Morphine and other opioids can provoke neuroinflammation by activating the innate immune system’s Toll-like receptor 4 (TLR4), but just how the compounds turn on TLR4 signaling has been unclear. A new paper suggests that opioids mimic microbial immunogens by binding the TLR4 accessory protein myeloid differentiation protein 2 (MD-2). The work offers a potential target for quelling opioid-induced inflammation, which has been linked to drug-induced pain, tolerance, and dependence. The study, from Hang (Hubert) Yin and Linda Watkins at the University of Colorado at Boulder, US, and Mark Hutchinson at the University of Adelaide, Australia, appeared online April 2 in the Proceedings of the National Academy of Sciences USA.
Toll-like receptors represent a first line of defense against microbial invaders: In the case of TLR4, binding of the bacterial cell wall component lipopolysaccharide (LPS, also known as endotoxin) to MD-2 causes TLR4 oligomerization, proinflammatory signaling, and cytokine release. TLRs respond to endogenous products of cell injury as well, and microglial TLR4 activation can contribute to chronic inflammatory and neuropathic pain in animal models (reviewed in Nicotra et al., 2011 and Liu et al., 2012).
In the setting of chronic pain, activation of TLR4 by morphine and other opioids may “put oil on the fire,” Hutchinson said, inciting neuroinflammation that undercuts the drugs’ analgesic effects. That has led to an interest in inhibitors of TLR4 as potential opioid adjuvants.
Based on computational modeling and other experiments, Watkins, Hutchinson, and colleagues previously proposed that the LPS-binding pocket on MD-2 could be a landing spot for opioids (Hutchinson et al., 2010). In the new paper, first author Xiaohui Wang and coworkers demonstrate that morphine indeed bound purified MD-2 in a concentration-dependent manner and competed with LPS for binding. In human cells overexpressing TLR4 and MD-2, co-immunoprecipitation showed that the morphine-MD-2 interaction triggered TLR4 oligomerization. And molecular dynamics simulations indicated that morphine binding has the potential to induce conformational changes in TLR4-MD-2 similar to those caused by LPS. Experiments in cultured microglia, primary CNS endothelial cells, and TLR4-deficient mice all supported the idea that morphine induces proinflammatory signaling exclusively via TLR4 and MD-2.
The Yin lab has developed novel drug-like small molecules that target either TLR4 or MD-2 and block receptor activation (Joce et al., 2010; Bevan et al., 2010). In the new study, they found that the compounds inhibited morphine-induced cytokine expression in cultured microglia. In vivo, both compounds enhanced the inhibitory effect of morphine on nociceptive reflex reactions to acute heat stimuli in healthy rats and mice.
Christoph Stein at the Freie Universität, Berlin, who was not involved in the work, said that the evidence that morphine binds and activates TLR4-MD-2 is convincing. “That is an interesting finding,” he said. However, “From a drug development perspective, I would definitely want to see more clinically relevant [animal] models. If they had included studies on an arthritis model, or a neuropathic pain model…I would have been much more excited,” he said. “At this point, it is very preliminary.”
Yin’s inhibitors may help researchers understand the complicated roles of TLR4 in the nervous system. Most work to date has focused on TLR4 in microglia, but the receptors have been found on neurons, too, where they contribute to pain sensation (Wadachi and Hargreaves, 2006; reviewed in Liu et al., 2012). Fletcher White, Indiana University, Indianapolis, US, told PRF that studies from his laboratory suggest that opioid-induced hyperalgesia, too, depends on TLR4 outside of microglia. White studies the mechanisms of opioid-induced hyperalgesia in primary afferents; he has used the Yin lab’s TLR4 inhibitors in his experiments, but was not involved in the current study.
Finally, the findings offer a reminder that opioids do a lot more than bind opioid receptors. “We can no longer say that, if we put an opioid into an in-vivo or in-vitro system, we are sure that the response is purely that of opioid receptor engagement,” Hutchinson told PRF. “We can’t just talk about opioids being opioids. We need to acknowledge their immunology.”
Image: Morphine (red) bound to MD-2 (yellow) as predicted by molecular simulation, compared to the structure of MD-2 alone (cyan). Image credit: Armando J. de Jesus, Yin lab.