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Extremely low-frequency electromagnetic stimulation (ELF-EMS) was demonstrated to be significantly beneficial in rodent models of permanent stroke. The mechanism involved enhanced cerebrovascular perfusion and endothelial cell nitric oxide production. However, the possible effect on the neuroinflammatory response and its efficacy in reperfusion stroke models remains unclear. To evaluate ELF-EMS effectiveness and possible immunomodulatory response, we studied neurological outcome, behavior, neuronal survival, and glial reactivity in a rodent model of global transient stroke treated with 13.5 mT/60 Hz. Next, we studied microglial cells migration and, in organotypic hippocampal brain slices, we assessed neuronal survival and microglia reactivity. ELF-EMS improved the neurological score and behavior in the ischemia-reperfusion model. It also improved neuronal survival and decreased glia reactivity in the hippocampus, with microglia showing the first signs of treatment effect. In vitro ELF-EMS decreased (Lipopolysaccharide) LPS and ATP-induced microglia migration in both scratch and transwell assay. Additionally, in hippocampal brain slices, reduced microglial reactivity, improved neuronal survival, and modulation of inflammation-related markers was observed. Our study is the first to show that an EMF treatment has a direct impact on microglial migration. Furthermore, ELF-EMS has beneficial effects in an ischemia/reperfusion model, which indicates that this treatment has clinical potential as a new treatment against ischemic stroke.

Microglial cells must act as the first line of defense of the central nervous system, but they can be exposed to various mechanical signals that may trigger their activation. While the impact of chemical signaling on brain cells has been studied in detail, our current understanding of the mechanical signaling in microglia is still limited. To address this challenge, we exposed microglial cells to a single mechanical stretch and compared their behavior to chemical activation by lipopolysaccharide treatment. Here we show that stretching microglial cells results in their activation, demonstrating a strong mechanosensitivity. Stretched microglial cells exhibited higher Iba1 protein levels, a denser actin cytoskeleton and migrated more persistently. In contrary to LPS-treated cells, stretched microglia maintain a robust secretory profile of chemokines and cytokines, except for TNF-α, highlighting the relevance of this model. Interestingly, a single stretch injury results in more compacted chromatin and DNA damage, suggesting possible long-term genomic instabilities in stretched microglia. Using neuronal networks in compartmentalized microfluidic chambers, we found that stretched microglial cells exhibit enhanced phagocytic and synaptic stripping activities. Altogether, our results propose that the immune potential of microglial cells can be unlocked by stretching events to maintain brain tissue homeostasis after mechanical injury.

p27

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

The Transient Receptor Potential Melastatin 3 (TRPM3) is a Ca2+-permeable non-selective cation channel activated by the neurosteroid pregnenolone sulfate (PS). This compound was previously shown to contract mouse aorta by activating TRPM3 in vascular smooth muscle cells (VSMC), and proposed as therapeutic modulator of vascular functions. However, PS effects and the role of TRPM3 in resistance arteries remain unknown. Thus, we aimed at determining the localization and physiological role of TRPM3 in mouse mesenteric arteries. Real-time qPCR experiments, anatomical localization using immunofluorescence microscopy and patch-clamp recordings in isolated VSMC showed that TRPM3 expression in mesenteric arteries is restricted to perivascular nerves. Pressure myography experiments in wild type (WT) mouse arteries showed that PS vasodilates with a concentration-dependence that was best fit by two Hill components (effective concentrations, EC50, of 14 and 100 μM). The low EC50 component was absent in preparations from Trpm3 knockout (KO) mice and in WT arteries in the presence of the CGRP receptor antagonist BIBN 4096. TRPM3-dependent vasodilation was partially inhibited by a cocktail of K+ channel blockers, and not mediated by β-adrenergic signaling. We conclude that, contrary to what was found in aorta, PS dilates mesenteric arteries, partly via an activation of TRPM3 that triggers CGRP release from perivascular nerve endings and a subsequent activation of K+ channels in VSMC. We propose that TRPM3 is implicated in the regulation of the tone of resistance arteries and that its activation by yet unidentified endogenous damage-associated molecules lead to protective vasodilation responses in mesenteric arteries., This work was supported by grants from the Fund for Scientific Research Flanders (FWO, G.0C68.15, Belgium) to KT, the Ministerio de Economía y Competitividad (BFU2013-45867-R and BFU2016-75360-R, Spain) to JRLL and MTPG and Junta de Castilla y León(VA114P17, Spain) to MTPG. Y.A.A. is a Postdoctoral Fellow of the FWO (Belgium).

Previously, we showed histamine-mediated sensitization of transient receptor potential (TRP) vanilloid 1 (TRPV1) in patients with irritable bowel syndrome (IBS). Sensitization of TRP ankyrin 1 (TRPA1) and TRP vanilloid 4 (TRPV4) are also involved in aberrant pain perception in preclinical models of somatic pain. Here, we hypothesize that in parallel with TRPV1, histamine sensitizes TRPA1 and TRPV4, contributing to increased visceral pain in patients with IBS. Rectal biopsies were collected from patients with IBS and healthy subjects (HS) to study neuronal sensitivity to TRPA1 and TRPV4 agonists (cinnamaldehyde and GSK1016790A) using intracellular Ca2+ imaging. In addition, the effect of supernatants of rectal biopsies on patients with IBS and HS was assessed on TRPA1 and TRPV4 responses in murine dorsal root ganglion (DRG) sensory neurons. Finally, we evaluated the role of histamine and histamine 1 receptor (H1R) in TRPA1 and TRPV4 sensitization. Application of TRPA1 and TRPV4 agonists evoked significantly higher peak amplitudes and percentage of responding submucosal neurons in biopsies of patients with IBS compared with HS. In HS, pretreatment with histamine significantly increased the Ca2+ responses to cinnamaldehyde and GSK1016790A, an effect prevented by H1R antagonism. IBS supernatants, but not of HS, sensitized TRPA1 and TRPV4 on DRG neurons. This effect was reproduced by histamine and prevented by H1R antagonism. We demonstrate that the mucosal microenvironment in IBS contains mediators, such as histamine, which sensitize TRPV4 and TRPA1 via H1R activation, most likely contributing to increased visceral pain perception in IBS. These data further underscore H1R antagonism as potential treatment for IBS. NEW & NOTEWORTHY We provide evidence for histamine-mediated transient receptor potential (TRP) ankyrin 1 and TRP vanilloid 4 sensitization in irritable bowel syndrome (IBS) via histamine 1 receptor (H1R) activation, most likely contributing to increased visceral pain perception. Our results reveal a general role of sensory TRP channels as histamine effectors in the pathophysiology of IBS and provide novel mechanistic insights into the therapeutic potential of H1R antagonism in IBS.

p27(kip1) is a multifunctional protein that promotes cell cycle exit by blocking the activity of cyclin/cyclin-dependent kinase complexes as well as migration and motility via signaling pathways that converge on the actin and microtubule cytoskeleton. Despite the broad characterization of p27(kip1) function in neural cells, little is known about its relevance in microglia. Here, we studied the role of p27(kip1) in microglia using a combination of in vitro and in situ approaches. While the loss of p27(kip1) did not affect microglial density in the cerebral cortex, it altered their morphological complexity in situ. However, despite the presence of p27(kip1) in microglial processes, as shown by immunofluorescence in cultured cells, loss of p27(kip1) did not change microglial process motility and extension after applying laser-induced brain damage in cortical brain slices. Primary microglia lacking p27(kip1) showed increased phagocytic uptake of synaptosomes, while a cell cycle dead variant negatively affected phagocytosis. These findings indicate that p27(kip1) plays specific roles in microglia. J.B. is a Ph.D. student supported by the Special Research Foundation from Hasselt University and Université de Liège. Y.A.A is supported by an FWO senior postdoctoral fellowship (12H8220N), and S.K. is a Ph.D. assistant supported by Hasselt University, J.-M.R. is a full Professor at Hasselt University, B.B. is an Associate Professor at Hasselt University and L.N. is Director from the F.R.S.-F.N.R.S. This work was supported by the Special Research Foundation (BOF17DOCLI01), the FWO research grant (1521619N), Funding Hercules (I000220N), the Sint-Gillis autism research grant, ROTARY Espoir en Tête—Hoofdzaak er is Hoop, the F.R.S.-F.N.R.S. (Synet; EOS 0019118F-RG36), the Fonds Leon Fredericq (L.N.), the Fondation Médicale Reine Elisabeth (L.N.), the Fondation Simone et Pierre Clerdent (L.N), the Belgian Science Policy (IAP-VII network P7/20 (L.N.)), and the ERANET Neuron STEM-MCD and NeuroTalk (L.N.). We sincerely thank Melissa Jans and Yennick Geuens for the maintenance of the mouse colonies at BIOMED and Petra Bex and Fanny Lepiemme for providing assistance in maintaining the mouse colonies at BIOMED or the GIGA research institute. We thank Rosette Beenaerts for technical assistance with RT-PCR and Sam Duwé from the BIOMED Imaging platform for his technical support. We also thank Sandra Ormenese, Jean-Jaques Goval (deceased), and Rafaat Stephan from the GIGA-Cell imaging and flow cytometry platforms for their technical guidance

The Transient Receptor Potential Ankyrin 1 cation channel (TRPA1) is a broadly-tuned chemosensor expressed in nociceptive neurons. Multiple TRPA1 agonists are chemically unrelated non-electrophilic compounds, for which the mechanisms of channel activation remain unknown. Here, we assess the hypothesis that such chemicals activate TRPA1 by inducing mechanical perturbations in the plasma membrane. We characterized the activation of mouse TRPA1 by non-electrophilic alkylphenols (APs) of different carbon chain lengths in the para position of the aromatic ring. Having discarded oxidative stress and the action of electrophilic mediators as activation mechanisms, we determined whether APs induce mechanical perturbations in the plasma membrane using dyes whose fluorescence properties change upon alteration of the lipid environment. APs activated TRPA1, with potency increasing with their lipophilicity. APs increased the generalized polarization of Laurdan fluorescence and the anisotropy of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH), also according to their lipophilicity. Thus, the potency of APs for TRPA1 activation is an increasing function of their ability to induce lipid order and membrane rigidity. These results support the hypothesis that TRPA1 senses non-electrophilic compounds by detecting the mechanical alterations they produce in the plasma membrane. This may explain how structurally unrelated non-reactive compounds induce TRPA1 activation and support the role of TRPA1 as an unspecific sensor of potentially noxious compounds. This work was funded by grants of the Research Council of the KU Leuven (C14/18/086) and the Fund for Scientific Research Flanders (FWO: G0D0417N and G0B2219N). J.B.S. has a Postdoctoral Mandate of the Research Council of the KU Leuven. R.N. is funded by a PhD Fellowship of the FWO. Y.A.A. has a Postdoctoral Fellowship of the FWO. Talavera, K (corresponding author), Katholieke Univ Leuven, Dept Cellular & Mol Med, Lab Ion Channel Res, B-3000 Leuven, Belgium ; VIB Ctr Brain & Dis Res, B-3000 Leuven, Belgium. justyna.startek@kuleuven.be; milicialina13@gmail.com; robbe.naert@kuleuven.be; andrei.segal@kuleuven.be; yeranddy.aguiaralpizar@uhasselt.be; thomas.voets@kuleuven.be; karel.talavera@kuleuven.be

Background: Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been associated with severe chronic rhinosinusitis with nasal polyps (CRSwNP). The pathogenic mechanism of SEB on epithelial barriers, however, is largely unexplored. Objective: We investigated the effect of SEB on nasal epithelial barrier function. Methods: SEB was apically administered to air-liquid interface (ALI) cultures of primary polyp and nasal epithelial cells of CRSwNP patients and healthy controls, respectively. Epithelial cell integrity and tight junction expression were evaluated. The involvement of Toll-like receptor 2 (TLR2) activation was studied in vitro with TLR2 monoclonal antibodies and in vivo in tlr2−/− knockout mice. Results: SEB applied to ALI cultures of polyp epithelial cells decreased epithelial cell integrity by diminishing occludin and zonula occludens (ZO)-1 protein expression. Antagonizing TLR2 prevented SEB-induced barrier disruption. SEB applied in the nose of control mice increased mucosal permeability and decreased mRNA expression of occludin and ZO-1, whereas mucosal integrity and tight junction expression remained unaltered in tlr2−/− mice. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6 and IL-8, which was prevented by TLR2 antagonization. Conclusion & Clinical relevance: SEB damages nasal polyp epithelial cell integrity by triggering TLR2 in CRSwNP. Our results suggest that SEB might represent a driving factor of disease exacerbation, rather than a causal factor for epithelial defects in CRSwNP. Interfering with TLR2 triggering might provide a way to avoid the pathophysiological consequences of S. aureus on inflammation in CRSwNP. Funding statement: The author’s laboratories are supported by grants from the Belgian Federal Government (IUAP P7/30), IWT (TBM project 130260), and the research council of the KU Leuven (GOA 14/011). RS and DMB are recipients of a senior research fellowship from the Fund of Scientific Research (FWO), Flanders, Belgium. BS is currently a Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO). Acknowledgements The authors would like to thank Ahmad Kasran, Lieve Coorevits and Ellen Dilissen for their insightful comments and technical advice, Dr. Camille Allonsius for helping with the transfection of HEK cells and Prof. Mark Jorissen (UZ Leuven) for collecting nasal tissue. Additionally, we thank Prof. Pieter Vanden Berghe for his assistance with confocal microscopy (Hercules AKUL/11/37 and FWO G.0929.15). We would like to thank Prof. Olivier Denis from Sciensano, Brussels, Belgium for the tlr2-/- mice. Author contributions: BS and KM performed experiments, acquired data, analyzed data and wrote the manuscript. YAA and SFS conducted experiments. CB, RS, DMB and SL discussed and interpreted findings, and critically revised the manuscript. JLC and BS designed research study and critically revised the manuscript. Funding: The author’s laboratories are supported by grants from the Belgian Federal Government (IUAP P7/30) and the research council of the KU Leuven (GOA 14/011). RS and DMB are recipients of a senior researcher fellowship from the Fund of Scientific Research (FWO), Flanders, Belgium. BS is currently a Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO).

ObjectiveResolvins (RvD1, RvD2 and RvE1) are endogenous anti-inflammatory lipid mediators that display potent analgesic properties in somatic pain by modulating transient receptor potential vanilloid 1 (TRPV1) activation. To what extent these molecules could also have a beneficial effect on TRPV1 sensitisation and visceral hypersensitivity (VHS), mechanisms involved in IBS, remains unknown.DesignThe effect of RvD1, RvD2 and RvE1 on TRPV1 activation and sensitisation by histamine or IBS supernatants was assessed on murine dorsal root ganglion (DRG) neurons using live Ca2+ imaging. Based on the results obtained in vitro, we further studied the effect of RvD2 in vivo using a murine model of post-infectious IBS and a rat model of post-inflammatory VHS. Finally, we also tested the effect of RvD2 on submucosal neurons in rectal biopsies of patients with IBS.ResultsRvD1, RvD2 and RvE1 prevented histamine-induced TRPV1 sensitisation in DRG neurons at doses devoid of an analgesic effect. Of note, RvD2 also reversed TRPV1 sensitisation by histamine and IBS supernatant. This effect was blocked by the G protein receptor 18 (GPR18) antagonist O-1918 (3–30 µM) and by pertussis toxin. In addition, RvD2 reduced the capsaicin-induced Ca2+ response of rectal submucosal neurons of patients with IBS. Finally, treatment with RvD2 normalised pain responses to colorectal distention in both preclinical models of VHS.ConclusionsOur data suggest that RvD2 and GPR18 agonists may represent interesting novel compounds to be further evaluated as treatment for IBS.

The transient receptor potential ankyrin (TRPA) channels are Ca2+-permeable nonselective cation channels remarkably conserved through the animal kingdom. Mammals have only one member, TRPA1, which is widely expressed in sensory neurons and in non-neuronal cells (such as epithelial cells and hair cells). TRPA1 owes its name to the presence of 14 ankyrin repeats located in the NH2 terminus of the channel, an unusual structural feature that may be relevant to its interactions with intracellular components. TRPA1 is primarily involved in the detection of an extremely wide variety of exogenous stimuli that may produce cellular damage. This includes a plethora of electrophilic compounds that interact with nucleophilic amino acid residues in the channel and many other chemically unrelated compounds whose only common feature seems to be their ability to partition in the plasma membrane. TRPA1 has been reported to be activated by cold, heat, and mechanical stimuli, and its function is modulated by multiple factors, including Ca2+, trace metals, pH, and reactive oxygen, nitrogen, and carbonyl species. TRPA1 is involved in acute and chronic pain as well as inflammation, plays key roles in the pathophysiology of nearly all organ systems, and is an attractive target for the treatment of related diseases. Here we review the current knowledge about the mammalian TRPA1 channel, linking its unique structure, widely tuned sensory properties, and complex regulation to its roles in multiple pathophysiological conditions. ispartof: PHYSIOLOGICAL REVIEWS vol:100 issue:2 pages:725-803 ispartof: location:United States status: published

Up to 20% of people worldwide develop gastrointestinal symptoms following a meal(1), leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H-1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders. In mice, oral tolerance to food antigens can break down after enteric infection, and this leads to food-induced pain resembling irritable bowel syndrome in humans.

BACKGROUND: Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells. RESULTS: Using fluorometric measurements of intracellular Ca2+ concentration ([Ca2+]i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca2+]i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells. CONCLUSIONS: Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs. Y.A.A. held a Postdoctoral Mandate of the KU Leuven and is currently a Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO). Research was supported by grants from the Research Foundation Flanders FWO (G076714), the Research Council of the KU Leuven (Grants GOA/14/011 and PF-TRPLe), The Spanish Ministry of Economy and Competitiveness (SAF2015-69762R and María de Maeztu Programme for Units of Excellence in R&D MDM-2014-0370), and the FEDER Funds.

The cation channel TRPA1 transduces a myriad of noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. Despite emergent evidence that TRPA1 is regulated by the membrane environment, it remains unknown whether this channel localizes in membrane microdomains or whether it interacts with cholesterol. Using total internal reflection fluorescence microscopy and density gradient centrifugation we found that mouse TRPA1 localizes preferably into cholesterol-rich domains and functional experiments revealed that cholesterol depletion decreases channel sensitivity to chemical agonists. Moreover, we identified two structural motifs in transmembrane segments 2 and 4 involved in mTRPA1-cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. We discuss the impact of such interactions on TRPA1 gating mechanisms, regulation by the lipid environment, and role of this channel in sensory membrane microdomains, all of which helps to understand the puzzling pharmacology and pathophysiology of this channel., SCOPUS: ar.j, info:eu-repo/semantics/published

Background: Staphylococcus aureus colonization and release of enterotoxin B (SEB) has been shown to contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP), but the pathogenic mechanisms are largely unknown. Methods: We investigated the effect of SEB on nasal epithelial function in vitro on primary polyp and nasal epithelial cells of patients with CRSwNP and healthy controls respectively, and in vivo in a mouse model by measuring epithelial integrity and tight junction expression. The involvement of TLR2 signaling was studied in vitro by using TLR2 monoclonal antibodies and in vivo in tlr2-/- transgenic mice. Findings: We found that in vitro stimulation with SEB decreased polyp epithelial integrity by reducing occludin and ZO-1 protein expression. This impaired barrier function was associated with elevated expression of epithelial TLR2 and antagonizing TLR2 triggering prevented SEB-induced barrier disruption in vitro. In wild type mice, SEB applied in the nose increased mucosal permeability and decreased occludin and ZO-1 mRNA expression, whereas tlr2-/- mice had an intact mucosal barrier and normal tight junction expression after SEB exposure. Furthermore, in vitro SEB stimulation resulted in epithelial production of IL-6, IL-8 and TNF, which was prevented when TLR2 triggering was antagonized. Interpretation: Our results indicate that SEB impairs epithelial barrier function in CRSwNP via TLR2 activation. Interfering with TLR2 activation may provide a way to avoid the pathophysiological consequences of S. aureus colonization in CRSwNP. Funding Statement: The author’s laboratories are supported by grants from the Belgian Federal Government (IUAP P7/30), IWT (TBM project 130260) and the research council of the KU Leuven (GOA 14/011). PWH, RS and DMB are recipients of a senior researcher fellowship from the Fund of Scientific Research (FWO), Flanders, Belgium. BS is currently a Postdoctoral Fellow of the Fund for Scientific Research Flanders (FWO). Declaration of Interests: The authors declare no competing interest related to this work. Ethics Approval Statement: All experiments were approved by the Medical Ethical Committee of the University Hospitals Leuven. Murine experiments were approved by the Ethical Committee for Animal Research at the KU Leuven.

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.DesignUsing Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1–5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.ResultsEFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.ConclusionEnteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.Trial registration numberNCT02425774.

Literature is convincing regarding the efficacy of capsaicin nasal treatment in idiopathic rhinitis (IR). However, up to 50% of IR patients do not meet the strict inclusion criteria of the trials conducted so far. As a consequence, the efficacy of capsaicin is unknown in a significant number of IR patients that do not meet the strict inclusion/exclusion criteria (J Allergy Clin Immunol. 2014;133:1332, J Allergy Clin Immunol. 2017; [Epub ahead of print]). "Mixed rhinitis" (MR) patients have more than one major etiologic factor involved in the mucosal pathology. We have no idea about the efficacy of capsaicin nasal spray in these patients nor about the time interval to seek a second application. We report here that capsaicin nasal spray is effective in a broader group of IR than the purely selected ones described before, that subjective nasal hyper-reactivity is a good predictor of positive outcome, and that the time interval for seeking a second treatment is likely to be shorter in MR patients than in the strictly selected IR patients.

Acute neurogenic inflammation and pain associated to bacterial infection have been traditionally ascribed to sensitization and activation of sensory nerve afferents secondary to immune cell stimulation. However, we recently showed that lipopolysaccharides (LPS) directly activate the Transient Receptor Potential channels TRPA1 in sensory neurons and TRPV4 in airway epithelial cells. Here we investigated whether LPS activates other sensory TRP channels expressed in sensory neurons. Using intracellular Ca2+ imaging and patch-clamp we determined the effects of LPS on recombinant TRPV1, TRPV2, TRPM3 and TRPM8, heterologously expressed in HEK293T cells. We found that LPS activates TRPV1, although with lower potency than for TRPA1. Activation of TRPV1 by LPS was not affected by mutations of residues required for activation by electrophilic agents or by diacylglycerol and capsaicin. On the other hand, LPS weakly activated TRPM3, activated TRPM8 at 25 °C, but not at 35 °C, and was ineffective on TRPV2. Experiments performed in mouse dorsal root ganglion (DRG) neurons revealed that genetic ablation of Trpa1 did not abolish the responses to LPS, but remain detected in 30% of capsaicin-sensitive cells. The population of neurons responding to LPS was dramatically lower in double Trpa1/Trpv1 KO neurons. Our results show that, in addition to TRPA1, other TRP channels in sensory neurons can be targets of LPS, suggesting that they may contribute to trigger and regulate innate defenses against gram-negative bacterial infections. ispartof: Cell Calcium vol:73 pages:72-81 ispartof: location:Netherlands status: published

Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan’s emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms. Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan’s emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms. ispartof: Scientific Reports vol:8 issue:1 ispartof: location:England status: published

Nasal hyperreactivity (NHR) is an important clinical feature of allergic rhinitis (AR). The efficacy of MP29-02 (azelastine hydrochloride (AZE) and fluticasone propionate (FP)) nasal spray on local inflammatory mediators and NHR in AR is unknown. We tested if MP29-02 decreases inflammatory mediators and NHR in AR and if this effect is due to restoration of nasal epithelial barrier function. A 4-week double-blinded placebo-controlled trial with MP29-02 treatment was conducted in 28 patients with house dust mite (HDM) AR. The presence of NHR was evaluated by measuring reduction of nasal flow upon cold dry air exposure. The effects of AZE+/-FP on barrier integrity and airway inflammation were studied in a murine model of HDM induced NHR and on reduced activation of murine sensory neurons and human mast cells. MP29-02 but not placebo reduced NHR (p

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction., LPS is a major component of gram-negative bacterial cell walls, and triggers immune responses in airway epithelium by activating TLR4. Here the authors show that LPS also activates TRPV4, thereby inducing fast defense responses such as nitric oxide production and increased ciliary beating in mice.

In order to survive, organisms must adapt to environmental conditions that may subject them to changes in physical variables such as temperature, pressure, light, concentration of chemicals, and so on. In addition, they must be able to react appropriately to their biological milieu and especially to the challenge presented by pathogens that colonize tissues, producing injury, depletion of resources, and interference with vital metabolic functions. The adaptation processes typically entail the initial detection of external stimuli and the posterior implementation of reflex reactions. While in the classical view the sensing of physical stimuli is carried out mainly by sensory neurons and the detection of pathogen-derived cues by the immune cells, in recent years it has become clear that there is a functional interplay between the nervous and immune systems. These neuroimmune interactions arise not only from an intense biochemical cross-talk between neurons and immune cells, but also from the overlap in the sensory functions of these cells.As exciting and important as it is, the study of neuroimmune interactions seems to be in its infancy, mainly due to insufficient interaction between immunologists and neuroscientists. There is, however, one concept—inflammation—that serves as a meeting point for these research communities. But again it is interesting to see that for immunologists, inflammation is mainly related to the action and regulation of immune cells, without much consideration given to neural functions. For neuroscientists, and especially for sensory neuroscientists, inflammation is mostly associated with “neurogenic inflammation.” This concept refers to the consequences of the local release of inflammatory mediators upon activation of sensory nerve endings, which include vasodilation, plasma extravasation, but also recruitment of leukocytes and degranulation of mast cells (Chiu et al., 2012; Engel et al., 2011a; Fernandes et al., 2009; Geppetti et al., 2008; Russell et al., 2014).Neuroimmune interactions can take place at systemic levels, as occurs when acetylcholine is released to the circulation by the parasympathetic innervation thus influencing the function of distal immune cells, but also at the level of close-range cross-talks between nerve and immune cells (Andersson and Tracey, 2012; Chiu et al., 2012; Ordovas-Montanes et al., 2015). Neuronal signals such as neurotransmitters and neuropeptides influence hematopoiesis, priming and migration of immune cells, while cytokines and histamine produced by the latter can lead to neuronal activation and sensitization. In the central nervous system, the two-sided interplay between neurons and immune cells is particularly evident for microglia and mast cells, which have important roles in neuroinflammatory conditions (Skaper et al., 2014).Several advances in the field of neuroimmunology have been driven by the need of considering, and in some cases reconsidering, the function of the nervous system to understand pathologies that were mostly defined as immune disorders (see, e.g., Bautista et al., 2014; Belvisi et al., 2016; Halliez and Buret, 2015; Hyland et al., 2014; Ji, 2015; Mayer et al., 2015; Mazzone and Undem, 2016; O'Malley, 2015; Undem and Taylor-Clark, 2014). Increasing evidence indicates that neuroimmune interactions are implicated in hypersensitivity and hyperreactivity conditions such as irritable bowel syndrome, rhinitis, and asthma. Additional momentum has been gained through the recent identification of some of the molecules and receptors implicated in the mechanisms of cellular neuroimmune cross-talk.In this chapter, we focus on transient receptor potential (TRP) proteins (Flockerzi and Nilius, 2014; Nilius and Flockerzi, 2014), which compose a superfamily of cation channels that play increasingly acknowledged roles in the pathophysiology of all of the vertebrate systems, including the nervous (Gerhold and Bautista, 2009; Julius, 2013; Mickle et al., 2015; Talavera et al., 2008; Vennekens et al., 2012) and immune (Parenti et al., 2016) systems. They are implicated in inflammatory responses, not only through the phenomenon of neurogenic inflammation (Geppetti et al., 2008; Xanthos and Sandkuhler, 2014), but also via their function in proinflammatory immune cells either resident in the nervous tissues or infiltrating, such as microglia (Echeverry et al., 2016; Eder, 2010; Sharma and Ping, 2014), macrophages (Isami et al., 2013), neutrophils (Gelderblom et al., 2014), and mast cells (Freichel et al., 2012). The two main protagonists are the capsaicin receptor TRPV1 and the broadly tuned noxious chemosensor TRPA1. The functional expression of these channels in nociceptive neurons and their roles in neurogenic inflammation are clearly established (Bevan et al., 2014; Nilius et al., 2012; Zygmunt and Hogestatt, 2014). In the following, we discuss how several neuronal TRP channels may serve to sense exogenous pathogen-derived cues, as well as endogenous effector molecules released by immune cells, and review the implication of these channels in neuroimmune interactions during inflammatory processes.

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a Ca(2+)-activated nonselective cation channel involved in the transduction of sweet, bitter, and umami tastes. We previously showed that TRPM5 is a locus for the modulation of taste perception by temperature changes, and by quinine and quinidine, 2 bitter compounds that suppress gustatory responses. Here, we determined whether other bitter compounds known to modulate taste perception also affect TRPM5. We found that nicotine inhibits TRPM5 currents with an effective inhibitory concentration of ~1.3mM at -50 mV. This effect may contribute to the inhibitory effect of nicotine on gustatory responses in therapeutic and experimental settings, where nicotine is often employed at millimolar concentrations. In addition, it implies the existence of a TRPM5-independent pathway for the detection of nicotine bitterness. Nicotine seems to act from the extracellular side of the channel, reducing the maximal whole-cell conductance and inducing an acceleration of channel closure that leads to a negative shift of the activation curve. TRPM5 currents were unaffected by nicotine's metabolite cotinine, the intensive sweetener saccharin or by the bitter xanthines caffeine, theobromine, and theophylline. We also tested the effects of bitter compounds on another essential element of the sweet taste transduction pathway, the type 3 IP3 receptor (IP3R3). We found that IP3R3-mediated Ca(2+) flux is slightly enhanced by nicotine, not affected by saccharin, modestly inhibited by caffeine, theobromine, and theophylline, and strongly inhibited by quinine. Our results demonstrate that bitter compounds have differential effects on key elements of the sweet taste transduction pathway, suggesting for heterogeneous mechanisms of bitter-sweet taste interactions.

The increase in cytosolic Ca2+ is essential in key effector functions of dendritic cells (DCs), including differentiation, maturation, cytokine expression, and phagocytosis. Although several Ca2+-permeable ion channels have been described in DCs, the contribution of transient receptor potential (TRP) channels remains poorly understood. Here, we investigated whether TRPV4 plays a role in the differentiation, maturation, and phagocytosis of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced mouse bone marrow-derived cells (BMDCs). Using intracellular Ca2+ imaging experiments, we found that TRPV4 was functionally expressed in the plasma membrane of immature CD11c+ BMDCs and that its activity and expression were downregulated in CD11c+ BMDCs matured with lipopolysaccharide (LPS). Comparative analysis of the GM-CSF-stimulated cells showed that Trpv4 knockout and wild-type bone marrow cultures had a similar distribution of differentiated cells, generating a heterogenous culture population rich in CD11c+, CD11b+ cells, and low levels of F4/80+ cells. The lack of TRPV4 did not prevent the LPS-induced nuclear translocation of NF-&kappa, B, the upregulation of the proinflammatory cytokines IL-6 and IL-12, or the upregulation of the maturation markers CD40, CD80, and CD86. In contrast, TRPV4-deficient CD11c+ BMDCs exhibited a significantly reduced endocytic capacity of IgG-coated beads, but the internalization of uncoated beads in the absence of TRPV4 was not affected. Taken together, our results demonstrate that TRPV4 was dispensable in the differentiation and maturation of mouse CD11c+ BMDCs but contributed to the mechanism underlying Fc receptor-mediated phagocytosis. Overall, our results further strengthen the role of TRPV4 in immune-related processes.

The powerful plant-derived irritant allyl isothiocyanate (AITC, aka mustard oil) induces hyperalgesia to heat in rodents and humans through mechanisms that are not yet fully understood. It is generally believed that AITC activates the broadly tuned chemosensory cation channel transient receptor potential cation channel subfamily A member 1 (TRPA1), triggering an inflammatory response that sensitizes the heat sensor transient receptor potential cation channel subfamily V member 1 (TRPV1). In the view of recent data demonstrating that AITC can directly activate TRPV1, we here explored the possibility that this compound sensitizes TRPV1 to heat stimulation in a TRPA1-independent manner. Patch-clamp recordings and intracellular Ca(2+) imaging experiments in HEK293T cells over-expressing mouse TRPV1 revealed that the increase in channel activation induced by heating is larger in the presence of AITC than in control conditions. The analysis of the effects of AITC and heat on the current-voltage relationship of TRPV1 indicates that the mechanism of sensitization is based on additive shifts of the voltage dependence of activation towards negative voltages. Finally, intracellular Ca(2+) imaging experiments in mouse sensory neurons isolated from Trpa1 KO mice yielded that AITC enhances the response to heat, specifically in the subpopulation expressing TRPV1. Furthermore, this effect was strongly reduced by the TRPV1 inhibitor capsazepine and virtually absent in neurons isolated from double Trpa1/Trpv1 KO mice. Taken together, these findings demonstrate that TRPV1 is a locus for cross sensitization between AITC and heat in sensory neurons and may help explaining, at least in part, the role of this channel in AITC-induced hyperalgesia to heat.

To study whether formulation influences biodistribution, necrosis avidity and tumoricidal effects of the radioiodinated hypericin, a necrosis avid agent for a dual-targeting anticancer radiotherapy.Iodine-123- and 131-labeled hypericin ((123)I-Hyp and (131)I-Hyp) were prepared with Iodogen as oxidant, and formulated in dimethyl sulfoxide (DMSO)/PEG400 (polyethylene glycol 400)/water (25/60/15, v/v/v) or DMSO/saline (20:80, v/v). The formulations with excessive Hyp were optically characterized. Biodistribution, necrosis avidity and tumoricidal effects were studied in rats (n = 42) without and with reperfused liver infarction and implanted rhabdomyosarcomas (R1). To induce tumor necrosis, R1-rats were pre-treated with a vascular disrupting agent. Magnetic resonance imaging, tissue-gamma counting, autoradiography and histology were used.The two formulations differed significantly in fluorescence and precipitation. (123)I-Hyp/Hyp in DMSO/PEG400/water exhibited high uptake in necrosis but lower concentration in the lung, spleen and liver (p 0.01). Tumor volumes of 0.9 ± 0.3 cm(3) with high radioactivity (3.1 ± 0.3% ID/g) were detected 6 days post-treatment. By contrast, (131)I-Hyp/Hypin DMSO/saline showed low uptake in necrosis but high retention in the spleen and liver (p 0.01). Tumor volumes reached 2.6 ± 0.7 cm(3) with low tracer accumulation (0.1 ± 0.04%ID/g).The formulation of radioiodinated hypericin/hypericin appears crucial for its physical property, biodistribution, necrosis avidity and tumoricidal effects.

Allyl isothiocyanate (AITC; aka, mustard oil) is a powerful irritant produced by Brassica plants as a defensive trait against herbivores and confers pungency to mustard and wasabi. AITC is widely used experimentally as an inducer of acute pain and neurogenic inflammation, which are largely mediated by the activation of nociceptive cation channels transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 (TRPV1). Although it is generally accepted that electrophilic agents activate these channels through covalent modification of cytosolic cysteine residues, the mechanism underlying TRPV1 activation by AITC remains unknown. Here we show that, surprisingly, AITC-induced activation of TRPV1 does not require interaction with cysteine residues, but is largely dependent on S513, a residue that is involved in capsaicin binding. Furthermore, AITC acts in a membrane-delimited manner and induces a shift of the voltage dependence of activation toward negative voltages, which is reminiscent of capsaicin effects. These data indicate that AITC acts through reversible interactions with the capsaicin binding site. In addition, we show that TRPV1 is a locus for cross-sensitization between AITC and acidosis in nociceptive neurons. Furthermore, we show that residue F660, which is known to determine the stimulation by low pH in human TRPV1, is also essential for the cross-sensitization of the effects of AITC and low pH. Taken together, these findings demonstrate that not all reactive electrophiles stimulate TRPV1 via cysteine modification and help understanding the molecular bases underlying the surprisingly large role of this channel as mediator of the algesic properties of AITC.

Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism. DOI: http://dx.doi.org/10.7554/eLife.13133.001, eLife digest An immune system can fight bacterial infections, ensuring an animal’s health and survival. However, mounting an immune response to a bacterial infection requires a lot of energy. It also can be potentially dangerous if the immune system becomes too active. Therefore, avoiding bacteria and not getting infected to begin with may be a better strategy to stay healthy. Fruit flies, like humans, can detect dangerous substances in the environment via their sense of smell, but it is not known whether they also detect disease-causing organisms through their sense of taste. Bacterial molecules called lipopolysaccharides (LPS) can alert the immune system to the presence of dangerous bacteria. Previous studies have found that when flies get in contact with LPS they begin cleaning themselves, which might help prevent infection. However it was not clear how the flies actually detected the LPS. Now, Soldano et al. show that fruit flies can taste LPS and avoid eating or laying eggs on food contaminated with LPS and bacteria. A series of experiments showed that when a fly tastes LPS it stimulates bitter-sensing neurons in the fly’s mouth and throat. The experiments also revealed that the protein that activates these neurons in response to LPS is the same protein that acts in humans as detector of pungent chemicals contained in ordinary food items like mustard, garlic and wasabi. This suggests this protein, called TRPA1, is part of a key survival mechanism that has been preserved in many species throughout evolution. Soldano et al. showed that a fly’s senses and nervous system are actively involved in protecting it from bacterial infection. This is particularly important to flies, because unlike humans they don’t develop resistance to future infections with the same bacteria. Future studies are needed to determine if flies use their sense of taste to detect other chemicals that are signs of infections. Additionally, studies are needed to determine if the activated bitter-sensing nerves alert the fly’s immune system to a potential infection. DOI: http://dx.doi.org/10.7554/eLife.13133.002

The TRPA1 ion channel functions as a broadly-tuned chemonociceptor in many species. In the fruit fly, Drosophila melanogaster, dTRPA1(A) is known to contribute to chemosensation for its expression in a subset of gustatory receptor neurons in the labral sense organ and in the labellum. These neurons also express a gustatory receptor Gr66a and mediate the avoidance of non-volatile compounds that are recognized by mammalian bitter taste receptors.In this study we aimed to further characterize the chemosensory properties of dTRPA1(A). This channel was reported to mediate the aversion towards citronellal, but to be activated indirectly through a G protein/PLC signalling cascade. We expressed the TRPA1(A) in a mammalian heterologous expression system. By use of the whole cell patch clamp technique, we were able to show that Citronellal activates TRPA1(A) in HEK293T cells.It has been recently shown that TRPA1 plays a role in the acute mammalian nocifensive responses to bacterial lipopolysaccharides (LPS). We hypothesized that dTRPA1 endows Drosophila fruit flies with the ability to detect LPS and to adopt avoiding behaviours towards this compound. Behavioural experiments showed that D. melanogaster flies avoid laying eggs on food contaminated with bacteria through mechanisms that are partly dependent on dTRPA1 activation. By use of the whole cell patch clamp technique we could illustrate the activation of dTRPA1(A) currents in HEK293T cells.Altogether, these experiments indicate for a direct role of TRPA1 in the detection and avoidance of a wide variety of noxious chemicals in D. melanogaster.

Background Lung cancer remains a leading cause of cancer mortality, and so the aim of the present study was to develop a therapeutic vaccine protocol. Methods We constructed a lentiviral vector (LV) expressing the extracellular domain (ECD) of murine Her1, an antigen associated with poor prognosis in lung cancer. Results A single LV injection, followed by two Her1 protein boosts, was effective in reducing the metastatic burden of Lewis lung carcinoma in mice. The Her1 LV immunisation generated CD8+ T cells that recognised Her1 ECD presented by dendritic cells, and that also homed to Her1-expressing tumours. Protein boosting further increased the CD8+ T cell response and generated anti-Her1 antibodies; in the antibody response, Her1 LV priming increased Th1-dependent immunoglobulin G2c production. Conclusions The ability of this vaccine protocol to break both T cell and B cell tolerance to a self-antigen likely explains its effectiveness. Copyright © 2012 John Wiley & Sons, Ltd.

EGFR (HER1) highlights as one of the most relevant tumor associated antigen in epithelial malignant cells. Monoclonal antibodies and tyrosine kinase inhibitors against EGFR remain as the most advanced approaches in clinical trials. More recently, an active immunotherapy using the HER1 extracellular domain (ECD) adjuvated in very small size proteoliposomes (VSSP) and emulsified in Montanide ISA-51 demonstrated its strength to inhibit tumor cell line proliferation by arresting cells in G(0)/G(1) stage and induction of apoptosis. In this study, we present a simpler HER1-ECD-based formulation, which is lacking the oily component Montanide ISA-51. Generated antibodies following non-emulsive formulation immunization recognized membrane EGFR; avoid EGF and TGFalpha coupling to EGFR leading to a marked abrogation of EGFR phosphorylation levels. Non-emulsive formulation also arrests cell cycle in G(0)/G(1) stage, demonstrating it preserves previous formulation quality in a newer and simpler formulation.

Asthma may be induced by chemical sensitisers,viamechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitrointracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using anin vivomodel, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, inTrpa1andTrpv1knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficientKitWsh/Wshmice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.

Background & Aims Histamine sensitizes the nociceptor transient reporter potential channel V1 (TRPV1) and has been shown to contribute to visceral hypersensitivity in animals. We investigated the role of TRPV1 in irritable bowel syndrome (IBS) and evaluated if an antagonist of histamine receptor H1 (HRH1) could reduce symptoms of patients in a randomized placebo-controlled trial. Methods By using live calcium imaging, we compared activation of submucosal neurons by the TRPV1 agonist capsaicin in rectal biopsy specimens collected from 9 patients with IBS (ROME 3 criteria) and 15 healthy subjects. The sensitization of TRPV1 by histamine, its metabolite imidazole acetaldehyde, and supernatants from biopsy specimens was assessed by calcium imaging of mouse dorsal root ganglion neurons. We then performed a double-blind trial of patients with IBS (mean age, 31 y; range, 18–65 y; 34 female). After a 2-week run-in period, subjects were assigned randomly to groups given either the HRH1 antagonist ebastine (20 mg/day; n = 28) or placebo (n = 27) for 12 weeks. Rectal biopsy specimens were collected, barostat studies were performed, and symptoms were assessed (using the validated gastrointestinal symptom rating scale) before and after the 12-week period. Patients were followed up for an additional 2 weeks. Abdominal pain, symptom relief, and health-related quality of life were assessed on a weekly basis. The primary end point of the study was the effect of ebastine on the symptom score evoked by rectal distension. Results TRPV1 responses of submucosal neurons from patients with IBS were potentiated compared with those of healthy volunteers. Moreover, TRPV1 responses of submucosal neurons from healthy volunteers could be potentiated by their pre-incubation with histamine; this effect was blocked by the HRH1 antagonist pyrilamine. Supernatants from rectal biopsy specimens from patients with IBS, but not from the healthy volunteers, sensitized TRPV1 in mouse nociceptive dorsal root ganglion neurons via HRH1; this effect could be reproduced by histamine and imidazole acetaldehyde. Compared with subjects given placebo, those given ebastine had reduced visceral hypersensitivity, increased symptom relief (ebastine 46% vs placebo 13%; P = .024), and reduced abdominal pain scores (ebastine 39 ± 23 vs placebo 62 ± 22; P = .0004). Conclusions In studies of rectal biopsy specimens from patients, we found that HRH1-mediated sensitization of TRPV1 is involved in IBS. Ebastine, an antagonist of HRH1, reduced visceral hypersensitivity, symptoms, and abdominal pain in patients with IBS. Inhibitors of this pathway might be developed as a new treatment approach for IBS. ClinicalTrials.gov no: NCT01144832.

Background: Further insight into the structural biology of TRP channels is crucial to explain molecular mechanisms of channel function. Results: We purified TRPV3, demonstrated its functional integrity, and used fluorescence spectroscopy to study ligand binding. Conclusion: TRPV3 ligands induce different conformational changes as observed by tryptophan fluorescence quenching. Significance: Availability of purified TRPV3 allows functional assays outside the cellular context and facilitates future structural studies., TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

Background The therapeutic action of capsaicin treatment in patients with idiopathic rhinitis (IR) is based on ablation of the transient receptor potential cation channel subfamily V, receptor 1 (TRPV1)–substance P nociceptive signaling pathway. However, the functional consequences of capsaicin treatment on nasal nerve activation and the association between the reduction in nasal hyperreactivity (NHR) and response to capsaicin treatment remain unknown. Objective We sought to study the effects of capsaicin nasal spray on the afferent innervation of the nasal mucosa by monitoring trigeminal nerve activity in patients with IR and healthy control (HC) subjects. Methods A double-blind, placebo-controlled randomized trial with capsaicin nasal spray was performed involving 33 patients with IR and 12 HC subjects. Before and at 4, 12, and 26 weeks after treatment, nasal mucosal potentials (NMPs) were measured while exposing the nasal mucosa of patients with IR and HC subjects to aerosols with increasing doses of the chemical irritants allyl isothiocyanate (AITC; also known as mustard oil) or capsaicin. The threshold for each compound was determined for each subject. The results of the NMP measurements were evaluated in parallel with the therapeutic response, visual analog scale scores for nasal symptoms, self-reported NHR, and mRNA expression of PGP9.5 ; TRPV1 ; transient receptor potential cation channel subfamily A, receptor 1 ( TRPA1 ); TRPV4 ; transient receptor potential cation channel subfamily M, member 8 (TRPM8) ; and nerve growth factor (NGF) in nasal biopsy specimens. Results AITC turned out to be the best stimulus because the coughing induced by capsaicin interfered with measurements. At baseline, the threshold for evoking changes in NMPs based on AITC was significantly lower for patients with IR compared with HC subjects ( P = .0423). Capsaicin treatment of IR patients increased the threshold for the response to AITC at 4 and 12 weeks compared with placebo ( P = .0406 and P = .0325, respectively), which returned to baseline by week 26 ( P = .0611). This increase correlated with changes in visual analog scale major symptom ( P = .0004) and total symptom ( P = .0018) scores. IR patients with self-reported NHR at baseline showed a trend to being better responders to capsaicin treatment compared with patients with IR but without NHR ( P = .10). Conclusion The lower threshold for AITC based on NMPs in patients with IR compared with HC subjects and the increased threshold for AITC after capsaicin treatment in patients with IR demonstrate the crucial role of TRPA1 and TRPV1 in IR pathophysiology. The strong correlation between the increase in AITC threshold in patients with IR and symptom reduction after capsaicin treatment demonstrates the clinical relevance of these findings.

Neurogenic inflammation and pain associated to bacterial infection have been ascribed to sensitization and activation of sensory nerve afferents. We have recently unveiled a role of Transient Receptor Potential (TRP) A1 channel as sensor of lipopolysaccharide (LPS) in nociceptive neurons. However, here we show that responses to LPS are still detected in 30% of dorsal root ganglion neurons isolated from Trpa1 KO mice. The proportion of cells responding to LPS was dramatically lower in double Trpa1/Trpv1 KO neurons. Using intracellular calcium imaging and patch-clamp in a recombinant expression system, we studied the effects of LPS on TRPV1, TRPV2, TRPM3 and TRPM8 heterologously expressed in HEK cells. In isolated sensory neurons, we compared the activation of TRPV1 and TRPA1 by LPS. In contrast, LPS was ineffective on TRPV2, weakly activated TRPM3 and activated TRPM8 at 25°C but not at 35°C. Our results indicate that, in addition to TRPA1, other TRP channels in sensory neurons can be targets of LPS, raising the possibility that they may also contribute to trigger and regulate innate immune responses.

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.-- et al., Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment. © 2014 Macmillan Publishers Limited. All rights reserved., O.F. and C.F.P. were predoctoral students of the Generalitat Valenciana. S.T., B.D., V.M. and J.A.M. were supported by predoctoral fellowships from the Spanish MINECO. Research was supported by Spanish public funds projects SAF2010-14990 and PROMETEO2010-046 to F.V., BFU2007-61524 to J.R.L.L., BFU2010-15898 to M.T.P.G., Instituto de Salud Carlos III PI12/00586 to R.S., BFU2005-08741 and CONSOLIDER-INGENIO 2010 CSD2007-00023 to C.B., ISCIII grants R006/009 (Red Heracles), the Spanish Fundación Marcelino Botín and Belgian Federal Government (IUAP P6/28 and P7/13), the Research Foundation-Flanders (F.W.O. G.0565.07, G.0686.09, G.A022.11N and G.0702.12), and the Research Council of the KU Leuven (GOA 2009/07, EF/95/010, PFV/10/006, OT/12/091 and GOA 14011).

It is often observed in intracellular Ca(2+) imaging experiments that the amplitudes of the Ca(2+) signals elicited by newly characterized TRP agonists do not correlate with the amplitudes of the responses evoked subsequently by a specific potent agonist. We investigated this rather controversial phenomenon by first testing whether it is inherent to the comparison of the effects of weak and strong stimuli. Using five well-characterized TRP channel agonists in commonly used heterologous expression systems we found that the correlation between the amplitudes of the Ca(2+) signals triggered by two sequentially applied stimuli is only high when both stimuli are strong. Using mathematical simulations of intracellular Ca(2+) dynamics we illustrate that the innate heterogeneity in expression and functional properties of Ca(2+) extrusion (e.g. plasma membrane Ca(2+) ATPase) and influx (TRP channels) pathways across a cellular population is a sufficient condition for low correlation between the amplitude of Ca(2+) signals elicited by weak and strong stimuli. Taken together, our data demonstrate that this phenomenon is an expected outcome of intracellular Ca(2+) imaging experiments that cannot be taken as evidence for lack of specificity of low-efficacy stimuli, or as an indicator of the need of other cellular components for channel stimulation.

Background Idiopathic rhinitis (IR) is a prevalent condition for which capsaicin nasal spray is the most effective treatment. However, the mechanisms underlying IR and the therapeutic action of capsaicin remain unknown. Objective We sought to investigate the molecular and cellular bases of IR and the therapeutic action of capsaicin. Methods Fourteen patients with IR and 12 healthy control subjects (HCs) were treated with intranasal capsaicin. The therapeutic effect was assessed in patients with IR by using visual analog scale and therapeutic response evaluation scores, and nasal hyperreactivity was evaluated by means of cold dry air provocation. Nasal samples served to measure the levels of neuromediators and expression of chemosensory cation channels, protein gene product 9.5 (PGP 9.5), and the mast cell marker c-kit. The effects of capsaicin were also tested in vitro on human nasal epithelial cells and mast cells. Results Patients with IR had higher baseline transient receptor potential cation channel subfamily V, receptor 1 (TRPV1) expression in the nasal mucosa and higher concentrations of substance P (SP) in nasal secretions than HCs. Symptomatic relief was observed in 11 of 14 patients with IR after capsaicin treatment. Expression of TRPV1; transient receptor potential cation channel subfamily M, receptor 8 (TRPM8); and PGP 9.5 was only reduced in patients with IR after capsaicin treatment. Capsaicin did not alter c-KIT expression or nasal epithelial morphology in patients with IR and HCs nor did it induce apoptosis or necrosis in cultured human nasal epithelial cells and mast cells. Conclusion IR features an overexpression of TRPV1 in the nasal mucosa and increased SP levels in nasal secretions. Capsaicin exerts its therapeutic action by ablating the TRPV1-SP nociceptive signaling pathway in the nasal mucosa.