Glucocorticoids Impair the 7α-Hydroxycholesterol-Enhanced Innate Immune Response

Abstract

Glucocorticoids suppress the vascular inflammation that occurs under hypercholesterolemia, as demonstrated in an animal model fed a high-cholesterol diet. However, the molecular mechanisms underlying these beneficial effects remain poorly understood. Because cholesterol is oxidized to form cholesterol oxides (oxysterols) that are capable of inducing inflammation, we investigated whether glucocorticoids affect the immune responses evoked by 7α-hydroxycholesterol (7αOHChol). The treatment of human THP-1 monocytic cells with dexamethasone (Dex) and prednisolone (Pdn) downregulated the expression of pattern recognition receptors (PRRs), such as TLR6 and CD14, and diminished 7αOHChol-enhanced response to FSL-1, a TLR2/6 ligand, and lipopolysaccharide, which interacts with CD14 to initiate immune responses, as determined by the reduced secretion of IL-23 and CCL2, respectively. Glucocorticoids weakened the 7αOHChol-induced production of CCL2 and CCR5 ligands, which was accompanied by decreased migration of monocytic cells and CCR5-expressing Jurkat T cells. Treatment with Dex or Pdn also reduced the phosphorylation of the Akt-1 Src, ERK1/2, and p65 subunits. These results indicate that both Dex and Pdn impair the expression of PRRs and their downstream products, chemokine production, and phosphorylation of signaling molecules. Collectively, glucocorticoids suppress the innate immune response and activation of monocytic cells to an inflammatory phenotype enhanced or induced by 7αOHChol, which may contribute to the anti-inflammatory effects in hypercholesterolemic conditions.

Keywords: 7α-Hydroxycholesterol; GlucocorticoidsMonocytes/macrophages; Inflammation; Pattern recognition receptors.

Figure 1. Downregulation of TLR6 and attenuated response to FSL-1.

(A) THP-1 cells are serum-starved for 24 h and treated for 48 h with 7αOHChol (5 µg/ml each) in the absence or presence of the indicated glucocorticoids (1 µM each). Total RNA isolated from the cells is reverse-transcribed, and quantitative real-time polymerase chain reaction is performed to determine the relative levels of TLR6 transcripts. The Y-axis values represent fold increases in the TLR6 messenger RNA levels normalized to the glyceraldehyde-3-phosphate dehydrogenase levels compared with those of control cells. (B) After treatment of serum-starved THP-1 cells with 7αOHChol in the absence or presence Dex or Pdn, cell surface TLR6 is immunostained, and fluorescence is analyzed by flow cytometry. (C) Serum-starved THP-1 cells are treated with 7αOHChol for 24 h and incubated for another 24 h after addition of FSL-1 (100 ng/ml) with or without the indicated glucocorticoids. The amount of IL-23 secreted into culture media is measured by enzyme-linked immunosorbent assay. Data are expressed as the means ± SDs (n=3 replicates/group). ***p<0.001 vs. control; ^##p<0.01 vs. 7αOHChol; ^###p<0.001 vs. 7αOHChol; ^§§§p<0.001 vs. control or 7αOHChol or FSL-1; ^¶¶p<0.01 vs. 7αOHChol+FSL-1.

Figure 2. Downregulation of CD14 and weakened LPS response.

(A, B) Serum-starved THP-1 cells are treated for 48 h with 7αOHChol (5 µg/ml) in the absence or presence of the indicated glucocorticoids (1 µM each). (A) Quantitative real-time polymerase chain reaction is performed to determine the relative levels of CD14 transcripts. (B) The mCD14 levels are measured by flow cytometry after immunostaining of surface CD14. (C) Following treatment of serum-starved THP-1 cells for 48 h with 7αOHChol in the absence or presence of Dex or Pdn, released sCD14 is measured by ELISA. (D) Serum-starved THP-1 cells are incubated for 24 h with or without 7αOHChol and then stimulated for 9 h with LPS (100 ng/ml) from Escherichia coli K12 in the absence or presence of Dex or Pdn. The amount of CCL2 in the culture media is determined by ELISA. Data are expressed as means ± SDs (n=3 replicates/group). 7αOHChol, 7α-hydroxycholesterol; Dex, dexamethasone; Pdn, prednisolone; LPS, lipopolysaccharide. ***p<0.001 vs. control; ^##p<0.01 vs. 7αOHChol; ^§§§p<0.001 vs. 7αOHChol or LPS; ^¶¶¶p< 0.001 vs. 7αOHChol+LPS.

Figure 3. Inhibition of CCL2 expression and monocytic cell migration.

Serum-starved THP-1 cells are treated for 48 h with 7αOHChol (5 µg/ml) in the absence or presence of the indicated glucocorticoids (1 µM each), (A) after which the levels of CCL2 transcripts are assessed by qRT-PCR and (B) the amount of secreted CCL2 protein in the culture media is measured by ELISA. (C) Monocytic cells are exposed to the conditioned media isolated from THP-1 cells stimulated with 7αOHChol (5 µg/ml) with or without the indicated glucocorticoids, and the numbers of migrated monocytic cells are counted. Data are expressed as means ± SDs (n=3 replicates for each group). ***p<0.001 vs. control; ^##p<0.01 vs. 7αOHChol; ^###p<0.001 vs. 7αOHChol.

Figure 4. Decreased production of CCR5 ligands and migration of CCR5-expressing T cells.

Following serum starvation, THP-1 cells are cultured for 48 h with 7αOHChol (5 µg/ml) in the presence or absence of the indicated glucocorticoids (1 µM each). (A) The transcript levels of CCL3 and CCL4 genes are assessed by qRT-PCR. (B) The amounts of CCL3 and CCL4 released from THP-1 cells are determined by ELISA. (C) CCR5-positive Jurkat T cells are exposed to conditioned media isolated from THP-1 cells stimulated with 7αOHChol with or without dexamethasone or prednisolone, and their ability to migrate is investigated by a chemotaxis assay. Data are expressed as means ± SDs (n=3 replicates for each group). **p<0.01 vs. control; ***p<0.001 vs. control; ^##p<0.01 vs. 7αOHChol; ^###p<0.001 vs. 7αOHChol.

Figure 5. Attenuated phosphorylation of multiple signaling molecules.

Cell lysates are obtained after exposure of THP-1 cells for 6 h to 7αOHChol (5 μg/ml) with dexamethasone or prednisolone (1 μM each). Following the determination of protein concentration, equal amounts of protein are analyzed by western blotting using the indicated Abs.

Figure 6. Pharmacological action of glucocorticoids in a milieu rich in 7αOHChol.

7αOHChol enhances the innate immune response by upregulating the surface expression of TLR6 and CD14, leading to increased cytokine and chemokine production. 7αOHChol also promotes the migration of monocytes and specific subtypes of CD4⁺ effector T cells. Glucocorticoids impair the effects of 7αOHChol on receptor expression, cytokine and chemokine production, and cell migration.