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Abstract: Opioids have been discovered to have Toll-like receptor (TLR) activity, beyond actions at classical opioid receptors. This raises the question whether other pharmacotherapies for pain control may also possess TLR activity, contributing to or opposing their clinical effects. We document that tricyclics can alter TLR4 and TLR2 signaling. In silico simulations revealed that several tricyclics docked to the same binding pocket on the TLR accessory protein, myeloid differentiation protein 2 (MD-2), as do opioids. Eight tricyclics were tested for effects on TLR4 signaling in HEK293 cells over-expressing human TLR4. Six exhibited mild (desipramine), moderate (mianserin, cyclobenzaprine, imiprimine, ketotifen) or strong (amitriptyline) TLR4 inhibition, and no TLR4 activation. In contrast, carbamazepine and oxcarbazepine exhibited mild and strong TLR4 activation, respectively, and no TLR4 inhibition. Amitriptyline but not carbamazepine also significantly inhibited TLR2 signaling in a comparable cell line. Live imaging of TLR4 activation in RAW264.7 cells and TLR4-dependent interleukin-1 release from BV-2 microglia revealed that amitriptyline blocked TLR4 signaling. Lastly, tricyclics with no (carbamazepine), moderate (cyclobenzeprine), and strong (amitriptyline) TLR4 inhibition were tested intrathecally (rats) and amitriptyline tested systemically in wildtype and knockout mice (TLR4 or MyD88). While tricyclics had no effect on basal pain responsivity, they potentiated morphine analgesia in rank-order with their potency as TLR4 inhibitors. This occurred in a TLR4/MyD88-dependent manner as no potentiation of morphine analgesia by amitriptyline occurred in these knockout mice. This suggests that TLR2 and TLR4 inhibition, possibly by interactions with MD2, contributes to effects of tricyclics in vivo. These studies provide converging lines of evidence that several tricyclics or their active metabolites may exert their biological actions, in part, via modulation of TLR4 and TLR2 signaling and suggest that inhibition of TLR4 and TLR2 signaling may potentially contribute to the efficacy of tricyclics in treating chronic pain and enhancing the analgesic efficacy of opioids. [Copyright &y& Elsevier]

Abstract: Spinal cord microglial toll-like receptor 4 (TLR4) has been implicated in enhancing neuropathic pain and opposing morphine analgesia. The present study was initiated to explore TLR4-mediated pain modulation by intrathecal lipopolysaccharide, a classic TLR4 agonist. However, our initial study revealed that intrathecal lipopolysaccharide failed to induce low-threshold mechanical allodynia in naive rats, suggestive that TLR4 agonism may be insufficient to enhance pain. These studies explore the possibility that a second signal is required; namely, heat shock protein-90 (HSP90). This candidate was chosen for study given its known importance as a regulator of TLR4 signaling. A combination of in vitro TLR4 cell signaling and in vivo behavioral studies of pain modulation suggest that TLR4-enhancement of neuropathic pain and TLR4-suppression of morphine analgesia each likely require HSP90 as a cofactor for the effects observed. In vitro studies revealed that dimethyl sulfoxide (DMSO) enhances HSP90 release, suggestive that this may be a means by which DMSO enhances TLR4 signaling. While 2 and 100 μg lipopolysaccharide intrathecally did not induce mechanical allodynia across the time course tested, co-administration of 1 μg lipopolysaccharide with a drug that enhances HSP90-mediated TLR4 signaling now induced robust allodynia. In support of this allodynia being mediated via a TLR4/HSP90 pathway, it was prevented or reversed by intrathecal co-administration of a HSP90 inhibitor, a TLR4 inhibitor, a microglia/monocyte activation inhibitor (as monocyte-derived cells are the predominant cell type expressing TLR4), and interleukin-1 receptor antagonist (as this proinflammatory cytokine is a downstream consequence of TLR4 activation). Together, these results suggest for the first time that TLR4 activation is necessary but not sufficient to induce spinally mediated pain enhancement. Rather, the data suggest that TLR4-dependent pain phenomena may require contributions by multiple components of the TLR4 receptor complex. [Copyright &y& Elsevier]