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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.

Cytokines and chemokines play diverse roles in different organ systems. Family with sequence similarity 19, member A1-5 (FAM19A1-A5; also known as TAFA1-5) is a group of conserved chemokine-like proteins enriched in the CNS of mice and humans. Their functions are only beginning to emerge. Here, we show that the expression of Fam19a1-a5 in different mouse brain regions are induced or suppressed by unfed and refed states. The striking nutritional regulation of Fam19a family members in the brain suggests a potential central role in regulating metabolism. Using a knockout (KO) mouse model, we show that loss of FAM19A1 results in sexually dimorphic phenotypes. In male mice, FAM19A1 deficiency alters food intake patterns during the light and dark cycle. Fam19a1 KO mice are hyperactive, and locomotor hyperactivity is more pronounced in female KO mice. Behavior tests indicate that Fam19a1 KO female mice have reduced anxiety and sensitivity to pain. Spatial learning and exploration, however, is preserved in Fam19a1 KO mice. Altered behaviors are associated with elevated norepinephrine and dopamine turnover in the striatum. Our results establish an in vivo function of FAM19A1 and highlight central roles for this family of neurokines in modulating animal physiology and behavior.-Lei, X., Liu, L., Terrillion, C. E., Karuppagounder, S. S., Cisternas, P., Lay, M., Martinelli, D. C., Aja, S., Dong, X., Pletnikov, M. V., Wong, G. W. FAM19A1, a brain-enriched and metabolically responsive neurokine, regulates food intake patterns and mouse behaviors.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene–related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an “itch cocktail” augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders., TGR5, MrgprA3, and MrgprC11, receptors normally associated with pruritis, are expressed by colonic afferents and, when activated, induce visceral hypersensitivity relevant to irritable bowel syndrome.

Cutaneous TRPV1(+) neurons directly sense noxious stimuli, inflammatory cytokines and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1(+) neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1(+) neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1(+) neuron activation was sufficient to elicit a local Type-17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited Type-17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1(+) neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.

Mas-related G-protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and has been actively investigated as a therapeutic target for the treatment of pain. By use of two HTS screening hit compounds, 4-(4-(benzyloxy)-3-methoxybenzylamino)benzimidamide (5a) and 4-(2-(butylsulfonamido)-4-methylphenoxy)benzimidamide (11a), as molecular templates, a series of human MRGPRX1 agonists were synthesized and evaluated for their agonist activity using HEK293 cells stably transfected with human MrgprX1. Conversion of the benzamidine moiety into a 1-aminoisoquinoline moiety carried out in the later stage of structural optimization led to the discovery of a highly potent MRGPRX1 agonist, N-(2-(1-aminoisoquinolin-6-yloxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (16), not only devoid of positively charged amidinium group but also with superior selectivity over opioid receptors. In mice, compound 16 displayed favorable distribution to the spinal cord, the presumed site of action for the MRGPRX1-mediated analgesic effects.

Asthma is a chronic allergic inflammatory airway disease caused by aberrant immune responses to inhaled allergens, which leads to airway hyperresponsiveness (AHR) to contractile stimuli and airway obstruction. Blocking T helper 2 (T(H)2) differentiation represents a viable therapeutic strategy for allergic asthma, and strong TCR-mediated ERK activation blocks T(H)2 differentiation. Here, we report that targeting diacylglycerol (DAG) kinase zeta (DGKζ), a negative regulator of DAG-mediated cell signaling, protected against allergic asthma by simultaneously reducing airway inflammation and AHR though independent mechanisms. Targeted deletion of DGKζ in T cells decreased type 2 inflammation without reducing AHR. In contrast, loss of DGKζ in airway smooth muscle cells decreased AHR but not airway inflammation. T cell–specific enhancement of ERK signaling was only sufficient to limit type 2 airway inflammation, not AHR. Pharmacological inhibition of DGK diminished both airway inflammation and AHR in mice and also reduced bronchoconstriction of human airway samples in vitro. These data suggest that DGK is a previously unrecognized therapeutic target for asthma and reveal that the inflammatory and AHR components of asthma are not as interdependent as generally believed.

Pseudomonas aeruginosa can cause serious persistent infections such as ventilator-associated pneumonia, sepsis, biofilm-related infections as in cystic fibrosis (CF) patients. Although CF lung infections can be treated with antibiotics, full clearance is difficult due to P. aeruginosa persistence. While antibiotic activity against growing P. aeruginosa is well documented, their activity against the non-growing persisters enriched in stationary phase cultures has not been well studied. Here, we systematically evaluated and ranked the six major classes of antibiotics, cell wall and cell membrane inhibitors, protein synthesis inhibitors, DNA synthesis inhibitors, RNA synthesis inhibitors, sulfa drugs, and nitrofurantoin, for their activity against both growing and persister forms of P. aeruginosa using colony forming count (CFU) and SYBR Green I/Propidium Iodide (PI) viability assay. Among the six major classes of antibiotics, cell wall and cell membrane inhibitors (Cefuroxime and Colistin), DNA synthesis inhibitors (Clinafloxacin) and sulfa drugs (Sulfamethoxazole) had good activity against stationary phase cells. In contrast, protein synthesis inhibitors (Gentamicin), RNA synthesis inhibitor (Rifampicin) and Nitrofurantoin had relatively poor activity against the stationary phase P. aeruginosa but relatively high activity against log phase P. aeruginosa. Clinafloxacin is the only single drug that could completely kill all (109 CFU) stationary phase cells in a 4 day drug exposure. The Cefuroxime + Gentamicin+ Clinafloxacin combination could kill all biofilm bacteria in 2 days whereas the clinically used drug combination Cefuroxime + Gentamicin + Colistin only partially killed the biofilm bacteria with 103 CFU remaining. In a murine persistent CF lung infection model, only Cefuroxime + Gentamicin+ Clinafloxacin cleared all bacteria in the infected lungs, whereas Clinafloxacin alone, or Cefuroxime + Clinafloxacin, or the current recommended drug combination Cefuroxime + Gentamicin, all failed to completely clear the bacterial load in the lungs. The complete sterilization of the bacterial load is a property of Clinafloxacin combination, as Cefuroxime + Gentamicin+ Levofloxacin combination was unable to clear the bacterial load in the lungs. Our findings demonstrate the importance of persister drug clinafloxacin, offer new therapeutic approaches for more effective treatment of persistent P. aeruginosa infections, and may have implications for treating other persistent infections.

Although joint pain in rheumatoid arthritis (RA) is conventionally thought to result from inflammation, arthritis pain and joint inflammation are at least partially uncoupled. This suggests that additional pain mechanisms in RA remain to be explored. Here we show that FcγRI, an immune receptor for IgG immune complex (IgG-IC), is expressed in a subpopulation of joint sensory neurons and that, under naive conditions, FcγRI crosslinking by IgG-IC directly activates the somata and peripheral terminals of these neurons to evoke acute joint hypernociception without obvious concurrent joint inflammation. These effects were diminished in both global and sensory neuron-specific Fcgr1 knockout mice. In murine models of inflammatory arthritis, FcγRI signaling was upregulated in joint sensory neurons. Acute blockade or global genetic deletion of Fcgr1 significantly attenuated arthritis pain and hyperactivity of joint sensory neurons without measurably altering joint inflammation. Conditional deletion of Fcgr1 in sensory neurons produced similar analgesic effects in these models. We therefore suggest that FcγRI expressed in sensory neurons contributes to arthritis pain independently of its functions in inflammatory cells. These findings expand our understanding of the immunosensory capabilities of sensory neurons and imply that neuronal FcγRI merits consideration as a target for treating RA pain.

Spinal pain is a major clinical problem, however, its origins and underlying mechanisms remain unclear. Here we report that in mice, osteoclasts induce sensory innervation in the porous endplates which contributes to spinal hypersensitivity in mice. Sensory innervation of the porous areas of sclerotic endplates in mice was confirmed. Lumbar spine instability (LSI), or aging, induces spinal hypersensitivity in mice. In these conditions, we show that there are elevated levels of PGE2 which activate sensory nerves, leading to sodium influx through Nav 1.8 channels. We show that knockout of PGE2 receptor 4 in sensory nerves significantly reduces spinal hypersensitivity. Inhibition of osteoclast formation by knockout Rankl in the osteocytes significantly inhibits LSI-induced porosity of endplates, sensory innervation, and spinal hypersensitivity. Knockout of Netrin-1 in osteoclasts abrogates sensory innervation into porous endplates and spinal hypersensitivity. These findings suggest that osteoclast-initiated porosity of endplates and sensory innervation are potential therapeutic targets for spinal pain., Spinal pain is a major clinical problem. Here the authors show that osteoclasts create porous area of endplates of the vertebrae and sensory innervation of porous endplates by Netrin-1 release from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

T cell cytokines contribute to immunity against Staphylococcus aureus , but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 . However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6 + T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6 + Vδ4 + T cells in immunity against S. aureus skin infections.

Summary Classical itch studies have focused on immunoglobulin E (IgE)-mediated mast cell activation and histamine release. Recently, members of the Mas-related G-protein-coupled receptor (Mrgpr) family have been identified as mast cell receptors, but their role in itch is unclear. Here, we report that mast cell activation via Mrgprb2 evoked non-histaminergic itch in mice independently of the IgE-Fc epsilon RI (FceRI)-histamine axis. Compared with IgE-FceRI stimulation, Mrgprb2 activation of mast cells was distinct in both released substances (histamine, serotonin, and tryptase) and the pattern of activated itch-sensory neurons. Mrgprb2 deficiency decreased itch in multiple preclinical models of allergic contact dermatitis (ACD), a pruritic inflammatory skin disorder, and both mast cell number and PAMP1-20 concentrations (agonist of the human Mrgprb2 homolog, MRGPRX2) were increased in human ACD skin. These findings suggest that this pathway may represent a therapeutic target for treating ACD and mast-cell-associated itch disorders in which antihistamines are ineffective.

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders like atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. Recent discoveries have implicated type 2 cell-associated cytokines such as IL-4, IL-13, and IL-31 in directly stimulating sensory neurons to promote chronic itch in AD. However, in addition to chronic itch, patients often experience intense acute itch exacerbations. Whether this form of itch represents amplification of known itch pathways or a different mechanism remains overlooked. Herein, we show that a large proportion of patients with AD harbor allergen-specific IgE and exhibit a propensity for acute itch flares. In mice, we found that while acute itch is mediated by the classical mast cell-histamine axis in steady-state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene receptor (CysLTR2) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a novel basophil-neuronal circuit that may underlie a variety of pathological neuroimmune processes.

Primary sensory neurons in the DRG play an essential role in initiating pain by detecting painful stimuli in the periphery. Tissue injury can sensitize DRG neurons, causing heightened pain sensitivity, often leading to chronic pain. Despite the functional importance, how DRG neurons function at a population level is unclear due to the lack of suitable tools. Here we developed an imaging technique that allowed us to simultaneously monitor the activities of >1,600 neurons/DRG in live mice and discovered a striking neuronal coupling phenomenon that adjacent neurons tend to activate together following tissue injury. This coupled activation occurs among various neurons and is mediated by an injury-induced upregulation of gap junctions in glial cells surrounding DRG neurons. Blocking gap junctions attenuated neuronal coupling and mechanical hyperalgesia. Therefore, neuronal coupling represents a new form of neuronal plasticity in the DRG and contributes to pain hypersensitivity by “hijacking” neighboring neurons through gap junctions.

Neurons derived from human pluripotent stem cells (hPSCs) are powerful tools for studying human neural development and diseases. Robust functional coupling of hPSC-derived neurons with target tissues in vitro is essential for modeling intercellular physiology in a dish and to further translational studies, but it has proven difficult to achieve. Here, we derive sympathetic neurons from hPSCs and show that they can form physical and functional connections with cardiac muscle cells. Using multiple hPSC reporter lines, we recapitulated human autonomic neuron development in vitro and successfully isolated PHOX2B::eGFP+ neurons that exhibit sympathetic marker expression and electrophysiological properties and norepinephrine secretion. Upon pharmacologic and optogenetic manipulation, PHOX2B::eGFP+ neurons controlled beating rates of cardiomyocytes, and the physical interactions between these cells increased neuronal maturation. This study provides a foundation for human sympathetic neuron specification and for hPSC-based neuronal control of organs in a dish.

Itch is described as an unpleasant or irritating skin sensation that elicits the desire or reflex to scratch. MrgprA3, one of members of the Mrgprs family, is specifically expressed in a subpopulation of dorsal root ganglion (DRG) in the peripheral nervous system (PNS). These MrgprA3-expressing DRG neurons have been identified as itch-specific neurons. They can be activated by the compound, chloroquine, which is used as a drug to treat malaria. In the present study, we labeled these itch-specific neurons using the method of molecular genetic markers, and then studied their electrophysiological properties. We also recorded the cutaneous MrgprA3(-) neurons retrogradely labeled by Dil dye (MrgprA3(-)-Dil). We first found that MrgprA3(+) neurons have a lower excitability than MrgprA3(-) neurons (MrgprA3(-)-non-Dil and MrgprA3(-)-Dil). The number of action potential (AP) was reduced more obviously in MrgprA3(+) neurons than that of in MrgprA3(-) neurons. In most cases, MrgprA3(+) neurons only generated single AP; however, in MrgprA3(-) neurons, the same stimulation could induce multiple AP firing due to the greater voltage-gated potassium (Kv) current existence in MrgprA3(+) than in MrgprA3(-) neurons. Thus, Kv current plays an important role in the regulation of excitability in itch-specific neurons.

Background Opioids have long been regarded as the most effective drugs for the treatment of severe acute and chronic pain. Unfortunately, their therapeutic efficacy and clinical utility have been limited because of central and peripheral side effects. Methods To determine the therapeutic value of peripheral μ-opioid receptors as a target for neuropathic pain treatment, the authors examined the effects of dermorphin [d-Arg2, Lys4] (1–4) amide (DALDA), a hydrophilic, peripherally acting μ-opioid receptor agonist, in male and female rats with spinal nerve ligation–induced neuropathic pain. The authors also utilized behavioral, pharmacologic, electrophysiologic, and molecular biologic tools to characterize DALDA’s possible mechanisms of action in male rats. Results DALDA, administered subcutaneously, had 70 times greater efficacy for inhibiting thermal (n = 8 to 11/group) than mechanical hypersensitivity (n = 6 to 8/group) in male rats. The pain inhibitory effects of DALDA on mechanical and heat hypersensitivity were abolished in animals pretreated with systemic methylnaltrexone (n = 7 to 9/group), a peripheral μ-opioid receptor antagonist. In the spinal wide-dynamic range neurons, systemic DALDA inhibited C-fiber–mediated, but not A-fiber–mediated, response in neuropathic male rats (n = 13). In primary sensory neurons, DALDA inhibited the capsaicin-induced [Ca2+] increase more than the β-alanine–induced [Ca2+] increase (n = 300); capsaicin and β-alanine activate subpopulations of neurons involved in the signaling of heat and mechanical pain, respectively. DALDA-treated rats (n = 5 to 8/group) did not exhibit motor deficits and locomotor impairment suggesting that it does not induce central side effects. Conclusions These findings suggest that DALDA may represent a potential alternative to current opioid therapy for the treatment of neuropathic pain and is likely to be associated with minimal adverse effects.

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.

Patients suffering from cholestasis often report experiencing a debilitating, unrelenting itch. In contrast to conditions, such as urticaria, in which histamine primarily drives itch (pruritus), cholestatic pruritus is multifactorial and more difficult to treat. Existing therapies are not always effective and have undesirable adverse effect profiles. Here, we conducted a systematic literature review to evaluate conventional treatment strategy, current pathophysiologic understanding, and the role of new therapies in the context of cholestatic pruritus. We discuss novel findings implicating bile acids, lysophosphatidic acid, and bilirubin as potential important mediators of cholestatic itch. New therapies that aim to remove or modulate pruritogens have been supported in observational cohort studies and randomized controlled trials. Although these new therapies show promise, further research is needed to confirm the pathophysiology of cholestatic pruritus so that targeted therapy can be developed.

Key points Sphingosine-1-phosphate (S1P) strongly activates mouse vagal C-fibres in the airways. Airway-specific nodose and jugular C-fibre neurons express mRNA coding for the S1P receptor S1PR3. S1P activation of nodose C-fibres is inhibited by a S1PR3 antagonist. S1P activation of nodose C-fibres does not occur in S1PR3 knockout mice. Abstract We evaluated the effect of sphingosine-1-phosphate (S1P), a lipid that is elevated during airway inflammatory conditions like asthma, for its ability to stimulate vagal afferent C-fibres in mouse lungs. Single cell RT-PCR on lung-specific vagal afferent neurons revealed that both TRPV1-expressing and TRPV1-non-expressing nodose neurons express mRNA coding for the S1P receptor S1PR3. TRPV1-expressing airway-specific jugular ganglion neurons also express S1PR3 mRNA. S1PR1 and S1PR2 mRNAs were also found to be expressed but only in a limited subset (32% and 22%, respectively) of airway-specific vagal sensory neurons; whereas S1PR4 and S1PR5 were rarely expressed. We used large scale two-photon imaging of the nodose ganglia from our ex vivo preparation isolated from Pirt-Cre;R26-GCaMP6s transgenic mice, which allows for simultaneous monitoring of calcium transients in ∼1000 neuronal cell bodies in the ganglia during tracheal perfusion with S1P (10 μM). We found that S1P in the lungs strongly activated 81.5% of nodose fibres, 70% of which were also activated by capsaicin. Single fibre electrophysiological recordings confirmed that S1P evoked action potential (AP) generation in a concentration-dependent manner (0.1-10 μM). Action potential generation by S1P in nodose C-fibres was effectively inhibited by the S1PR3 antagonist TY 52156 (10 μM). Finally, in S1PR3 knockout mice, S1P was not able to activate any of the airway nodose C-fibres analysed. These results support the hypothesis that S1P may play a role in evoking C-fibre-mediated airway sensations and reflexes that are associated with airway inflammatory diseases.

Neuropathic pain remains a therapeutic challenge because of its complicated mechanisms. Mas-related GPCR D (MrgprD) is specifically expressed in small-diameter, nociceptive neurons of dorsal root ganglia (DRGs) and is implicated in pain modulation. However, the underlying mechanism of MrgprD involved in neuropathic pain remains elusive. In this study, we used behavioral experiments and physiologic examination methods to investigate the role of MrgprD in chronic constriction injury (CCI)-induced neuropathic pain. We found that MrgprD is necessary for the initiation of mechanical hypersensitivity and cold allodynia, but not for heat allodynia. Moreover, we demonstrated that transient receptor potential cation channel (TRP)-A1 was the ion channel downstream of MrgprD, and the β-alanine-induced calcium signal was attributed mostly to TRP-A1 function. We further showed that PKA serves as a downstream mediator of β-alanine-activated MrgprD signaling to activate TRP-A1 in DRG neurons and in human embryonic kidney 293 cells, to coexpress MrgprD and TRP-A1 plasmids. Finally, we found that the β-alanine-induced pain behavior was increased, whereas the itching behavior was unchanged in CCI models compared with sham-injured animals. Knockout of TRPA1 also attenuated the β-alanine-induced pain behavior in CCI models. In conclusion, MrgprD is essential in cold allodynia in CCI-induced neuropathic pain through the PKA-TRP-A1 pathway. TRP-A1 facilitates MrgprD to development of neuropathic pain. Our findings reveal a novel mechanism of neuropathic pain formation and highlight MrgprD as a promising drug target for the treatment of neuropathic pain.-Wang, C., Gu, L., Ruan, Y., Geng, X., Xu, M., Yang, N., Yu, L., Jiang, Y., Zhu, C., Yang, Y., Zhou, Y., Guan, X., Luo, W., Liu, Q., Dong, X., Yu, G., Lan, L., Tang, Z. Facilitation of MrgprD by TRP-A1 promotes neuropathic pain.

Sophorae Flavescentis Radix (SFR) is a medicinal herb with many functions that are involved in anti-inflammation, antinociception, and anticancer. SFR is also used to treat a variety of itching diseases. Matrine (MT) is one of the main constituents in SFR and also has the effect of relieving itching, but the antipruritic mechanism is still unclear. Here, we investigated the effect of MT on anti-pruritus. In acute and chronic itch models, MT significantly inhibited the scratching behavior not only in acute itching induced by histamine (His), chloroquine (CQ) and compound 48/80 with a dose-depended manner, but also in the chronic pruritus models of atopic dermatitis (AD) and acetone-ether-water (AEW) in mice. Furthermore, MT could be detected in the blood after intraperitoneal injection (i.p.) and subcutaneous injection (s.c.). Finally, electrophysiological and calcium imaging results showed that MT inhibited the excitatory synaptic transmission from dorsal root ganglion (DRG) to the dorsal horn of the spinal cord by suppressing the presynaptic N-type calcium channel. Taken together, we believe that MT is a novel drug candidate in treating pruritus diseases, especially for histamine-independent and chronic pruritus, which might be attributed to inhibition of the presynaptic N-type calcium channel.

The μ-opioid receptor (MOR) agonist morphine is commonly used for pain management, but it has severe adverse effects and produces analgesic tolerance. Thus, alternative ways of stimulating MOR activity are needed. We found that MrgC11, a sensory neuron-specific G protein-coupled receptor, may form heteromeric complexes with MOR. Peptide-mediated activation of MrgC11 enhanced MOR recycling by inducing coendocytosis and sorting of MOR for membrane reinsertion. MrgC11 activation also inhibited the coupling of MOR to β-arrestin-2 and enhanced the morphine-dependent inhibition of cAMP production. Intrathecal coadministration of a low dose of an MrgC agonist potentiated acute morphine analgesia and reduced chronic morphine tolerance in wild-type mice but not in Mrg-cluster knockout (Mrg KO) mice. BAM22, a bivalent agonist of MrgC and opioid receptors, enhanced the interaction between MrgC11 and MOR and produced stronger analgesia than did the individual monovalent agonists. Morphine-induced neuronal and pain inhibition was reduced in Mrg KO mice compared to that in wild-type mice. Our results uncover MrgC11-MOR interactions that lead to positive functional modulation of MOR. MrgC shares genetic homogeneity and functional similarity with human MrgXI. Thus, harnessing this positive modulation of MOR function by Mrg signaling may enhance morphine analgesia in a sensory neuron- specific fashion to limit central side effects.

Itch (pruritis) and pain represent two distinct sensory modalities; yet both have evolved to alert us to potentially harmful external stimuli. Compared with pain, our understanding of itch is still nascent. Here, we report a new clinical case of debilitating itch and altered pain perception resulting from the heterozygous de novo p.L811P gain-of-function mutation in Na(V)1.9, a voltage-gated sodium (Na(V)) channel subtype that relays sensory information from the periphery to the spine. To investigate the role of Na(V)1.9 in itch, we developed a mouse line in which the channel is N-terminally tagged with a fluorescent protein, thereby enabling the reliable identification and biophysical characterization of Na(V)1.9-expressing neurons. We also assessed Na(V)1.9 involvement in itch by using a newly created Na(V)1.9(–/–) and Na(V)1.9(L799P/WT) mouse model. We found that Na(V)1.9 is expressed in a subset of nonmyelinated, nonpeptidergic small-diameter dorsal root ganglia (DRGs). In WT DRGs, but not those of Na(V)1.9(–/–) mice, pruritogens altered action potential parameters and Na(V) channel gating properties. Additionally, Na(V)1.9(–/–) mice exhibited a strong reduction in acute scratching behavior in response to pruritogens, whereas Na(V)1.9(L799P/WT) mice displayed increased spontaneous scratching. Altogether, our data suggest an important contribution of Na(V)1.9 to itch signaling.

Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete netrin-1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, dorsal root ganglion neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for netrin-1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g., netrin-1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.

High-voltage-activated (HVA) calcium channels play an important role in synaptic transmission. Activation of Mas-related G-protein-coupled receptor subtype C (MrgC; mouse MrgC11, rat homolog rMrgC) inhibits HVA calcium current (ICa) in small-diameter dorsal root ganglion (DRG) neurons, but the intracellular signaling cascade underlying MrgC agonist-induced inhibition of HVA ICa in native DRG neurons remains unclear. To address this question, we conducted patch-clamp recordings in MrgA3-eGFP-wild-type mice, in which most MrgA3-eGFP(+) DRG neurons co-express MrgC11 and can be identified for recording. We found that the inhibition of HVA ICa by JHU58 (0.001-100nM, a dipeptide, MrgC-selective agonist) was significantly reduced by pretreatment with a phospholipase C (PLC) inhibitor (U73122, 1μM), but not by its inactive analog (U73343) or vehicle. Further, in rats that had undergone spinal nerve injury, pretreatment with intrathecal U73122 nearly abolished the inhibition of mechanical hypersensitivity by intrathecal JHU58. The inhibition of HVA ICa in MrgA3-eGFP(+) neurons by JHU58 (100nM) was partially reduced by pretreatment with a Gβγ blocker (gallein, 100μM). However, applying a depolarizing prepulse and blocking the Gαi and Gαs pathways with pertussis toxin (PTX) (0.5μg/mL) and cholera toxin (CTX) (0.5μg/mL), respectively, had no effect. These findings suggest that activation of MrgC11 may inhibit HVA ICa in mouse DRG neurons through a voltage-independent mechanism that involves activation of the PLC, but not Gαi or Gαs, pathway.

Electrical stimulation of low-threshold Aβ-fibers (Aβ-ES) is used clinically to treat neuropathic pain conditions that are refractory to pharmacotherapy. However, it is unclear how Aβ-ES modulates synaptic responses to high-threshold afferent inputs (C-, Aδ-fibers) in superficial dorsal horn. Substantia gelatinosa (SG) (lamina II) neurons are important for relaying and modulating converging spinal nociceptive inputs. We recorded C-fiber-evoked excitatory postsynaptic currents (eEPSCs) in spinal cord slices in response to paired-pulse test stimulation (500 μA, 0.1 millisecond, 400 milliseconds apart). We showed that 50-Hz and 1000-Hz, but not 4-Hz, Aβ-ES (10 μA, 0.1 millisecond, 5 minutes) induced prolonged inhibition of C-fiber eEPSCs in SG neurons in naive mice. Furthermore, 50-Hz Aβ-ES inhibited both monosynaptic and polysynaptic forms of C-fiber eEPSC in naive mice and mice that had undergone spinal nerve ligation (SNL). The paired-pulse ratio (amplitude second eEPSC/first eEPSC) increased only in naive mice after 50-Hz Aβ-ES, suggesting that Aβ-ES may inhibit SG neurons by different mechanisms under naive and nerve-injured conditions. Finally, 50-Hz Aβ-ES inhibited both glutamatergic excitatory and GABAergic inhibitory interneurons, which were identified by fluorescence in vGlut2-Td and glutamic acid decarboxylase-green fluorescent protein transgenic mice after SNL. These findings show that activities in Aβ-fibers lead to frequency-dependent depression of synaptic transmission in SG neurons in response to peripheral noxious inputs. However, 50-Hz Aβ-ES failed to induce cell-type-selective inhibition in SG neurons. The physiologic implication of this novel form of synaptic depression for pain modulation by Aβ-ES warrants further investigation.

The skin epidermis is densely innervated by peripheral sensory nerve endings. Nociceptive neurons, whose terminals are in close contact with epidermal keratinocytes, can be activated directly by noxious physical and chemical stimuli to trigger pain. However, whether keratinocytes can signal acutely to sensory nerve terminals to initiate pain in vivo remains unclear. Here, using the keratin 5 promoter to selectively express the capsaicin receptor TRPV1 in keratinocytes of TRPV1-knockout mice, we achieved specific stimulation of keratinocytes with capsaicin. Using this approach, we found that keratinocyte stimulation was sufficient to induce strong expression of the neuronal activation marker, c-fos, in laminae I and II of the ipsilateral spinal cord dorsal horn and to evoke acute paw-licking nocifensive behavior and conditioned place aversion. These data provide direct evidence that keratinocyte stimulation is sufficient to evoke acute nociception-related responses.

Rodents are often used for studying chronic pain mechanisms and developing new pain therapeutics, but objectively determining the animal’s pain state is a major challenge. To improve the precision of using reflexive withdrawal behaviors for interpreting the mouse pain state, we adopted high-speed videography to capture sub-second movement features of mice upon hind paw stimulation. We identified several parameters that are significantly different between behaviors evoked by innocuous and noxious stimuli, and combined them to map the mouse pain state through statistical modeling and machine learning. To test the utility of this approach, we determined the pain state triggered by von Frey hairs (VFHs) and optogenetic activation of two nociceptor populations. Our method reliably assesses the “pain-like” probability for each mouse paw withdrawal reflex under all scenarios, highlighting the improved precision of using this high resolution behavior-centered composite methodology to determine the mouse pain state from reflexive withdrawal assays.

Pseudo-allergic reactions-adverse, non-immunologic, anaphylaxis-like sudden onset reactions mediated through an IgE-independent pathway—are activated by various basic compounds and occur at least as frequently as IgE-mediated reactions to drugs. A large family of G protein coupled receptors (Mas-related genes; Mrgprs) is closely related to pseudo-allergies. However, few therapies can directly target pseudo-allergies and related Mrgprs. Saikosaponin A (SSA) is effective in the treatment of passive cutaneous anaphylaxis (PCA), adjuvant arthritis, and delayed hypersensitiveness. In this study, we investigated the anti-pseudo-allergy effect of SSA and its underlying mechanism. We examined the effect of SSA on both IgE-independent and IgE-dependent responses using PCA and active systemic anaphylaxis models, as well as in vitro-cultured mast cells. We also evaluated whether the anti-allergy effect is related to Mrgprs by using in vitro Mrgprx2-expressing HEK293 cells. SSA dose dependently suppressed compound 48/80 (C48/80)-induced PCA and mast cell degranulation in mice. When SSA and C48/80 were administered together through the vein, C48/80-induced systemic anaphylaxis did not occur, and C48/80-induced shock ratio decreased dose-dependently upon SSA treatment. However, SSA did not affect IgE-dependent allergy. When administered topically 24 h before antigen challenge, Evans blue leakage and paw swelling were induced in the SSA-treated group and the vehicle group. Our in vitro studies revealed that SSA reduced C48/80-induced calcium flux and suppressed degranulation in LAD2 cells. SSA could also dose-dependently inhibit C48/80-induced Mrgprx2-expressing HEK293 cell activation. As a conclusion, SSA could inhibits IgE-independent allergy, but not IgE-dependent allergy, and this effect involves the Mrgprx2 pathway. This study provided a new sight on pseudo-allergy and its therapy.

Anaphylactoid reactions are common clinical acute adverse drug reactions that can exacerbate a patient’s condition and produce effects that may become life-threatening. Therefore, it is important to establish a novel method to evaluate drugs for anaphylactoid reactions. In this study, we developed a sensitive and rapid method to detect histamine release from LAD2 cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and constructed a relative release index based on various release curve parameters, including allergen release time and sudden change rate, to evaluate the potential and strength of allergen-induced anaphylactoid reactions. This LAD2 release model was used to evaluate anaphylactoid reactions induced by ciprofloxacin, norfloxacin, lomefloxacin, moxifloxacin, and baicalin. The results positively correlated with those obtained with an Evans blue ear test and negatively correlated with the Ca2+ influx EC50. In summary, the current study established a novel in vitro method to analyze the properties of histamine release from LAD2 cells and characterize the sensitization and strength of sensitization of drugs or components that may induce anaphylactoid reactions.

Mast cells are unique immunocytes that function as sentinel cells in host defense reactions such as immediate hypersensitivity responses and anaphylactic responses. The mast cell specific receptor MRGPRX2 (Mas-related G protein-coupled receptor X2) triggers mast cell degranulation-a key process in anaphylactic reactions. We sought to better understand anaphylactic reaction induced by sinomenine hydrochloride (SH). MRGPRX2-related pseudo-allergic reactions induced by SH were investigated using the hindpaw swelling and extravasation assay in vivo and mast cell degranulation assays in vitro. MrgprB2 knockout mice exhibit a reduced SH-induced inflammation effect. Furthermore, MRGPRX2 (the orthologous gene of MrgprB2) related human mast cells are activated by SH in a dose-dependent manner; however, MRGPRX2 knockdown mast cells showed reduced degranulation. The results showed a kind of mechanism that SH-induced anaphylactoid reactions were mediated by MRGPRX2 via activating PLC molecular signaling pathways to provoke mast cells Ca2+ mobilization and degranulation.

ObjectiveThe purpose of this study was to develop a method for analyzing sensory neuron responses to mechanical stimuli in vivo, and to evaluate whether these neuronal responses change after destabilization of the medial meniscus (DMM).MethodsDMM or sham surgery was performed in 10-week old male C57BL/6 wild-type or Pirt-GCaMP3+/− mice. All experiments were performed eight weeks after surgery. Knee and hind paw hyperalgesia were assessed in wild-type mice. The retrograde label DiI was injected into the ipsilateral knee to quantify the number of knee-innervating neurons in the L4 dorsal root ganglion (DRG) in wild-type mice. In vivo calcium imaging was performed on the ipsilateral L4 DRG of Pirt-GCaMP3+/− mice as mechanical stimuli (paw pinch, knee pinch, knee twist) were applied to the ipsilateral hind limb.ResultsEight weeks after surgery, DMM mice had more hyperalgesia in the knee and hind paw compared to sham mice. Intra-articular injection of DiI labeled similar numbers of neurons in the L4 DRG of sham and DMM mice. Increased numbers of sensory neurons responded to all three mechanical stimuli in DMM mice, as assessed by in vivo calcium imaging. The majority of responses in sham and DMM mice were in small-to-medium-sized neurons, consistent with the size of nociceptors. The magnitude of responses was similar between sham and DMM mice.ConclusionsWe demonstrated that increased numbers of small-to-medium sized DRG neurons respond to mechanical stimuli 8 weeks after DMM surgery, suggesting that nociceptors have become sensitized by lowering the response threshold.

According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.

Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons’ intrinsic axonal growth competence are not well understood. Here we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5- hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.

SummaryNeural crest (NC) generates diverse lineages including peripheral neurons, glia, melanocytes, and mesenchymal derivatives. Isolating multipotent human NC has proven challenging, limiting our ability to understand NC development and model NC-associated disorders. Here, we report direct reprogramming of human fibroblasts into induced neural crest (iNC) cells by overexpression of a single transcription factor, SOX10, in combination with environmental cues including WNT activation. iNC cells possess extensive capacity for migration in vivo, and single iNC clones can differentiate into the four main NC lineages. We further identified a cell surface marker for prospective isolation of iNCs, which was used to generate and purify iNCs from familial dysautonomia (FD) patient fibroblasts. FD-iNC cells displayed defects in cellular migration and alternative mRNA splicing, providing insights into FD pathogenesis. Thus, this study provides an accessible platform for studying NC biology and disease through rapid and efficient reprogramming of human postnatal fibroblasts.

Previous studies have shown that activation of mouse MrgC11, a G-protein coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large scale screen has been carried out to isolate small molecule allosteric agonists of MrgX1, the human homologue of MrgC11. The goal of this study is to improve the efficacy and potency of the positive allosteric modulators with therapeutic implications of anti-chronic pain. Here, we report an iterative parallel synthesis effort and a structure-activity relationship of a series of arylsulfonamides, which led to the discovery of the first positive allosteric modulator (PAM) of MrgX1, ML382.

Mas-related G-protein-coupled receptor subtype C (mouse MrgC11 and rat rMrgC), expressed specifically in small-diameter primary sensory neurons, may constitute a novel pain inhibitory mechanism. We have shown previously that intrathecal administration of MrgC-selective agonists can strongly attenuate persistent pain in various animal models. However, the underlying mechanisms for MrgC agonist-induced analgesia remain elusive. Here, we conducted patch-clamp recordings to test the effect of MrgC agonists on high-voltage-activated (HVA) calcium current in small-diameter dorsal root ganglion (DRG) neurons. Using pharmacological approaches, we show for the first time that an MrgC agonist (JHU58) selectively and dose-dependently inhibits N-type, but not L- or P/Q-type, HVA calcium channels in mouse DRG neurons. Activation of HVA calcium channels is important to neurotransmitter release and synaptic transmission. Patch-clamp recordings in spinal cord slices showed that JHU58 attenuated the evoked excitatory postsynaptic currents in substantia gelatinosa (SG) neurons in wild-type mice, but not in Mrg knockout mice, after peripheral nerve injury. These findings indicate that activation of endogenously expressed MrgC receptors at central terminals of primary sensory fibers may decrease peripheral excitatory inputs onto SG neurons. Together, these results suggest potential cellular and molecular mechanisms that may contribute to intrathecal MrgC agonist-induced analgesia. Because MrgC shares substantial genetic homogeneity with human MrgX1, our findings may suggest a rationale for developing intrathecally delivered MrgX1 receptor agonists to treat pathological pain in humans and provide critical insight regarding potential mechanisms that may underlie its analgesic effects.

The itch-scratch reflex serves as a protective mechanism in everyday life. However, chronic persistent itching can be devastating. Despite the clinical importance of the itch sensation, its mechanism remains elusive. In the past decade, substantial progress has been made to uncover the mystery of itching. Here, we review the molecules, cells, and circuits known to mediate the itch sensation, which, coupled with advances in understanding the pathophysiology of chronic itching conditions, will hopefully contribute to the development of new anti-itch therapies.

Chronic neuropathic pain is often refractory to current pharmacotherapies. The rodent Mas-related G-protein-coupled receptor subtype C (MrgC) shares substantial homogeneity with its human homologue, MrgX1, and is located specifically in small-diameter dorsal root ganglion neurons. However, evidence regarding the role of MrgC in chronic pain conditions has been disparate and inconsistent. Accordingly, the therapeutic value of MrgX1 as a target for pain treatment in humans remains uncertain. Here, we found that intrathecal injection of BAM8-22 (a 15-amino acid peptide MrgC agonist) and JHU58 (a novel dipeptide MrgC agonist) inhibited both mechanical and heat hypersensitivity in rats after an L5 spinal nerve ligation (SNL). Intrathecal JHU58-induced pain inhibition was dose dependent in SNL rats. Importantly, drug efficacy was lost in Mrg-cluster gene knockout (Mrg KO) mice and was blocked by gene silencing with intrathecal MrgC siRNA and by a selective MrgC receptor antagonist in SNL rats, suggesting that the drug action is MrgC dependent. Further, in a mouse model of trigeminal neuropathic pain, microinjection of JHU58 into ipsilateral subnucleus caudalis inhibited mechanical hypersensitivity in wild-type but not Mrg KO mice. Finally, JHU58 attenuated the miniature excitatory postsynaptic currents frequency both in medullary dorsal horn neurons of mice after trigeminal nerve injury and in lumbar spinal dorsal horn neurons of mice after SNL. We provide multiple lines of evidence that MrgC agonism at spinal but not peripheral sites may constitute a novel pain inhibitory mechanism that involves inhibition of peripheral excitatory inputs onto postsynaptic dorsal horn neurons in different rodent models of neuropathic pain.

SUMMARY Rodents are the main model systems for pain research, but determining their pain state is challenging. To develop an objective method to assess pain sensation in mice, we adopt high-speed videography to capture sub-second behavioral features following hind paw stimulation with both noxious and innocuous stimuli and identify several differentiating parameters indicating the affective and reflexive aspects of nociception. Using statistical modeling and machine learning, we integrate these parameters into a single index and create a “mouse pain scale,” which allows us to assess pain sensation in a graded manner for each withdrawal. We demonstrate the utility of this method by determining sensations triggered by three different von Frey hairs and optogenetic activation of two different nociceptor populations. Our behavior-based “pain scale” approach will help improve the rigor and reproducibility of using withdrawal reflex assays to assess pain sensation in mice., Graphical Abstract, In Brief Abdus-Saboor et al. develop a behavior-centered “mouse pain scale” using high-speed videography, statistical modeling, and machine learning. With this method, they assess the sensation induced by noxious, innocuous, and optogenetic stimuli. This method will improve the reliability of using the mouse hind paw withdrawal to measure pain.

International audience; Quorum sensing molecules (QSMs) are secreted by bacteria to signal population density. Upon reaching a critical concentration, QSMs induce transcriptional alterations in bacteria that enable virulence factor expression and biofilm formation. It is unclear whether mammalian hosts can recognize QSMs to trigger responsive antibacterial immunity. We report that mouse mast cell-specific G protein-coupled-receptor Mrgprb2, and its human homologue MRGPRX2, are receptors for Gram-positive QSMs, including competence-stimulating peptide (CSP)-1. CSP-1 activates Mrgprb2/MRGPRX2, triggering mast cell degranulation which inhibits bacterial growth and prevents biofilm formation. Such antibacterial functions are reduced in Mrgprb2-deficient mast cells, while wildtype mast cells fail to inhibit the growth of bacterial strains lacking CSP-1. Mrgprb2-knockout mice exhibit reduced bacterial clearance, while pharmacologically activating Mrgprb2 in vivo eliminates bacteria and improves disease score. These findings identify a host defense mechanism that uses QSMs as an “Achilles heel” and suggest MRGPRX2 as a potential therapeutic target for controlling bacterial infections.

In this study, we sought to elucidate the molecular mechanism underlying human Mas-related G protein-coupled receptor X1 (MrgprX1) mediated itch sensation. We found that activation of MrgprX1 by BAM8-22 triggered robust action potential discharges in dorsal root ganglion (DRG) neurons. This neuronal excitability is not mediated by Transient receptor potential (TRP) cation channels, M-type potassium channels, or chloride channels. Instead, activation of MrgprX1 lowers the activation threshold of TTX-resistant sodium channels and induces inward sodium currents. These MrgprX1-elicited action potential discharges can be blocked by Pertussis toxin (PTX) and a Gβγ inhibitor – Gallein. Behavioral results showed that Nav1.9 knockout but not Trpa1 knockout significantly reduced BAM8-22 evoked scratching behavior. Collectively, these data suggest that activation of MrgprX1 triggers itch sensation by increasing the activity of TTX-resistant voltage-gated sodium channels.