes in cytoplasmic complexity (granularity) and cellular auto-fluorescence during macrophage differentiation. Forward light scatter (FSC-A) and sidelight scatter (SSC-A) plots and histograms of auto-fluorescence of Mo and MDM (a), U937- treated cells (DMSO-, VD3-, PMA-, PMA + VD3-, DMSOr-, VD3r-, PMAr- and PMAr + VD3) (b and c). MFI is shown in the upper right hand corner. The data are representatives of at least three independent experiments. Figure 3 CD14 and TLR2 expression with macrophage differentiation. Histograms of the relative expression of CD14+ (a) and TLR2 (b), graphs of the percentage of positive cells and MFI of CD14+ (c) and the percentage of positive cells and MFI of TLR2 (d) expressed on the cell surface of U937-treated cells are presented. Each histogram is representative of at least four independent experiments; gray areas (a and b) correspond to an appropriate isotype-control reaction. n = 4. Differences were calculated using the Friedman test with Dunn’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001. All data were recorded as the mean ± standard deviation. In the case of the conventional differentiation proto- cols of these cell lines to macrophages, it was reported that expression of CD14 is up-regulated in differentiated macrophages [19,42,46], as we observed in PMA + VD3- treated cells. However, this behavior differed from the expression pattern observed in differentiation of primary Mean of the percentage of positive cells and MFI of CD14, TLR2, CD11b, Cd11c, CD68 and CD86 expressed on the cell surface and/or inside of U937 cells. n = 3—4. T: treatment; MM: molecular marker. Figure 4 CD11b and CD11c expression with macrophage differentiation. Histograms of the relative expression of CD11b (a) and CD11c (b). Graphs of the percentage of positive cells and MFI of CD11b (c) and CD11c (d) expressed on the cell surface of U937- treated cells. The histograms are representatives of at least four independent experiments; gray areas (a and b) correspond to an appropriate isotype-control reaction. n = 4. Differences were calculated using the Friedman test with Dunn’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001. All data were recorded as the mean ± standard deviation. Mo to macrophages, since the expression of this marker was down-regulated [6,8]. Furthermore, we found a syner- gic effect between PMA and VD3 in the expression of CD14 and CD11b in PMA + VD3- and PMAr + VD3-treated cells that had not been previously described. The expression level of CD11b is under critical control during cell differentiation and inflammation because this integrin is responsible for the adhesion of monocytes to activated endothelial cells [47]. We suggest that the synergic effect of PMA and VD3 in CD11b expression occurs early in U937 differentiation, since a similar expression level was observed in PMA + VD3-treated cells. According to our results, an increased expression of surface markers such as CD11b, CD11c and CD68 dur- ing macrophage differentiation was previously reported [1]. Another surface marker that we find increased in PMA or PMAr-treated cells was CD86, but in PMAr + VD3-treated cells a decreased expression was observed, suggesting that VD3 down-regulates the expression of these markers as has been reported in DCs and macrophages, promoting the development of tolerogenic activation profiles in these cell populations [48—50]. Changes in the expression of TLR2 and TLR4 are frequently used as markers of monocyte differentiation to macrophages since the expression of both TLRs decreases during this process [6]. Here, although we did not find statistically significant decreases in the TLR2 expression level in PMAr- and PMAr + VD3-treated cells, the percentage of positive TLR2 cells was lower compared with PMA- and PMA + VD3-treated cells. Previously it was reported that treatment of freshly isolated human monocytes with VD3 only slightly inhibited TLR2 expression; in contrast, the expression of TLR2 significantly decreased in macrophages treated with VD3 [51]. Yet, other reports have shown that VD3 down-regulates the expression of TLR2 in monocytes [52]. In contrast, we observed that VD3-treated cells increase the expression of TLR2 in a time-dependent manner in U937 cells. This unexpected effect of VD3 on the expression of TLR2 could be due to intrinsic character- istics of the cells influenced by the state of their cellular differentiation. Figure 5 CD68 expression with macrophage differentiation. Histograms of relative expression of CD68 (a). Graphs of the per- centage of positive cells (b) and MFI (c) of CD68 expressed inside of U937-treated cells. The histograms are representatives of at least four independent experiments, gray areas (a) correspond to an appropriate isotype-control reaction; n = 4. Differences were calculated using the Friedman test with Dunn’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001. All data were recorded as the mean ± standard deviation. VD3 has immunomodulator activity and in monocytes, macrophages and DCs, it was reported that VD3 down- regulates the production of pro-inflammatory cytokines such as TNF-α and IL-1β [48,49,53]. However, the effect of VD3 on IL-6 production is controversial since some reports show that VD3 down-regulates their production of IL-6 in monocytes, macrophages and DCs, whereas others reach different conclusions [48,50,52,54,55]. Here, we observed that PMAr + VD3 treatment down-regulated the production of IL-1β, but up-regulated the production of IL-6 in comparison to PMAr-treated cells, indicating a role of VD3 in modulating the secretion of both cytokines. Our results contrast with what was previously reported in THP-1, a human monocytic cell line with characteristics of promonocyte [56], in which it was reported that the VD3 treatment enhanced the NLRP3-dependent secretion of IL- 1β [57]. Furthermore, PMAr + VD3-treated cells responded better to LPS stimuli than PMAr-treated cells, possibly because PMAr + VD3-treated cells expressed higher amounts of CD14 surface marker than PMAr-treated cells. Previous reports had shown that VD3-induced the differentiation of different monocytic cell lines, including U937 cells, to monocyte-like cells [6,17,58,59]. However, our results show that although treatment of U937 cells with VD3- induced an increase in the expression of monocytic surface markers, VD3 did not induce changes in cell morphology, generating a possible intermediate phenotype between monoblast and monocyte. In contrast, U937 cells treated with PMA + VD3 for 2 days acquired morphological and phenotypic features similar to those observed in primary monocytes. Figure 6 CD86 expression with macrophage differentiation. Histograms of relative expression of CD86 (a). Graphs of the percent- age of positive cells (b) and MFI (c) of CD86 expressed on the cell surface of U937-treated cells. The histograms are representatives of at least four independent experiments, gray areas (A) correspond to an appropriate isotype-control reaction; n = 4. Differences were calculated using the Friedman test with Dunn’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001. All data were recorded as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, and the Friedman test with Dunn’s multiple comparisons test. All data were recorded as the mean ± standard deviation. Conclusion Our findings show that when U937 cells are exposed to PMA and VD3 for two days, they generate cells with a phenotype and morphology more closely related to monocytes. How- ever, when U937 cells are exposed to PMA and VD3 for two days, followed by 6 d of resting in culture without PMA but in the presence of VD3, they generate macrophage-like cells with a phenotype and morphology more closely related to MDMs. These cells provide an additional tool to further inves- tigate the biology of monocytes and tissue macrophages and await further studies. Figure 7 Differential cytokine expression with macrophage differentiation. The IL-β and IL-6 productions were measured from supernatants of PMAr and PMAr + VD3 culture (a). PMAr- and PMAr + VD3-treated cells were unstimulated (Control) or stimulated with Pam2CSK4, Poly (I:C), LPS and R848, agonists of TLR2/6, TLR3, TLR4, and TLR7/8, respectively. IL-β (b) and IL-6 (c) were quantified from supernatants. n = between 4 and 5. 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