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Phosphodiesterase 4 (PDE4) is highly expressed in keratinocytes and immune cells and promotes pro-inflammatory responses upon activation. The activity of PDE4 has been attributed to various inflammatory conditions, leading to the development and approval of PDE4 inhibitors as host-directed therapeutics in humans. For example, the topical PDE4 inhibitor, crisaborole, is approved for the treatment of mild-to-moderate atopic dermatitis and has shown efficacy in patients with psoriasis. However, the role of crisaborole in regulating the immunopathogenesis of inflammatory skin diseases and infection is not entirely known. Therefore, we evaluated the effects of crisaborole in multiple mouse models, including psoriasis-like dermatitis, AD-like skin inflammation with and without filaggrin mutations, and S. aureus skin infection. We discovered that crisaborole dampens myeloid cells and itch in the skin during psoriasis-like dermatitis. Furthermore, crisaborole was effective in reducing skin inflammation in the context of filaggrin deficiency. Importantly, crisaborole reduced S. aureus skin colonization during AD-like skin inflammation. However, crisaborole was not efficacious in treating S. aureus skin infections, even as adjunctive therapy to antibiotics. Taken together, we found that crisaborole reduced itch during psoriasis-like dermatitis and decreased S. aureus skin colonization upon AD-like skin inflammation, which act as additional mechanisms by which crisaborole dampens the immunopathogenesis in mouse models of inflammatory skin diseases. Further examination is warranted to translate these preclinical findings to human disease.

There is an unmet need to identify new therapeutic targets for temporomandibular disorder (TMD) pain because current treatments are limited and unsatisfactory. TMEM100, a two-transmembrane protein, was recently identified as a regulator to weaken the TRPA1-TRPV1 physical association, resulting in disinhibition of TRPA1 activity in sensory neurons. Recent studies have also shown that Tmem100, Trpa1, and Trpv1 mRNAs were upregulated in trigeminal ganglion (TG) after inflammation of the temporomandibular joint (TMJ) associated tissues. These findings raise a critical question regarding whether TMEM100 in TG neurons is involved in TMD pain via regulating the TRPA1-TRPV1 functional interaction. Here, using two mouse models of TMD pain induced by TMJ inflammation or masseter muscle injury, we found that global knockout or systemic inhibition of TRPA1 and TRPV1 attenuated pain. In line with their increased genes, mice exhibited significant upregulation of TMEM100, TRPA1, and TRPV1 at the protein levels in TG neurons after TMD pain. Importantly, TMEM100 co-expressed with TRPA1 and TRPV1 in TG neurons-innervating the TMJ and masseter muscle and their co-expression was increased after TMD pain. Moreover, the enhanced activity of TRPA1 in TG neurons evoked by TMJ inflammation or masseter muscle injury was suppressed by inhibition of TMEM100. Selective deletion of Tmem100 in TG neurons or local administration of TMEM100 inhibitor into the TMJ or masseter muscle attenuated TMD pain. Together, these results suggest that TMEM100 in TG neurons contributes to TMD pain by regulating TRPA1 activity within the TRPA1-TRPV1 complex. TMEM100 therefore represents a potential novel target-of-interest for TMD pain.

Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represents a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. Yet, the role of TRPC3 in acute and chronic itch is still not well defined. Here, we show that, among mouse trigeminal ganglion (TG) neurons, Trpc3 mRNA is predominantly expressed in nonpeptidergic small diameter TG neurons of mice. Moreover, Trpc3 mRNA signal was present in most presumptively itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch-like and pain-like behaviors in naive mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of contact hypersensitivity (CHS), the Trpc3 mRNA expression level and function were upregulated in the TG after CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch through a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca 2+ -activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of Ano1 in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca 2+ imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of Ano1 in DRG neurons of Ano1 -deficient mice rescued the impaired itching observed in Ano1 -deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.

Mas-related G protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and potential target for the treatment of pain. Positive allosteric modulators (PAMs) of MRGPRX1 have the potential to preferentially activate the receptors at the central terminals of primary sensory neurons and minimize itch side effects caused by peripheral activation. Using a high-throughput screening (HTS) hit, a series of thieno[2,3-d]pyrimidine-based molecules were synthesized and evaluated as human MRGPRX1 PAMs in HEK293 cells stably transfected with human MrgprX1 gene. An iterative process to improve potency and metabolic stability led to the discovery of orally available 6-(tert-butyl)-5-(3,4-dichlorophenyl)-4-(2-(trifluoromethoxy)phenoxy)thieno[2,3-d]pyrimidine (1t), which can be distributed to the spinal cord, the presumed site of action, following oral administration. In a neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI), compound 1t (100 mg/kg, po) reduced behavioral heat hypersensitivity in humanized MRGPRX1 mice, demonstrating the therapeutic potential of MRGPRX1 PAMs in treating neuropathic pain.

Primary sensory neurons in dorsal root ganglia (DRG) are wrapped by satellite glial cells (SGCs), and neuron-SGC interaction may affect somatosensation, especially nociceptive transmission. P2-purinergic receptors (P2Rs) are key elements in the two-way interactions between DRG neurons and SGCs. However, because the cell types are in such close proximity, conventional approaches such as in vitro culture and electrophysiologic recordings are not adequate to investigate the physiologically relevant responses of these cells at a population level. Here, we performed in vivo calcium imaging to survey the activation of hundreds of DRG neurons in Pirt-GCaMP6s mice and to assess SGC activation in GFAP-GCaMP6s mice in situ. By combining pharmacologic and electrophysiologic techniques, we investigated how ganglionic purinergic signaling initiated by α,β-methyleneadenosine 5'-triphosphate (α,β-MeATP) modulates neuronal activity and excitability at a population level. We found that α,β-MeATP induced robust activation of small neurons-likely nociceptors-through activation of P2X3R. Large neurons, which are likely non-nociceptive, were also activated by α,β-MeATP, but with a delay. Blocking pannexin 1 channels attenuated the late phase response of DRG neurons, indicating that P2R stimulation may subsequently induce paracrine ATP release, which could further activate cells in the ganglion. Moreover, ganglionic α,β-MeATP treatment in vivo sensitized small neurons and enhanced responses of spinal wide-dynamic-range neurons to subsequent C-fiber inputs, suggesting that modulation via ganglionic P2R signaling could significantly affect nociceptive neuron excitability and pain transmission. Therefore, targeting functional P2Rs within ganglia may represent an important new strategy for pain modulation.

Mast cells (MCs) are heterogenous tissue-resident immune cells associated with blood vessels and neurons. However, the extent to which specialized MCs can exhibit micro-environment-specific functions is unclear. Using single cell RNA sequencing, we characterize connective tissue-type and mucosal MCs across organs and found that they can be discriminated based on MrgprB2 expression. While MrgprB2+ connective MCs are enriched in neuroreceptors, develop during the embryogenesis and renew independently of the bone marrow (BM); MrgprB2neg mucosal MCs arise postnatally, are sensitive to signals from the microbiome and renewed by BM progenitors. Importantly, MrgprB2+, but not MrgprB2neg MCs are required for food-induced anaphylactic shock. In the gut, MrgprB2+ MCs occur in contact with substance P-secreting enteric neurons and the specific depletion of either MrgprB2+ MCs or substance P results in dysregulation of gut contractions. Thus, we demonstrate that two independent MC populations exist in multiple organs, with different developmental dynamics and biological programming.

Trigeminal neuralgia, historically dubbed the “suicide disease,” is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)–approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.

Itch sensation provokes the scratch reflex to protect us from harmful stimuli in the skin. Although scratching transiently relieves acute itch through activation of mechanoreceptors, it propagates the vicious itch-scratch cycle in chronic itch by further aggravating itch over time. Although well recognized clinically, the peripheral mechanisms underlying the itch-scratch cycle remain poorly understood. Here, we show that mechanical stimulation of the skin results in activation of the Piezo2 channels on Merkel cells that pathologically promotes spontaneous itch in experimental dry skin. Three-dimensional reconstruction and immunoelectron microscopy revealed structural alteration of MRGPRA3 + pruriceptor nerve endings directed toward Merkel cells in the setting of dry skin. Our results uncover a functional miswiring mechanism under pathologic conditions, resulting in touch receptors triggering the firing of pruriceptors in the skin to drive the itch-scratch cycle.

BackgroundAllergic drug reaction or drug allergy is an immunologically mediated drug hypersensitivity reaction (DHR). G-protein coupled receptors (GPCRs) are common drug targets and communicate extracellular signals that initiate cellular responses. Recent evidence shows that GPCR MRGPRX2 is of major importance in IgE-independent pseudo-allergic DHRs based on the suspected interactions between many FDA-approved peptidergic compounds and MRGPRX2.ObjectiveOur aim was to uncover novel MRGPRX2-selective and -potent agonists as drug candidates responsible for clinical features of pseudo-allergic DHRs.MethodsWe conducted a primary high-throughput screening (HTS), coupled with mutagenesis targeting the MRGPRX2 N62S mutation, on a panel of 3,456 library compounds. We discovered pharmacologically active hit compounds as agonists of the MRGPRX2 protein according to high degrees of potency evaluated by the calcium response and validated by the degranulation assay. Using the molecular tool Forge, we also characterized the structure-activity relationship shared by identified hit compounds.ResultsThe alternative allele of single nucleotide polymorphism rs10833049 (N62S) in MRGPRX2 demonstrated loss-of-function property in response to substance P and antineoplastic agent daunorubicin hydrochloride. We applied a unique assay system targeting the N62S mutation to the HTS and identified 84 MRGPRX2-selective active hit compounds representing diverse classes according to primary drug indications. The top five highly represented groups included fluoroquinolone and non-fluoroquinolone antibiotics; antidepressive/antipsychotic; antihistaminic and antineoplastic agents. We classified hit compounds into 14 clusters representing a variety of chemical and drug classes beyond those reported, such as opioids, neuromuscular blocking agents, and fluoroquinolones. We further demonstrated MRGPRX2-dependent degranulation in the human mast cell line LAD2 cells induced by three novel agonists representing the non-fluoroquinolone antibiotics (bacitracin A), anti-allergic agents (brompheniramine maleate) and tyrosine-kinase inhibitors (imatinib mesylate).ConclusionOur findings could facilitate the development of interventions for personalized prevention and treatment of DHRs, as well as future pharmacogenetic investigations of MRGPRX2 in relevant disease cohorts.

Migraine pain is characterized by an intense, throbbing pain in the head area and possesses complex pathological and physiological origins. Among the various factors believed to contribute to migraine are mast cells (MCs), resident tissue immune cells that are closely associated with pain afferents in the meninges. In this review, we aim to examine and discuss recent findings on the individual roles of MCs and the trigeminal nerve in migraine, as well as the various connections between their mechanisms with an emphasis on the contributions those relationships make to migraine. This is seen through MC release of histamine, among other compounds, and trigeminal nerve release of calcitonin-gene-related-peptide (CGRP) and pituitary adenylate cyclase activating peptide-38 (PACAP-38), which are peptides that are thought to contribute to migraine. Secondly, we illustrate the bi-directional relationship of neurogenic inflammation as well as highlight the role of MCs and their effect on the trigeminal nerve in migraine mechanisms. Lastly, we discuss potential new targets for clinical interventions of MC- and trigeminal nerve-mediated migraine, and present future perspectives of mechanistic and translational research.

Pruritus in the setting of cholestatic liver disease is difficult to treat and occurs in patients ranging in age from infancy to adulthood. Likely multifactorial in etiology, this symptom often involves multimodal therapy targeting several pathways and mechanisms proposed in the underlying etiology of cholestatic pruritus. Many patients in both the pediatric and adult populations continue to experience unrelenting pruritus despite maximal conventional therapy. Options are further limited in treating pediatric patients due to sparse data regarding medication safety and efficacy in younger patients. Conventional therapies for the treatment of cholestatic pruritus in children include ursodeoxycholic acid, cholestyramine, hydroxyzine, and rifampin. Certain therapies are more routinely used in the adult populations but with limited data available for use in child and adolescent patients, including opioid antagonists and selective serotonin reuptake inhibitors. Recently, ileal bile acid transport inhibitors have been shown to alleviate pruritus in many children with Alagille syndrome and progressive familial intrahepatic cholestasis and is an additional therapy available for consideration for these patients. Ultimately, surgical options such as biliary diversion or liver transplantation are considered in specific circumstances when medical therapies have been exhausted and pruritus remains debilitating. While further investigation regarding underlying etiologies and effective therapies are needed to better understand itch pathogenesis and treatment in pediatric cholestasis, current considerations beyond conventional management include the use of opioid antagonists, selective serotonin reuptake inhibitors, ileal bile acid transport inhibitors, and surgical intervention.

[Image: see text] The present study describes our continued efforts in the discovery and characterization of a series of 2-sulfonamidebenzamides as allosteric modulators of MrgX1. MrgX1 has been shown to be an attractive target as a nonopioid receptor for the potential treatment of chronic pain. Working from our original compound, ML382, and utilizing iterative medicinal chemistry, we have identified key halogen substituents that improve MrgX1 potency by ∼8-fold. In addition, we have evaluated the compounds in Tier 1 drug metabolism and pharmacokinetics assays and have identified key compounds that impart improved potency and microsomal stability.

Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Type I interferon receptors are expressed by the majority of vagal C-fibre neurons innervating the respiratory tract Interferon alpha and beta acutely and directly activate vagal C-fibers in the airways. The interferon-induced activation of C-fibers occurs secondary to stimulation of type 1 interferon receptors Type 1 interferons may contribute to the symptoms as well as the spread of respiratory viral infections by causing coughing and other defensive reflexes associated with vagal C-fibre activation ABSTRACT: We evaluated the ability of type I interferons to acutely activate airway vagal afferent nerve terminals in mouse lungs. Using single cell RT-PCR of lung-specific vagal neurons we found that IFNAR1 and IFNAR2 were expressed in 70% of the TRPV1-positive neurons (a marker for vagal C-fibre neurons) and 44% of TRPV1-negative neurons. We employed an ex vivo vagal innervated mouse trachea-lung preparation to evaluate the effect of interferons in directly activating airway nerves. Utilizing 2-photon microscopy of the nodose ganglion neurons from Pirt-Cre;R26-GCaMP6s mice we found that applying IFNα or IFNβ to the lungs acutely activated the majority of vagal afferent nerve terminals. When the type 1 interferon receptor, IFNAR1, was blocked with a blocking antibody the response to IFNβ was largely inhibited. The type 2 interferon, IFNγ, also activated airway nerves and this was not inhibited by the IFNAR1 blocking antibody. The Janus kinase inhibitor GLPG0634 (1 μm) virtually abolished the nerve activation caused by IFNβ. Consistent with the activation of vagal afferent C-fibers, infusing IFNβ into the mouse trachea led to defensive breathing reflexes including apneas and gasping. These reflexes were prevented by pretreatment with an IFN type-1 receptor blocking antibody. Finally, using whole cell patch-clamp electrophysiology of lung-specific neurons we found that IFNβ (1000 U ml-1 ) directly depolarized the membrane potential of isolated nodose neurons, in some cases beyond to action potential threshold. This acute non-genomic activation of vagal sensory nerve terminals by interferons may contribute to the incessant coughing that is a hallmark of respiratory viral infections.

The discovery of Mas-related G protein-coupled receptors (MRGPRs) in itch sensation promised a search for novel therapeutics of itch that ultimately met with little success. Recent structural determination of these receptors by Roth and Sun marks a big step forward in the search for therapeutics of debilitating itch.

Background: Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represent a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. However, the role of TRPC3 in acute and chronic itch is still not well defined. Methods: RNAscope in situ hybridization and immunohistochemical staining were performed on mouse trigeminal ganglion (TG) neurons. Fura-2 calcium imaging was used to characterize the function of TRPC3 in dissociated TG neurons. In native mice, the TRPC3 agonist and pruritogens were subcutaneously injected to the cheek and nape of the neck of mice, respectively. Site directed scratching and/or wiping behaviors were video recorded. Contact hypersensitivity (CHS) model was induced in mouse ears by topical application of SADBE or DNCB. Spontaneous scratching behaviors were recorded by video monitoring. Global and conditional Trpc3 knockout mice were employed to determine the contribution of TRPC3 to acute and chronic itch. The mRNA expression levels of Trpc3 and proinflammatory cytokines were assayed by quantitative real-time PCR. H&E. staining was used for the evaluation of the thickness of mouse ears. Flow cytometry was performed to assess immune cell infiltration in mouse ear tissues. Results: Among mouse TG neurons, RNAscope assay revealed that Trpc3 mRNA was predominantly expressed in nonpeptidergic small diameter neurons. Moreover, Trpc3 mRNA signal was present in the majority of itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch- and pain-like behaviors in naïve mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of CHS, Trpc3 mRNA expression level and function were upregulated in the TG following CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. Conclusions: These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch via a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1β and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Nociceptors detect noxious capsaicin (CAPS) via the transient receptor potential vanilloid 1 (TRPV1) ion channel, but coding mechanisms for relaying CAPS concentration [CAPS] remain obscure. Prolonged (up to 1h.) exposure to CAPS is used clinically to desensitise sensory fibres for treatment of neuropathic pain, but its signalling has typically been studied in cultures of dissociated sensory neurons employing low cell numbers and very short exposure times. Thus, it was pertinent to examine responses to longer CAPS exposures in large populations of adult neurons.Confocal fluorescence microscopy was used to monitor the simultaneous excitation by CAPS of neuronal populations in intact L3/4 dorsal root ganglia (DRG) explants from adult pirt-GCaMP3 mice that express a cytoplasmic, genetically-encoded CaExposure for 5 min. to CAPS activated plasmalemmal TRPV1 and led to increased fluorescence due to CaIndividual neurons within DRG differed extensively in the dynamics of response to CAPS, but systematic changes elicited by elevating [CAPS] increased signal density in a graded manner, unveiling a possible mechanism for population coding of responses to noxious chemicals. Signal density is sustained during prolonged and repeated exposure to CAPS, despite profound tachyphylaxis in some neurons, by signal facilitation in others. This may explain the burning sensation that persists for several hours when CAPS is used clinically.

International audience; Allergic skin diseases, such as atopic dermatitis, are clinically characterized by severe itching and type 2 immunity-associated hypersensitivity to widely distributed allergens, including those derived from house dust mites (HDMs). Here we found that HDMs with cysteine protease activity directly activated peptidergic nociceptors, which are neuropeptide-producing nociceptive sensory neurons that express the ion channel TRPV1 and Tac1, the gene encoding the precursor for the neuropeptide substance P. Intravital imaging and genetic approaches indicated that HDM-activated nociceptors drive the development of allergic skin inflammation by inducing the degranulation of mast cells contiguous to such nociceptors, through the release of substance P and the activation of the cationic molecule receptor MRGPRB2 on mast cells. These data indicate that, after exposure to HDM allergens, activation of TRPV1+Tac1+ nociceptor–MRGPRB2+ mast cell sensory clusters represents a key early event in the development of allergic skin reactions.

Intrathecal morphine infusion is often applied to treat chronic pain related to cancer and other conditions. However, persistent pain can be caused by nerve compression because of granuloma formation. In this study, a mouse model of morphine-induced granuloma formation by intrathecal catheterization morphine infusion into the atlanto-occipital membrane of the foramen magnum was established in wild-type mice, MrgprB2 mutant (MrgprB2−/−) mice, and in mast cell–deficient W-sash c-kit mutant (KitW-sh/W-sh) mice. Heat-related pain after surgery was performed to investigate the antipain effect of morphine. H&E staining and immunofluorescence staining of the spinal cord were applied to analyze the mechanism of granuloma formation. Morphine-induced mast cell degranulation was assessed by measuring the Ca2+ influx and mediator release. Anaphylactoid reactions were measured after s.c. morphine infusion to the paws. Chemokine release by mast cells was determined by Human XL Cytokine Array. Experiments with wild-type, MrgprB2 mutant, and mast cell–deficient W-sash c-kit mutant mice demonstrated that morphine activated mast cells and inflammatory cell aggregation through MrgprB2 in intrathecal infusion sites. The chemokine production of human mast cells demonstrated that granuloma formation is correlated with chemokines release. In addition, morphine activated mouse primary mast cells and de novo chemokine synthesis via the MRGPRX2 in human LAD2 cells. We concluded that granuloma formation during intrathecal morphine infusion was associated with MrgprB2/X2. Reducing MRGPRX2 potentially blocks morphine-induced side effects, including granuloma formation.

The continuous advancement of microscopic imaging techniques combined with the discovery and use of more powerful calcium indicators has made calcium imaging technology much more effective and has increased its use in the study of pain circuitry. Using calcium imaging to study spinal pain mechanisms causes less damage to animals compared to electrophysiological techniques and is also able to observe the firing pattern of spinal neurons and the connections between them on a large scale. These advantages allow any changes in spinal cord circuits caused by pain transmission to be observed more effectively. This review will discuss the development of calcium indicators over the past decades as well as the various applications of calcium imaging, from in vitro to in vivo spinal cord experiments, in the study of pain circuits. We will also discuss possible directions for the study of spinal pain circuits in the future.

Patients suffering from cholestasis, the slowing or stoppage of bile flow, commonly report experiencing an intense, chronic itch. Numerous pruritogens are up-regulated in cholestatic patient sera, including bile acids (BAs). Acute injection of BAs results in itch in both mice and humans, and BA-modulating therapy is effective in controlling patient itch. Here, we present evidence that human sensory neuron-expressed Mas-related G protein-coupled receptor X4 (MRGPRX4), an orphan member of the Mrgpr family of GPCRs, is a BA receptor. Using Ca(2+) imaging, we determined that pathophysiologically relevant levels of numerous BAs activated MRGPRX4. No mouse Mrgpr orthologs were activated by BAs. To assess the in vivo relevance of BA activation of MRGPRX4, we generated a humanized mouse with targeted expression of MRGPRX4 in itch-encoding sensory neurons. BAs activated MRGPRX4(+) sensory neurons at higher levels compared with WT neurons. Compared with control animals, MRGPRX4(+) mice scratched more upon acute injection of BAs and in a model of cholestatic itch. Overall, these data suggest that targeting MRGPRX4 is a promising strategy for alleviating cholestatic itch.

Mammals possess a remarkable ability to sense subtle temperature deviations from the thermoneutral skin temperature of ~33 °C, which ensures precise warm sensation. However, the underlying mechanisms remain unclear. Here we show that STIM1, an endoplasmic reticulum (ER) resident transmembrane protein that responds to both ER Ca2+ depletion and heat, mediates temperature-induced Ca2+ influx in skin keratinocytes via coupling to Orai Ca2+ channels in plasma membrane. Behaviorally, the keratinocyte-specific knockout of STIM1 shifts the optimal preference temperature (OPT) of mice from ~32 °C to ~34 °C, resulting in a strikingly reversed preference between 32 °C and 34 °C. Importantly, the thermally inactive STIM1-ΔK knock-in mice show altered OPT and warm preference behaviors as well, demonstrating the requirement of STIM1 thermosensitivity for warm sensation. Furthermore, the wild-type and mutant mice prefer temperatures closer to their respective OPTs, but poorly distinguish temperatures that are equally but oppositely deviated from their OPTs. Mechanistically, keratinocyte STIM1 affects the in vivo warm responses of sensory neurons by likely involving TRPA1 as a downstream transduction channel. Collectively, our data suggest that STIM1 serves as a novel in vivo thermosensor in keratinocytes to define the OPT, which might be utilized as a peripheral reference temperature for precise warm sensation.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that epithelial cell-derived antimicrobial peptides defensins activate orphan GPCRs Mrgpra2a/b on neutrophils. This signaling axis is required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Furthermore, we found that defensins and Mrgpra2 are critical for combatting S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggers neutrophils to release the pro-inflammatory cytokine IL-1β, which is vital for proper amplification and propagation of the antibacterial immune response. This study demonstrates the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

Chronic kidney disease is a disease with decreased, irreversible renal function. Pruritus is the most common skin symptom in patients with chronic kidney disease, especially in end-stage renal disease (AKA chronic kidney disease-associated pruritus [CKD-aP]); however, the underlying molecular and neural mechanism of the CKD-aP in patients remains obscure. Our data show that the level of allantoin increases in the serum of CKD-aP and CKD model mice. Allantoin could induce scratching behavior in mice and active DRG neurons; the calcium influx and the action potential were significantly reduced in DRG neurons of MrgprD KO or TRPV1 KO mice. U73122, an antagonist of PLC, could also block calcium influx in DRG neurons induced by allantoin. Thus, our results concluded that allantoin plays an important role in CKD-aP, mediated by MrgprD and TrpV1, in CKD patients.

Significance Odorant sensing GPCRs are the largest gene family in the human genome. We previously found multiple olfactory receptors and their obligate downstream effectors expressed in the smooth muscle of human bronchi. However, the extent to which odorant-sensing receptors (and the ligands to which they respond) on airway smooth muscle (ASM) are physiologically relevant is not established. Here we show that a monoterpene nerol activates the odorant receptor OR2W3 to relax ASM in both cell and tissue models. Surprisingly, the mechanism of action of OR2W3-mediated ASM relaxation involves paradoxical increases in [Ca2+]i that invoke a cooperative activation of TMEM16A and CFTR to compartmentalize calcium and regulate excitation-contraction coupling in human ASM cells., The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases.

We present heightened skin reactivity in vivo to two MRGPRX2 drug ligands in patients with chronic spontaneous urticaria. Selectivity for the MRGPRX2 pathway by drug ligands was demonstrated by the loss of drug activation in MRGPRX2-KO LAD2 cells.

Background Mas gene–related G protein–coupled receptors (MRGPRs) are a G protein–coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. Objectives Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. Methods To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. Results We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. Conclusion Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.

Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Pain is the most prominent symptom of osteoarthritis (OA) progression. However, the relationship between pain and OA progression remains largely unknown. Here we report osteoblast secret prostaglandin E2 (PGE2) during aberrant subchondral bone remodeling induces pain and OA progression in mice. Specific deletion of the major PGE2 producing enzyme cyclooxygenase 2 (COX2) in osteoblasts or PGE2 receptor EP4 in peripheral nerve markedly ameliorates OA symptoms. Mechanistically, PGE2 sensitizes dorsal root ganglia (DRG) neurons by modifying the voltage-gated sodium channel NaV1.8, evidenced by that genetically or pharmacologically inhibiting NaV1.8 in DRG neurons can substantially attenuate OA. Moreover, drugs targeting aberrant subchondral bone remodeling also attenuates OA through rebalancing PGE2 production and NaV1.8 modification. Thus, aberrant subchondral remodeling induced NaV1.8 neuronal modification is an important player in OA and is a potential therapeutic target in multiple skeletal degenerative diseases., eLife digest Many people will suffer from joint pain as they age, particularly in their knees. The most common cause of this pain is osteoarthritis, a disease that affects a tissue inside joints called cartilage. In a healthy knee, cartilage acts as a shock absorber. It cushions the ends of bones and enables them to move smoothly against one another. But in osteoarthritis, cartilage gradually wears away. As a result, the bones within a joint rub against each other whenever a person moves. This makes activities such as running or climbing stairs painful. But how does this pain arise? Previous work has implicated cells called osteoblasts. Osteoblasts are found in the area of the bone just below the cartilage. They produce new bone tissue throughout our lives, enabling our bones to regenerate and repair. Each time we move, forces acting on the knee joint activate osteoblasts. The cells respond by releasing a key molecule called PGE2, which is a factor in pain pathways. The joints of people with osteoarthritis produce too much PGE2. But exactly how this leads to increased pain sensation has been unclear. Zhu et al. now complete this story by working out how PGE2 triggers pain. Experiments in mice reveal that PGE2 irritates the nerve fibers that carry pain signals from the knee joint to the brain. It does this by activating a channel protein called Nav1.8, which allows sodium ions through the membranes of those nerve fibers. Zhu et al. show that, in a mouse model of osteoarthritis, Nav1.8 opens too widely in response to binding of PGE2, so the nerve cells become overactive and transmit a stronger pain sensation. This means that even small movements cause intense pain signals to travel from the joints to the brain. Building on their findings, Zhu et al. developed a drug that acts directly on bone to reduce PGE2 production, and show that this drug reduces pain in mice with osteoarthritis. At present, there are no treatments that reverse the damage that occurs during osteoarthritis, but further testing will determine whether this new drug could one day relieve joint pain in patients.

Itch is a unique sensation that helps organisms scratch away external threats; scratching itself induces an immune response that can contribute to more itchiness. Itch is induced chemically in the peripheral nervous system via a wide array of receptors. Given the superficial localization of itch neuron terminals, cells that dwell close to the skin contribute significantly to itch. Certain mechanical stimuli mediated by recently discovered circuits also contribute to the itch sensation. Ultimately, in the spinal cord, and likely in the brain, circuits that mediate touch, pain, and itch engage in cross modulation. Much of itch perception is still a mystery, but we present in this review the known ligands and receptors associated with itch. We also describe experiments and findings from investigations into the spinal and supraspinal circuitry responsible for the sensation of itch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Summary Spontaneous pain refers to pain occurring without external stimuli. It is a primary complaint in chronic pain conditions and remains difficult to treat. Moreover, the mechanisms underlying spontaneous pain remain poorly understood. Here we employed in vivo imaging of dorsal root ganglion (DRG) neurons and discovered a distinct form of abnormal spontaneous activity following peripheral nerve injury: clusters of adjacent DRG neurons firing synchronously and sporadically. The level of cluster firing correlated directly with nerve injury-induced spontaneous pain behaviors. Furthermore, we demonstrated that cluster firing is triggered by activity of sympathetic nerves, which sprout into DRGs after injury, and identified norepinephrine as a key neurotransmitter mediating this unique firing. Chemogenetic and pharmacological manipulations of sympathetic activity and norepinephrine receptors suggest that they are necessary and sufficient for DRG cluster firing and spontaneous pain behavior. Therefore, blocking sympathetically mediated cluster firing may be a new paradigm for treating spontaneous pain.

Intraperitoneal administration of the melanocortin agonist melanotan II (MTII) to mice causes a profound, transient hypometabolism/hypothermia. It is preserved in mice lacking any one of melanocortin receptors 1, 3, 4, or 5, suggesting a mechanism independent of the canonical melanocortin receptors. Here we show that MTII-induced hypothermia was abolished in KitW-sh/W-sh mice, which lack mast cells, demonstrating that mast cells are required. MRGPRB2 is a receptor that detects many cationic molecules and activates mast cells in an antigen-independent manner. In vitro, MTII stimulated mast cells by both MRGPRB2-dependent and -independent mechanisms, and MTII-induced hypothermia was intact in MRGPRB2-null mice. Confirming that MTII activated mast cells, MTII treatment increased plasma histamine levels in both wild-type and MRGPRB2-null, but not in KitW-sh/W-sh, mice. The released histamine produced hypothermia via histamine H1 receptors because either a selective antagonist, pyrilamine, or ablation of H1 receptors greatly diminished the hypothermia. Other drugs, including compound 48/80, a commonly used mast cell activator, also produced hypothermia by both mast cell-dependent and -independent mechanisms. These results suggest that mast cell activation should be considered when investigating the mechanism of drug-induced hypothermia in mice.

Several reports support the idea that μ- and δ-opioid receptors (ORs) may exist as heterodimers in brain regions involved in pain signaling. The unique pharmacology of these heteromers may present a novel analgesic target. However, the role of μ-δ heteromers in sensory neurons involved in pain and opioid analgesia remains unclear, particularly during neuropathic pain. We examined the effects of spinal nerve injury on μ-δ heteromer expression in dorsal root ganglion (DRG) neurons and the effects of a μ-δ heteromer–targeting agonist, CYM51010, on neuropathic pain behavior in rats and mice. An L5 spinal nerve ligation (SNL) in rats significantly decreased μ-δ heteromer expression in L5 DRG, but increased heteromer levels in uninjured L4 DRG. Importantly, in SNL rats, subcutaneous (s.c.) injection of CYM51010 inhibited mechanical hypersensitivity in a dose-related manner (EC(50): 1.09 mg/kg) and also reversed heat hyperalgesia and attenuated ongoing pain (2 mg/kg, s.c.). HEK-293T cells surface-labeled with μ- and δ-ORs internalized both receptors after exposure to CYM51010. In contrast, in cells transfected with μ-OR alone, CYM51010 was significantly less effective at inducing receptor internalization. Electrophysiologic studies showed that CYM51010 inhibited the C-component and windup phenomenon in spinal wide-dynamic range neurons of SNL rats. The pain inhibitory effects of CYM51010 persisted in morphine-tolerant rats, but was markedly attenuated in μ-OR knockout mice. Our studies show that spinal nerve injury may increase μ-δ heterodimerization in uninjured DRG neurons and that μ-δ heteromers may be a potential therapeutic target for relieving neuropathic pain, even under conditions of morphine tolerance.

International audience; Recent studies revealed the existence of unique functional links between mast cells and nociceptors in the skin. Here, we propose that mast cells and nociceptors form a single regulatory unit in both physiology and disease. In this model, MrgprB2/X2 signaling is a primary mechanism by which mast cells functionally interact with nociceptors to form specialized neuroimmune clusters that regulate pain, inflammation, and itch.

Itch is a unique sensory experience that is encoded by genetically distinguishable neurons both in the peripheral nervous system (PNS) and central nervous system (CNS) to elicit a characteristic behavioral response (scratching). Itch interacts with the other sensory modalities at multiple locations, from its initiation in a particular dermatome to its transmission to the brain where it’s finally perceived. In this review, we summarize the current understanding of the molecular and neural mechanisms of itch by starting in the periphery, where itch is initiated and perceived, and discussing the circuits involved in itch processing in the CNS.

Asthma, accompanied by lung inflammation, bronchoconstriction and airway hyper-responsiveness, is a significant public health burden. Here we report that Mas-related G protein-coupled receptors (Mrgprs) are expressed in a subset of vagal sensory neurons innervating the airway and mediates cholinergic bronchoconstriction and airway hyper-responsiveness. These findings provide insights into the neural mechanisms underlying the pathogenesis of asthma.