Yuzhen Tian

Functional significance of macrophage-derived exosomes in inflammation and pain

Summary Macrophage‐derived exosomes attenuated complete Freunds adjuvant‐induced thermal hyperalgesia in mice. Exosomal microRNA signature from patients with complex regional pain syndrome suggests a potential therapeutic and biomarker utility for exosomes. ABSTRACT Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome‐mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide‐stimulated cells were sufficient to cause nuclear factor‐&kgr;B activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage‐derived exosomes. Macrophage‐derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility.

Central Mechanisms of Menthol-induced Analgesia

Menthol is one of the most commonly used chemicals in our daily life, not only because of its fresh flavor and cooling feeling but also because of its medical benefit. Previous studies have suggested that menthol produces analgesic action in acute and neuropathic pain through peripheral mechanisms. However, the central actions and mechanisms of menthol remain unclear. Here, we report that menthol has direct effects on the spinal cord. Menthol decreased both ipsilateral and contralateral pain hypersensitivity induced by complete Freunds adjuvant in a dose-dependent manner. Menthol also reduced both first and second phases of formalin-induced spontaneous nocifensive behavior. We then identified the potential central mechanisms underlying the analgesic effect of menthol. In cultured dorsal horn neurons, menthol induced inward and outward currents in a dose-dependent manner. The menthol-activated current was mediated by Cl− and blocked by bicuculline, suggesting that menthol activates γ-aminobutyric acid type A receptors. In addition, menthol blocked voltage-gated sodium channels and voltage-gated calcium channels in a voltage-, state-, and use-dependent manner. Furthermore, menthol reduced repetitive firing and action potential amplitude, decreased neuronal excitability, and blocked spontaneous synaptic transmission of cultured superficial dorsal horn neurons. Liquid chromatography/tandem mass spectrometry analysis of brain menthol levels indicated that menthol was rapidly concentrated in the brain when administered systemically. Our results indicate that menthol produces its central analgesic action on inflammatory pain probably via the blockage of voltage-gated Na+ and Ca2+ channels. These data provide molecular and cellular mechanisms by which menthol decreases neuronal excitability, therefore contributing to menthol-induced central analgesia.

Potent analgesic effects of a store-operated calcium channel inhibitor.

Summary The SOC channel inhibitor YM‐58483 has analgesic actions in chronic pain, produces antinociceptive effects in acute pain and prevents the development of chronic pain in mice. Abstract Chronic pain often accompanies immune responses and immune cells are known to be involved in chronic pain. Store‐operated calcium (SOC) channels are calcium‐selective cation channels and play an important role in the immune system. YM‐58483, a potent SOC channel inhibitor, has been shown to inhibit cytokine production from immune cells and attenuate antigen‐induced hypersensitivity reactions. Here, we report that YM‐58483 has analgesic actions in chronic pain and produces antinociceptive effects in acute pain and prevents the development of chronic pain in mice. Oral administration of 10 mg/kg or 30 mg/kg YM‐58483 dramatically attenuated complete Freund adjuvant (CFA)‐induced thermal hyperalgesia and prevented the development of thermal and mechanical hypersensitivity in a dose‐dependent manner. Analgesic effects were observed when YM‐58483 was administered systemically, intrathecally and intraplantarly. YM‐58483 decreased spared nerve injury (SNI)‐induced thermal and mechanical hypersensitivity and prevented the development of SNI‐induced pain hypersensitivity. Pretreatment with YM‐58483 strongly reduced both the first and second phases of formalin‐induced spontaneous nocifensive behavior in a dose‐dependent manner. YM‐58483 produced antinociception in acute pain induced by heat or chemical or mechanical stimuli at a dose of 30 mg/kg. YM‐58483 diminished CFA‐induced paw edema, and reduced production of TNF‐&agr;, IL‐1&bgr; and PGE2 in the CFA‐injected paw. In vitro, SOC entry in nociceptors was more robust than in nonnociceptors, and the inhibition of SOC entry by YM‐58483 in nociceptors was much greater than in nonnociceptors. Our findings indicate that YM‐58483 is a potent analgesic and suggest that SOC channel inhibitors may represent a novel class of therapeutics for pain.

A store‐operated calcium channel inhibitor attenuates collagen‐induced arthritis

Store‐operated calcium (SOC) channels are thought to play a critical role in immune responses, inflammatory diseases and chronic pain. The aim of this study was to explore the potential role and mechanisms of SOC channels in collagen‐induced arthritis (CIA).

Effect of Histone Deacetylase Inhibitor JNJ-26481585 in Pain

Recent studies have shown that histone deacetylase (HDAC) inhibitors can alleviate inflammatory and neuropathic pain. We investigated the effects of JNJ-26481585, a pan-HDAC inhibitor on basal mechanical sensitivity. Unlike previous reports for HDAC inhibitors, JNJ-26481585 induced mechanical hypersensitivity in mice. This effect was reversible with gabapentin. Voltage-dependent calcium channel subunit alpha-2/delta-1, one of the putative targets for gabapentin, was upregulated in the spinal cord from JNJ-26481585-treated mice. Transcriptional profiling of spinal cord from JNJ-26481585-treated mice showed significant alterations in pathways involved in axon guidance, suggesting overlap in mechanisms underlying neurotoxicity caused by other known chemotherapeutic agents. To investigate the mechanisms underlying the development of pain, RAW 264.7 mouse macrophage cells were treated with JNJ-26481585. There was a dose- and time-dependent activation of nuclear factor-kappaB and interleukin-1β increase. Thus, alterations in the axon guidance pathway, increase in voltage-dependent calcium channel alpha(2)delta-1 subunit, and the induction of proinflammatory mediators by JNJ-26481585 could all contribute to increased mechanical sensitivity. Our data indicate that the effect of HDAC inhibitors may be unique to the compound studied and highlights the potential to develop chemotherapy-induced peripheral neuropathy with the use of a pan-HDAC inhibitor for cancer treatment, and this pain may be alleviated by gabapentin.

MicroRNAs downregulated in neuropathic pain regulate MeCP2 and BDNF related to pain sensitivity.

Nerve injury induces chronic pain and dysregulation of microRNAs in dorsal root ganglia (DRG). Several downregulated microRNAs are predicted to targetMecp2. MECP2 mutations cause Rett syndrome and these patients report decreased pain perception. We confirmed MeCP2 upregulation in DRG following nerve injury and repression of MeCP2 by miRNAsin vitro. MeCP2 regulates brain‐derived neurotrophic factor (BDNF) and downregulation of MeCP2 by microRNAs decreasedBdnf in vitro. MeCP2 T158A mice exhibited reduced mechanical sensitivity andMecp2‐null and MeCP2 T158A mice have decreasedBdnf in DRG. MeCP2‐mediated regulation ofBdnf in the DRG could contribute to altered pain sensitivity.

Neuronal P2X7 receptor-induced reactive oxygen species production contributes to nociceptive behavior in mice

ATP can activate a variety of pathways through P2 purinoreceptors, leading to neuroprotection and pathology in the CNS. Among all P2X receptors, the P2X7 receptor (P2X7R) is a well-defined therapeutic target for inflammatory and neuropathic pain. Activation of P2X7R can generate reactive oxygen species (ROS) in macrophages and microglia. However, the role of ROS in P2X7R–induced pain remains unexplored. Here, we investigated the downstream effects of neuronal P2X7R activation in the spinal cord. We found that ATP induces ROS production in spinal cord dorsal horn neurons, an effect eliminated by ROS scavenger N-tert-butyl-α-phenylnitrone (PBN) and P2X7R antagonist A438079. A similar effect was observed with a P2X7R agonist, BzATP, and was attenuated by a NADPH oxidase inhibitor apocynin. Intrathecal administration of BzATP resulted in ROS production in the spinal cord and oxidative DNA damage in dorsal horn neurons. BzATP also induced robust biphasic spontaneous nociceptive behavior. Pre-treatment with A438079 abolished all BzATP-induced nociceptive behaviors, while ROS scavengers dose-dependently attenuated the secondary response. Here, we provide evidence that neuronal P2X7R activation leads to ROS production and subsequent nociceptive pain in mice. Together, the data indicate that P2X7R-induced ROS play a critical role in the P2X7R signaling pathway of the CNS.

Orai1 plays a crucial role in central sensitization by modulating neuronal excitability

Pathological pain is a common and debilitating condition that is often poorly managed. Central sensitization is an important mechanism underlying pathological pain. However, candidate molecules involved in central sensitization remain unclear. Store-operated calcium channels (SOCs) mediate important calcium signals in nonexcitable and excitable cells. SOCs have been implicated in a wide variety of human pathophysiological conditions, including immunodeficiency, occlusive vascular diseases, and cancer. However, the role of SOCs in CNS disorders has been relatively unexplored. Orai1, a key component of SOCs, is expressed in the human and rodent spinal cord dorsal horn, but its functional significance in dorsal horn neurons is poorly understood. Here we sought to explore a potential role of Orai1 in the modulation of neuronal excitability and A-type potassium channels involved in pain plasticity. Using both male and female Orai1 knock-out mice, we found that activation of Orai1 increased neuronal excitability and reduced A-type potassium channels via the protein kinase C–extracellular signal-regulated protein kinase (PKC–ERK) pathway in dorsal horn neurons. Orai1 deficiency significantly decreased acute pain induced by noxious stimuli, nearly eliminated the second phase of formalin-induced nociceptive response, markedly attenuated carrageenan-induced ipsilateral pain hypersensitivity and abolished carrageenan-induced contralateral mechanical allodynia. Consistently, carrageenan-induced increase in neuronal excitability was abolished in the dorsal horn from Orai1 mutant mice. These findings uncover a novel signaling pathway involved in the pain process and central sensitization. Our study also reveals a novel link among Orai1, ERK, A-type potassium channels, and neuronal excitability. SIGNIFICANCE STATEMENT Orai1 is a key component of store-operated calcium channels (SOCs) in many cell types. It has been implicated in such pathological conditions as immunodeficiency, autoimmunity, and cancer. However, the role of Orai1 in CNS disorders remains poorly understood. The functional significance of Orai1 in neurons is elusive. Here we demonstrate that activation of Orai1 modulates neuronal excitability and Kv4-containing A-type potassium channels via the protein kinase C–extracellular signal-regulated protein kinase (PKC–ERK) pathway. Genetic knock-out of Orai1 nearly eliminates the second phase of formalin-induced pain and markedly attenuates carrageenan-induced pain hypersensitivity and neuronal excitability. These findings reveal a novel link between Orai1 and neuronal excitability and advance our understanding of central sensitization.

miR-34a-mediated regulation of XIST in female cells under inflammation

Background Evidence is overwhelming for sex differences in pain, with women representing the majority of the chronic pain patient population. There is a need to explore novel avenues to elucidate this sex bias in the development of chronic inflammatory pain conditions. Complex regional pain syndrome (CRPS) is a chronic neuropathic pain disorder, and the incidence of CRPS is greater in women than in men by ~4:1. Since neurogenic inflammation is a key feature of CRPS, dysregulation of inflammatory responses can be a factor in predisposing women to chronic pain. Methods Our studies investigating alterations in circulating microRNAs (miRNAs) in whole blood from female CRPS patients showed significant differential expression of miRNAs between responders and poor responders to ketamine treatment. Several of these miRNAs are predicted to target the long noncoding RNA, X-inactive-specific transcript (XIST). XIST mediates X-chromosome inactivation and is essential for equalizing the expression of X-linked genes between females and males. Based on the well-established role in inflammatory process, we focused on miR-34a, one of the miRNAs predicted to target XIST, and downregulated in CRPS patients responding poorly to ketamine. Results Our in vitro and in vivo models of acute inflammation and data from patients with CRPS showed that miR-34a can regulate XIST under inflammation directly, and through pro-inflammatory transcription factor Yin-Yang 1 (YY1). XIST was significantly upregulated in a subset of CRPS patients responding poorly to ketamine. Conclusion Since dysregulation of XIST can result in genes escaping inactivation or reactivation in female cells, further investigations on the role of XIST in the predominance of chronic inflammatory and pain disorders in women is warranted.