Side-Chain Immune Oxysterols Induce Neuroinflammation by Activating Microglia

Abstract

In individuals with Alzheimer's disease, the brain exhibits elevated levels of IL-1β and oxygenated cholesterol molecules (oxysterols). This study aimed to investigate the effects of side-chain oxysterols on IL-1β expression using HMC3 microglial cells and ApoE-deficient mice. Treatment of HMC3 cells with 25-hydroxycholesterol (25OHChol) and 27-hydroxycholesterol (27OHChol) led to increased IL-1β expression at the transcript and protein levels. Additionally, these oxysterols upregulated the surface expression of MHC II, a marker of activated microglia. Immunohistochemistry performed on the mice showed increased microglial expression of IL-1β and MHC II when fed a high-cholesterol diet. However, cholesterol and 24s-hydroxycholesterol did not increase IL-1β transcript levels or MHC II expression. The extent of IL-1β increase induced by 25OHChol and 27OHChol was comparable to that caused by oligomeric β-amyloid, and the IL-1β expression induced by the oxysterols was not impaired by polymyxin B, which inhibited lipopolysaccharide-induced IL-1β expression. Both oxysterols enhanced the phosphorylation of Akt, ERK, and Src, and inhibition of these kinase pathways with pharmacological inhibitors suppressed the expression of IL-1β and MHC II. The pharmacological agents chlorpromazine and cyclosporin A also impaired the oxysterol-induced expression of IL-1β and upregulation of MHC II. Overall, these findings suggest that dysregulated cholesterol metabolism leading to elevated levels of side-chain oxysterols, such as 25OHChol and 27OHChol, can activate microglia to secrete IL-1β through a mechanism amenable to pharmacologic intervention. The activation of microglia and subsequent neuroinflammation elicited by the immune oxysterols can contribute to the development of neurodegenerative diseases.

Keywords: immune oxysterol; interleukin-1β; microglia; neuroinflammation.

Figure 1

The effects of side chain oxysterols, Aβ_1–42, and LPS on the microglial expression of IL-1β. HMC3 cells (1 × 10⁶ cells/dish) were treated for 48 h with cholesterol or the indicated oxysterols (1 μg/mL each). (A) IL-1β transcripts were detected by RT-PCR and levels of IL-1β transcripts were analyzed by qPCR. The y-axis values represent fold increases in IL-1β mRNA levels normalized to GAPDH levels relative to that of the untreated microglia (control). * p < 0.05 vs. control; *** p < 0.001 vs. control. (B) The concentrations of IL-1β secreted from cells to the culture medium were quantitatively detected by ELISA. *** p < 0.001 vs. control. (C) The cells were stimulated with 5 and 10 μM of Aβ_1–42 or with 25OHChol or 27OHChol (1 μg/mL each) for 48 h. Using total RNA isolated from cells, IL-1β transcripts were detected by RT-PCR, and levels of IL-1β transcripts were assessed by qPCR. (D) The amounts of IL-1β released into culture media were quantitatively measured by ELISA. *** p < 0.001 vs. control; ** p < 0.01 vs. control. Data are expressed as the mean ± SD (n = 3 replicates for each group). (E) The cells were treated with the indicated oxysterols (1 μg/mL each) and LPS (100 ng/mL) in the absence or presence of polymyxin B (10 μg/mL) for 48 h. IL-1β transcripts were detected and assessed by RT-PCR and qPCR, respectively, and (F) the amounts of IL-1β secreted to culture media were measured by ELISA. *** p < 0.001 vs. control; ^## p < 0.01 vs. LPS. Data are representative of three independent experiments and are expressed as mean ± SD (n = 3 replicates for each group).

Figure 2

Concentration and time-course effects of 25OHChol and 27OHChol on IL-1β expression. Microglia were treated with the indicated concentrations of 25OHChol and 27OHChol for 48 h. Levels of IL-1β transcripts were assessed by qPCR (A), and concentrations of IL-1β protein secreted to the medium were quantitatively detected by ELISA (B). *** p < 0.001 vs. control; ** p < 0.01 vs. control; and * p < 0.05 vs. control. Data are expressed as the mean ± SD (n = 3 replicates for each group). Following treatment of microglia with 25OHChol and 27OHChol (1 μg/mL each) for the indicated periods, levels of IL-1β transcripts and secreted IL-1β protein were measured by qPCR (C) and by ELISA (D), respectively. *** p < 0.001 vs. control; ** p < 0.01 vs. control; and * p < 0.05 vs. control. Data are expressed as the mean ± SD (n = 3 replicates for each group).

Figure 3

Effects of protein kinase inhibitors on IL-1β expression induced by the oxysterols. (A) Cell lysates were obtained after treatment of microglia with 25OHChol or 27OHChol (1 μg/mL) for the indicated periods. Following the determination of protein concentrations, an equal amount of protein was analyzed by Western blotting using Abs against phosphorylated and unphosphorylated forms of Akt, ERk1/2, and Src. Results are representative of three independent experiments. Microglia were treated with 25OHChol and 27OHChol for 48 h in the absence or presence of the indicated kinase inhibitors (10 μM each). Levels of IL-1β transcripts were assessed by qPCR (B), and the amounts of IL-1β protein released from cells were measured by ELISA (C). *** p < 0.001 vs. control; ^### p < 0.001 vs. 25OHChol or 27OHChol; and ^## p < 0.01 vs. 25OHChol. Data are expressed as the mean ± SD (n = 3 replicates for each group).

Figure 4

Enhanced MHC II expression on the microglial surface following treatment with side-chain oxysterols. HMC3 microglial cells were seeded on coverslips and treated for 48 h with cholesterol, 24sOHChol, 25OHCho1, and 27OHChol (1 μg/mL each). The cells were immunostained with a fluorescence-conjugated antibody against MHC II (green). The nuclei were stained with DAPI (blue). The fluorescence signal representing MHC II was visualized using a confocal microscope at a magnification of 200×. The data presented are representative of three independent experiments.

Figure 5

Microglial activation in the brain of ApoE-deficient mice fed HFD (n = 10). The brain sections prepared from wild-type (WT) and ApoE-deficient (ApoE-KO) mice were immunostained with fluorescence-conjugated antibodies. After staining the nuclei with DAPI (blue), the fluorescence was visualized by confocal microscopy. (A) The sections were labeled with antibodies against anti-Iba-1 (red) and anti-IL-1β (green). Co-localized regions are indicated by white arrows. (B) The sections were labeled with anti-Iba-1 (red) and anti-MHC II (green) antibodies. Co-localized images are indicated by white arrows. The images presented are representative of three samples, and the results are representative of three independent experiments. Scale bars represent 100 μm.

Figure 6

Impaired microglial expression of IL-1β by treatment with CsA and CPZ. Microglial cells (1 × 10⁶ cell/dish) were stimulated for 48 h with 1 μg/mL of the indicated oxysterol in the presence of CPZ (2 μM), CsA (50 nM), or Df (25 μg/mL). (A) Total RNA was isolated from the cells, and levels of IL-1β transcripts were measured by qPCR. (B) The amounts of secreted IL-1β in culture media were quantitatively detected by ELISA. *** p < 0.01 vs. control; ^### p < 0.01 vs. oxysterols. After treatment of the cells for 48 h with 1 μg/mL of 25OHChol or 27OHChol in the presence of the indicated concentrations of CsA (C) and CPZ (D), IL-1β transcript levels were assessed by qPCR to calculate the IC₅₀ values. The results are representative of three independent experiments.