The present series of studies utilized a combination of in vitro, in vivo, and in silico techniques to explore whether opioids may potentially influence TLR4 signaling. Evidence was found suggestive that TLR4 signaling can occur in response to clinically-employed opioid agonists, their non-opioid (+)-isomers, and the opioid-inactive metabolite morphine-3-glucuronide, but not other classes of typical/atypical analgesics or glial attenuators. Also, the opioid-inactive (+)-naloxone and opioid-active (−)-naloxone block activation of TLR4 signaling by opioids and LPS. Due to the LPS-RS sensitivity of opioid-induced TLR4 responses, and in silico to in vitro correlations, it appears that these xenobiotics (chemicals not endogenous to the organism) can potentially have TLR4 action possibly via interaction with MD-2. The in vivo consequence of xenobiotic induced-MD-2/TLR4 signaling appears to be opposition of acute and chronic opioid analgesia, and contribution to development of hyperalgesia and dependence. Each of these opioid-induced effects has previously been hypothesized to be mediated, in part, via proinflammatory responses induced by unknown mechanisms. Whilst glia appear a likely cellular candidate as the cellular source of opioid-induced proinflammation, the contributory and/or permissive role of other cell types and cofactors within the CNS remains to be elucidated. Together, these data suggest a departure from classical views of opioid pharmacology, and also may provide a novel explanation for several key phenomena, such as effects of M3G.
Prior publications support our conclusion of opioid-induced MD-2/TLR4 signaling and non-stereoselective MD-2/TLR4 signaling inhibition. Firstly, (+)-opioid antagonists block suppression of morphine analgesia by LPS-induced proinflammatory glial activation (Wu et al., 2006
). Secondly, naloxone nonstereoselectively prevents LPS-induced microglial activation (Liu et al., 2000
). Thirdly, naloxone non-stereoselectively decreases microglial LPS binding (Liu et al., 2000
). Last, TLR2 and MyD88-dependent apoptosis in HEK293 cells is induced by morphine (Li et al., 2009
). Given TLR4/MD-2 is the endogenous receptor complex for LPS, this suggests a role of TLR4/MD-2 in the non-stereoselective action of both opioid agonists and antagonists. The activity of morphine in TLR2-expressing cells demonstrates that small molecules can have TLR activity, with the possibility that MD-2 may facilitate this for both TLR4 and TLR2, owing to the role of MD-2 in also enabling TLR2 signaling (Dziarski et al., 2001
). An alternative hypothesis of (+)-isomer inhibition of a disparate enzyme has been postulated (Liu et al., 2000
), but this line of evidence cannot explain the proinflammatory glial activation caused by opioid agonists, as it is unlikely opioid agonists cause activation of the enzyme postulated by Liu et al. (2000)
Acute blockade of the TLR4 receptor, genetic knockout of TLR4, or blockade of TLR4 downstream signaling, was observed to potentiate the magnitude and duration of (−)morphine analgesia. These data suggest a rapid (within minutes) TLR4-dependent opposition of analgesia. A spinal site of action exists since systemic morphine analgesia is potentiated by intrathecal (+)-naloxone. A role for TLR4 in pain enhancement following chronic opioids is suggested since (+)-naloxone blunts development of analgesic tolerance and blocks opioid-induced hyperalgesia. These findings extend previous reports that opioid-induced proinflammatory cytokines and chemokines oppose acute and chronic opioid analgesia (Hutchinson et al., 2007
; Hutchinson et al., 2008
; Hutchinson et al., 2008
; Johnston et al., 2004
; Shavit et al., 2005
; Watkins et al., 2005
). Moreover, evidence was obtained that opioid-TLR4-induced signaling may contribute to opioid dependence, in agreement with previous findings that attenuation of opioid-induced glial activation blunts opioid dependence and withdrawal-induced allodynia (Hutchinson et al., 2009
; Hutchinson et al., 2008
; Johnston et al., 2004
). Previous studies of opioids have used the spontaneous TLR4 mutant, LPS-nonresponsive mouse strain C3H/HeJ (Liang et al., 2006
; Rady and Fujimoto, 2001
). Differences in opioid action in this strain have been observed, although conclusions are clouded by design issues and lack of control strains, unlike in the present study where knockouts and wildtypes were Balb/c. C3H.HeJ mice are insensitive to dynorphin induced anti-analgesia (Rady and Fujimoto, 2001
) and develop less morphine tolerance (Liang et al., 2006
). Our direct examination of morphine dose responses, and lack of effect of (+)-naloxone, in TLR4 knockout versus wildtype animals are supportive of a potential importance of morphine-induced TLR4 signaling in morphine’s actions.
The structure activity relationship of xenobiotic MD-2/TLR4 signaling activation differs from that of opioid receptors. Firstly, the similarities are that opioid agonists are MD-2/TLR4 signaling activators, with the exception of M6G; and opioid antagonists are MD-2/TLR4 signaling inhibitors. However, the similarities end here, as the MD-2/TLR4 activity of these xenobiotics is non-stereoselective. Both opioid-active (−)-isomer agonists and opioid-inactive (+)-isomer agonists display MD-2/TLR4 activity. For MD-2/TLR4 activity, there is a significant departure from reliance on the 3′OH of the 4,5-epoxymorphinan, which is required for opioid receptor activity (Chen et al., 1991
). This is typified by the MD-2/TLR4 signaling activation of M3G and lack of activity of M6G. Interestingly, the 6′ position of the 4,5-epoxymorphinan is important as (+)-nalmefene loses all activity whilst (+)-naltrexone is TLR4 active. In silico
docking suggests that the valine 48 residue of MD-2 is important in this disparity, although the significance of this is unknown. The disparity in the opioid receptor agonist and antagonist structure activity relationship versus that of MD-2/TLR4 is a fortuitous and pharmacologically exploitable one. Importantly, not all xenobiotics display MD-2/TLR4 activity, with a range of other glial attenuators, typical and atypical analgesics being devoid of any such TLR4 responses. Therefore, TLR4 activity is a property of select xenobiotics. Given this, in silico
to in vitro
prediction tools would be useful. Here, such models were implemented with sizable success of predicting in vitro
activity of opioids and non-opioids based on MD-2 in silico
docking data. Further development and refinement of this model is underway.
Opioid induced Akt1 phosphorylation has been demonstrated previously using both small molecule (Gupta et al., 2002
) and peptide (Polakiewicz et al., 1998
) opioid agonists. This action was thought to be mediated exclusively via classical μ opioid receptors. However, the previous non-classical opioid literature, plus the data newly presented here, are consistent with a role of TLR4 in mediating such responses, as well. Moreover, MD-2/TLR4 activity is not limited to neuronally active opioids since the opioid receptor-inactive (+)-isomers also possess the same properties as their opioid active (−)-isomer counterparts. Rather a range of small molecule xenobiotics more broadly may also act at MD-2/TLR4 in either activator or inhibitor fashions (Hutchinson et al., 2007
Tentative speculations about the mechanism of action at MD-2/TLR4 of small molecules are possible, owing to the diverse techniques employed. Firstly, it appears that the TLR4 signaling activation of (−)-morphine may involve recruitment of MyD88-dependent and MyD88-independent signaling cascades since (−)-morphine induced cytosolic Akt1 clearance and the TIRAP inhibitor potentiated (−)-morphine analgesia. TIRAP is a pivotal adapter protein that interacts with the TIR domain of TLR4 (Schilling et al., 2002
) that enables recruitment of MyD88 and the activation of the Toll-Interleukin-1 signaling cascade. In contrast, Akt1 is recruited by TLR4 activation via the TRIF/TRAM complex activation of PI3K (Okun et al., 2008
). Since the common component to these two signaling cascades is TLR4, morphine may plausibly be exerting an action at or near TLR4 or at parallel multi-site actions within multiple downstream signaling cascades, which would appear a far less likely possibility than a direct action at or near TLR4. The blockade of morphine’s actions by LPS-RS is in agreement with this possibility as it is a selective TLR4 competitive antagonist. Secondly, a similar conclusion can be made for the TLR4 signaling blockade by (+)-naloxone. (+)-Naloxone blocks (−)- and (+)-morphine, and LPS-induced Akt1 cytosolic clearance, TLR4-induced SEAP expression, and TLR4-mediated actions in vivo
. Given this, it appears plausible that (+)-naloxone may inhibit above Akt1 in the TLR4 signaling cascade as discussed previously and blocks MyD88-dependent TLR4 signaling as complete inhibition of LPS and (−)-morphine-induced hTLR4 SEAP expression was observed. The in silico
data pointing toward MD-2 are consistent with these hypotheses.
In conclusion, these in vitro, in vivo, and in silico data suggest a potential for non-stereoselective TLR4 agonist and antagonist activity of opioids. The consequence of such MD-2/TLR4 signaling includes opposition of acute opioid analgesia, and development of opioid tolerance, hyperalgesia and dependence, all previously associated with opioid-induced proinflammatory glial activation. If substantiated by future investigations, such non-classical opioid action of MD-2/TLR4 signaling and identification of novel inhibitors of this xenobiotic-induced signaling may suggest new pharmacological avenues to separate the beneficial neuronal analgesic actions of opioids, from the detrimental MD-2/TLR4-mediated glial unwanted side effects.