Astrocytes Assemble Thalamocortical Synapses by Bridging NRX1α and NL1 via Hevin.

Proper establishment of synapses is critical for constructing functional circuits. Interactions between presynaptic neurexins and postsynaptic neuroligins coordinate the formation of synaptic adhesions. An isoform code determines the direct interactions of neurexins and neuroligins across the synapse. However, whether extracellular linker proteins can expand such a code is unknown. Using a combination of in vitro and in vivo approaches, we found that hevin, an astrocyte-secreted synaptogenic protein, assembles glutamatergic synapses by bridging neurexin-1alpha and neuroligin-1B, two isoforms that do not interact with each other. Bridging of neurexin-1alpha and neuroligin-1B via hevin is critical for the formation and plasticity of thalamocortical connections in the developing visual cortex. These results show that astrocytes promote the formation of synapses by modulating neurexin/neuroligin adhesions through hevin secretion. Our findings also provide an important mechanistic insight into how mutations in these genes may lead to circuit dysfunction in diseases such as autism.

Dectin-1 limits autoimmune neuroinflammation and promotes myeloid cell-astrocyte crosstalk via Card9-independent expression of Oncostatin M.

Pathologic roles of innate immunity in neurologic disorders are well described, but their beneficial aspects are less understood. Dectin-1, a C-type lectin receptor (CLR), is largely known to induce inflammation. Here, we report that Dectin-1 limited experimental autoimmune encephalomyelitis (EAE), while its downstream signaling molecule, Card9, promoted the disease. Myeloid cells mediated the pro-resolution function of Dectin-1 in EAE with enhanced gene expression of the neuroprotective molecule, Oncostatin M (Osm), through a Card9-independent pathway, mediated by the transcription factor NFAT. Furthermore, we find that the Osm receptor (OsmR) functioned specifically in astrocytes to reduce EAE severity. Notably, Dectin-1 did not respond to heat-killed Mycobacteria, an adjuvant to induce EAE. Instead, endogenous Dectin-1 ligands, including galectin-9, in the central nervous system (CNS) were involved to limit EAE. Our study reveals a mechanism of beneficial myeloid cell-astrocyte crosstalk regulated by a Dectin-1 pathway and identifies potential therapeutic targets for autoimmune neuroinflammation.

A monoclonal antibody that targets a NaV1.7 channel voltage sensor for pain and itch relief.

Voltage-gated sodium (NaV) channels control the upstroke of the action potentials in excitable cells. Multiple studies have shown distinct roles of NaV channel subtypes in human physiology and diseases, but subtype-specific therapeutics are lacking and the current efforts have been limited to small molecules. Here, we present a monoclonal antibody that targets the voltage-sensor paddle of NaV1.7, the subtype critical for pain sensation. This antibody not only inhibits NaV1.7 with high selectivity, but also effectively suppresses inflammatory and neuropathic pain in mice. Interestingly, the antibody inhibits acute and chronic itch despite well-documented differences in pain and itch modulation. Using this antibody, we discovered that NaV1.7 plays a key role in spinal cord nociceptive and pruriceptive synaptic transmission. Our studies reveal that NaV1.7 is a target for itch management, and the antibody has therapeutic potential for suppressing pain and itch. Our antibody strategy may have broad applications for voltage-gated cation channels.

STING controls nociception via type I interferon signalling in sensory neurons.

The innate immune regulator STING is a critical sensor of self- and pathogen-derived DNA. DNA sensing by STING leads to the induction of type-I interferons (IFN-I) and other cytokines, which promote immune-cell-mediated eradication of pathogens and neoplastic cells1,2. STING is also a robust driver of antitumour immunity, which has led to the development of STING activators and small-molecule agonists as adjuvants for cancer immunotherapy3. Pain, transmitted by peripheral nociceptive sensory neurons (nociceptors), also aids in host defence by alerting organisms to the presence of potentially damaging stimuli, including pathogens and cancer cells4,5. Here we demonstrate that STING is a critical regulator of nociception through IFN-I signalling in peripheral nociceptors. We show that mice lacking STING or IFN-I signalling exhibit hypersensitivity to nociceptive stimuli and heightened nociceptor excitability. Conversely, intrathecal activation of STING produces robust antinociception in mice and non-human primates. STING-mediated antinociception is governed by IFN-Is, which rapidly suppress excitability of mouse, monkey and human nociceptors. Our findings establish the STING-IFN-I signalling axis as a critical regulator of physiological nociception and a promising new target for treating chronic pain.

Specialized Pro-Resolving Mediators as Resolution Pharmacology for the Control of Pain and Itch.

Specialized pro-resolving mediators (SPMs), including resolvins, protectins, and maresins, are endogenous lipid mediators that are synthesized from omega-3 polyunsaturated fatty acids during the acute phase or resolution phase of inflammation. Synthetic SPMs possess broad safety profiles and exhibit potent actions in resolving inflammation in preclinical models. Accumulating evidence in the past decade has demonstrated powerful analgesia of exogenous SPMs in rodent models of inflammatory, neuropathic, and cancer pain. Furthermore, endogenous SPMs are produced by sham surgery and neuromodulation (e.g., vagus nerve stimulation). SPMs produce their beneficial actions through multiple G protein-coupled receptors, expressed by immune cells, glial cells, and neurons. Notably, loss of SPM receptors impairs the resolution of pain. I also highlight the emerging role of SPMs in the control of itch. Pharmacological targeting of SPMs or SPM receptors has the potential to lead to novel therapeutics for pain and itch as emerging approaches in resolution pharmacology.

Astrocytes in chronic pain and itch.

Astrocytes are critical for maintaining the homeostasis of the CNS. Increasing evidence suggests that a number of neurological and neuropsychiatric disorders, including chronic pain, may result from astrocyte 'gliopathy'. Indeed, in recent years there has been substantial progress in our understanding of how astrocytes can regulate nociceptive synaptic transmission via neuronal-glial and glial-glial cell interactions, as well as the involvement of spinal and supraspinal astrocytes in the modulation of pain signalling and the maintenance of neuropathic pain. A role of astrocytes in the pathogenesis of chronic itch is also emerging. These developments suggest that targeting the specific pathways that are responsible for astrogliopathy may represent a novel approach to develop therapies for chronic pain and chronic itch.

Microglial STING activation alleviates nerve injury-induced neuropathic pain in male but not female mice.

Microglia, resident immune cells in the central nervous system, play a role in neuroinflammation and the development of neuropathic pain. We found that the stimulator of interferon genes (STING) is predominantly expressed in spinal microglia and upregulated after peripheral nerve injury. However, mechanical allodynia, as a marker of neuropathic pain following peripheral nerve injury, did not require microglial STING expression. In contrast, STING activation by specific agonists (ADU-S100, 35 nmol) significantly alleviated neuropathic pain in male mice, but not female mice. STING activation in female mice leads to increase in proinflammatory cytokines that may counteract the analgesic effect of ADU-S100. Microglial STING expression and type I interferon-ß (IFN-ß) signaling were required for the analgesic effects of STING agonists in male mice. Mechanistically, downstream activation of TANK-binding kinase 1 (TBK1) and the production of IFN-ß, may partly account for the analgesic effect observed. These findings suggest that STING activation in spinal microglia could be a potential therapeutic intervention for neuropathic pain, particularly in males.

Intrathecal administration of conditioned serum from different species resolves Chemotherapy-Induced neuropathic pain in mice via secretory exosomes.

Chemotherapy-induced peripheral neuropathy (CIPN) is the most prevalent neurological complication of chemotherapy for cancer, and has limited effective treatment options. Autologous conditioned serum (ACS) is an effective biologic therapy used by intra-articular injection for patients with osteoarthritis. However, ACS has not been systematically tested in the treatment of peripheral neuropathies such as CIPN. It has been generally assumed that the analgesic effect of this biologic therapy results from augmented concentrations of anti-inflammatory cytokines and growth factors. Here we report that a single intrathecal injection of human conditioned serum (hCS) produced long-lasting inhibition of paclitaxel chemotherapy-induced neuropathic pain (mechanical allodynia) in mice, without causing motor impairment. Strikingly, the analgesic effect of hCS in our experiments was maintained even 8 weeks after the treatment, compared with non-conditioned human serum (hNCS). Furthermore, the hCS transfer-induced pain relief in mice was fully recapitulated by rat or mouse CS transfer to mice of both sexes, indicating cross-species and cross-sex effectiveness. Mechanistically, CS treatment blocked the chemotherapy-induced glial reaction in the spinal cord and improved nerve conduction. Compared to NCS, CS contained significantly higher concentrations of anti-inflammatory and pro-resolving mediators, including IL-1Ra, TIMP-1, TGF-ß1, and resolvins D1/D2. Intrathecal injection of anti-TGF-ß1 and anti-Il-1Ra antibody transiently reversed the analgesic action of CS. Nanoparticle tracking analysis revealed that rat conditioned serum contained a significantly greater number of exosomes than NCS. Importantly, the removal of exosomes by high-speed centrifugation largely diminished the CS-produced pain relief, suggesting a critical involvement of small vesicles (exosomes) in the beneficial effects of CS. Together, our findings demonstrate that intrathecal CS produces a remarkable resolution of neuropathic pain mediated through a combination of small vesicles/exosomes and neuroimmune/neuroglial modulation.

Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis.

Astrocytes are complex glial cells with numerous fine cellular processes that infiltrate the neuropil and interact with synapses. The mechanisms that control the establishment of astrocyte morphology are unknown, and it is unclear whether impairing astrocytic infiltration of the neuropil alters synaptic connectivity. Here we show that astrocyte morphogenesis in the mouse cortex depends on direct contact with neuronal processes and occurs in parallel with the growth and activity of synaptic circuits. The neuroligin family cell adhesion proteins NL1, NL2, and NL3, which are expressed by cortical astrocytes, control astrocyte morphogenesis through interactions with neuronal neurexins. Furthermore, in the absence of astrocytic NL2, the formation and function of cortical excitatory synapses are diminished, whereas inhibitory synaptic function is enhanced. Our findings highlight a previously undescribed mechanism of action for neuroligins and link astrocyte morphogenesis to synaptogenesis. Because neuroligin mutations have been implicated in various neurological disorders, these findings also point towards an astrocyte-based mechanism of neural pathology.

Inflammatory changes in the plasma and cerebrospinal fluid of patients with persistent pain and postpartum depression after elective Cesarean delivery: an exploratory prospective cohort study.

PURPOSE: Severe acute pain after Cesarean delivery increases the risk of developing persistent pain (~20% incidence) and postpartum depression (PPD) (~15% incidence). Both conditions contribute to maternal morbidity and mortality, yet early risk stratification remains challenging. Neuroinflammation has emerged as a key mechanism of persistent pain and depression in nonobstetric populations. Nevertheless, most studies focus on plasma cytokines, and the relationship between plasma and cerebrospinal fluid (CSF) cytokine levels is unclear. Our primary aim was to compare inflammatory marker levels between patients who developed the composite outcome of persistent pain and/or PPD vs those who did not. METHODS: We recruited term patients with singleton pregnancies undergoing elective Cesarean delivery under neuraxial anesthesia into an exploratory prospective cohort study. We collected baseline demographic, obstetric, and Edinburgh Postnatal Depression Scale information, and performed quantitative sensory tests. Plasma was collected preoperatively and 48 hr postoperatively. In the operating room, 10 mL of CSF was collected, followed by a standardized anesthetic. Intra- and postoperative management were according to standard practice. We obtained Edinburgh Postnatal Depression Scale and pain scores at six weeks and three months after delivery. The primary outcome was persistent pain and/or PPD at three months. We analyzed the difference in inflammatory marker levels between the groups (primary aim) using two-sided Mann-Whitney tests. RESULTS: Eighty participants were enrolled, and 63 patients completed the study; 23 (37%) experienced the primary outcome at three months. Preoperative plasma transforming growth factor beta 1 (TGF-ß1) concentration was higher in patients who developed the primary outcome compared with those who did not (median [interquartile range (IQR)], 2,879 [2,241-5,494] vs 2,292 [1,676-2,960] pg·mL-1; P = 0.04), while CSF IL-1ß concentration was higher in patients who developed the primary outcome than in those who did not (median [IQR], 0.36 [0.29-0.39] vs 0.30 [0.25-0.35] pg·mL-1; P = 0.03). CONCLUSIONS: We observed differential levels of plasma and CSF inflammatory biomarkers in patients who developed persistent pain and PPD compared with those who did not. We showed the feasibility of collecting plasma and CSF samples at Cesarean delivery, which may prove useful for future risk-stratification. STUDY REGISTRATION: ClinicalTrials.gov (NCT04271072); registered 17 February 2020.

RéSUMé: OBJECTIF: La douleur aiguë sévère après un accouchement par césarienne augmente le risque de douleur persistante (~20 % d'incidence) et de dépression post-partum (DPP) (~15 % d'incidence). Ces deux conditions contribuent à la morbidité et à la mortalité maternelles, mais la stratification précoce des risques demeure difficile. La neuroinflammation est apparue comme un mécanisme clé de la douleur persistante et de la dépression dans les populations non obstétricales. Néanmoins, la plupart des études se concentrent sur les cytokines plasmatiques, et la relation entre les taux de cytokines plasmatiques et de liquide céphalorachidien (LCR) n'est pas claire. Notre objectif principal était de comparer les taux de marqueurs inflammatoires entre les patient·es qui ont eu un résultat composite de douleur persistante et/ou de DPP vs les personnes qui n'en ont pas eu. MéTHODE: Nous avons recruté des patient·es à terme avec des grossesses uniques bénéficiant d'une césarienne programmée sous anesthésie neuraxiale dans une étude de cohorte prospective exploratoire. Nous avons recueilli des informations démographiques de base, obstétricales et tirées de l'Échelle de dépression postnatale d'Édimbourg, et effectué des tests sensoriels quantitatifs. Le plasma a été prélevé avant l'opération et 48 heures après l'opération. En salle d'opération, 10 mL de LCR ont été recueillis, suivis d'un anesthésie standardisée. La prise en charge per- et postopératoire était conforme à la pratique courante. Nous avons obtenu les scores sur l'Échelle de dépression postnatale d'Édimbourg et les scores de douleur six semaines et trois mois après l'accouchement. Le critère d'évaluation principal était la douleur persistante et/ou la DPP à trois mois. Nous avons analysé la différence dans les niveaux de marqueurs inflammatoires entre les groupes (objectif principal) en utilisant des tests bilatéraux de Mann-Whitney. RéSULTATS: Quatre-vingts personnes ont été recrutées et 63 patient·es ont terminé l'étude; 23 (37 %) ont rapporté le critère d'évaluation principal à trois mois. Le facteur TGF-ß1 (transforming growth factor beta 1) plasmatique préopératoire était plus élevé chez les patient·es qui ont manifesté le critère d'évaluation principal par rapport aux personnes qui ne l'ont pas manifesté (médiane [écart interquartile (ÉIQ)], 2879 [2241-5494] vs 2292 [1676­2960] pg·mL−1; P = 0,04), tandis que le IL-1ß dans le LCR était plus élevé chez les patient·es qui ont manifesté le critère d'évaluation principal que chez les personnes qui ne l'ont pas manifesté (médiane [ÉIQ], 0,36 [0,29-0,39] vs 0,30 [0,25­0,35] pg·mL−1; P = 0,03). CONCLUSION: Nous avons observé des taux différentiels de biomarqueurs inflammatoires plasmatiques et de LCR chez les patient·es qui ont manifesté une douleur persistante et une DPP par rapport aux personnes qui n'en ont pas manifesté. Nous avons montré la faisabilité de la collecte d'échantillons de plasma et de LCR lors de l'accouchement par césarienne, ce qui pourrait s'avérer utile pour la stratification future des risques. ENREGISTREMENT DE L'éTUDE: clinicaltrials.gov (NCT04271072); enregistrée le 17 février 2020.

Epithelia-Sensory Neuron Cross Talk Underlies Cholestatic Itch Induced by Lysophosphatidylcholine.

BACKGROUND & AIMS: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus. METHODS: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a. RESULTS: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1+ sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates. CONCLUSIONS: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1+ pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception.

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition.

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.

Functional selection of protease inhibitory antibodies.

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli-an antibody clone, a protease of interest, and a ß-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified ß-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), ß-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aß40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

Emerging Roles of Type-I Interferons in Neuroinflammation, Neurological Diseases, and Long-Haul COVID.

Interferons (IFNs) are pleiotropic cytokines originally identified for their antiviral activity. IFN-α and IFN-ß are both type I IFNs that have been used to treat neurological diseases such as multiple sclerosis. Microglia, astrocytes, as well as neurons in the central and peripheral nervous systems, including spinal cord neurons and dorsal root ganglion neurons, express type I IFN receptors (IFNARs). Type I IFNs play an active role in regulating cognition, aging, depression, and neurodegenerative diseases. Notably, by suppressing neuronal activity and synaptic transmission, IFN-α and IFN-ß produced potent analgesia. In this article, we discuss the role of type I IFNs in cognition, neurodegenerative diseases, and pain with a focus on neuroinflammation and neuro-glial interactions and their effects on cognition, neurodegenerative diseases, and pain. The role of type I IFNs in long-haul COVID-associated neurological disorders is also discussed. Insights into type I IFN signaling in neurons and non-neuronal cells will improve our treatments of neurological disorders in various disease conditions.

Inflammation and Infection in Pain and the Role of GPR37.

Inflammation is known to cause pain, and pain is of one of the cardinal signs of inflammation. Mounting evidence suggests that acute inflammation also resolves pain through specialized pro-resolving mediators (SPMs) and macrophage signaling. GPR37 is expressed by neurons and oligodendrocytes in the brain and has been implicated in multiple disorders, such as demyelination, Parkinson's disease, stroke, and cancer. Recent studies have demonstrated that GPR37 is expressed by macrophages and confers protection against infection by bacteria and parasites. Furthermore, GPR37 promotes the resolution of inflammatory pain and infection-induced pain, as the duration of pain after tissue injury and infection is prolonged in mice lacking Gpr37. Mechanistically, activation of GPR37 enhances macrophage phagocytosis, and Gpr37-deficient macrophages exhibit dysregulations of pro-inflammatory and anti-inflammatory cytokines, switching from M2- to M1-like phenotypes. We also discuss novel ligands of GPR37, including neuroprotectin D1 (NPD1), a SPM derived from docosahexaenoic acid (DHA), and bone-derived hormone osteocalcin (OCN), which can suppress oligodendrocyte differentiation and myelination. NPD1 stimulates macrophage phagocytosis via GPR37 and exhibits potent analgesic actions in various animal models of inflammatory and neuropathic pain. Targeting GPR37 may lead to novel therapeutics for treating inflammation, infection, pain, and neurological diseases.

Lysophospholipids Contribute to Oxaliplatin-Induced Acute Peripheral Pain.

Oxaliplatin, a platinum-based chemotherapeutic drug, which is used as first-line treatment for some types of colorectal carcinoma, causes peripheral neuropathic pain in patients. In addition, an acute peripheral pain syndrome develop in almost 90% of patients immediately after oxaliplatin treatment, which is poorly understood mechanistically but correlates with incidence and severity of the later-occurring neuropathy. Here we investigated the effects of acute oxaliplatin treatment in a murine model, showing that male and female mice develop mechanical hypersensitivity 24 h after oxaliplatin treatment. Interestingly, we found that the levels of several lipids were significantly altered in nervous tissue during oxaliplatin-induced acute pain. Specifically, the linoleic acid metabolite 9,10-EpOME (epoxide of linoleic acid) as well as the lysophospholipids lysophosphatidylcholine (LPC) 18:1 and LPC 16:0 were significantly increased 24 h after oxaliplatin treatment in sciatic nerve, DRGs, or spinal cord tissue as revealed by untargeted and targeted lipidomics. In contrast, inflammatory markers including cytokines and chemokines, ROS markers, and growth factors are unchanged in the respective nervous system tissues. Importantly, LPC 18:1 and LPC 16:0 can induce Ca2+ transients in primary sensory neurons, and we identify LPC 18:1 as a previously unknown endogenous activator of the ligand-gated calcium channels transient receptor potential V1 and M8 (transient receptor potential vanilloid 1 and transient receptor potential melastatin 8) in primary sensory neurons using both pharmacological inhibition and genetic knockout. Additionally, a peripheral LPC 18:1 injection was sufficient to induce mechanical hypersensitivity in naive mice. Hence, targeting signaling lipid pathways may ameliorate oxaliplatin-induced acute peripheral pain and the subsequent long-lasting neuropathy.SIGNIFICANCE STATEMENT The first-line cytostatic drug oxaliplatin can cause acute peripheral pain and chronic neuropathic pain. The former is causally connected with the chronic neuropathic pain, but its mechanisms are poorly understood. Here, we performed a broad unbiased analysis of cytokines, chemokines, growth factors, and ∼200 lipids in nervous system tissues 24 h after oxaliplatin treatment, which revealed a crucial role of lysophospholipids lysophosphatidylcholine (LPC) 18:1, LPC 16:0, and 9,10-EpOME in oxaliplatin-induced acute pain. We demonstrate for the first time that LPC 18:1 contributes to the activation of the ion channels transient receptor potential vanilloid 1 and transient receptor potential melastatin 8 in sensory neurons and causes mechanical hypersensitivity after peripheral injection in vivo These findings suggest that the LPC-mediated lipid signaling is involved in oxaliplatin-induced acute peripheral pain.

A new synthetic protectin D1 analog 3-oxa-PD1n-3 DPA reduces neuropathic pain and chronic itch in mice.

The resolution of inflammation is a biosynthetically active process controlled by the interplay between oxygenated polyunsaturated mediators and G-protein coupled receptor-signaling pathways. These enzymatically oxygenated polyunsaturated fatty acids belong to distinct families of specialized pro-resolving autacoids. The protectin family of mediators has attracted an interest because of their potent pro-resolving and anti-inflammatory actions verified in several in vivo disease models. Herein, we present the stereoselective synthesis and biological evaluations of 3-oxa-PD1n-3 DPA, a protectin D1 analog. Results from mouse models indicate that the mediators protectin D1, PD1n-3 DPA and the new analog 3-oxa-PD1n-3 DPA all relieved streptozotocin-induced diabetic neuropathic pain at doses of 90 and 300 pmol, equivalent to 30 and 100 ng, respectively, following intrathecal (I.T.) injection. Of interest, at a low dose of only 30 pmol (10 ng; I.T.) only 3-oxa PD1n-3 DPA was able to alleviate neuropathic pain, directly compared to vehicle controls. Moreover, using a chronic itch model of cutaneous T-cell lymphoma (CTCL), all three compounds at 300 pmol (100 ng) showed a significant reduction in itching for several hours. The biomolecular information on the structure-functions of the protectins and the new synthetic analog 3-oxa-PD1n-3 DPA is of interest towards developing new immunoresolvents.

Macrophage Toll-like Receptor 9 Contributes to Chemotherapy-Induced Neuropathic Pain in Male Mice.

Chemotherapy-induced peripheral neuropathy (CIPN) remains a pressing clinical problem; however, our understanding of sexual dimorphism in CIPN remains unclear. Emerging studies indicate a sex-dimorphic role of Toll-like receptor 4 (TLR4) in driving neuropathic pain. In this study, we examined the role of TLR9 in CIPN induced by paclitaxel in WT and Tlr9 mutant mice of both sexes. Baseline pain sensitivity was not affected in either Tlr9 mutant male or female mice. Intraplantar and intrathecal injection of the TLR9 agonist ODN 1826 induced mechanical allodynia in both sexes of WT and Tlr4 KO mice but failed to do so in Tlr9 mutant mice. Moreover, Trpv1 KO or C-fiber blockade by resiniferatoxin failed to affect intraplantar ODN 1826-induced mechanical allodynia. Interestingly, the development of paclitaxel-evoked mechanical allodynia was attenuated by TLR9 antagonism or Tlr9 mutation only in male mice. Paclitaxel-induced CIPN caused macrophage infiltration to DRGs in both sexes, and this infiltration was not affected by Tlr9 mutation. Paclitaxel treatment also upregulated TNF and CXCL1 in macrophage cultures and DRG tissues in both sexes, but these changes were compromised by Tlr9 mutation in male animals. Intraplantar adoptive transfer of paclitaxel-activated macrophages evoked mechanical allodynia in both sexes, which was compromised by Tlr9 mutation or by treatment with TLR9 inhibitor only in male animals. Finally, TLR9 antagonism reduced paclitaxel-induced mechanical allodynia in female nude mice (T-cell and B-cell deficient). Together, these findings reveal sex-dimorphic macrophage TLR9 signaling in chemotherapy-induced neuropathic pain.SIGNIFICANCE STATEMENT Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect in cancer patients undergoing clinical chemotherapy treatment regimens. The role of sex dimorphism with regards to the mechanisms of CIPN and analgesia against CIPN remains unclear. Previous studies have found that the infiltration of immune cells, such as macrophages into DRGs and their subsequent activation promote CIPN. Interestingly, the contribution of microglia to CIPN appears to be limited. Here, we show that macrophage TLR9 signaling promotes CIPN in male mice only. This study suggests that pathways in macrophages may be sex-dimorphic in CIPN. Our findings provide new insights into the role of macrophage signaling mechanisms underlying sex dimorphism in CIPN, which may inspire the development of more precise and effective therapies.

Is Optogenetic Activation of Vglut1-Positive Aß Low-Threshold Mechanoreceptors Sufficient to Induce Tactile Allodynia in Mice after Nerve Injury?

Mechanical allodynia is a cardinal feature of pathological pain. Recent work has demonstrated the necessity of Aß-low-threshold mechanoreceptors (Aß-LTMRs) for mechanical allodynia-like behaviors in mice, but it remains unclear whether these neurons are sufficient to produce pain under pathological conditions. We generated a transgenic mouse in which channelrhodopsin-2 (ChR2) is conditionally expressed in vesicular glutamate transporter 1 (Vglut1) sensory neurons (Vglut1-ChR2), which is a heterogeneous population of large-sized sensory neurons with features consistent with Aß-LTMRs. In naive male Vglut1-ChR2 mice, transdermal hindpaw photostimulation evoked withdrawal behaviors in an intensity- and frequency-dependent manner, which were abolished by local anesthetic and selective A-fiber blockade. Surprisingly, male Vglut1-ChR2 mice did not show significant differences in light-evoked behaviors or real-time aversion after nerve injury despite marked hypersensitivity to punctate mechanical stimuli. We conclude that optogenetic activation of cutaneous Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice.SIGNIFICANCE STATEMENT Mechanical allodynia, in which innocuous touch is perceived as pain, is a common feature of pathological pain. To test the contribution of low-threshold mechanoreceptors (LTMRs) to nerve-injury-induced mechanical allodynia, we generated and characterized a new transgenic mouse (Vglut1-ChR2) to optogenetically activate cutaneous vesicular glutamate transporter 1 (Vglut1)-positive LTMRs. Using this mouse, we found that light-evoked behaviors were unchanged by nerve injury, which suggests that activation of Vglut1-positive LTMRs alone is not sufficient to produce pain. The Vglut1-ChR2 mouse will be broadly useful for the study of touch, pain, and itch.