Effect of Propofol on the Production of Inflammatory Cytokines by Human Polarized Macrophages

Abstract

Macrophages are key immune system cells involved in inflammatory processes. Classically activated (M1) macrophages are characterized by strong antimicrobicidal properties, whereas alternatively activated (M2) macrophages are involved in wound healing. Severe inflammation can induce postoperative complications during the perioperative period. Invasive surgical procedures induce polarization to M1 macrophages and associated complications. As perioperative management, it is an important strategy to regulate polarization and functions of macrophages during inflammatory processes. Although propofol has been found to exhibit anti-inflammatory activities in monocytes and macrophages, it is unclear whether propofol regulates the functions of M1 and M2 macrophages during inflammatory processes. This study therefore investigated the effects of propofol on human macrophage polarization. During M1 polarization, propofol suppressed the production of IL-6 and IL-1β but did not affect TNF-α production. In contrast, propofol did not affect the gene expression of M2 markers, such as IL-10, TGF-β, and CD206, during M2 polarization. Propofol was similar to the GABAA agonist muscimol in inducing nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and inhibiting IL-6 and IL-1β, but not TNF-α, production. Knockdown of Nrf2 using siRNA significantly reduced the effect of propofol on IL-6 and IL-1β production. These results suggest that propofol prevents inflammatory responses during polarization of human M1 macrophages by suppressing the expression of IL-6 and IL-1β through the GABAA receptor and the Nrf2-mediated signal transduction pathway.

Figures

Figure 1

Propofol had no effect on CD86 mRNA and cell surface expression in M1 macrophages. M0 macrophages were polarized to M1 macrophages in the presence of 0.05% DMSO (solvent control) or propofol (1–5 μM). (a) qRT-PCR assays of CD86 mRNA levels. Data were normalized relative to β-actin mRNA (internal control) and presented as mean ± SD (n = 3 per group). (b) Flow cytometric analysis of CD86 surface expression on M1 macrophages treated with propofol (thick line) or DMSO (thin line) and on M1 macrophages incubated with isotype-matched control IgG and propofol (gray-filled line) or DMSO (dashed line).

Figure 2

Propofol reduced IL-6 and IL-1β gene expression and protein production in M1 macrophages. M0 macrophages were polarized to M1 macrophages in the presence of 0.05% DMSO (solvent control) or propofol (1–5 μM). (a–c) qRT-PCR assays of IL-6, IL-1β, and TNF-α mRNA levels in M1 macrophages. Data were normalized relative to β-actin mRNA (internal control) and presented as mean ± SD (n = 3 per group). (d–f) ELISA measurements of IL-6 (d), IL-1β (e), and TNF-α (f) secreted by M1 macrophages. Data are presented as mean ± SD (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared with control cells by one-way ANOVA with Bonferroni's post hoc test.

Figure 3

Muscimol reduced IL-6 and IL-1β gene expression and protein production in M1 macrophages. M0 macrophages were polarized to M1 macrophages in the absence or presence of muscimol (100 μM). (a–d) qRT-PCR assays of IL-6, IL-1β, TNF-α, and CD86 mRNA. Data were normalized relative to β-actin mRNA (internal control) and presented as mean ± SD (n = 4 per group). (e–g) ELISA measurements of IL-6 (e), IL-1β (f), and TNF-α (g) secreted by M1 macrophages. Data are presented as mean ± SD (n = 4 per group). ∗P < 0.05 compared with control cells by Wilcoxon-Mann-Whitney test.

Figure 4

Propofol had no effect on IL-10, TGF-β1, and CD206 gene expression and protein production in M2 macrophages. M0 macrophages were polarized to M2 macrophages in the presence of 0.05% DMSO (solvent control) or propofol (1–5 μM). (a–c) qRT-PCR assays of IL-10, TGF-β1, and CD206 mRNA. Data were normalized relative to β-actin mRNA (internal control) and presented as mean ± SD (n = 3 per group). ELISA measurements of IL-10 (d) and TGF-β1 (e), secreted by M2 macrophages. Data are presented as mean ± SD (n = 3 per group). Statistical comparisons were analyzed using one-way ANOVA with Bonferroni's post hoc test.

Figure 5

Propofol and muscimol enhanced nuclear translocation of Nrf2 in M1 THP-1 cells. (a) Immunoblotting analysis of the effects of propofol (50 μM) on nuclear translocation of Nrf2. (b) Densitometric analysis of bands in (a). (c) Immunoblotting analysis of the effects of muscimol (100 μM) on nuclear translocation of Nrf2. (d) Densitometric analysis of bands in (c). Data were normalized relative to lamin C (internal control for nuclear proteins) and presented as mean ± SD of four independent experiments. ∗P < 0.05 compared with control cells by one-way ANOVA with Bonferroni's post hoc test or by the Wilcoxon-Mann-Whitney test.

Figure 6

siRNA knockdown of Nrf2 significantly reduced the anti-inflammatory effects of propofol in M1 THP-1 cells. (a) Immunoblotting analysis of Nrf2 expression. THP-1 cells were transfected with control nontarget or Nrf2 siRNA and treated with PMA for 72 hr. (b) Densitometric analysis of bands in (a). Data were normalized relative to β-actin (internal control) and presented as mean ± SD of four independent experiments. ∗P < 0.05 compared with control cells by the Wilcoxon-Mann-Whitney test. (c–e) Effects of Nrf2 siRNA on IL-6, IL-1β, and TNF-α production by M1 THP-1 cells in the absence or presence of propofol. PMA-differentiated siRNA-transfected cells were further polarized into M1 macrophages in the presence of 0.05% DMSO (solvent control) or propofol (50 μM). IL-6, IL-1β, and TNF-α concentrations in supernatants were measured by ELISA. ∗P < 0.05 compared with cells transfected with nontarget siRNA by one-way ANOVA with Bonferroni's post hoc test. NS = not significant.