Fentanyl enhances immune cell response through TLR4/MD-2 complex

Abstract

Introduction: Opioids have been shown to induce neuroinflammation and immune cell activation, that might contribute to some of the opioid side effects, such as opioid-induced tolerance and paradoxical hyperalgesia. In this context, TLR4/MD-2 complex has been proposed as an off-target site for opioid action. This study was aimed at investigating the effect of fentanyl on lipopolysaccharide (LPS)-induced TLR4/MD-2 activation in rat primary microglia and human monocyte-derived macrophages (MDM).

Materials and methods: The effect of fentanyl was first explored by measuring the expression and release of different proinflammatory mediators in primary rat microglia and human MDM by real-time PCR and ELISA. Then, the involvement of TLR4/MD-2 signaling was investigated studying NF-κB activation in HEK293 cells stably transfected with human TLR4, MD-2, and CD14 genes (HEK-Blue hTLR4 cells) and in human MDM.

Results: Fentanyl increased mRNA levels, as well as the LPS-induced secretion of proinflammatory mediators in primary microglia and MDM. Two inhibitors of TLR4/MD-2 signaling, namely the oxazoline derivative of N-palmitoylethanolamine (PEA-OXA) and CLI-095, blocked the production and release of proinflammatory cytokines by microglia stimulated with LPS and fentanyl, suggesting that TLR4/MD-2 could be the target of the proinflammatory activity of fentanyl. Finally, we showed that fentanyl in combination with LPS activated NF-κB signaling in human MDM and in HEK-Blue hTLR4 cells and this effect was blocked by inhibitors of TLR4/MD-2 complex.

Discussion: These results provide new insight into the mechanism of the proinflammatory activity of fentanyl, which involves the activation of TLR4/MD-2 signaling. Our findings might facilitate the development of novel inhibitors of TLR4/MD-2 signaling to combine with opioid-based analgesics for effective and safe pain management.

Keywords: TLR4/MD-2 complex; fentanyl; inflammatory cytokines; macrophages; microglia.

FIGURE 1

Effect of fentanyl on proinflammatory gene expression in primary cortical microglia. Microglia were cultured in 10% serum-containing medium, which was replaced with serum-free medium before treatment with 10 μM fentanyl (F) for (A-D) 6 h or (E-H) 24 h in the absence (white bars) or presence (gray bars) of 10 ng/mL LPS. Gene expression was quantified by real-time PCR. Data are presented as means ± SEM (n = 3) and analyzed by one-way ANOVA followed by Holm-Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control cells (CTR); °p < 0.05, °°°p < 0.01, °°°p < 0.001 vs. LPS stimulation.

FIGURE 2

Effect of fentanyl on proinflammatory mediator release from primary cortical microglia. Microglia were cultured in 10% serum-containing medium, which was replaced with serum-free medium before treatment with 10 μM fentanyl (F) for 24 h in the absence (white bars) or presence (gray bars) of 10 ng/mL LPS. Supernatants were collected and analyzed for (A) IL-1β, (B) TNF-α, and (C) NO release. Results are shown as means ± SEM (n = 3) and analyzed by one-way ANOVA followed by Holm-Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control cells (CTR); °p < 0.01 vs. LPS stimulation.

FIGURE 3

Effect of naloxone on proinflammatory cytokine release from primary cortical microglia. Microglia were cultured in 10% serum-containing medium, which was replaced with serum-free medium before treatments for 24 h. Supernatants were collected and analyzed for (A) IL-1β and (B) TNF-α release. Left panels: cells were treated with naloxone alone (0.001–10 μM). Middle panels: cells were pre-treated for 30 min with naloxone (0.001–10 μM) and the stimulated with 10 ng/mL LPS. Right panels: cells were pre-treated for 30 min with naloxone (0.001–10 μM) and then stimulated with 10 ng/mL LPS and fentanyl (F, 10 μM). White bars show the effect of control treatment for each panel. Results are shown as means ± SEM (n = 3) and analyzed by one-way ANOVA.

FIGURE 4

Effect of TLR4/MD-2 inhibition on the proinflammatory activity of fentanyl. Microglia were cultured in 10% serum-containing medium, which was replaced with serum-free medium before treatment with fentanyl (F, 10 μM) and LPS (10 ng/mL), in the presence of (A, B) PEA-OXA (30 μM) or (C, D) CLI-095 (0.5 μg/mL) for 24 h (A, C) IL-1β mRNA levels were quantified by real-time PCR. (B, D) Supernatant were collected and analyzed for IL-1β content. Data are means ± SEM (n = 3). ***p ˂ 0.001 vs. control cells (CTR); °°p < 0.01 and °°°p < 0.001 vs. LPS; ^###p < 0.001 vs. LPS + fentanyl. One-way ANOVA followed by Holm-Sidak’s test.

FIGURE 5

Effect of fentanyl on TLR4/MD-2 activation. (A) HEK-Blue hTLR4 cells were incubated with fentanyl (0.1–100 μM) and the amount of SEAP released into the culture medium was quantified after 24 h (B) HEK-Blue hTLR4 cells were treated with LPS alone (from 10⁻¹¹ to 10⁻⁶ g/mL) or co-treated with fentanyl (F) at increasing concentrations (1–100 µM) and the amount of SEAP released into the culture medium was quantified after 24 h. EC₅₀ and E_max values are given in Table 4. (C) HEK-Blue hTLR4 cells were co-treated with 100 μM fentanyl and 5 ng/mL LPS, in the absence or presence of 30 μM PEA-OXA or 0.5 μg/mL CLI-095 for 24 h. Data are shown as OD₆₃₀ and are means ± SEM (n = 4). **p < 0.01 and ***p < 0.001 vs. control cells (CTR); °°p < 0.01 and °°°p < 0.001 vs. LPS; ^###p < 0.001 vs. LPS + fentanyl. One-way ANOVA followed by Holm-Sidak’s test.

FIGURE 6

Effect of fentanyl on LPS-induced MDM inflammatory response. Human MDM were cultured in 10% serum-containing medium, which was replaced with serum-free medium before treatment with 10 μM fentanyl (F) in the absence (white bars) or presence (gray bars) of 100 ng/mL LPS. (A, B) IL-1β and TNF-α mRNA levels were quantified by real-time PCR after 3-h stimulation. (C) TNF-α release was measured by ELISA in MDM supernatants collected after 24-h stimulation. (D–F) pIkB and IkB protein levels were analyzed by Western blot after 90-min stimulation. GAPDH was used as loading control. (G) The opioid receptor gene expression was quantified by real-time PCR and shown relative to NOP receptor mRNA levels set to 1. Data are presented as means ± SEM of 3–5 independent experiments and analyzed by one-way ANOVA followed by Sidak’s multiple comparison test. **p < 0.01 and ***p < 0.001 compared to control (CTR); °p < 0.05 vs. LPS stimulation. O.D., optical density in arbitrary units.