<p>Spinal TLR4/P2X7 Receptor-Dependent NLRP3 Inflammasome Activation Contributes to the Development of Tolerance to Morphine-Induced Antinociception</p>

Abstract

Background: Long-term use of morphine induces antinociceptive tolerance and limits its clinical efficacy. Neuroinflammation in the spinal cord is thought to play a pivotal role in the development of morphine tolerance. Toll-like receptor 4 (TLR4) and P2X7 receptor (P2X7R) are key modulators of neuroinflammation. Recent studies show that the Nod-like receptor protein 3 (NLRP3) inflammasome play a crucial role in microglia-mediated neuroinflammation. Thus far, the mechanism underlying NLRP3 inflammasome activation during morphineinduced tolerance is not yet fully understood. Therefore, we sought to investigate the mechanisms of NLRP3 inflammasome activation and its role in the development of morphine-induced tolerance. Methods: Repeated morphine treatment through intrathecal injection (15 μg once daily for 7 days) was given to establish antinociceptive tolerance in mice. Tail-flick latency was used to evaluate morphine-induced antinociception. NLRP3 knockout mice were used to assess the role of NLRP3 inflammasome in morphine tolerance. TLR4 knockout mice and A438079, a P2X7R antagonist, were used to assess the role of TLR4 and P2X7R in chronic morphine-induced NLRP3 inflammasome activation. Western blot and immunofluorescence were used for quantitative comparison. Results: Repeated morphine treatment increased the expression of NLRP3. Knockout of NLRP3 attenuated morphine-induced tolerance and suppressed morphine-induced activation of microglia. Knockout of TLR4 alleviated morphine tolerance and chronic morphineinduced upregulation of spinal NLRP3. Inhibition of spinal P2X7R with A438079 not only prevented the development of morphine-induced tolerance but also inhibited repeated morphine treatment-induced upregulation of spinal NLRP3. Furthermore, spinal NLRP3, TLR4 and P2X7R were collectively colocalized with the microglia marker Iba1. Conclusion: This study demonstrates that the NLRP3 inflammasome in microglia plays a crucial role in morphine tolerance and that both TLR4-and P2X7R-dependent pathways are required for NLRP3 inflammasome activation over the course of the development of morphine-induced tolerance. Our results provide a new perspective for the targeted treatment of morphine-induced tolerance.

Molecular mechanisms of opioid tolerance: From opioid receptors to inflammatory mediators (Review)

“…Inflammatory immune responses are considered a critical contributor to the regulation of the opioid receptors. Numerous studies have suggested that suppression of the TLR4/NLRP3-mediated inflammasome activation and the inflammatory response in microglia lead to an important attenuation of morphine tolerance ( 18 ). Taken together, these results could reveal novel therapeutic targets of anti-opioid tolerance, from opioid receptors to inflammatory factors.…”

“…Since IL-1β and IL-18 are both classical cytokines released during inflammasome activation, TLR4 and NLRP3 signalling may be critical for opioid tolerance and should be investigated more thoroughly ( 18 ). TLR4, a member of the toll-like receptor family, can recognise specific danger-associated molecular patterns and initiate an immune response ( 82 , 83 ).…”

“…Morphine treatment upregulates IL-1β production, and antagonisation of the IL-1 receptor reverses morphine tolerance ( 14 ). Thus, both the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, a major signalling pathway involved in IL-1β release, and TLR4 signalling are receiving increasing attention for the regulation of morphine tolerance ( 18 ).…”

The NLRP3 inflammasome: an emerging therapeutic target for chronic pain

“…These signs are two major adverse effects of prolonged morphine or fentanyl treatment, which severely reduced their clinical usages [ 105 ]. Prolonged morphine treatment can induce significant NLRP3 inflammasome activation in spinal cord of an animal model of morphine-induced analgesic tolerance and hyperalgesia [ 106 , 107 ]. Further evidence demonstrates morphine treatment can induce a persistent release of DMAPs (including HMGB1, biglycan, heat shock protein 90) in spinal cord [ 108 ].…”

Morphine-3-Glucuronide, Physiology and Behavior

“…Several studies have implicated TLR4 in dampening morphine antinociceptive effects or in some side effects, such as antinociceptive tolerance ( Hutchinson et al, 2010 ; Liu et al, 2011 ; Wang et al, 2012 , 2020 , 2021 ; Eidson and Murphy, 2013 ; Bai et al, 2014 ; Grace et al, 2014 ; Eidson et al, 2017 ; Thomas et al, 2022 ). For instance, a recent study revealed that antinociceptive tolerance was prevented in TLR2 and TLR4 null mutants, but not in MyD88 –/– animals ( Thomas et al, 2022 ).…”