Evidence that opioids may have toll-like receptor 4 and MD-2 effects

Abstract

Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Figure 1. (+)-Naloxone, like (−)-Naloxone and the classic TLR competitive inhibitor LPS-RS, inhibits TLR4 Akt1 signaling

LPS (200 ng/ml) plus vehicle (■) causes significant membrane localization of GFP-Akt1 quantified by cytosolic clearance of GFP-Akt1 in a stably expressing RAW264.7 cell line. Pretreatment with the competitive TLR4 antagonist LPS-RS (200 ng/ml, ●, panel A) or the novel TLR4 inhibitors (+)-naloxone (200 μM, ▼, panel B) and (−)-naloxone (200 μM, ▲, panel C) all significantly attenuate subsequent LPS-induced GFP-Akt1 membrane localization. To ensure the TLR4-selectivity of the inhibition the Akt1 blockade, cells were then stimulated with C5a (25 ng/ml), which utilizes a non-TLR4 pathway to activate Akt1. Notably, C5a triggers significant GFP-Akt1 membrane localization. Given how early Akt1 is activated in the TLR4 cascade, this reveals a non-classical opioid effect at or very close to TLR4 itself. n = 10 cells/group from a minimum of 4 separate plates.

Figure 2. Morphine non-stereoselectively activates (+)- and (−)-naloxone sensitive TLR4 Akt1 signaling

(−)-Morphine (200 μM; A,C,E) and (+)-morphine (200 μM; B,D,F) plus vehicle (■) causes significant membrane localization of GFP-Akt1 quantified by cytosolic clearance of GFP-Akt1 in a stably expressing RAW264.7 cell line. Pretreatment with characterized TLR4 antagonist LPS-RS (200 ng/ml, ●, A,B) or the novel TLR4 inhibitors (+)-naloxone (200 μM, ▼, C,D) and (−)-naloxone (200 μM, ▲, E,F) all significantly attenuate subsequent non-stereoselective morphine-induced GFP-Akt1 membrane localization. To ensure the TLR4-selectivity of the inhibition the blockaded cells were then stimulated with C5a (25 ng/ml), which triggers significant GFP-Akt1 membrane localization. n = 10 cells/group from a minimum of 4 separate plates.

Figure 3. TLR4 blockade potentiates morphine analgesia

Intrathecal (i.t.) co-administration of morphine (15 μg) with LPS antagonist (A; 20 μg; msbB E. coli mutant), LPS-RS (B; 40 μg), a TIRAP inhibitory peptide (C; 50 μM; TIRAP is a secondary signaling molecule required to for a TLR4 signaling complex), (+)-naloxone (D; 20 μg), or (+)-naltrexone (E; 20 μg) all lead to significant potentiation of morphine tailflick analgesia (morphine + vehicle black; morphine in combination with TLR4 signaling attenuators in grey). Coadministration with (+)-nalmefene (F: 20 μg) failed to potentiate morphine analgesia. Systemic morphine (4 mg/kg) is also potentiated by coadministration of systemic (G; 8 mg/kg) or intrathecal (H; 20 μg) (+)-naloxone. n = 6/group * = P < 0.05, ** = P < 0.01, *** = P < 0.001; n = 6/group

Figure 4. Morphine is a more effective analgesic in TLR4 knockout mice compared to wildtype controls

A: A morphine dose response in TLR4 knockout is shifted significantly to the left (EC₅₀ 4.0 mg/kg) compared to the wildtype mice (EC₅₀ 11.9 mg/kg) assessed by hotplate latency. B: Pharmacological blockade of TLR4 with (+)-naloxone (60 mg/kg i.p.) or microglial activation attenuation with minocycline (50 mg/k i.p) only potentiates morphine analgesia in wildtype but not TLR4 knockout animals. n = 6/group

Figure 5. Non-stereoselective TLR4 agonism by opioid agonists and antagonism by (+)-naloxone

A: Opioid agonists (10 μM) non-stereoselectively produced significant TLR4 signaling in the HEK293-hTLR4 cell line, except for morphine-6-glucuronide (M6G), which failed to produce any significant TLR4 response. Notably, TLR4 agonism was observed for representative members of every clinically relevant class of opioids (4,5-epoxymorphinans: morphine, oxycodone, buprenorphine; 3,3,-diphenylpropylamine: methadone; 4-phenylpiperidine: pethadine/meperadine; 4-amilinopiperidine: fentanyl). Other glial attenuators (minocycline, propentofylline and AV411) and non-opioid typical/atypical analgesics (acetaminophen, gabapentin, dextromethorphan and clonidine) had no TLR4 agonist activity. B: (−)-Morphine and morphine-3-glucuronide (M3G) TLR4 signaling is dose dependently inhibited by (+)-naloxone. * = P < 0.05, n = 3 experiments/group.

Figure 6. in silico MD-2 docking to in vitro TLR4 signaling prediction model

A: 10 opioid ligands were used to build the in silico to in vitro prediction model which was subsequently tested on 7 opioid and 3 non-opioid ligands, with the predicted and actual in vitro scores displayed. B: The modified complete 20 ligands in silico to in vitro model retested on the structurally diverse TLR4 signaling inhibitor AV411, with the predicted and actual in vitro scores displayed.