Pharmacological characterization of LPS and opioid interactions at the toll-like receptor 4

Abstract

Background and purpose: Previous work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells.

Experimental approach: NF-κB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-κB activation, a pathway activated by TLR4 stimulation.

Key results: LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides). A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-κB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective μ opioid receptor antagonist, β-FNA. Indeed, both naloxone and β-FNA also inhibited NF-κB activation in reporter cells. Further examination of fentanyl and β-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion.

Conclusions and implications: These results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation.

Figure 1

Stimulation of TLR4 signalling by LPS and inhibition by LPS-RS. (A) LPS concentration–response curve of stimulation of TLR4 activity. HEK-Blue4 cells were treated as described in Methods with LPS alone (from 10⁻¹² to 10⁻⁶ g·mL⁻¹) or co-treated with the LPS antagonist (RS) at increasing concentrations (in legend as ng·mL⁻¹). EC₅₀ and E_max values are given in Table 1. Data points are given as mean + SEM; error bars not visible are within the symbol. Experiments were carried out in triplicate with n = 9 for each treatment group. (B) Schild plot of the LPS-RS antagonism for LPS stimulation of TLR4 signalling shown in panel A. The regression line was not different than 1.0, suggesting a single site competitive antagonism and the apparent affinity (K_D) of LPS-RS at the competitive site is found at the concentration where the line crosses the abscissa (−8.867 log or 1.36 ng·mL⁻¹).

Figure 2

(A) Effect of morphine on TLR4 activity. Left panel: Treatment groups were unstimulated controls (US) and morphine concentrations from 1–100 μM. Asterisks denote significantly different than US control. Middle panel: Cells were co-treated with LPS (100 ng·mL⁻¹) and morphine concentrations from 1–100 μM. Asterisks denote significantly different than LPS only-stimulated cells. Right panel: Concurrent treatment consisted of LPS (100 ng·mL⁻¹), naltrexone (100 μM) and morphine concentrations from 1–100 μM. Asterisks denote significantly different than LPS only-stimulated cells. Data points are given as mean + SEM; error bars not visible are within the symbol. Experiments were carried out in triplicate with n = 9 for each treatment group. Hashed bars show effects of control treatments for each panel. (B) Effect of fentanyl on TLR4 activity. Left panel: Treatment groups were unstimulated controls (US) and fentanyl concentrations from 0.1–100 μM. Asterisks denote significantly different than US control. Middle panel: Cells were co-treated with LPS (30 ng·mL⁻¹) and fentanyl concentrations from 0.1–100 μM. Asterisks denote significantly different than LPS only-stimulated cells. Right panel: Concurrent treatment consisted of LPS (30 ng·mL⁻¹), β-FNA (30 μM) and fentanyl concentrations from 0.1–100 μM. Plus signs denote significantly different than corresponding LPS and fentanyl-treated cells in middle panel. Data points are given as mean + SEM; error bars not visible are within the symbol. Experiments were carried out in triplicate with n = 9 for each treatment group. Hashed bars show effects of control treatments for each panel.

Figure 3

(A) Effect of naltrexone on TLR4 activity. Left panel: Treatment groups were unstimulated controls (US) and naltrexone concentrations from 3–1000 μM. Right panel: Cells were co-treated with LPS (100 ng·mL⁻¹) and naltrexone concentrations from 3–1000 μM. Asterisks denote significantly different than LPS only-stimulated cells. Data points are given as mean + SEM; error bars not visible are within the symbol. Experiments were carried out in triplicate with n = 9 for each treatment group. (B) Effect of β-FNA on TLR4 activity. Left panel: Treatment groups were unstimulated controls (US) and β-FNA concentrations from 3–30 μM. Right panel: Cells were co-treated with LPS (30 ng·mL⁻¹) and β-FNA concentrations from 3 to 30 μM. Asterisks denote significantly different than LPS only-stimulated cells. Data points are given as mean + SEM; error bars not visible are within the symbol. Experiments were carried out in triplicate with n = 9 for each treatment group. Hashed bars show effects of control treatments for each panel.

Figure 4

(A) Effect of fentanyl on the LPS concentration–response curve of TLR4 activity. LPS was given alone (from 10⁻¹² to 10⁻⁶ g·mL⁻¹), with the LPS antagonist (RS; 10 ng·mL⁻¹), with fentanyl (FENT; 3.0 μM), or with fentanyl plus the LPS antagonist and LPS concentrations ranging from 10⁻¹² to 10⁻⁶ g·mL⁻¹. (B) Effect of β-FNA on the LPS concentration–response curve of TLR4 activity. LPS was given alone (concentrations ranging from 10⁻¹¹ to 10⁻⁶ g·mL⁻¹), with the LPS antagonist (RS; 10 ng·mL⁻¹), with β-FNA (30 μM) or with β-FNA plus the LPS antagonist and LPS (concentrations ranging from 10⁻¹¹ to 10⁻⁶ g·mL⁻¹). For both panels, EC₅₀ and E_max values are given in Table 2. Data points are given as mean ± SEM; error bars not visible are within the symbol. Experiments were carried out in triplicate with n = 9 or greater for each treatment group. Hashed bars show effects of control treatments for each panel.