The Mechanism of Pertussis Cough Revealed by the Mouse-Coughing Model.

Pertussis, also known as whooping cough, is a contagious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. This disease is characterized by severe and uncontrollable coughing, which imposes a significant burden on patients. However, its etiological agent and the mechanism are totally unknown because of a lack of versatile animal models that reproduce the cough. Here, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components and demonstrate that lipooligosaccharide (LOS), pertussis toxin (PTx), and Vag8 of the bacterium cooperatively function to cause coughing. Bradykinin induced by LOS sensitized a transient receptor potential ion channel, TRPV1, which acts as a sensor to evoke the cough reflex. Vag8 further increased bradykinin levels by inhibiting the C1 esterase inhibitor, the major downregulator of the contact system, which generates bradykinin. PTx inhibits intrinsic negative regulation systems for TRPV1 through the inactivation of Gi GTPases. Our findings provide a basis to answer long-standing questions on the pathophysiology of pertussis cough. IMPORTANCE The Gram-negative bacterium Bordetella pertussis causes a respiratory disease called whooping cough, or pertussis. This disease is characterized by paroxysmal coughing, the mechanism of which has not been intensively studied because of a lack of versatile animal models that reproduce the cough. In this study, we present a mouse model that reproduces coughing after intranasal inoculation with the bacterium or its components. Using this model, we demonstrate that lipooligosaccharide, Vag8, and pertussis toxin of the bacteria cooperatively function to cause coughing. Our results also indicate that bradykinin, an inflammatory mediator, and TRPV1, an ion channel linked to nociceptive signaling, are host factors involved in the coughing mechanism.

Inhibition of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 by mosquito and mouse saliva.

ABSTRACT: Arthropods are the largest group of living organisms, and among them, mosquitoes spread parasites and viruses causing deadly diseases. They can easily spread these pathogens because of their painless skin piercing. Although the lack of pain is mainly due to the thinness of their fascicle, it is possible that mosquito saliva, which is discharged during their piercing, might also contribute to it. If mosquito saliva contains antinociceptive substances, it should act on the sensory neurons innervating the epidermis where there are several ion channels that can detect noxious stimuli, such as the transient receptor potential (TRP) channels. We found that mosquito head homogenates and mouse saliva inhibit TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) channels, either heterologously expressed in HEK293T cells or endogenously expressed in native mouse sensory neurons. Among the different substances contained in mosquito head homogenates or mouse saliva, we have also identified sialorphin as a candidate antinociceptive peptide because it showed similar inhibition effects on TRPV1 and TRPA1. Finally, we confirmed the antinociceptive effects of mosquito head homogenates, mouse saliva, and sialorphin in vivo by observing decreased pain-related behaviors in mice coinjected with these substances. Similar inhibitory effects of mosquito head homogenates and mouse saliva on TRPV1 and TRPA1 suggest that the antinociceptive effects of saliva are universal, which could explain why many animals including humans often lick their wounds. These findings would lead to the development of novel and safe antinociceptive agents.

Thermosensitive TRPV4 channels mediate temperature-dependent microglia movement.

Microglia maintain central nervous system homeostasis by monitoring changes in their environment (resting state) and by taking protective actions to equilibrate such changes (activated state). These surveillance and protective roles both require constant movement of microglia. Interestingly, induced hypothermia can reduce microglia migration caused by ischemia, suggesting that microglia movement can be modulated by temperature. Although several ion channels and transporters are known to support microglia movement, the precise molecular mechanism that regulates temperature-dependent movement of microglia remains unclear. Some members of the transient receptor potential (TRP) channel superfamily exhibit thermosensitivity and thus are strong candidates for mediation of this phenomenon. Here, we demonstrate that mouse microglia exhibit temperature-dependent movement in vitro and in vivo that is mediated by TRPV4 channels within the physiological range of body temperature. Our findings may provide a basis for future research into the potential clinical application of temperature regulation to preserve cell function via manipulation of ion channel activity.

5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid accelerates the healing of colitis by inhibiting transient receptor potential vanilloid 4-mediated signaling.

5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid (5,6-DiHETE) is an eicosapentaenoic acid-derived lipid metabolite, which we previously detected in inflamed mouse colon. In this study, we investigated the pathophysiological roles of 5,6-DiHETE in murine colitis and its underlying mechanisms of action, focusing on the effects on transient receptor potential vanilloid (TRPV) channel activity. Oral administration of dextran sodium sulfate (DSS, 2%, for 4 days) caused colon inflammation, which peaked on day 7 and gradually declined by day 18. 5,6-DiHETE concentration in colon tissue was significantly increased during the healing phase of colitis (days 9 to 18). In vitro study showed that pretreatment with 5,6-DiHETE (0.1-1 µM, 30 minutes) significantly inhibited endothelial barrier disruption induced by a TRPV4 agonist (GSK1016790A, 50 nM). Intracellular Ca2+ imaging also showed that pretreatment with 5,6-DiHETE (1 µM, 10 minutes) reduced GSK1016790A-induced intracellular Ca2+ increase in HEK293T cells overexpressing TRPV4. In vivo, intraperitoneal administration of 5,6-DiHETE (50 µg kg-1  day-1 ) during the healing phase accelerated the recovery from DSS-induced colitis. Pathological studies showed that the administration of 5,6-DiHETE inhibited edema formation and leukocyte infiltration in inflamed colon tissue. In conclusion, we identified 5,6-DiHETE as a novel endogenous TRPV4 antagonist, and we also demonstrated that its administration promotes the healing of colitis by inhibiting inflammatory responses.

Emerging Perspectives on Pain Management by Modulation of TRP Channels and ANO1.

Receptor-type ion channels are critical for detection of noxious stimuli in primary sensory neurons. Transient receptor potential (TRP) channels mediate pain sensations and promote a variety of neuronal signals that elicit secondary neural functions (such as calcitonin gene-related peptide [CGRP] secretion), which are important for physiological functions throughout the body. In this review, we focus on the involvement of TRP channels in sensing acute pain, inflammatory pain, headache, migraine, pain due to fungal infections, and osteo-inflammation. Furthermore, action potentials mediated via interactions between TRP channels and the chloride channel, anoctamin 1 (ANO1), can also generate strong pain sensations in primary sensory neurons. Thus, we also discuss mechanisms that enhance neuronal excitation and are dependent on ANO1, and consider modulation of pain sensation from the perspective of both cation and anion dynamics.

TRPV4 heats up ANO1-dependent exocrine gland fluid secretion.

Several ion channels and transporters regulate fluid secretion in salivary and lacrimal glands. In salivary glands, the major anion channel involved in fluid secretion is the calcium-activated chloride channel anoctamin 1 (ANO1). Several members of the transient receptor potential (TRP) channel superfamily regulate ANO1 activity. Here, we report a functional interaction between thermosensitive TRP vanilloid (TRPV)4 and ANO1 in acinar cells isolated from mouse salivary and lacrimal glands. TRPV4 activation induced chloride currents and shrinkage of acinar cells by increasing intracellular calcium concentrations. The chloride currents evoked by a TRPV4-specific activator (GSK1016790A) were identified as ANO1-mediated currents. Moreover, TRPV4 activation by an inositol 1,4,5-trisphosphate (IP3)-dependent mechanism was found to contribute to the muscarinic pathway of fluid secretion. Muscarinic stimulation of saliva and tear secretion was down-regulated in both TRPV4-deficient mice and in acinar cells treated with a TRPV4-specific antagonist (HC-067047). Furthermore, the temperature dependence of muscarinic salivation was shown to depend mainly on TRPV4. Our results suggest that TRPV4 interacts with IP3 receptors and ANO1 to regulate the muscarinic pathway that mediates salivation and lacrimation.-Derouiche, S., Takayama, Y., Murakami, M., Tominaga, M. TRPV4 heats up ANO1-dependent exocrine gland fluid secretion.

Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF).

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis.

Activation of TRPA1 Channel by Antibacterial Agent Triclosan Induces VEGF Secretion in Human Prostate Cancer Stromal Cells.

Accruing evidence indicates that exposure to environmental compounds may adversely affect human health and promote carcinogenesis. Triclosan (TCS), an antimicrobial agent widely used as a preservative in personal care products, has been shown to act as an endocrine disruptor in hormone-dependent tissues. Here, we demonstrate a new molecular mechanism by which TCS stimulates the secretion by human prostate cancer stromal cells of vascular endothelial growth factor (VEGF), a factor known to promote tumor growth. This mechanism involves an increase in intracellular calcium levels due to the direct activation of a membrane ion channel. Using calcium imaging and electrophysiology techniques, we show for the first time that environmentally relevant concentrations of TCS activate a cation channel of the TRP family, TRPA1 (Transient Receptor Potential Ankirin 1), in primary cultured human prostate cancer stromal cells. The TCS-induced TRPA1 activation increased basal calcium in stromal cells and stimulated the secretion of VEGF and epithelial cells proliferation. Interestingly, immunofluorescence labeling performed on formalin-fixed paraffin-embedded prostate tissues showed an exclusive expression of the TRPA1 channel in prostate cancer stromal cells. Our data demonstrate an impact of the environmental factor TCS on the tumor microenvironment interactions, by activating a tumor stroma-specific TRPA1 ion channel. Cancer Prev Res; 10(3); 177-87. ©2017 AACR.

Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Testes of most male mammals present the particularity of being externalized from the body and are consequently slightly cooler than core body temperature (4-8°C below). Although, hypothermia of the testis is known to increase germ cells apoptosis, little is known about the underlying molecular mechanisms, including cold sensors, transduction pathways, and apoptosis triggers. In this study, using a functional knockout mouse model of the cold and menthol receptors, dubbed transient receptor potential melastatine 8 (TRPM8) channels, we found that TRPM8 initiated the cold-shock response by differentially modulating cold- and heat-shock proteins. Besides, apoptosis of germ cells increased in proportion to the cooling level in control mice but was independent of temperature in knockout mice. We also observed that the rate of germ cell death correlated positively with the reactive oxygen species level and negatively with the expression of the detoxifying enzymes. This result suggests that the TRPM8 sensor is a key determinant of germ cell fate under hypothermic stimulation.-Borowiec, A.-S., Sion, B., Chalmel, F., Rolland, A. D., Lemonnier, L., De Clerck, T., Bokhobza, A., Derouiche, S., Dewailly, E., Slomianny, C., Mauduit, C., Benahmed, M., Roudbaraki, M., Jégou, B., Prevarskaya, N., Bidaux, G. Cold/menthol TRPM8 receptors initiate the cold-shock response and protect germ cells from cold-shock-induced oxidation.

Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats from Argentina, French Guyana, and Mexico.

BACKGROUND: Histoplasma capsulatum and Pneumocystis organisms cause host infections primarily affecting the lung tissue. H. capsulatum is endemic in the United States of America and Latin American countries. In special environments, H. capsulatum is commonly associated with bat and bird droppings. Pneumocystis-host specificity has been primarily studied in laboratory animals, and its ability to be harboured by wild animals remains as an important issue for understanding the spread of this pathogen in nature. Bats infected with H. capsulatum or Pneumocystis spp. have been found, with this mammal serving as a probable reservoir and disperser; however, the co-infection of bats with both of these microorganisms has never been explored. To evaluate the impact of H. capsulatum and Pneumocystis spp. infections in this flying mammal, 21 bat lungs from Argentina (AR), 13 from French Guyana (FG), and 88 from Mexico (MX) were screened using nested-PCR of the fragments, employing the Hcp100 locus for H. capsulatum and the mtLSUrRNA and mtSSUrRNA loci for Pneumocystis organisms. RESULTS: Of the 122 bats studied, 98 revealed H. capsulatum infections in which 55 of these bats exhibited this infection alone. In addition, 51 bats revealed Pneumocystis spp. infection of which eight bats exhibited a Pneumocystis infection alone. A total of 43 bats (eight from AR, one from FG, and 34 from MX) were found co-infected with both fungi, representing a co-infection rate of 35.2% (95% CI = 26.8-43.6%). CONCLUSION: The data highlights the H. capsulatum and Pneumocystis spp.co-infection in bat population's suggesting interplay with this wild host.

Functional coupling between large-conductance potassium channels and Cav3.2 voltage-dependent calcium channels participates in prostate cancer cell growth.

It is strongly suspected that potassium (K(+)) channels are involved in various aspects of prostate cancer development, such as cell growth. However, the molecular nature of those K(+) channels implicated in prostate cancer cell proliferation and the mechanisms through which they control proliferation are still unknown. This study uses pharmacological, biophysical and molecular approaches to show that the main voltage-dependent K(+) current in prostate cancer LNCaP cells is carried by large-conductance BK channels. Indeed, most of the voltage-dependent current was inhibited by inhibitors of BK channels (paxillin and iberiotoxin) and by siRNA targeting BK channels. In addition, we reveal that BK channels constitute the main K(+) channel family involved in setting the resting membrane potential in LNCaP cells at around -40 mV. This consequently promotes a constitutive calcium entry through T-type Cav3.2 calcium channels. We demonstrate, using single-channel recording, confocal imaging and co-immunoprecipitation approaches, that both channels form macromolecular complexes. Finally, using flow cytometry cell cycle measurements, cell survival assays and Ki67 immunofluorescent staining, we show that both BK and Cav3.2 channels participate in the proliferation of prostate cancer cells.

Bisphenol A stimulates human prostate cancer cell migration via remodelling of calcium signalling.

Bisphenol A (BPA), the principal constituent of reusable water bottles, metal cans, and plastic food containers, has been shown to be involved in human prostate cancer (PCa) cell proliferation. The aim of the present study was to explore the effect of BPA on PCa cell migration and the pathways involved in these processes. Using the transwell technique, we clearly show for the first time that the pre-treatment of the cells with BPA (1-10 nM) induces human PCa cell migration. Using a calcium imaging technique, we show that BPA pre-treatment induces an amplification of Store-Operated Calcium Entry (SOCE) in LNCaP cells. RT-PCR and Western blot experiments allowed the identification of the ion channel proteins which are up-regulated by BPA pre-treatments. These include the Orai1 protein, which is known as an important SOCE actor in various cell systems, including human PCa cells. Using a siRNA strategy, we observed that BPA-induced amplification of SOCE was Orai1-dependent. Interestingly, the BPA-induced PCa cell migration was suppressed when the calcium entry was impaired by the use of SOCE inhibitors (SKF96365, BTP2), or when the extracellular calcium was chelated. Taken together, the results presented here show that BPA induces PCa cells migration via a modulation of the ion channel protein expression involved in calcium entry and in cancer cell migration. The present data provide novel insights into the molecular mechanisms involved in the effects of an environmental factor on cancer cells and suggest both the necessity of preventive measures and the possibility of targeting ion channels in the treatment of PCa cell metastasis.

Characterizing Pneumocystis in the lungs of bats: understanding Pneumocystis evolution and the spread of Pneumocystis organisms in mammal populations.

Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.