ABSTRACT:The pathophysiology of primary burning mouth syndrome (BMS) remains controversial. Targeted analyses or "omics" approach of saliva provide diagnostic or pathophysiological biomarkers. This pilot study's primary objective was to explore the pathophysiology of BMS through a comparative analysis of the salivary metabolome among 26 BMS female cases and 25 age- and sex-matched control subjects. Secondary objectives included comparative analyses of inflammatory cytokines, neuroinflammatory markers, and steroid hormones among cases and control subjects, and among BMS patients according to their clinical characteristics. Salivary metabolome, neuroinflammatory markers, cytokines, and steroids were, respectively, analysed by liquid chromatography coupled with mass spectrometry, ELISA and protease activity assay, and multiparametric Luminex method. Among the 166 detected metabolites, univariate analysis did not find any discriminant metabolite between groups. Supervised multivariate analysis divided patients into 2 groups with an accuracy of 60% but did not allow significant discrimination (permutation test, P = 0.35). Among the metabolites contributing to the model, 3 belonging to the tyrosine pathway ( l -dopa, l -tyrosine, and tyramine) were involved in the discrimination between cases and control subjects, and among BMS patients according to their levels of pain. Among the detectable molecules, levels of cytokines, steroid hormones, and neuroinflammatory markers did not differ between cases and control subjects and were not associated with characteristics of BMS patients. These results do not support the involvement of steroid hormones, inflammatory cytokines, or inflammatory neurogenic mediators in the pathophysiology of pain in BMS, whereas the observed shift in tyrosine metabolism may indicate an adaptative response to chronic pain or an impaired dopaminergic transmission.

Pruritus (or itch) is an unpleasant sensation leading to a desire to scratch. In the epidermis, there are selective C or Aδ epidermal nerve endings that are pruriceptors. At their other ends, peripheral neurons form synapses with spinal neurons and interneurons. Many areas in the central nervous system are involved in itch processing. Although itch does not occur solely because of parasitic, allergic, or immunologic diseases, it is usually the consequence of neuroimmune interactions. Histamine is involved in a minority of itchy conditions, and many other mediators play a role: cytokines (eg, IL-4, IL-13, IL-31, IL-33, and thymic stromal lymphopoietin), neurotransmitters (eg, substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, NBNP, endothelin 1, and gastrin-releasing peptide), and neurotrophins (eg, nerve growth factor and brain-derived neurotrophic factor). Moreover, ion channels such as voltage-gated sodium channels, transient receptor potential vanilloid 1, transient receptor ankyrin, and transient receptor potential cation channel subfamily M (melastatin) member 8 play a crucial role. The main markers of nonhistaminergic pruriceptors are PAR-2 and MrgprX2. A notable phenomenon is the sensitization to pruritus, in which regardless of the initial cause of pruritus, there is an increased responsiveness of peripheral and central pruriceptive neurons to their normal or subthreshold afferent input in the context of chronic itch.

Introduction: Seaweeds are a rich source of elements such as iodine, and are also able to accumulate contaminants such as trace elements. Methods: The aim of this study was to assess the dietary exposure as well as the risk from iodine and trace elements in edible seaweeds for the French population using current consumption data. The contribution of seaweeds to overall dietary exposure to trace elements and iodine was evaluated, and for those substances with minimal contribution to overall dietary exposure, simulations were performed to propose increased maximal limits in seaweeds. Results: Cadmium, inorganic arsenic and mercury in seaweeds were very low contributors to total dietary exposure to these contaminants (0.7 % 1.1 % and 0.1 % on average, respectively). Dietary exposure to lead via seaweed may contribute up to 3.1 % of total dietary exposure. Dietary consumption of iodine via seaweed may contribute up to 33 % of total exposure to iodine, which makes seaweeds the strongest contributor to iodine in diet. Discussion: New maximal values in seaweeds are proposed for the very low contributors to total dietary exposure: 1 mg/kg dw for cadmium, 10 mg/kg dw for inorganic arsenic and 0.3 mg/kg dw for mercury.

Seaweeds accumulate toxic contaminants present in the surrounding waters such as trace elements, ammonium, dioxins and pesticides. Seaweed consumption data are scarce in France as in Europe. Given that seaweed consumption data are essential to assess exposure and the risks for human health linked to toxic substances, it would appear essential to generate these data. The aim of the study was to assess the current consumption of seaweed foodstuffs by the French population via an online survey conducted on 780 adults (seaweed foodstuffs consumed, percentage of consumers and frequency of consumption). The daily consumption of seaweeds was assessed. Enquiries at points of purchase were also performed to reference the type of foodstuffs found on the French market, the seaweed species present, and the percentage of seaweed in the product. These new data generated in this work will be useful for safety assessors and for safety agencies.

Until recently, itch pathophysiology was poorly understood and treatments were poorly effective in relieving itch. Current progress in our knowledge of the itch processing, the numerous mediators and receptors involved has led to a large variety of possible therapeutic pathways. Currently, inhibitors of IL-31, IL-4/13, NK1 receptors, opioids and cannabinoids, JAK, PDE4 or TRP are the main compounds involved in clinical trials. However, many new targets, such as Mas-related GPCRs and unexpected new pathways need to be also explored.

Ciguatera fish poisoning (CFP) and neurotoxic shellfish poisoning syndromes are induced by the consumption of seafood contaminated by ciguatoxins and brevetoxins. Both toxins cause sensory symptoms such as paresthesia, cold dysesthesia and painful disorders. An intense pruritus, which may become chronic, occurs also in CFP. No curative treatment is available and the pathophysiology is not fully elucidated. Here we conducted single-cell calcium video-imaging experiments in sensory neurons from newborn rats to study in vitro the ability of Pacific-ciguatoxin-2 (P-CTX-2) and brevetoxin-1 (PbTx-1) to sensitize receptors and ion channels, (i.e., to increase the percentage of responding cells and/or the response amplitude to their pharmacological agonists). In addition, we studied the neurotrophin release in sensory neurons co-cultured with keratinocytes after exposure to P-CTX-2. Our results show that P-CTX-2 induced the sensitization of TRPA1, TRPV4, PAR2, MrgprC, MrgprA and TTX-r NaV channels in sensory neurons. P-CTX-2 increased the release of nerve growth factor and brain-derived neurotrophic factor in the co-culture supernatant, suggesting that those neurotrophins could contribute to the sensitization of the aforementioned receptors and channels. Our results suggest the potential role of sensitization of sensory receptors/ion channels in the induction or persistence of sensory disturbances in CFP syndrome.

Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G protein-coupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2-evoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2-elicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway.

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

En France, de nombreuses toxines non reglementees sont suivies dans le cadre du reseau de veille d’emergence des biotoxines marines dans les coquillages (EMERGTOX) dont le comite de pilotage est preside par la DGAL et inclut la DGS, la Direction des peches maritimes et de l'aquaculture (DPMA), Sante publique France, l’Anses, l’Ifremer et la Direction de l’eau et de la biodiversite (DEB). Les brevetoxines sont incluses dans ce reseau depuis janvier 2018. Elles ont ete mises en evidence pour la premiere fois en France en novembre 2018 dans des moules en Corse (117 μg/kg de glande digestive). Le prelevement d’eau associe pour le suivi du phytoplancton a montre la presence de Karenia spp. (genre de microalgues dont certaines especes produisent des brevetoxines). Il n’existe pas de limite maximale pour ces toxines dans le reglement (CE) n°853/2004. Les USA, l’Australie/Nouvelle-Zelande et le Mexique appliquent un seuil a 800 μg d'equivalent BTX-2/kg de chair de coquillages. Dans la norme CODEXSTAN 292–2008 (rev.2015) du Codex alimentarius, la limite maximale pour les brevetoxines est de 200 unites souris ou equivalent par kg de chair de mollusque vivant. Dans ce contexte, il a ete demande a l’Anses de fournir des elements de reponse aux questions suivantes : Question 1. Quelles sont les donnees toxicologiques disponibles pour les brevetoxines ? Permettent-elles a l’Anses de proposer des valeurs toxicologiques de reference aigue et chronique par voie orale ? Question 2. Sur la base des donnees toxicologiques identifiees par l’Anses, est-il possible de proposer une valeur guide dans les coquillages au-dela de laquelle des investigations complementaires seraient demandees dans le cadre du reseau EMERGTOX ? Question 3. Quelles modalites de suivi des brevetoxines dans le milieu marin pourraient etre recommandees dans le cadre du reseau EMERGTOX ? Quelles investigations complementaires seraient a realiser en cas de depassement de la valeur guide ? Question 4. Compte tenu des donnees toxicologiques et au vu du contexte, existe-t-il une preoccupation de sante publique aux niveaux de contamination identifies dans certaines zones marines francaises liee a la consommation de coquillages ? Question 5. Existe-t-il une preoccupation sanitaire liee a l’exposition aux brevetoxines par un contact direct avec de l’eau contaminee notamment en cas de baignade ou de pratique d’activites nautiques ou en cas d’exposition par inhalation a des embruns ?

Les ciguatoxines (CTXs) et les brévétoxines (PbTxs) sont des neurotoxines impliquées respectivement dans la ciguatéra et l’intoxication neurologique par les fruits de mer, deux syndromes consécutifs à l’ingestion de poissons ou de mollusques contaminés. Les symptômes communs mais d’intensité plus marquée dans la ciguatéra, sont des troubles neuro-cutanés (prurit, paresthésies et dysesthésies au froid) qui peuvent persister plusieurs semaines à plusieurs années. Les cibles primaires de ces toxines sont le canal sodium dépendant du potentiel de membrane (Nav) dont elles entraînent l’ouverture. Il s’ensuit une hyperexcitabilité membranaire des neurones périphériques. Cependant, les événements moléculaires en aval restent peu connus et aucun traitement n’existe pour soulager ces troubles neuro-cutanés persistants et invalidants. Récemment, nous avons mis en évidence que la Pacific-ciguatoxine-2 (P-CTX-2), appliquée à une co-culture de kératinocytes/neurones sensoriels, induit la libération des neuropeptides de l’inflammation neurogène (CGRP et substance P). Chez la souris, il a été montré que l’allodynie au froid induite par les CTXs implique la sensibilisation (i.e. l’abaissement du seuil d’activation) Nav-dépendante de TRPA1. Nos travaux ont pour but d’étudier la sensibilisation de plusieurs récepteurs sensoriels cutanés par ces neurotoxines. Des cultures de neurones sensoriels de rats nouveaux-nés ont été incubés pendant 24 h avec la P-CTX-2 ou la brévétoxine-1 (PbTx-1). À l’aide de la vidéo-microscopie en fluorescence sur cellule unique, nous avons étudié la modulation par ces toxines de la réponse calcique à un agoniste spécifique du récepteur sensoriel d’intérêt. Dans un second temps, nous avons dosé dans le surnageant de co-cultures de kératinocytes et neurones sensoriels traitée par la P-CTX-2 plusieurs médiateurs susceptibles de contribuer à la sensibilisation de récepteurs sensoriels. Nos résultats montrent que la P-CTX-2 et la PbTx-1 sensibilisent différents récepteurs sensoriels de la peau. De plus, ils mettent en évidence que la P-CTX-2 augmente, dans la coculture la libération de plusieurs médiateurs, suggérant leur rôle dans la sensibilisation des récepteurs sensoriels cutanés induite par les neurotoxines. Ce travail permet de mieux comprendre la physiopathologie des troubles sensoriels induits par les CTXs et les PbTxs et le prurit de manière générale. Cette étude suggère que le mécanisme de sensibilisation mis en évidence pourrait participer à l’initiation et la persistance des troubles neuro-cutanées induits par la P-CTX-2 et la PbTx-1. Nos résultats et la caractérisation des mécanismes impliqués dans la sensibilisation pourraient permettre d’identifier des voies thérapeutiques ciblées. Ce travail a été réalisé avec le soutien de la Société française de dermatologie.

Ciguatera fish poisoning (CFP), the most prevalent seafood poisoning worldwide, is caused by the consumption of tropical and subtropical fish contaminated with potent neurotoxins called ciguatoxins (CTXs). Ciguatera is a complex clinical syndrome in which peripheral neurological signs predominate in the acute phase of the intoxication but also persist or reoccur long afterward. Their recognition is of particular importance in establishing the diagnosis, which is clinically-based and can be a challenge for physicians unfamiliar with CFP. To date, no specific treatment exists. Physiopathologically, the primary targets of CTXs are well identified, as are the secondary events that may contribute to CFP symptomatology. This review describes the clinical features, focusing on the sensory disturbances, and then reports on the neuronal targets and effects of CTXs, as well as the neurophysiological and histological studies that have contributed to existing knowledge of CFP neuropathophysiology at the molecular, neurocellular and nerve levels.

Introduction: Ciguatera fish poisoning (CFP), the most common seafood poisoning worldwide, is caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Pruritus is one of the most distressing symptoms, associated with other cutaneous sensory disorders, including paresthesia and cold dysesthesia. No specific treatment exists. CTXs are known to primarily activate voltage-gated sodium channels, but the downstream molecular events that lead to sensory disturbances remain poorly defined. Peptidergic sensory neurons were recently identified as major players in CFP sensory disturbances. Methods: In this study, we examined the role of molecular actors in 2 effects induced by Pacific CTX-2 (P-CTX-2): the increase in cytosolic calcium levels in rat primary sensory neurons; and the release of the neuropeptide substance P (SP) in sensory neurons co-cultured with keratinocytes. Results: Our results (i) rule out the involvement of the Na+/Ca2+ exchanger (NCX) and the transient receptor potential channels transient receptor potential ankyrin 1 and and transient receptor potential vanilloid 1; (ii) show that N-type voltage-gated calcium (Cav) channels contribute to the initiation of the calcium signal elicited by P-CTX-2 in rat sensory neurons, while N-type and L-type Cav channels play equal parts in the SP release in the co-culture; and (iii) identify store-operated calcium entry supported by Orai calcium release-activated calcium modulator 1 (ORAI1) as a critical effector of the late phase of the calcium signal and the subsequent SP release elicited by P-CTX-2. Discussion: Our in vitro findings indicate that Cav and ORAI1 channels may be promising pharmacological targets for specifically relieving the sensory effects of CTXs.

Red tides involving Karenia brevis expose humans to brevetoxins (PbTxs). Oral exposition triggers neurotoxic shellfish poisoning, whereas inhalation induces a respiratory syndrome and sensory disturbances. No curative treatment is available and the pathophysiology is not fully elucidated. Protease-activated receptor 2 (PAR2), cathepsin S (Cat-S) and substance P (SP) release are crucial mediators of the sensory effects of ciguatoxins (CTXs) which are PbTx analogs. This work explored the role of PAR2 and Cat-S in PbTx-1-induced sensory effects and deciphered the signaling pathway involved. We performed calcium imaging, PAR2 immunolocalization and SP release experiments in monocultured sensory neurons or co-cultured with keratinocytes treated with PbTx-1 or P-CTX-2. We demonstrated that PbTx-1-induced calcium increase and SP release involved Cat-S, PAR2 and transient receptor potential vanilloid 4 (TRPV4). The PbTx-1-induced signaling pathway included protein kinase A (PKA) and TRPV4, which are compatible with the PAR2 biased signaling induced by Cat-S. Internalization of PAR2 and protein kinase C (PKC), inositol triphosphate receptor and TRPV4 activation evoked by PbTx-1 are compatible with the PAR2 canonical signaling. Our results suggest that PbTx-1-induced sensory disturbances involve the PAR2-TRPV4 pathway. We identified PAR2, Cat-S, PKA, and PKC that are involved in TRPV4 sensitization induced by PbTx-1 in sensory neurons.

Objective Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons. Methods Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies. Results Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic‐like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic‐like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE‐mediated (syntaxin1) vesicle release. Interpretation By providing selective communication between keratinocytes and sensory neurons, synaptic‐like contacts are the hubs of a 2‐site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long‐held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte–sensory neuron synaptic‐like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch. ANN NEUROL 2020;88:1205–1219

Le paclitaxel est une des chimiothérapies les plus utilisées dans le cadre des cancers solides, notamment le sarcome de Kaposi. Les neuropathies périphériques sont des complications fréquentes associés à des symptômes sensoriels neuro-cutanés (prurit, douleur, paresthésies, dysesthésies) et parfois à une baisse de la densité des fibres nerveuses intra-épidermiques. La physiopathologie n'est pas clairement établie. Des données obtenues in vivo suggèrent que l'activation et/ou la sensibilisation des canaux/récepteurs sensoriels jouent un rôle-clé. Le but de cette étude était d'évaluer in vitro la capacité du paclitaxel à sensibiliser les canaux/récepteurs TRPV1, TRPV4, TRPA1, NaV, PAR2 et TRL4 sur un modèle de neurones sensoriels en culture. Les cultures de neurones étaient issues de ganglions extraits des racines dorsales de ratons. Après un pré-traitement (20 h) des cultures avec du paclitaxel, son solvant (kolliphor) ou aucun traitement, l'imagerie calcique a été employée pour évaluer la capacité du paclitaxel à moduler la réponse des neurones à chaque agoniste des récepteurs/canaux étudiés. Des expériences d'immunocytochimie et de RT-qPCR ont été réalisées pour mesurer les niveaux d'expression des récepteurs/canaux après exposition au paclitaxel, au solvant ou au témoin sans traitement. Les résultats étaient appariés par expérience et normalisés par rapport au témoin sans traitement avant analyse statistique. Nos résultats montrent que nos cultures de neurones sensoriels expriment PAR2, TLR4, TRPA1, TRPV1 et TRPV4. Les résultats d'imagerie calcique indiquent que le paclitaxel à 100 nM, sans altérer la viabilité cellulaire, augmente le pourcentage de neurones répondant à la capsaïcine (agoniste de TRPV1 ; 141,3 % ± 41,2) et l'amplitude de cette réponse (1,53 u.a. ± 0,48 vs contrôle), l'amplitude de la réponse à l'AITC (agoniste de TRPA1 ; 2,00 u.a. ± 0,10) et le pourcentage de neurones répondant à la vératridine (agoniste des NaV ; 163,7 % ± 64,9). Le paclitaxel a aussi augmenté l'expression des ARNm de tous les sous-types de NaV, particulièrement NaV1.2 (d'un facteur 1,90 ± 0,52). Nos cultures de neurones expriment les récepteurs/canaux sensoriels impliqués in vivo dans la neuropathie induite par le paclitaxel. Celui-ci sensibilise à 100 nM les canaux TRPA1, TRPV1 et les NaV. La sensibilisation des canaux NaV a été associée à une expression accrue de tous les sous-types de NaV, particulièrement NaV1.2. L'étude de l'influence du paclitaxel sur l'expression des autres récepteurs/canaux sensoriels, et des mécanismes à l'origine de leur sensibilisation, est en cours. Les kératinocytes, qui expriment aussi TRPV1, TRPA1 et NaV pourraient aussi jouer un rôle dans la neuropathie induite par le paclitaxel. Le paclitaxel induit des symptômes sensoriels en sensibilisant des canaux ioniques neuronaux, mais peut-être aussi kératinocytaires.

Skin is constantly subjected to pressure at different levels. Pressure-induced vasodilation (PIV) is one of the response mechanisms to low pressure that maintains the homeostasis of the skin. PIV results from the interaction of primary afferent nerves and vascular endothelium of skin vessels. Thanks to this cutaneous neuro-vascular interaction, the cutaneous blood flow increase allows the maintenance of an optimal level of oxygenation and minimizes the lack of vascularization of the skin tissue under low pressure. It seems to be associated with the cutaneous protection mechanisms to prevent pressure ulcers. In some contexts, where microangiopathy and neuropathy can occur, such as aging and diabetes, PIV is impaired, leading to a dramatic early decrease in local skin blood flow when low pressure is applied. In aging, PIV alteration is due to endothelial dysfunction, essentially from an alteration of the nitric oxide pathway. In the inflamm-aging context, oxidative stress increases leading to endothelial cell and nerve damages. An age-related sensory neuropathy will exacerbate the alteration of PIV during the aging process. In diabetes, non-controlled hyperglycaemia leads to an increase in several pathological biochemical pathways that involve oxidative stress and can affect PIV. Sorbinil, alagebrium and alpha-lipoic acid are able individually to restore PIV through a possible oxidative stress reduction. Candesartan, an angiotensin II type 1 receptor blocker, is also able to restore PIV and prevent pressure ulcer formation. The possibility of preventing pressure ulcer associated to diabetes and/or aging with the restoration of PIV seems to be a promising research path.

Ciguatera Fish Poisoning (CFP) is a widespread tropical intoxication consecutive to ciguatoxin (CTX) ingestion, which is characterized by persistent neuro-cutaneous disturbances, including an intense pruritus (itch). Currently, there is no specific treatment. The primary target of CTXs is the voltage dependent sodium channel (Nav), which is largely expressed in sensory nerves and, to a lesser extent, in keratinocytes. By activating Nav, CTXs induce neuronal hyper-excitability but the downstream molecular mechanisms leading to sensory disorders are poorly understood. Recent advances in dermatology reveal protease-activated receptor-2 (PAR-2) involvement in itch pathophysiology. Interestingly, this receptor is expressed in sensory neurons and keratinocytes. To better understand the pathophysiology of CFP pruritus, the purpose of the present study was to identify cellular and molecular actors involved in the substance P (SP) release elicited by P-CTX-2 from co-cultured sensory neurons and keratinocytes. First, P-CTX-2 is able to induce calcium signal in both sensory neurons and keratinocytes. We show that antagonism of PAR-2 significantly inhibited the P-CTX-2-evoked calcium signal in both cells. Then, our results demonstrate the synergistic role of keratinocytes in the SP release elicited by P-CTX-2 in the coculture. The P-CTX-2-induced SP was almost completely abolished by PAR-2 or cathepsin S (Cat S) antagonists, and Cat S activity was significantly increased after P-CTX-2 treatment. Finally, P-CTX-2 is able to internalise PAR-2 in keratinocytes. Taken together, this work reveals that keratinocytes, PAR-2 and Cat S are novel actors in the P-CTX-2-induced release of SP, suggesting those are promising pharmacological targets for specifically treating CFP neuro-cutaneous disorders.

The skin is densely innervated to transmit all sensations (touch, temperature, pressure, pain, and pruritus) but not only it. Indeed, innervation plays a major role in the structuration of the epidermis, in its renewal, and in the process as wound healing. There are increasing evidences that skin cells and cutaneous nerve endings are in close interactions each other. So, to study them is an important issue to better understand the behavior of the skin and its both physiological and pathological processes. However, due to scientific, technical, ethical, or economic reasons, the study of these interactions in human or animals in vivo remains quite impossible. So, the development of in vitro models is crucial to better understand them. Since several years, all the actors of these interactions, skin cells such as keratinocytes, fibroblasts, melanocytes, Merkel cells or stem cells, and sensory neurons, could be extracted and cultured independently or together so named 2-D cocultures. Other cocultures, the 3-D cocultures, could also be considered by the use of the epidermis or dermis or whole portions of native or reconstructed skin. These 3-D models offer also an alternative by the use of compartmented cocultures to only analyze the biochemical communication between the different types of cells. After a description of the different models available, this chapter will give some clues to define the best model(s) depending of the applications and, finally, will discuss of the advantages and the limitations of these types of cultures to study cutaneous innervation mechanisms.