Dexamethasone inhibits activation of monocytes/macrophages in a milieu rich in 27-oxygenated cholesterol

Molecular mechanisms underlying the decreased number of macrophages and T cells in the arteries of cholesterol-fed-rabbits following dexamethasone administration are unknown. We investigated the possibility that dexamethasone could affect activation of monocytic cells induced by oxygenated derivatives of cholesterol (oxysterols) using THP-1 monocyte/macrophage cells. 27-Hydroxycholesterol (27OHChol), an oxysterol elevated with hypercholesterolemia, enhanced production of CCL2, known as MCP1, chemokine from monocytes/macrophages and migration of the monocytic cells, but the CCL2 production and the cell migration were reduced by treatment with dexamethasone. Dexamethasone inhibited superproduction of CCL2 induced by 27OHChol plus LPS and attenuated transcription of matrix metalloproteinase 9 as well as secretion of its active gene product induced by 27OHChol. The drug downregulated cellular and surface levels of CD14 and blocked release of soluble CD14 without altering transcription of the gene. Dexamethasone also inhibited expression and phosphorylation of the NF-κB p65 subunit enhanced by 27OHChol. Collectively, these results indicate that dexamethasone inhibits activation of monocytes/macrophages in response to 27OHChol, thereby leading to decreased migration of inflammatory cells in milieu rich in oxygenated derivatives of cholesterol.


Introduction
Dexamethasone, a synthetic glucocorticoid with anti-inflammatory and immunosuppressant effects, is used to treat many inflammatory conditions, including allergic disorders, skin conditions, ulcerative colitis, arthritis, lupus, psoriasis, and breathing disorders [1]. However, prolonged corticosteroid therapy can induce or exacerbate coronary risk factors, such as hypertension, hypercholesterolemia, hypertriglyceridemia, and impairment of glucose tolerance [2][3][4]. In animal experiments, dexamethasone has been reported to inhibit cell migration, thereby reducing development of atherosclerosis. Administration of dexamethasone results in a decreased number of immune cells, such as macrophages and T lymphocytes, in the intima PLOS  of cholesterol-fed rabbits [5][6][7]. These findings suggest that inhibitory effects of the drug on cellular responses to high cholesterol may occur, but it is unknown how it exerts such effects in a milieu rich in cholesterol molecules. Cholesterol molecules accumulate in the artery where they undergo oxidation to oxysterols, the oxygenated derivatives of cholesterol. Oxysterols are biologically active molecules affecting multiple types of cells. Oxysterols oxidized at C7 of cholesterol differentially activate human umbilical venous endothelial cells and play a key role in apoptosis and IL-1β secretion [8]. Oxysterol-induced IL-8 secretion is a calcium-dependent phenomenon leading to the IL-8 gene activation via the MEK/ERK1/2 and AP-1 (c-fos) [9]. , the most abundant oxysterol in atherosclerotic arteries and in sera of patients suffering hypercholesterolemia, is synthesized by sterol 27-hydroxylase or formed by non-enzymatic oxidation [10,11], and the levels of 27OHChol are two orders of magnitude higher than circulating levels in the arteries [12]. 27OHChol promotes inflammatory process in vivo [13], presumably via activation of monocytic cells. The oxysterol increases expression of pattern recognition receptors and enhances migration of immune cells by inducing secretion of chemoattractants such as CCL2, which is widely known as MCP1, CCL3 and CCL4 from monocyte/macrophage cells [14][15][16][17]. These findings indicate that 27OHCHol is a key oxygenated cholesterol derivative that activates monocytic cells.
We investigated whether dexamethasone affects monocytes/macrophages activation in a milieu rich in 27-oxygenated derivative of cholesterol to understand the molecular mechanisms underlying its anti-migration effect in cholesterol-fed animals.

Cell treatments
Human monocytic THP-1 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in 10% FBS-supplemented RPMI 1640 medium. THP-1 cells (2.5 × 10 5 cells/ml) were serum-starved by incubating overnight in RPMI medium supplemented with 0.1% BSA (endotoxin-free), and the serum-starved cells were treated with 27OHChol in the presence of indicated concentrations of dexamethasone. 27OHChol and dexamethasone were dissolved in ethanol and dimethyl sulfoxide, respectively. In experiment of CCL2 superinduction, serum-starved THP-1 cells were cultured for 24 h with 27OHChol in the presence of dexamethasone, followed by stimulation for 9 h with LPS (100 ng/ml) dissolved in endotoxin-free water.

Chemotaxis assay
Migration of THP-1 cells was measured using Transwell Permeable Supports (Costar, Cambridge, MA, USA) as previously described [16]. Cells (5 × 10 5 cells in 100 μL of 0.1% BSA) were loaded into the top chamber of 5-μm-pore polycarbonate transwell inserts. Transwell chambers were inserted into wells filled with a supernatant isolated from THP-1 cells treated with 27OHChol with or without dexamethasone. After incubation for 3 h at 37˚C, the number of cells that migrated to the bottom chamber was counted using a Vi-Cell cell counter (Beckman Coulter, Inc. Brea, CA, USA).

MMP-9 gelatinolytic activity in supernatants
Supernatants isolated from cultured THP-1 cells were collected and concentrated 30-fold using Vivaspin 2 Centricon as previously described [18]. The concentrated medium was then electrophoretically separated onto an 8% polyacrylamide gel containing 0.15% gelatin. After electrophoresis, the gel was washed, activated, and stained with 0.2% Coomassie brilliant blue R-250 prior to destaining. Clear zones against the blue background indicated gelatinolytic activity.

Flow cytometric analysis
THP-1 cells were harvested by centrifugation and incubated for 40 min with anti-CD14 antibody conjugated with a green fluorescent dye (Santa Cruz Biotechnology Inc.) at 4˚C. After washing twice with PBS, cells were resuspended in 1% paraformaldehyde in phosphate-buffered saline (PBS). Fluorescence was analyzed by flow cytometry.

Enzyme-linked immunosorbent assay
The levels of CCL2, sCD14, and MMP-9 secreted into the culture media were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

Western blot analysis
Cell lysates were separated by 10% SDS-PAGE followed by transfer to nitrocellulose membranes. After blocking for 1 h in 1% skim milk (in TBS containing 0.05% Tween-20), membranes were incubated with primary antibodies against CD14, phosphorylated p65, p65 subunit or β-actin at 4˚C overnight. After three washes with TBS-T, membranes were incubated for 1 h with HRP-conjugated secondary antibodies at room temperature. Bands were detected using chemiluminescent detection reagents.

Statistical analysis
One-way ANOVA followed by Dunnett's multiple comparison tests was performed using PRISM (version 5.0) (GraphPad Software Inc., San Diego, CA, USA). Null hypotheses of no difference were rejected if p-values were less than 0.05.

Inhibitory effects on expression of CCL2 and migration of monocytic cells
We investigated whether dexamethasone affected expression of CCL2 chemokine. Transcription of the CCL2 gene was induced in the presence of 27OHChol, and the induction was attenuated by treatment with dexamethasone in a dose-dependent manner ( Fig 1A). CCL2 transcript levels were increased by 13.4-fold in the presence of 27OHChol, but reduced to 9.5-, 5.3-, and 3.8-folds by treatment with 0.01, 0.1, and 1 μM of dexamethasone, respectively. Dexamethasone influenced CCL2 secretion in a pattern similar to that observed with transcription of the CCL2 gene ( Fig 1B). The amount of CCL2 secreted from THP-1 cells increased significantly in the presence of 27OHChol compared with that secreted from unstimulated THP-1 cells. The levels of CCL2 secreted from 27OHCHol-treated THP-1 cells were dose-dependently reduced by treatment with dexamethasone. The reduction was not due to cytotoxicity because dexamethasone did not affect cell viability (S1 Fig).
We conducted a chemotaxis assay to determine if dexamethasone alters migration of monocytic cells (Fig 1C). Migration of monocytic cells was blocked by treatment with high concentrations of dexamethasone. Monocytic cell migration was increased in response to the supernatant isolated from cells stimulated with 27OHChol. The migration was slightly reduced by treatment with 0.01 and 0.1 μM of dexamethasone and even further reduced to basal levels by 1 μM dexamethasone. Overall, these results indicate that dexamethasone inhibited CCL2 expression and monocytic cell migration induced by 27OHChol.

Inhibition of CCL2 superinduction
Expression of CCL2 is super-induced when LPS is added to monocytic cells stimulated with 27OHChol [18]. We investigated whether dexamethasone influences super-induction of CCL2. Transcript levels of the CCL2 gene were increased by 16.5-and 3.4-folds in the presence of 27OHChol and LPS, respectively (Fig 2A). The addition of LPS to 27OHChol-stimulated cells resulted in a 30.8-fold elevation in CCL2 transcript levels, but the elevation was blocked by treatment with dexamethasone. Dexamethasone influenced secretion of CCL2 in a pattern similar to that of transcription ( Fig 2B). 27OHChol increased secretion of CCL2, and the addition of LPS to 27OHChol-stimulated cells resulted in further enhanced secretion of CCL2. The enhanced CCL2 secretion was significantly attenuated by treatment with dexamethasone. These results indicate that dexamethasone suppressed super-production of CCL2 induced by LPS in combination with 27OHChol.

Downregulation of 27OHChol-induced expression of CD14 at the protein level
Because LPS responses depend on the expression of membrane CD14 (mCD14) by monocytic cells [19], we investigated whether dexamethasone downregulates mCD14 on the surface of monocytic cells. The percentage of cells expressing mCD14 was increased from 38.6% to 79.1% in the presence of 27OHChol, but it was reduced to 41.5%, 21.8%, and 19.3% by treatment with 0.01, 0.1, and 1 μM of dexamethasone, respectively (Fig 3A). We also evaluated the effects of dexamethasone on CD14 protein via Western blot analysis (Fig 3B), and found that the level of total CD14 protein was increased in the presence of 27OHChol. However, the expression of CD14 protein was reduced by treatment with 0.01 and 0.1 μM of dexamethasone and further decreased to below the basal level by 1 μM dexamethasone. We also investigated whether dexamethasone affected transcription of CD14. Transcript levels of the CD14 were A soluble form of the CD14 molecule (sCD14) is capable of activating monocytic cells independently of mCD14 [20]. Therefore, we determined whether dexamethasone alters generation of sCD14. THP-1 cells released a basal level of sCD14 of 651.2 ± 79.7 pg/ml to the media, and the level increased significantly to 1450.8 ± 222.2 pg/ml in the presence of 27OHChol ( Fig  3C). However, the increased release of sCD14 was blocked by dexamethasone. The amounts of sCD14 released from 27OHChol-treated THP-1 cells were reduced to the basal level by treatment with 0.01, 0.1 or 1 μM of dexamethasone. These findings indicate that dexamethasone suppresses expression of CD14 protein and production of sCD14.

Attenuation of 27OHChol-induced expression of MMP-9
Proteolytic cleavage of mCD14 by MMP-9 is one of the mechanisms generating sCD14 [21]. Therefore, we examined the effects of dexamethasone on MMP-9 expression. Transcript levels of the MMP-9 gene were increased by 5.8-fold in the presence of 27OHChol. The increase, however, was reduced to 4.6-, 2.9-, and 2.2-folds in response to treatment with 0.01, 0.1, and 1 μM of dexamethasone, respectively (Fig 4A).
We determined the effects of dexamethasone on production of MMP-9 protein by evaluating the total (latent and active) levels of MMP-9 secreted into the medium (Fig 4B). THP-1 cells secreted a low basal level of MMP-9, and the amount of secreted MMP-9 protein was significantly increased to 3.1 ± 0.3 ng/ml in the presence of 27OHChol, which was reduced to 1.0 ± 0.1, 0.7 ± 0.1, and 0.7 ± 0.09 ng/ml by treatment with 0.01, 0.1, and 1 μM of dexamethasone, respectively. We next examined whether dexamethasone affected the activity of MMP-9 by zymography. Stimulation of THP-1 cells with 27OHChol resulted in increased MMP-9 activity in supernatant, but this increase in activity was attenuated by treatment with dexamethasone (Fig 4C). These results mean that dexamethasone inhibits the monocytic cell expression of MMP-9 induced by 27OHCHol at the mRNA and protein levels. Suppression of NF-κB p65 expression The inducible transcription factor NF-κB plays a central role in regulation of inflammatory genes [22]. Therefore, we investigated the effects of 27OHChol and dexamethasone on expression of the NF-κB p65 subunit. Transcript levels of the p65 gene were elevated by 1.28-, 2.04-, and 1.77-folds after incubation for 3 h, 6 h, and 9 h with 27OHChol, but these increases were  reduced to 0.94-, 0.46-, and 0.95-folds by treatment with dexamethasone, respectively ( Fig 5A). We also determined whether dexamethasone affected expression of p65 protein (Fig 5B). The amount of total p65 protein increased in proportion to incubation periods with 27OHChol for up to 9 h, but the expression was inhibited by treatment with dexamethasone. 27OHChol and dexamethasone affected phosphorylation of p65 in a similar fashion as that of expression of total p65 protein (Fig 5B). Taken together, these results indicate that dexamethasone reduces expression of the p65 subunit at the transcript and protein levels.

Discussion
Monocytes migrate into the arterial wall during progression of atherosclerosis and migrated monocytes differentiate into macrophages, which is activated and secrete inflammatory mediators [23]. This migration is orchestrated by chemokine CCL2, which triggers firm adhesion of monocytes to the endothelium and directs their migration into intima [24]. Administration of dexamethasone reduces the number of macrophages in the aortas of cholesterol-fed-rabbits [5,6]. Therefore, we determined the inhibitory effects of dexamethasone on 27OHChol-induced activation by measuring CCL2. Treatment with dexamethasone resulted in impaired production of CCL2, as well as migration of monocytic cells. We believe that these results may explain the observation of reduced macrophages in the aortas of rabbits fed a high-cholesterol diet. Infection with gram-negative bacteria such as Porphyromonas gingivalis leads to accelerated development of atherosclerotic lesions in mice after a high-cholesterol diet, and administration of LPS to ApoE-deficient mice results in aggravation of inflammation [25,26]. LPS is recognized by CD14, which transfers LPS to toll-like receptor 4, initiating an inflammatory responses by enhancing the production of chemokine, including CCL2 [27]. We demonstrated downregulation of mCD14 and sCD14 and inhibition of LPS-induced superinduction of CCL2 in the presence of dexamethasone. These results are in line with Bhattacharyya's finding that LPSmediated expression of inflammatory molecules is inhibited by treatment with dexamethasone [28]. In sum, these findings suggest that dexamethasone inhibits further activation of macrophages by microbial pathogens under an environment rich in oxygenated cholesterol derivatives via downregulation of CD14 molecules at the protein level because dexamethasone did not affect transcription of the CD14 gene.
Activated macrophages show dysregulated synthesis and secretion of matrix metalloproteases (MMPs) [29]. Among MMPs, expression of MMP-9 is linked to macrophage recruitment in vivo because deficiency in MMP-9 results in reduced macrophage infiltration in ApoE-knockout mice [30]. In agreement with the results of a previous study [30], MMP-9 production was substantially reduced in concurrence with decreased migration of monocytic cells. The reduced production of MMP-9 is in line with reports that intravenous administration with glucocorticoid results in markedly reduced levels of MMP-9 in patients [31,32]. Though we demonstrated inhibitory effects of dexamethasone on MMP-9 expression induced by 27OHChol at the mRNA and protein levels, it is possible that dexamethasone downregulates MMP-9 activity at the post-translational level because it can reduce protease activity by increasing tissue inhibitor of metalloproteinase-1 (TIMP-1) [33].
NF-κB is composed of homo-and heterodimeric complexes of members of the Rel family. The most common and best-characterized form of NF-κB is the p65/p50 heterodimer. Upon phosphorylation at the serine residues, the NF-κB dimer translocates to the nucleus where it induces expression of a variety of genes involved in inflammation [22]. Of the two subunits, the p65 subunit is responsible for the strong transcription activating potential of NF-κB [34]. The results of enhanced expression and phosphorylation of the p65 subunit mean that 27OHChol will potentiate transcriptional activity of NF-κB, which is in agreement with the results of a study that reported increased phosphorylation and activation of the subunit in the presence of oxygenated cholesterol derivatives [35]. Moreover, a decreased level of the p65 subunit coincided with those of inflammatory mediators after dexamethasone treatment. Taken together, these results suggest that NF-κB activated in the presence of 27OHChol is likely to play a significant role in inflammation in diseases in which oxygenated cholesterol molecules are involved in their pathogenesis.
In this study, we report new pharmacological effects of dexamethasone; namely, inhibition of monocytic cells activation induced by 27OHChol. We think that the inhibitory effects contribute to anti-inflammatory activity of the drug in a milieu rich in oxygenated cholesterol molecules. In contrast to experimental animal models of atherosclerosis, cardiovascular risk factors are aggravated following high doses and long-term administration of glucocorticoids in clinical studies, which increases cardiovascular events. Therefore, local delivery of dexamethasone by using nanoparticles targeting atherosclerotic lesions may be used as a therapeutic approach against inflammation caused by oxygenated cholesterol molecules in atherosclerosis.