IntroductionB-type natriuretic peptide (BNP) is a well-known cardiac hormone and biomarker of heart failure, but emerging evidence suggests that it also possesses immunomodulatory properties, including a role in inflammatory skin conditions like atopic dermatitis (AD). Langerhans cells (LCs), specialized epidermal antigen-presenting cells, orchestrate cutaneous immunity and are targets of neuropeptides. In the present study, we investigated how BNP treatment during differentiation affects the activation, cytokine profile, and interaction of immune cells with moLCs subsequently activated via Toll-like receptors (TLRs).MethodsMoLCs were differentiated in the presence or absence of BNP, followed by 24-hour activation with the TLR7/8 agonist CL075 and/or the TLR3 agonist polyinosinic:polycytidylic acid (poly(I:C)). Cell surface markers of moLCs were assessed using flow cytometry. ELISA was used to analyze the production of cytokines. The T cell proliferation-inducing ability of moLCs was detected through T cell coculture. Transwell migration experiments were conducted to elucidate the migratory capacity of moLCs, as well as the migration of other lymphocytes toward moLCs.ResultsBNP treatment during the differentiation of moLCs did not alter the expression of activation markers; however, it significantly counteracted the robust increase in both pro- and anti-inflammatory cytokine production induced by TLR activation. Combined TLR activation significantly increased T cell proliferation capacity, and this effect was significantly diminished in moLCs differentiated in the presence of BNP. Functionally, BNP pre-treated moLCs exhibited significantly enhanced chemotaxis towards the lymph node chemokines, supporting the previously observed migratory phenotype. Transcriptomic analysis further supported this finding, demonstrating that BNP pre-treatment attenuated the inflammatory gene signature induced by TLR agonism. Furthermore, the supernatant from TLR-activated, BNP-treated moLCs showed a marked reduction in the ability to induce the migration of peripheral blood CD56+ Natural Killer (NK) cells.DiscussionWe found that BNP primes moLCs toward a migratory phenotype and, upon subsequent TLR activation, exerts a potent inhibitory effect on their cytokine and chemokine production, thereby limiting their capacity to drive T cell proliferation and NK cell migration. This dual effect suggests that BNP may play a context-dependent role in skin immunity, potentially restraining inflammation while promoting LC transit to the draining lymph nodes.

IntroductionThe interaction between the nervous and immune systems is crucial for maintaining homeostasis and can influence disease progression in inflammatory skin diseases, such as atopic dermatitis (AD). Sensory neurons in the skin can secrete neuropeptides that modulate immune cell activity, including Langerhans cells (LCs), one of the primary antigen-presenting cells in the epidermis. In our study we investigated the effects of neuropeptides on the differentiation of monocyte-derived LCs (moLCs), specifically the neuropeptides with the most profound effect, i.e. atrial- and B-type natriuretic peptides (ANP and BNP, respectively).MethodsRNA sequencing and RT-qPCR were used to analyze neuropeptide receptor expression in moLCs and immature dendritic cells (iDCs), and the most translationally relevant, natriuretic peptide receptor A (NPR1) was validated on the protein level using western blotting. Cell surface markers of moLCs were assessed using flow cytometry, and NPR1 functionality was confirmed through intracellular cGMP assays. Confocal microscopy was used to confirm the expression of NPR1 in situ in healthy and AD skin. RNA-Seq analysis was also employed to characterize the phenotypic changes in moLCs differentiated in the presence of BNP.ResultsNPR1 expression was significantly higher in moLCs compared to iDCs, and treatment with ANP and BNP enhanced moLC differentiation, increasing CD207, CD1a, and HLA-DQ expression, while other tested neuropeptides (calcitonin gene-related peptide [CGRP], neurotensin) had no significant effect. NPR1 was functionally active, as evidenced by increased intracellular cGMP levels upon ligand binding. Confocal microscopy revealed NPR1 expression on LC cell bodies in both healthy and AD skin, with reduced intensity in AD. RNA-Seq analysis of BNP-treated moLCs indicated a shift toward a migratory LC phenotype, marked by upregulation of genes associated with cell motility (e.g., CCR7, LAMP3).DiscussionThese findings demonstrate that NPR1 activation promotes a migratory LC phenotype, highlighting the role of neuropeptides in shaping cutaneous immune responses. The reduced number of LCs in AD skin suggests a potential link between neuropeptide signaling and disease pathology.

IntroductionThe endocannabinoid system (ECS), named after the chemical compounds found in the cannabis plant, is a regulatory network of neurotransmitters, receptors, and enzymes that plays crucial roles in skin health and disease. Endogenous ligands of the ECS, called endocannabinoids, have proven to be important regulators of immune responses. One of the most prevalent endocannabinoids, arachidonoylethanolamide (also known as anandamide), is known for its anti-inflammatory effects. Langerhans cells (LCs) are the sole antigen-presenting cells present in the human epidermis. They serve as the first line of defense against pathogens and are essential for the skin’s specific immune responses and play a critical role in maintaining tissue homeostasis; however, little is known about the effect of endocannabinoids on these cells. Our research aimed to provide the connection between monocyte-derived Langerhans cells (moLCs) and the ECS, shedding light on their collaborative roles in immune homeostasis and inflammation.MethodsHuman monocytes were differentiated into moLCs using established protocols. Anandamide was applied during the differentiation process to test its effect on the viability, marker expression, and cytokine production of the cells, as well as in short term treatments for intracellular calcium measurement. TLR ligands applied after the differentiation protocol were used to activate moLCs. The impact of anandamide on the functionality of moLCs was further assessed using differential gene expression analysis of bulk RNA-Seq data, moLC-T cell cocultures, while ELISpot was employed to determine polarization of T cells activated in the aforementioned cocultures.ResultsAnandamide did not significantly affect the viability of moLCs up to 10 µM. When applied during the differentiation process it had only a negligible effect on CD207 expression, the prototypic marker of LCs; however, there was an observed reduction in CD1a expression by moLCs. Anandamide had no significant effects on the maturation status of moLCs, nor did it affect the maturation induced by TLR3 and TLR7/8 agonists. MoLCs differentiated in the presence of anandamide did however show decreased production of CXCL8, IL-6, IL-10 and IL-12 cytokines induced by TLR3 and TLR7/8 activation. Anandamide-treated moLCs showed an increased capability to activate naïve T cells; however, not to the level seen with combined TLR agonism. RNA sequencing analysis of moLCs differentiated with anandamide showed modest changes compared to control cells but did reveal an inhibitory effect on oxidative phosphorylation specifically in activated moLCs. Anandamide also promoted the polarization of naïve T cells towards a Th1 phenotype.DiscussionOur results show that anandamide has nuanced effects on the differentiation, maturation, cytokine secretion, metabolism and function of activated moLCs. Among these changes the decrease in CD1a expression on moLCs holds promise to selectively dampen inflammation induced by CD1a restricted T cells, which have been implicated as drivers of inflammation in common inflammatory skin conditions such as psoriasis, atopic dermatitis and contact dermatitis.

Dendritic cells (DCs) act as central coordinators of innate and adaptive immunity. Their fundamental function is to take up antigens and present them to T cells, and they therefore function as the main messenger between the innate and adaptive immune systems. Recently, there has been an exponential increase in the production and use of Cannabis sativa medicinal products. Phytocannabinoids (cannabinoids derived from the cannabis plant) have been observed to have beneficial effects in several inflammatory disease models. In the present study, we aimed to investigate the role of the most popular non-psychoactive cannabinoid, cannabidiol (CBD), on human monocyte-derived dendritic cells (MoDCs). After excluding the possibility of early apoptotic or necrotic processes, we investigated the effect of CBD on the differentiation and maturation of moDCs. We detected moDCs with a more mature phenotype as a result of high doses of CBD, and lipopolysaccharide (LPS)-induced maturation was not affected by CBD. Immature DCs showed reduced endocytotic activity upon treatment with CBD. Treatment with CBD induced a low level of active IL-6, IL-8 secretion by moDCs alone, whereas it was enhanced in the presence of LPS. The results from moDS - naïve T cell co-culture indicate that CBD-treated moDCs enhance naïve T cell activation. When controlling for CBD-activated secondary messenger pathways, we found that CBD in immature moDS form had a more pronounced stimulatory effect on phosphorylation levels of p38, ERK. RNA sequencing revealed that the expression of several genes was significantly altered by CBD treatment. Taken together, we conclude that CBD may affect the immunological functions of moDCs, which may be a key target for future research in the treatment of inflammatory skin diseases.

The immune system is strongly linked to the nervous system, to the point that in many diseases neuroimmune crosstalk is essential for the development of skin lesions. Inside the epidermis, the only professional antigen-presenting cells capable of initiating adaptive immune responses underlying chronic inflammatory skin diseases under the steady-state are Langerhans cells (LCs). LCs are anatomically associated with neurons that produce neuropeptides, providing a putative mechanism of neuro-immune crosstalk in the epidermal compartment. B-type natriuretic peptide (BNP) is a key mediator of itch on all levels of pruritic signal transduction, including skin cells, sensory neurons, and the spinal cord. The receptor activated by BNP, Natriuretic Peptide Receptor 1 (NPR1), is also activated atrial natriuretic peptide (ANP). We aimed to elucidate NPR1 receptor expression in our monocyte-derived LCs model, and to treat moLCs with NPR1 agonists. We validated previously performed RNASeq analysis of these cells on the protein and mRNA level with qPCR and western blot respectively, as well as performing immunohistochemical staining on human skin samples to detect the NPR1 receptor. Neither NPR1 agonist had any effect on maturation induced by combined TLR3 and 7/8 activation, although HLA-DQ was increased by ANP. T cells stimulated by moLCs activated with TLR agonists showed increased proliferation compared to controls, and this effect was decreased by both tested treatments. ELISA experiments showed that IL6 and IL8 production induced by TLR agonists was significantly decreased by NPR1 activation. Building on these results we next performed RNASeq analysis of BNP treated moLCs, and found using GSEA that the observed effects were downstream of KRAS and STAT5 signaling, and that IDO1 and 2 upregulation could underlie decreased T cell proliferation. Our results show that NPR1 signaling stimulates LC differentiation, and modulates the function of the cells.

Introduction:Extracts and compounds isolated from hemp (Cannabis sativa) are increasingly gaining popularity in the treatment of a number of diseases, with topical formulations for dermatological conditions leading the way. Phytocannabinoids such as ( )-cannabidiol, ( )-cannabinol and ( )-Δ9-tetrahydrocannabivarin (CBD, CBN, and THCV, respectively), are present in variable amounts in the plant, and have been shown to have mostly anti-inflammatory effects both in vitro and in vivo, albeit dominantly in murine models. The role of phytocannabinoids in regulating responses of dendritic cells (DCs) remains unclear.Methods:Our research aimed to investigate the effects of CBD, CBN, and THCV on human DCs differentiated from monocytes (moDCs). moDCs were treated with up to 10 μM of each phytocannabinoid, and their effects on viability, differentiation, and maturation were assessed both alone, and in conjunction with TLR agonists. The effects of CBD on cytokine production, T cell activation and polarization as well as the transcriptome of moDCs was also determined.Results:Phytocannabinoids did not influence the viability of moDCs up to 10 μM, and only CBD had effects on maturational markers of moDCs, and neither compound influenced LPS-induced activation at 10 μM. Since only CBD had measurable effects on moDCs, in our subsequent experiments we tested the effect only of that pCB. On moDCs differentiated in the presence of CBD subsequent activation by LPS induced a markedly different, much more tolerogenic response. CBD-treated moDCs also produced significantly more interleukin (IL)-6, TNFα and, importantly, IL-10 in response to LPS, which shows a shift toward anti-inflammatory signaling, as well as a more robust secretory response in general. To rule out the possibility that these effects of CBD are specific to TLR4 signaling, we determined the effect of CBD on TLR7/8-induced maturation as well, and saw similar, although less marked responses. CBD-treated moDCs were also less efficient at activating naïve T cells after LPS stimulation, further supporting the tolerogenic effect of this phytocannabinoid on moDCs. Reactome pathway analysis showed an inflammatory response to LPS in moDCs, and to a lesser extent to CBD as well. In contrast CBD-treated moDCs responded to LPS with a shift towards a more tolerogenic phenotype, as IL-10 signaling was the most prominently induced pathway in this group.Discussion:Our results show that CBD achieves an anti-inflammatory effect on adaptive immune responses only in the presence of an activating stimuli on moDCs by reprogramming cells during long-term treatment, and not through acute, short-term effects.

The only professional antigen-presenting cells in the human epidermis are Langerhans cells (LC). They function as the first line of defense against pathogens but are also crucial for the maintenance of tissue homeostasis. One of the most well-known endocannabinoids, anandamide (arachidonoyl ethanolamide, AEA), is characterized by a general anti-inflammatory effect, as it reduces the production of pro-inflammatory cytokines in many immune cells, the strength of the humoral and cellular immune response. In our current research, we investigated the effect of AEA in regulating responses of monocyte-derived LCs by monitoring changes in their maturation markers, viability, differentiation, cytokine production, naïve T cell activation capacity. First, to exclude the possibility of necrotic processes, we performed viability tests using 7-ADD, which demonstrated that AEA did not cause any cell death even when applied at high concentrations to moLCs. Moreover, we could also demonstrate that AEA mostly induced cell maturation at high concentrations, whereas TLR3-7/8 agonist-induced maturation was not affected. We investigated the secretome of moLCs and activated moLCs using a cytokine array, and found that activated cells produce IL8, MMP-9 and CCL17 among others. AEA treatment decreased the quantity of secreted interleukins compared to that of activated moLCs. AEA also enhanced moLC-induced naïve T cell proliferation compared to the control. To explore the secondary messenger systems activated by AEA treatment we determined the relative levels of human protein kinase phosphorylation with the help of Proteome Profiler Human Phospho-Kinase Array. Taken together, our data suggest that AEA may play varied roles on the immunological function of moLCs. Collectively, our findings introduce the AEA may act as a potent immune regulator of human monocyte-derived Langerhans cells, and in these cells might be more pro- rather than anti-inflammatory in some aspects.

Biological treatments targeting the IL-17A pathway have been assessed in patients with a long-term history of plaque psoriasis (PsO). We hypothesize that early systemic treatment with secukinumab may prolong treatment-free remission and alter the clinical outcome of PsO. The mechanistic sub-study of the STEPIn trial (NCT03020199) assessed molecular changes in the skin of patients with new-onset (≤12 months, naïve to systemic treatment) vs chronic (≥5 years since first symptoms) moderate to severe PsO who were randomized to receive secukinumab 300 mg or narrow-band ultraviolet B treatment. Results presented here focus on lesional skin biopsies taken at Baseline, Week 16, and Week 52 from patients with new-onset or chronic PsO treated with secukinumab, with never-lesional samples taken only at Week 52 as a reference. RNA-sequencing was performed on enzymatically separated epidermis and dermis. Gene expression analysis in the epidermis revealed that lesional transcriptomes at Baseline were largely similar between new-onset and chronic PsO. Following secukinumab treatment, gene expression signatures related to inflammation and PsO were normalized to never-lesional levels in both cohorts, including a "residual disease genomic profile (RDGP)" previously described to persist in PsO following etanercept treatment. Importantly, while comparable clinical responses were observed in both groups, normalization at the molecular level occurred considerably faster in new-onset (already at Week 16) than in chronic (at Week 52) PsO patients. In addition, secukinumab normalized pathways related to epigenetic control of transcription in new-onset, but not in chronic PsO lesional skin. These results suggest that early intervention with secukinumab is beneficial for PsO patients.

TRPV3 (transient receptor potential vanilloid 3) is a pro-inflammatory ion channel mostly expressed by keratinocytes of the human skin. Previous studies have shown that the expression of TRPV3 is markedly upregulated in the lesional epidermis of atopic dermatitis (AD) patients suggesting a potential pathogenetic role of the ion channel in the disease. In the current study, we aimed at defining the molecular and functional expression of TRPV3 in non-lesional skin of AD patients as previous studies implicated that healthy-appearing skin in AD is markedly distinct from normal skin with respect to terminal differentiation and certain immune function abnormalities. By using multiple, complementary immunolabelling and RT-qPCR technologies on full-thickness and epidermal shave biopsy samples from AD patients (lesional, non-lesional) and healthy volunteers, we provide the first evidence that the expression of TRPV3 is markedly upregulated in non-lesional human AD epidermis, similar to lesional AD samples. Of further importance, by using the patch-clamp method on cultured healthy and non-lesional AD keratinocytes, we also show that this upregulation is functional as determined by the significantly augmented TRPV3-specific ion current (induced by agonists) on cultured non-lesional AD keratinocytes when compared to healthy ones.

Langerhans cells (LCs) are the sole professional antigen-presenting cell normally found in the human epidermal compartment. Research into their physiological role is hindered by the fact that they are invariably activated during isolation from the skin. To overcome this challenge, we turned to a monocyte-derived LC (moLC) model, which we characterized with RNA sequencing, and compared the transcriptome of moLCs with that of donor-matched immature dendritic cells. We found that moLCs express markers characteristic of LC2 cells as well as TRPV4. TRPV4 is especially important in the skin because it has been linked to the conservation of the skin barrier, immunological responses, as well as acute and chronic itch, but we know little about its function on LCs. Our results show that TRPV4 activation increased the expression of Langerin and led to increased intracellular calcium concentration in moLCs. Regarding the functionality of moLCs, we found that TRPV4 agonism had a mitigating effect on their inflammatory responses because it decreased their cytokine production and T-cell activating capability. Because TRPV4 has emerged as a potential therapeutic target in dermatological conditions, it is important to highlight LCs as, to our knowledge, a previously unreported target of these therapies.

The immune system is strongly linked to the nervous system, to the point that in many diseases they cannot be discussed as separate entities. Specifically, inflammatory skin diseases such as atopic dermatitis and psoriasis have been reported to be intensified by stress, and, interestingly the skin lesions in these diseases sometimes resolve after nerve injury. The putative cause underlying this empirical observation is that sensory nerves secrete neuropeptides (NPs) and other mediators that are important members of neuro-immune communication. Inside the epidermis, the only dedicated resident immune cells under steady-state are the antigen-presenting cells called Langerhans cells (LCs). LCs have been shown to be anatomically associated with neurons that produce Calcitonin Gene-Related Peptide (CGRP), supporting the hypothesis that NPs possibly play a role in cutaneous inflammation. Previous reports have shown that CGRP reduces LC antigen-presentation to a Th1 clone while simultaneously, enhancing antigen-presentation for Th2 clones in mice, but little is known in humans. As a first, exploratory step we utilized RNA sequencing from monocyte-derived LCs samples from five donors and we detected the expression of neuropeptide receptor genes. We found high expression of CGRP receptor and its coreceptor RAMP1 and also one of the neurotensin receptors (SORT1, also known as NTR3) and the Brain Natriuretic peptide receptor NPR1, and validated these with RT-qPCR. We also determined the effect of all three neuropeptides on the differentiation of monocyte-derived LCs with flow cytometry and found that NPR1 activation significantly increased the differentiation of LCs, while the others had no effect. Interestingly, BNP had only minimal effect on the T cell stimulatory capability of LCs, or on their cytokine (IL-6, IL-8, IL-10 and IL-12) production. These data suggest that neuroimmune communication might be an important prerequisite of LC differentiation.

During the molecular transduction of itch, the stimulation of pruriceptors on sensory fibers leads to the activation or sensitization of ion channels, which results in a consequent depolarization of the neurons. These ion channels mostly belong to the transient receptor potential (TRP) channels, which are involved in nociception and thermosensation. In particular, TRPV1 and TRPA1 were described in the transduction of both thermal nociception as well as histaminergic and non-histaminergic itch. The thermosensitive TRPM3 plays an indispensable role in heat nociception together with TRPV1 and TRPA1. However, the role of TRPM3 in the development of pruritus has not been studied yet. Therefore, in this study we aimed at investigating the potential role of TRPM3 in the transduction of pruritus and pain by investigating itch- and nociception-related behavior of Trpm3+/+ and Trpm3-/- mice, and by studying the activation of somatosensory neurons isolated from trigeminal ganglia upon application of algogenic and pruritogenic substances. Activators of TRPM3 evoked only nocifensive responses, but not itch in Trpm3+/+ animals, and these nocifensive responses were abolished in the Trpm3-/- strain. Histamine and endogenous non-histaminergic pruritogens induced itch in both Trpm3+/+ and Trpm3-/- mice to a similar extent. Genetic deletion or pharmacological blockade diminished TRPM3 mediated Ca2+ responses of sensory neurons, but did not affect responses evoked by pruritogenic substances. Our results demonstrate that, in contrast to other thermosensitive TRP channels, TRPM3 selectively mediates nociception, but not itch sensation, and suggest that TRPM3 is a promising candidate to selectively target pain sensation.

Dendritic cells (DCs) act as a major link between the innate and adaptive immune systems. Nowadays, as the use of herbal active ingredients from the cannabis plant (Cannabis sativa) is gaining in popularity, the use of these products has shown an exponential increase, even in topical formulations that can directly influence the main function of DCs in skin. Up to now more than 100 different cannabinoids have been isolated from the Cannabis plant, which are called phytocannabinoids (PCs). PCs, especially the non-psychoactive compounds, have been shown to have beneficial effects in multiple inflammatory disease models; nevertheless, they were mostly investigated in mouse models and not on human cells. In our current work, we investigated the effect of four non-psychoactive PCs (cannabidiol, cannabinol, cannabigerol, tetrahydrocannabivarin, in regulating responses of monocyte-derived DCs by monitoring changes in their maturation markers. First, to exclude the possibility of the onset of early apoptotic or necrotic processes, we performed viability tests (PrestoBlue and G6PDH release assay), which demonstrated that our PCs did not cause any cell death of moDC. Moreover, we found that our PCs generally induced cell maturation at high concentrations on moDC, whereas LPS-induced maturation was not affected. Furthermore, we also found that CBG and THCV lead to an increase in the endocytotic capability of iDCs. Interestingly the increased maturation of the cells upon PC treatment did not result in inflammatory mediator production, however LPC-induced mediator production was enhanced by PCs. PCs also increased DC-induced T cell proliferation. Taken together, our data suggest that PCs may play varied roles on the immunological function of moDCs, and their use as potent anti-inflammatory treatments must be tested extensively.

Photodamage-induced and viral keratitis could benefit from treatment with novel nonsteroid anti-inflammatory agents. Therefore, we determined whether human corneal epithelial cells (HCECs) express members of the endocannabinoid system (ECS), and examined how the endocannabinoid anandamide (AEA, N-arachidonoyl ethanolamine) influences the Toll-like receptor 3 (TLR3) agonism- or UVB irradiation-induced inflammatory response of these cells. Other than confirming the presence of cannabinoid receptors, we show that endocannabinoid synthesizing and catabolizing enzymes are also expressed in HCECs in vitro, as well as in the epithelial layer of the human cornea in situ, proving that they are one possible source of endocannabinoids. p(I:C) and UVB irradiation was effective in promoting the transcription and secretion of inflammatory cytokines. Surprisingly, when applied alone in 100 nM and 10 μM, AEA also resulted in increased pro-inflammatory cytokine production. Importantly, AEA further increased levels of these cytokines in the UVB model, whereas its lower concentration partially prevented the transcriptional effect of p(I:C), while not decreasing the p(I:C)-induced cytokine release. HCECs express the enzymatic machinery required to produce endocannabinoids both in vitro and in situ. Moreover, our data show that, despite earlier reports about the anti-inflammatory potential of AEA in murine cornea, its effects on the immune phenotype of human corneal epithelium may be more complex and context dependent.

We have shown previously that endocannabinoids promote sebaceous lipogenesis, and sebocytes are involved in the metabolism of the endocannabinoid-like substance oleoylethanolamide (OEA). OEA is an endogenous activator of GPR119, a recently deorphanized receptor, which currently is being investigated as a promising antidiabetic drug target. In this study, we investigated the effects of OEA as well as the expression and role of GPR119 in human sebocytes. We found that OEA promoted differentiation of human SZ95 sebocytes (elevated lipogenesis, enhanced granulation, and the induction of early apoptotic events), and it switched the cells to a proinfiammatory phenotype (increased expression and release of several proinflammatory cytokines). Moreover, we could also demonstrate that GPR119 was expressed in human sebocytes, and its small interfering RNA-mediated gene silencing suppressed OEA-induced sebaceous lipogenesis, which was mediated via cillun N-terminal kinase, extracellular signal-regulated kinase 1/2, protein kinase B, and CRE-binding protein activation. Finally, our pilot data demonstrated that GPR119 was downregulated in the sebaceous glands of patients with acne, arguing that GPR119 signaling may indeed be disturbed in acne. Collectively, our findings introduce the OEA/GPR119 signaling as a positive regulator of sebocyte differentiation and highlight the possibility that dysregulation of this pathway may contribute to the development of seborrhea and acne.

Background" Volatile anaesthetics (VAs) are the most widely used compounds to induce reversible loss of consciousness and maintain general anaesthesia during surgical interventions. Although the mechanism of their action is not yet fully understood, it is generally believed, that VAs depress central nervous system functions mainly through modulation of ion channels in the neuronal membrane, including 2 -pore-domain K+ channels, GABA and NMDA receptors. Recent research also reported their action on nociceptive and thermosensitive TRP channels expressed in the peripheral nervous system, including TRPV1, TRPA1, and TRPM8. Here, we investigated the effect of VAs on TRPM3, a less characterized member of the thermosensitive TRP channels playing a central role in noxious heat sensation. Methods: We investigated the effect of VAs on the activity of recombinant and native TRPM3, by monitoring changes in the intracellular Ca2+ concentration and measuring TRPM3-mediated transmembrane currents. Results: All the investigated VAs (chloroform, halothane, isoflurane, sevoflurane) inhibited both the agonistinduced (pregnenolone sulfate, CIM0216) and heat-activated Ca2+ signals and transmembrane currents in a concentration dependent way in HEK293T cells overexpressing recombinant TRPM3. Among the tested VAs, halothane was the most potent blocker (IC50 = 0.52 +/- 0.05 mM). We also investigated the effect of VAs on native TRPM3 channels expressed in sensory neurons of the dorsal root ganglia. While VAs activated certain sensory neurons independently of TRPM3, they strongly and reversibly inhibited the agonist-induced TRPM3 activity. Conclusions: These data provide a better insight into the molecular mechanism beyond the analgesic effect of VAs and propose novel strategies to attenuate TRPM3 dependent nociception.

Transient receptor potential (TRP) ion channels, originally described mostly in neuronal cells, are now known to be expressed in numerous tissues, including immune cells. They affect many cellular functions, such as migration, cytokine secretion, and phagocytosis. TRP vanilloid (TRPV) channels were shown to be expressed on dendritic cells, where they decreased their differentiation and maturation. We have no information however, about their putative role on Langerhans cells (LCs). Our previous work highlighted the role of TRPV4 on monocyte-derived LCs (moLCs), where we showed that TRPV4 activation increased their differentiation, and led to an increase in intracellular calcium concentration, which effects could be abrogated by the specific antagonist of TRPV4. In our current work we further investigated the role of TRPV4 on moLCs. To support our functional data we showed that TRPV4 is expressed on the protein level. We next wanted to investigate the role of TRPV4 on the maturation of moLCs, which we induced by the addition of peptidoglycan (PGN). The TRPV4 agonist GSK1016790A when applied with PGN did not further stimulate the maturation of LCs, showing that TPRV4 influences the differentiation, but not the activation of moLCs. To determine what role LCs might play in the epidermal intracellular communication network we investigated the proteins secreted by our moLCs using a cytokine array. Our results showed that these cells produce CXCL8 and MIF among others, and that the transcription of these genes was not influenced by TRPV4 activation. Our results show that while TRPV4 stimulates the differentiation of moLCs, it does not affect the maturation of the cells.