Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is involved in phagocytosis activation during THP-1 cell differentiation

Abstract

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase that plays a major role in cell metabolism. Although mTORC1 inhibitors are known to exert immunosuppressive effects, their effects on immune cells are not fully understood. In the present study, we examined the involvement of mTORC1 in the differentiation and functions of macrophages using THP-1 cells, which are derived from human monocytic leukemia and differentiate into macrophage-like cells upon treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We also examined the effects of two mTOR inhibitors, Torin 1 and rapamycin, on TPA-stimulated THP-1 cells. Although mTORC1 activation was observed upon TPA stimulation, mTOR inhibitors did not affect TPA-induced morphological changes or expression of the general macrophage marker, CD11b. In contrast, phagocytosis and fluid endocytosis were significantly impaired by the mTOR inhibitors. Endocytosis suppression was observed when mTOR inhibitors were added during differentiation, but not before or after differentiation, suggesting that endocytosis suppression altered the direction of differentiation. Furthermore, mTOR inhibitors altered the expression of M1/M2 polarization markers. These results suggest that the immunosuppressive effects of mTOR inhibitors may involve the suppression of macrophage endocytosis caused by abnormal cell differentiation.

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in integrating environmental cues and regulating cell metabolism [26]. mTOR functions as the catalytic subunit of two canonical mTOR complexes: complex 1 (mTORC1) and complex 2 (mTORC2). These mTOR complexes exhibit different substrate specificities. For example, mTORC1 phosphorylates substrates, such as S6K and 4E-BP1, leading to the activation of mRNA translation [23, 26]. Dysregulation of mTOR signaling is suggested to contribute to the abnormal proliferation of tumor cells, and mTOR inhibitors have been approved for cancer treatment [1]. In addition, mTOR inhibitors exert immunosuppressive effects, and the mTORC1-specific inhibitor, rapamycin, and its analogs have been used as immunosuppressants after organ transplant surgery [24]. Although the immunosuppressive abilities of mTORC1 inhibitors are suggested to involve T cell anergy induced by mTORC1 inhibition during antigen presentation, it also regulates various aspects of innate and adaptive immunity, including immune cell proliferation, differentiation, and activation [24, 26, 30]. For example, mTORC1 promotes the production of Th1 and Th17 cells, whereas mTORC2 promotes that of Th2 cells. When mTORC1 and mTORC2 are absent, regulatory T (Treg) cells increase [24]. Regarding antigen-presenting cells, dendritic cells matured in the presence of the mTORC1 inhibitor rapamycin showed decreased uptake of extracellular materials [12] and promoted T cell tolerance [24]. Despite the complexity of mTORC1 activity, understanding the effects of mTORC1 inhibition on immunity is important for the clinical application of its immunosuppressive and anticancer properties.

In this study, we investigated the involvement of mTORC1 signaling in macrophage differentiation and function. Macrophages derived from monocytes and other progenitor cells maintain tissue homeostasis by eliminating foreign agents, including pathogens, dead cells, and cell debris, and facilitating and resolving inflammatory reactions [30, 31]. In this study, we used THP-1 cells derived from human monocytic leukemic cells, which are widely used in studies on macrophage differentiation and function [6]. THP-1 cells differentiate into macrophage-like cells when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), also known as phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C [2, 4]. TPA treatment results in mTORC1 activation [20, 25]. Although this suggests that mTORC1 is involved in macrophage-like differentiation of THP-1 cells, the importance of mTORC1 in THP-1 differentiation and its underlying mechanism remain to be elucidated.

To examine the effects of mTOR complexes, two mTOR inhibitors, Torin 1 and rapamycin, were used in this study. Torin 1 is a potent ATP-competitive mTOR kinase inhibitor that inhibits both mTORC1 and mTORC2. On the other hand, rapamycin specifically inhibits mTORC1 by simultaneously binding to 12-kDa FK506 binding protein (FKBP12) and FKBP−rapamycin binding (FRB) domain of mTORC1. The mTORC1-suppressing ability of rapamycin is weaker than that of Torin 1, and it can cause dephosphorylation of only a subset of mTORC1-dependent phosphorylation sites [13, 26]. Particular attention has been paid to the effects of mTOR inhibitors on endocytosis/phagocytosis in relation to macrophage differentiation.

MATERIALS AND METHODS

Cells

THP-1 cells (RCB1189, derived from a human patient with acute monocytic leukemia) were obtained from RIKEN BioResource Research Center (Tsukuba, Japan). THP-1 cells were maintained in the Roswell Park Memorial Institute 1640 medium (FujiFilm-Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 units/mL), and streptomycin (100 µg/mL) (FujiFilm-Wako).

Reagents

The mTORC1/2 inhibitor, Torin 1, was purchased from Tocris Bioscience (Bristol, UK). The mTORC1-specific inhibitor, rapamycin, was purchased from Sigma-Aldrich (St. Louis, MO, USA). TPA was purchased from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor^TM 594-conjugated wheat germ agglutinin (WGA) was purchased from Thermo Fisher Scientific.

Western blotting analysis

The cells were seeded in a 12-well plate at a density of 1 × 10⁵ cells/mL (1 mL/well). After incubation for 22–26 hr, the cells were treated with DMSO or an mTOR inhibitor. At the time points indicated in the figures, cells were harvested as follows: for harvesting adherent cells (differentiated THP-1), cells were washed with phosphate-buffered saline (PBS) and lysed with cold lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂-EDTA, 1 mM sodium pyrophosphate, 1 mM disodium β-glycerophosphate pentahydrate, 10 mM sodium orthovanadate, 1% Triton X-100, and a proteinase inhibitor cocktail (cOmplete; Roche Diagnostics, Basel, Switzerland). The floating cells (undifferentiated THP-1) were collected and centrifuged at 1,500 rpm (200 × g) for 3 min at room temperature, and the pelleted cells were lysed with cold lysis buffer. Lysates were then centrifuged at 14,000 rpm (18,000 × g) for 10 min at 4°C, and the supernatants were collected and mixed with 6 × sodium dodecyl sulfate sample buffer. Samples were boiled for 5 min and stored at −20°C.

Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% acrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P; Merck Millipore, Burlington, MA, USA). Membranes were soaked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 2% skim milk for blocking. The membranes were then incubated with one of the following primary antibodies: mouse-anti-CD11b (1:1,000; ProteinTech, Rosemont, IL, USA, 66519-1-lg), rabbit anti-phospho-S6K (Thr389) (1:1,000; Cell Signaling Technology, #9234), rabbit anti-S6K (1:1,000; Cell Signaling Technology, #2708), and rabbit anti-GAPDH (1:5,000; Cell Signaling Technology, #5174) antibodies. Membranes were washed with TBS-T and incubated with goat anti-mouse IgG (H+L) or goat anti-rabbit IgG (H+L) antibody conjugated with horseradish peroxidase (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) as a secondary antibody. After incubation with the substrate solution (EzWestLumi plus; Atto, Tokyo, Japan), chemiluminescent signals were detected using LuminoGraph I (Atto) and quantified using the CS Analyzer 4 software (Atto).

Induction of THP-1 differentiation without mTOR inhibitors

THP-1 cells were plated in a 6-well or 12-well plate at a density of 5 × 10⁵ or 1 × 10⁵ cells/mL (1 mL/well). After incubation for 22–26 hr, TPA (100 nM) was added. After 24 hr of incubation, the culture medium was removed, and the cells were washed with the medium. Then, the cells were incubated for an additional 24 hr. Differentiation of THP-1 cells was confirmed by the morphological change from floating cells to macrophage-like adherent cells.

Preparation of fluorescent beads

Polystyrene fluorescent beads (2.07 µm mean diameter, FSFR005) were purchased from Bangs Laboratories Inc. (Fishers, IN, USA). Beads were added to 1 mL ice-cold PBS, centrifuged at 10,000 rpm (9,200 × g) for 5 min at 4°C. Then, PBS was removed and fresh ice-cold PBS containing the bovine serum albumin (BSA; Fujifilm-Wako, 0.3 mg/mL) or human IgG (Sigma-Aldrich, 0.3 mg/mL) was added, and the beads were incubated and shaken at 4°C overnight. After incubation, beads were centrifuged at 10,000 rpm (9,200 × g) for 5 min at 4°C and washed twice with ice-cold PBS.

Measurement of endocytosis activity using flow cytometry

THP-1 cells were plated in a 6-well plate at a density of 5 × 10 cells/mL (1 mL/well). After incubation for 22–26 hr, the cells were subjected to two different experiments. (1) Cells were pretreated with DMSO or an mTOR inhibitor for 30 min. Cells were then treated with 100 nM TPA (in the presence of the reagent used for pretreatment) and incubated for 24 hr. After 24 hr of incubation, the culture medium was removed, and the cells were washed with the medium. Then, a new medium containing the vehicle or an mTOR inhibitor was added, and cells were incubated for an additional 24 hr. (2) Cells were first differentiated by 24-hr incubation with 100 nM TPA, the culture medium was removed, and cells were washed with the medium. Then, new medium was added and the cells were incubated for an additional 23 hr. After 23 hr of incubation, cells were treated with DMSO or an mTOR inhibitor for 1 hr. For both experiments, the cells were incubated with fluorescein isothiocyanate (FITC)-dextran (FD10S; Sigma-Aldrich) for 30 min or with beads coated with BSA or human IgG for 1 hr. After incubation, the cells were collected and centrifuged at 1,500 rpm (200 × g) for 3 min, washed with PBS, and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The cells were centrifuged at 2,500 rpm (600 × g) for 2 min at 4°C, washed with PBS, and resuspended in PBS. The cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). At least 20,000 cells were counted in each sample.

Gentamicin protection assay

THP-1 cells were pretreated with an mTOR inhibitor for 30 min, differentiated with 100 nM TPA for 24 hr in the presence of the mTOR inhibitor, and treated with the mTOR inhibitor without TPA for an additional 24 hr. Cells were washed twice with the growth medium. Then, the cells were incubated with Escherichia coli (E. coli; ATCC #25922) at a multiplicity of infection of 50 and incubated for 1 hr. Cells were washed twice with PBS and incubated for 1 hr in growth medium containing 100 µg/mL of gentamicin. The cells were washed with PBS and incubated for 15 min at room temperature in PBS containing 0.1% Triton X-100. All samples were spread on Luria-Bertani agar plates, incubated for 16 hr at 37°C, and the colonies were counted.

Confocal microscopy

THP-1 cells were plated in a 6-well plate containing coverslips and pretreated with an mTOR inhibitor for 30 min. The cells were then differentiated with 100 nM TPA for 24 hr in the presence of the mTOR inhibitor before being treated with the mTOR inhibitor without TPA for an additional 24 hr. Cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and washed twice with PBS. Cell shape was visualized with Alexa Fluor^TM 594-conjugated WGA, which labels glycosylated proteins on the plasma membrane, using the manufacturer’s recommended protocol. After staining, the cells were washed twice with PBS, rinsed with pure water, and sealed on glass slides using ProLong^TM Diamond Antifade Mountant containing 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Fluorescent images were captured using an LSM710 confocal microscope (ZEISS, Oberkochen, Germany) equipped with a Plan-Apo 63×/1.40 objective lens.

Quantitative polymerase chain reaction (qPCR)

THP-1 cells were pretreated with an mTOR inhibitor for 30 min, differentiated with 100 nM TPA for 24 hr in the presence of the mTOR inhibitor, and treated with the mTOR inhibitor without TPA for an additional 24 hr. Total RNA was isolated from the cells using ISOGEN (Nippon Gene, Tokyo, Japan), according to the manufacturer’s protocol. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). qPCR was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the QuantiTect SYBR Green PCR Kit (Qiagen), and the expression of mRNA was normalized to that of β-actin (ACTB). The primer sequences used in this study were as follows.

ACTB Forward 5′-ATTGCCGACAGGATGCAGAA-3′

ACTB Reverse 5′-GCTGATCCACATCTGCTGGAA-3′

NOS2 Forward 5′-CCTTCAACCCCAAGGTTGTCT-3′

NOS2 Reverse 5′-CCATTGCCAAACGTACTGGTC-3′

CD80 Forward 5′-TCTCAGAAGTGGAGTCTTACCCT-3′

CD80 Reverse 5′-GTGGATTTAGTTTCACAGCTTGC-3′

CD206 Forward 5′-GCTGTTCTCCTACTGGACACC-3′

CD206 Reverse 5′-TTCGGACACCCATCGGAATTT-3′

CD204 Forward 5′-AGTGCTGCTTTCTTTAGGACGA-3′

CD204 Reverse 5′-AGGGCTGTTTTTAGGATTCGGA-3′

The thermocycling parameters were as follows: i) initial denaturation at 95°C for 15 min, followed 45 cycles of 94°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec. The results were analyzed using the 2^−ΔΔCt method.

Statistical analyses

Statistical significance was determined using GraphPad Prism 7 (version 7.0e; GraphPad Software, San Diego, CA, USA) by performing Welch’s t-test (for pairwise comparisons) and one-way analysis of variance, followed by Dunnett’s test (for comparisons of more than two groups).

RESULTS

mTORC1 activation and CD11b expression are induced by TPA

TPA treatment increases mTORC1 activity in cultured cells [20, 25]. First, we attempted to confirm whether this result could be reproduced in our experimental setting. To this end, we examined the phosphorylation of threonine 389 in S6K, which is a direct target of mTORC1 and is often used as an indicator of mTORC1 activity (Fig. 1). In this experiment, THP-1 cells were treated with 100 nM TPA for 24 hr, followed by an additional 24-hr resting period without TPA. Western blotting analysis confirmed that TPA treatment increased the phosphorylation of S6K at Thr389 (Fig. 1A). In addition, the protein expression of the macrophage differentiation marker, CD11b, was induced by TPA (Fig. 1B). Furthermore, the morphology of THP-1 cells changed from floating to adherent upon TPA treatment (Fig. 2C). These results confirmed that THP-1 cells successfully differentiated into macrophage-like cells under our experimental conditions (24-hr treatment with 100 nM TPA, followed by 24-hr rest without TPA).

Fig. 1.

Mechanistic/mammalian target of rapamycin complex 1 (mTORC1) activation and CD11b expression are induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). THP-1 cells were treated with 100 nM TPA for 24 hr and incubated in a TPA-free medium for another 24 hr. Phosphorylation of the mTORC1 substrate (S6K at Thr389) and expression levels of the macrophage marker, CD11b, were determined using western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (A, top) Representative western blot images from ten independent experiments. (A, bottom) Expression levels of S6K were quantified from the western blot images shown in (A, top), and the values of the control (dimethyl sulfoxide, DMSO) were standardized to 100. n=10. (B, top) Representative images from three independent experiments. (B, bottom) Expression levels of CD11b were quantified from the western blot images shown in (B, top), and the values of the TPA-treated cells were standardized to 100. n=3. Each bar indicates the mean ± SEM. *P<0.05, ****P<0.0001.

Fig. 2.

Mechanistic/mammalian target of rapamycin (mTOR) inhibitors do not affect CD11b expression levels. THP-1 cells were pretreated with mTOR inhibitors for 30 min, treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of mTOR inhibitors (250 nM Torin 1 or 10 nM rapamycin) for 24 hr, and incubated in a TPA-free mTOR inhibitor-containing medium for another 24 hr. S6K and CD11b levels were determined using western blotting. (A and B, top) Representative western blot images from three independent experiments. (A and B, bottom) Expression levels of proteins were quantified from the western blot images (A and B, top), and the values of the TPA-treated cells were standardized to 100. Each bar indicates the mean ± SEM. ns, not significant; ***P<0.001. n=3. (C) THP-1 cells treated with mTOR inhibitors and TPA were fixed and stained with WGA and DAPI to visualize the cell membrane and nuclei, respectively. Scale bar=10 μm.

mTOR inhibitors do not affect CD11b expression levels

Next, we investigated the effects of mTOR inhibitors on TPA-induced differentiation. THP-1 cells were pretreated with an mTOR inhibitor (250 nM Torin 1 or 10 nM rapamycin) for 30 min, stimulated with TPA treatment for 24 hr, and rested for 24 hr. TPA stimulation and resting were performed in the presence of the mTOR inhibitor. mTOR inhibitor treatment almost completely suppressed the phosphorylation of S6K at Thr389 (Fig. 2A). In contrast, induction of CD11b expression by TPA was largely unaffected by mTOR inhibitors (Fig. 2B). Similarly, TPA-induced morphological changes in adherent cells were not apparently altered by the mTOR inhibitors (Fig. 2C). Taken together, these results indicate that mTOR inhibitors do not globally affect TPA-induced THP-1 differentiation into macrophage-like cells, at least in terms of the macrophage marker, CD11b, and morphology.

mTOR inhibition during cell differentiation suppresses phagocytosis and fluid endocytosis

Next, we investigated the effects of mTOR inhibitors on macrophage function. Active phagocytosis of large particles, including bacteria and dead cells, is the primary function of macrophages. In addition, macrophages exhibit high endocytosis activity, which engulfs the extracellular fluid [8, 17]. We examined the uptake of FITC-conjugated dextran to measure the activity of fluid endocytosis and phagocytosis of fluorescent latex beads coated with BSA or human IgG, as well as live bacteria (E. coli) to measure phagocytosis activity. IgG-coated beads were used to imitate opsonized antigens that are phagocytosed in an Fcγ receptor-dependent manner.

Undifferentiated THP-1 cells without TPA treatment were incubated with FITC-dextran, BSA-coated beads, or IgG-coated beads. Treatment with an mTOR inhibitor did not suppress the uptake of these substances in undifferentiated THP-1 cells (Fig. 3A). In contrast, when we performed experiments using mTOR inhibitors during TPA-induced differentiation (the same treatment as shown in Fig. 2), mTOR inhibitors significantly suppressed the uptake of FITC-dextran and beads (Fig. 3B, i-iii). Additionally, the phagocytic ability of live bacteria was examined using a gentamicin protection assay. We found that E. coli uptake was significantly inhibited by mTOR inhibitors (Fig. 3B, iv). Taken together, these results indicate that mTOR inhibitors significantly suppress fluid endocytosis and bead/bacterial phagocytosis during TPA-induced differentiation, but not in undifferentiated THP-1 cells.

Fig. 3.

Timing-dependent effects of mechanistic/mammalian target of rapamycin (mTOR) inhibitors on endocytosis in THP-1 cells. (A) Undifferentiated THP-1 cells were incubated in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-free mTOR inhibitor-containing medium for 48 hr. Fluorescein isothiocyanate (FITC)-dextran (0.5 mg/mL) or fluorescent beads (10 particles/cell) were then added to the cells and cultured for 30 min or 1 hr. The cells were fixed and the amount of FITC-dextran incorporated into the beads was measured using flow cytometry. (i) FITC-dextran uptake. The values for the DMSO-treated (vehicle) cells were standardized to 100. (ii) Uptake of the bovine serum albumin (BSA)-coated beads. The values for the DMSO-treated (vehicle) cells were standardized to 100. (iii) Uptake by human IgG-coated beads. The values for the DMSO-treated (vehicle) were standardized to 100. Each bar indicates the mean ± SEM. ns: not significant. n=3. (B) THP-1 cells were pretreated with mTOR inhibitors for 30 min, differentiated with 100 nM TPA in the presence of mTOR inhibitors for 24 hr, and incubated in a TPA-free mTOR inhibitor-containing medium for another 24 hr. Endocytic activity was analyzed as described in (A). (i) FITC-dextran uptake. The values for the TPA-treated cells were standardized to 100. (ii) Uptake of the BSA-coated beads. The values for the TPA-treated cells were standardized to 100. (iii) Uptake by human IgG-coated beads. The values for the TPA-treated cells were standardized to 100. (iv) After treatment with mTOR inhibitors and TPA, the cells were incubated for 1 hr in a medium containing E. coli at a multiplicity of infection of 50, followed by gentamicin treatment for 1 hr and cell extraction. The number of colonies on the Luria–Bertani plates was counted, and the number of TPA-treated cells was standardized to 100. Each bar indicates the mean ± SEM. ****: P<0.0001. n=4.

mTOR inhibitors do not suppress the phagocytosis and fluid endocytosis after TPA-induced differentiation

Previous results indicated that mTOR inhibitors suppress endocytosis in TPA-stimulated THP-1 cells (Fig. 3B). It is possible that mTOR inhibitors affected TPA-induced differentiation, resulting in cells with defective endocytosis. Another possibility is that the mTOR inhibitor directly suppressed the endocytic machinery without affecting differentiation. To validate these possibilities, we treated THP-1 cells with mTOR inhibitors for 1 hr after TPA-induced differentiation. mTORC1 expression was almost completely suppressed by the 1-hr treatment with mTOR inhibitors (Fig. 4A and 4B). However, the uptake of FITC-dextran and fluorescent beads was not suppressed by the mTOR inhibitors (Fig. 4C–E). This suggests that mTOR inhibitors suppress endocytosis, not by direct inhibition of the endocytosis machinery, but by the induction of abnormal differentiation into endocytosis-deficient cells.

Fig. 4.

Mechanistic/mammalian target of rapamycin (mTOR) inhibitors do not suppress phagocytosis and fluid endocytosis after 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation. (A) THP-1 cells were treated with 100 nM TPA for 24 hr, rested for another 23 hr, and treated with the mTOR inhibitors for 1 hr. (B, upper) Representative images of three independent western blotting experiments. (B, lower) Expression levels of S6K were quantified from the western blot images shown in (B, upper), and the values of the TPA-treated cells were standardized to 100. (C–E) THP-1 cells were treated as shown in (A). After treatment, the cells were then incubated with the FITC-dextran (0.5 mg/mL) or fluorescent beads (10 particles/cell) for another 30 min or 1 hr, respectively. The cells were fixed and analyzed by flow cytometry. The values of the TPA-treated cells were standardized to 100. Each bar indicates the mean ± SEM. ****: P<0.0001, ns: not significant. n=3.

Effects of mTOR inhibitors on the direction of macrophage polarization

Finally, we examined the mechanism by which the mTOR inhibitors affected the TPA-induced differentiation of THP-1 cells. The expression levels of macrophage markers were quantified by qPCR. The mRNA expression levels of NOS2/iNOS (M1 marker), CD80 (M1 marker), CD206 (M2 marker), and CD204 (M2 marker) were measured. The expression of the M1 marker, NOS2, was not significantly altered by TPA treatment, but was upregulated by Torin 1 (Fig. 5A). In contrast, the expression of another M1 marker, CD80, was significantly increased by TPA treatment and decreased by mTOR inhibitors (Fig. 5B). The expression levels of the M2 markers CD206 and CD204 showed expression patterns similar to CD80. They were significantly upregulated by TPA treatment and downregulated by mTOR inhibitors (Fig. 5C and 5D). These results suggest that although mTOR inhibitors affect THP-1 polarization, their effects on M1/M2 markers may be complex; as demonstrated by the inconsistent effects on the two M1 markers, NOS2 and CD80.

Fig. 5.

Effects of mechanistic/mammalian target of rapamycin (mTOR) inhibitors on the direction of macrophage polarization. cDNA samples obtained from THP-1 cells were analyzed using real-time PCR. The mRNA levels of nitric oxide synthase (NOS) 2/ iNOS (M1 marker), CD80 (M1 marker), CD206 (M2 marker), and CD204 (M2 marker) were examined. The values for TPA-treated cells were standardized to 100. THP-1 cells were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 hr, rested for another 24 hr (TPA) or pretreated with mTOR inhibitors for 30 min, treated with TPA in the presence of mTOR inhibitors for 24 hr, and incubated in a TPA-free mTOR inhibitor-containing medium for another 24 hr (TPA + Torin 1 and TPA + Rapamycin). Each bar indicates the mean ± SEM. ns: not significant, **P<0.01, ***P<0.001, ****P<0.0001. n=3.

DISCUSSION

In this study, we investigated the involvement of mTORC1 in endocytosis in relation to the differentiation of THP-1 cells. TPA-induced differentiation was accompanied by mTORC1 activation (Fig. 1), suggesting that mTORC1 is involved in THP-1 differentiation. Although we confirmed the induction of the general macrophage marker, CD11b (Fig. 1), mTOR inhibitor treatment did not affect the expression of CD11b (Fig. 2B) or morphological changes from floating to adherent cells (Fig. 2C). In contrast, when THP-1 cells were treated with mTOR inhibitors during a 30-min pretreatment, 24-hr TPA treatment, and 24-hr post-TPA resting period; endocytosis of FITC-dextran, latex beads, and live bacteria was significantly impaired (Fig. 3B). Dextran is a water-soluble molecule that is frequently used as an indicator of fluid endocytosis (pinocytosis), including macropinocytosis [11]. FITC-dextran with a molecular weight of approximately 10 kDa was used in this study. Cellular uptake of dextran with this relatively small molecular weight is known to reflect not only micropinocytosis but also clathrin/dynamin-dependent pinocytosis [19]. A previous study showed that dendritic cells that matured in the presence of rapamycin exhibited decreased pinocytosis [12]. This result is consistent with our finding that mTOR inhibitors suppress pinocytosis (Fig. 3B).

mTORC1 inhibition suppresses phagocytosis induction by a lipid mediator, resolvin E1, in human monocyte-derived macrophages [22]. Another study demonstrated that when using human monocyte-derived macrophages and TPA-stimulated THP-1 cells, mTORC1 inhibition abrogated the increase in phagocytosis caused by altered pressure but did not suppress basal phagocytosis [27]. These results are consistent with our finding that mTORC1 inhibition does not affect phagocytosis before differentiation induction or after differentiation completion (Figs. 3A and 4C). Although the detailed mechanism of mTOR inhibitor-induced endocytosis suppression requires further investigation, our current data indicates that mTOR inhibitors affect endocytosis only when used during TPA-induced differentiation (Fig. 3B). Therefore, it is likely that mTOR inhibitors affect the differentiation process of THP-1, leading to the production of endocytosis-deficient cells rather than directly inhibiting the endocytosis machinery.

Macrophages differentiate into different subtypes in response to various stimuli [14, 30, 31]. Differentiated macrophages are classified into M1 and M2 subtypes based on their function and expressed markers. M1 macrophages are induced by lipopolysaccharides, interferon-γ, and granulocyte-macrophage colony-stimulating factor, secrete pro-inflammatory cytokines, and promote inflammatory responses. In addition, M1 macrophages promote pathogen elimination via the production of nitrogen monoxide by inducing the expression of NOS2/iNOS. In contrast, M2 macrophages are induced by the macrophage colony-stimulating factor and interleukin-4 and produce anti-inflammatory cytokines. M2 macrophages contribute to the suppression of inflammatory responses, wound healing, and tumor promotion [8, 14].

It has been suggested that macrophage polarization of TPA-treated THP-1 cells is initially biased in the M1 direction and later in the M2 direction with prolonged TPA treatment [18]. Additionally, after TPA stimulation, the degree of differentiation of THP-1 cells is altered during the resting period in TPA-free medium [28]. Under our experimental condition, 24-hr TPA treatment followed by 24-hr rest without TPA, TPA treatment alone significantly induced the expression of the M2 markers CD206 and CD204 (Fig. 5C and 5D) and the M1 marker CD80 (Fig. 5B), without affecting the M1 marker NOS2 (Fig. 5A). mTOR inhibitors significantly decreased the expression of CD206, CD204, and CD80 whereas they increased NOS2 expression (Fig. 5). Despite the discrepancy between the two M1 markers NOS2 and CD80, mTOR inhibitors appear to affect polarization (Fig. 3B). Although the underlying mechanisms are unclear, M1/M2 polarization status has been linked to endocytosis activity. Previous studies have shown that M2 macrophages exhibit high activities of fluid endocytosis, including macropinocytosis, and phagocytosis [8, 15, 29].

Macrophage M1/M2 polarization is affected by external signals, including cytokines and growth factors, as well as environmental cues such as nutrients and metabolites [30]. The PI3K-AKT-mTOR pathway, which plays a major role in nutrient or growth factor sensing, has been suggested to influence M1/M2 polarization. For example, the activation of PI3K by myeloid-specific knockout of Pten or Inpp5d (Ship1) enhances M2 polarization; with elevated activation of the transcription factor STAT6, a well-known M2-polarizing protein [30]. Previous studies using human blood-derived macrophages have also shown that mTORC1 suppression by rapamycin during macrophage differentiation enhanced M1 polarization [5, 21]. However, the effects of mTOR manipulation on M1/M2 polarization are sometimes inconsistent and may, therefore, be context-dependent. For example, unlike rapamycin, mTORC1 suppression by myeloid-specific knockout of Raptor, an mTORC1 subunit, results in impaired M1 polarization and enhanced M2 polarization [16]. mTORC1 activation by myeloid-specific knockout of Tsc1, an mTORC1 suppressor, enhanced M1 polarization but showed inconsistent results regarding M2 polarization; with enhanced [9] or impaired [3, 32] M2 polarization. These studies have suggested that enhanced M1 polarization by Tsc1 knockout is independent of mTOR and dependent on the RAS-RAF-MEK-ERK pathway [32] whereas impaired M2 polarization in Tsc1 knockout macrophages is dependent on mTOR [3, 32], CCAAT/enhancer-binding protein-β [32], and AKT [3]. In addition, mTORC2 suppression by myeloid-specific knockout of Rictor, a mTORC2 subunit, promotes M1 polarization [10]. Therefore, the regulation of M1/M2 polarization by mTORC1 and mTORC2 is complex and requires further investigation to elucidate the precise mechanism.

mTORC1, often in combination with mTORC2, is activated in human and mouse monocytes, macrophages, and dendritic cells in response to stimulation with Toll-like receptor ligands, growth factors including granulocyte-macrophage colony-stimulating factor, and cytokines such as IL-4 [30]. Activated mTORC1 induces a variety of pathways including protein synthesis and cell growth. Consistent with previous studies, our results showed that mTOR affects the expression of M1/M2 polarization markers (Fig. 5). In the early stages of the immune response, M1 macrophages generate proinflammatory cytokines and contribute to the induction of inflammation. Subsequently, M2 macrophages suppress inflammation and promote wound healing and tissue repair [7]. mTORC1 may play an important role in this coordinated and temporally ordered polarization process. As M1/M2 polarity is known to be plastic and macrophages are thought to even switch between cell types (M1 to M2 or vice versa) [7], it is possible that mTORC1 activity induces the switch between M1 and M2 in a time-dependent manner.

In summary, this study demonstrates that in TPA-stimulated THP-1 cells, mTOR inhibitors reduce endocytosis, including phagocytosis, a major function of macrophages. mTOR inhibitors do not directly suppress the endocytic machinery; rather, macrophage polarization is likely to be affected, as shown by the altered expression of M1/M2 polarization markers. These data suggest that mTOR inhibitors induce differentiation of THP-1 cells into endocytosis-deficient macrophages. Our findings contribute to a better understanding of coordinated macrophage polarization in response to environmental cues and suggest that abnormal macrophage differentiation may be associated with the immunosuppressive effects of mTOR inhibitors. Since most studies on macrophage differentiation and function have been conducted using human and mouse macrophages, it is important that future studies identify any differences from these results in other animal species.

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest regarding the contents of this study.