Amelioration of injury-induced tissue acidosis by a nonsteroidal analgesic attenuates antinociceptive effects of the pH-dependent opioid agonist NFEPP.

Opioid agonists are powerful drugs for managing pain. However, their central side effects are limiting their use and drugs with similar potency, but a lower risk profile are needed. (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenylpropionamide (NFEPP) is a novel opioid agonist that preferentially activates opioid receptors at acidic extracellular pH. NFEPP was designed to activate peripheral opioid receptors in injured tissue, therefore precluding side effects elicited at normal pH in brain or intestinal wall. Considering the common combination of opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) in multimodal analgesia, we investigated the interaction between NFEPP and a widely prescribed prototypical NSAID, diclofenac (DCF), in a rat model of unilateral hindpaw inflammation induced by complete Freund's adjuvant. We evaluated the effects of systemically applied DCF on the paw tissue pH, on the expression of inflammatory mediators in immune cells from inflamed paws and on the expression of opioid receptors in dorsal root ganglia. Additionally, we investigated the antinociceptive efficacy of NFEPP injected into the inflamed paws after DCF treatment. We found that DCF reduced inflammation-induced nociceptive responses and tissue acidosis, but did not change the mRNA expression of IL-1ß, TNF-α, IL-6, IL-4, NGF, or of mu-, delta-, or kappa-opioid receptors. The treatment with DCF moderately reduced the antinociceptive efficacy of NFEPP, suggesting a correlation between an increase in local tissue pH and the decreased antinociceptive effect of this pH-sensitive opioid agonist.

Interleukin-4 Induces the Release of Opioid Peptides from M1 Macrophages in Pathological Pain.

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), ß-endorphin and dynorphin A 1-17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca2+ chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca2+-mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue.SIGNIFICANCE STATEMENT Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1ß, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca2+-dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4-opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects.

Immune cell-mediated opioid analgesia.

Pathological pain is regulated by a balance between pro-algesic and analgesic mechanisms. Interactions between opioid peptide-producing immune cells and peripheral sensory neurons expressing opioid receptors represent a powerful intrinsic pain control in animal models and in humans. Therefore, treatments based on general suppression of immune responses have been mostly unsuccessful. It is highly desirable to develop strategies that specifically promote neuro-immune communication mediated by opioids. Promising examples include vaccination-based recruitment of opioid-containing leukocytes to painful tissue and the local reprogramming of pro-algesic immune cells into analgesic cells producing and secreting high amounts of opioid peptides. Such approaches have the potential to inhibit pain at its origin and be devoid of central and systemic side effects of classical analgesics. In support of these concepts, in this article, we describe the functioning of peripheral opioid receptors, migration of opioid-producing immune cells to inflamed tissue, opioid peptide release, and the consequent pain relief. Conclusively, we provide clinical evidence and discuss therapeutic opportunities and challenges associated with immune cell-mediated peripheral opioid analgesia.

Opioid Receptors in Immune and Glial Cells-Implications for Pain Control.

Opioid receptors comprise µ (MOP), δ (DOP), κ (KOP), and nociceptin/orphanin FQ (NOP) receptors. Opioids are agonists of MOP, DOP, and KOP receptors, whereas nociceptin/orphanin FQ (N/OFQ) is an agonist of NOP receptors. Activation of all four opioid receptors in neurons can induce analgesia in animal models, but the most clinically relevant are MOP receptor agonists (e.g., morphine, fentanyl). Opioids can also affect the function of immune cells, and their actions in relation to immunosuppression and infections have been widely discussed. Here, we analyze the expression and the role of opioid receptors in peripheral immune cells and glia in the modulation of pain. All four opioid receptors have been identified at the mRNA and protein levels in immune cells (lymphocytes, granulocytes, monocytes, macrophages) in humans, rhesus monkeys, rats or mice. Activation of leukocyte MOP, DOP, and KOP receptors was recently reported to attenuate pain after nerve injury in mice. This involved intracellular Ca2+-regulated release of opioid peptides from immune cells, which subsequently activated MOP, DOP, and KOP receptors on peripheral neurons. There is no evidence of pain modulation by leukocyte NOP receptors. More good quality studies are needed to verify the presence of DOP, KOP, and NOP receptors in native glia. Although still questioned, MOP receptors might be expressed in brain or spinal cord microglia and astrocytes in humans, mice, and rats. Morphine acting at spinal cord microglia is often reported to induce hyperalgesia in rodents. However, most studies used animals without pathological pain and/or unconventional paradigms (e.g., high or ultra-low doses, pain assessment after abrupt discontinuation of chronic morphine treatment). Therefore, the opioid-induced hyperalgesia can be viewed in the context of dependence/withdrawal rather than pain management, in line with clinical reports. There is convincing evidence of analgesic effects mediated by immune cell-derived opioid peptides in animal models and in humans. Together, MOP, DOP, and KOP receptors, and opioid peptides in immune cells can ameliorate pathological pain. The relevance of NOP receptors and N/OFQ in leukocytes, and of all opioid receptors, opioid peptides and N/OFQ in native glia for pain control is yet to be clarified.

IL-4 induces M2 macrophages to produce sustained analgesia via opioids.

IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80+ macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, ß-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (δ, µ, κ) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80+ macrophages from IL-4-treated donors. Together, IL-4-induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain.

Advances in Achieving Opioid Analgesia Without Side Effects.

Opioids are the most effective drugs for the treatment of severe pain, but they also cause addiction and overdose deaths, which have led to a worldwide opioid crisis. Therefore, the development of safer opioids is urgently needed. In this article, we provide a critical overview of emerging opioid-based strategies aimed at effective pain relief and improved side effect profiles. These approaches comprise biased agonism, the targeting of (i) opioid receptors in peripheral inflamed tissue (by reducing agonist access to the brain, the use of nanocarriers, or low pH-sensitive agonists); (ii) heteromers or multiple receptors (by monovalent, bivalent, and multifunctional ligands); (iii) receptor splice variants; and (iv) endogenous opioid peptides (by preventing their degradation or enhancing their production by gene transfer). Substantial advancements are underscored by pharmaceutical development of new opioids such as peripheral κ-receptor agonists, and by treatments augmenting the action of endogenous opioids, which have entered clinical trials. Additionally, there are several promising novel opioids comprehensively examined in preclinical studies, but also strategies such as biased agonism, which might require careful rethinking.

Mu-Opioid Receptor Agonist Induces Kir3 Currents in Mouse Peripheral Sensory Neurons - Effects of Nerve Injury.

Neuropathic pain often arises from damage to peripheral nerves and is difficult to treat. Activation of opioid receptors in peripheral sensory neurons is devoid of respiratory depression, sedation, nausea, and addiction mediated in the brain, and ameliorates neuropathic pain in animal models. Mechanisms of peripheral opioid analgesia have therefore gained interest, but the role of G protein-coupled inwardly rectifying potassium (Kir3) channels, important regulators of neuronal excitability, remains unclear. Whereas functional Kir3 channels have been detected in dorsal root ganglion (DRG) neurons in rats, some studies question their contribution to opioid analgesia in inflammatory pain models in mice. However, neuropathic pain can be diminished by activation of peripheral opioid receptors in mouse models. Therefore, here we investigated effects of the selective µ-opioid receptor (MOR) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) on potassium conductance in DRG neurons upon a chronic constriction injury (CCI) of the sciatic nerve in mice. For verification, we also tested human embryonic kidney (HEK) 293 cells transfected with MOR and Kir3.2. Using patch clamp, we recorded currents at -80 mV and applied voltage ramps in high extracellular potassium concentrations, which are a highly sensitive measures of Kir3 channel activity. We found a significantly higher rate of HEK cells responding with potassium channel blocker barium-sensitive inward current (233 ± 51 pA) to DAMGO application in transfected than in untransfected group, which confirms successful recordings of inward currents through Kir3.2 channels. Interestingly, DAMGO induced similar inward currents (178 ± 36-207 ± 56 pA) in 15-20% of recorded DRG neurons from naïve mice and in 4-27% of DRG neurons from mice exposed to CCI, measured in voltage clamp or voltage ramp modes. DAMGO-induced currents in naïve and CCI groups were reversed by barium and a more selective Kir3 channel blocker tertiapin-Q. These data indicate the coupling of Kir3 channels with MOR in mouse peripheral sensory neuron cell bodies, which was unchanged after CCI. A comparative analysis of opioid-induced potassium conductance at the axonal injury site and peripheral terminals of DRG neurons could clarify the role of Kir3 channel-MOR interactions in peripheral nerve injury and opioid analgesia.

Adoptive transfer of M2 macrophages reduces neuropathic pain via opioid peptides.

BACKGROUND: During the inflammation which occurs following nerve damage, macrophages are recruited to the site of injury. Phenotypic diversity is a hallmark of the macrophage lineage and includes pro-inflammatory M1 and anti-inflammatory M2 populations. Our aim in this study was to investigate the ability of polarized M0, M1, and M2 macrophages to secrete opioid peptides and to examine their relative contribution to the modulation of neuropathic pain. METHODS: Mouse bone marrow-derived cells were cultured as unstimulated M0 macrophages or were stimulated into an M1 phenotype using lipopolysaccharide and interferon-γ or into an M2 phenotype using interleukin-4. The macrophage phenotypes were verified using flow cytometry for surface marker analysis and cytokine bead array for cytokine profile assessment. Opioid peptide levels were measured by radioimmunoassay and enzyme immunoassay. As a model of neuropathic pain, a chronic constriction injury (CCI) of the sciatic nerve was employed. Polarized M0, M1, and M2 macrophages (5 × 105 cells) were injected perineurally twice, on days 14 and 15 following CCI or sham surgery. Mechanical and heat sensitivity were measured using the von Frey and Hargreaves tests, respectively. To track the injected macrophages, we also transferred fluorescently stained polarized cells and analyzed the surface marker profile of endogenous and injected cells in the nerves ex vivo. RESULTS: Compared to M0 and M1 cells, M2 macrophages contained and released higher amounts of opioid peptides, including Met-enkephalin, dynorphin A (1-17), and ß-endorphin. M2 cells transferred perineurally at the nerve injury site reduced mechanical, but not heat hypersensitivity following the second injection. The analgesic effect was reversed by the perineurally applied opioid receptor antagonist naloxone methiodide. M2 cells did not affect sensitivity following sham surgery. Neither M0 nor M1 cells altered mechanical and heat sensitivity in CCI or sham-operated animals. Tracing the fluorescently labeled M0, M1, and M2 cells ex vivo showed that they remained in the nerve and preserved their phenotype. CONCLUSIONS: Perineural transplantation of M2 macrophages resulted in opioid-mediated amelioration of neuropathy-induced mechanical hypersensitivity, while M1 macrophages did not exacerbate pain. Therefore, rather than focusing on macrophage-induced pain generation, promoting opioid-mediated M2 actions may be more relevant for pain control.

Distinct roles of exogenous opioid agonists and endogenous opioid peptides in the peripheral control of neuropathy-triggered heat pain.

Neuropathic pain often results from peripheral nerve damage, which can involve immune response. Local leukocyte-derived opioid peptides or exogenous opioid agonists inhibit neuropathy-induced mechanical hypersensitivity in animal models. Since neuropathic pain can also be augmented by heat, in this study we investigated the role of opioids in the modulation of neuropathy-evoked heat hypersensitivity. We used a chronic constriction injury of the sciatic nerve in wild-type and opioid peptide-knockout mice, and tested opioid effects in heat and mechanical hypersensitivity using Hargreaves and von Frey tests, respectively. We found that although perineural exogenous opioid agonists, including peptidergic ligands, were effective, the endogenous opioid peptides ß-endorphin, Met-enkephalin and dynorphin A did not alleviate heat hypersensitivity. Specifically, corticotropin-releasing factor, an agent triggering opioid peptide secretion from leukocytes, applied perineurally did not attenuate heat hypersensitivity in wild-type mice. Exogenous opioids, also shown to release opioid peptides via activation of leukocyte opioid receptors, were equally analgesic in wild-type and opioid peptide-knockout mice, indicating that endogenous opioids do not contribute to exogenous opioid analgesia in heat hypersensitivity. Furthermore, exogenously applied opioid peptides were ineffective as well. Conversely, opioid peptides relieved mechanical hypersensitivity. Thus, both opioid type and sensory modality may determine the outcome of neuropathic pain treatment.

Leukocyte opioid receptors mediate analgesia via Ca(2+)-regulated release of opioid peptides.

Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, ß-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, µ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gßγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gßγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the Gαi/o-Gßγ protein-PLC-IP3 receptors-intracellular Ca(2+) pathway. Our findings suggest that opioid receptors in immune cells are important targets for the control of pathological pain.

Recent advances in understanding neuropathic pain: glia, sex differences, and epigenetics.

Neuropathic pain results from diseases or trauma affecting the nervous system. This pain can be devastating and is poorly controlled. The pathophysiology is complex, and it is essential to understand the underlying mechanisms in order to identify the relevant targets for therapeutic intervention. In this article, we focus on the recent research investigating neuro-immune communication and epigenetic processes, which gain particular attention in the context of neuropathic pain. Specifically, we analyze the role of glial cells, including microglia, astrocytes, and oligodendrocytes, in the modulation of the central nervous system inflammation triggered by neuropathy. Considering epigenetics, we address DNA methylation, histone modifications, and the non-coding RNAs in the regulation of ion channels, G-protein-coupled receptors, and transmitters following neuronal damage. The goal was not only to highlight the emerging concepts but also to discuss controversies, methodological complications, and intriguing opinions.