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1.
Front Immunol ; 12: 772941, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34925342

RESUMO

Dry skin is a symptom of skin barrier dysfunction that evokes pruritus; however, the cutaneous neuroimmune interactions underlying dry skin-induced pruritus remain unclear. Therefore, we aimed to elucidate the mechanisms underlying dry skin-induced pruritus. To this end, an acetone/ethanol/water (AEW)-induced mouse model of dry skin was used in this study. We observed that the production of thymic stromal lymphopoietin (TSLP) significantly increased in the keratinocytes of AEW mice. Importantly, treatment with an antagonist of transient receptor potential cation channel subfamily V member 4 (TRPV4), HC067047, ameliorated dry skin conditions in AEW mice. The symptoms of dry skin were significantly reduced in Trpv4 knockout (KO) mice following treatment with AEW. The increase in the intracellular calcium levels by TSLP in the dorsal root ganglia (DRG) of Trpv4 KO mice was also significantly attenuated. The spontaneous scratching bouts were significantly decreased in both the HC067047-treated and Trpv4 KO AEW mice. Importantly, the TSLP-dependent release of tryptase from the mast cells was significantly reduced in both the HC067047-treated mice and Trpv4 KO AEW mice. Notably, inhibition of the TSLP-induced signaling pathway in DRG selectively reduced the spontaneous scratching bouts in AEW mice. Overall, the results demonstrated that the cutaneous neuroimmune interactions of TSLP and TRPV4 play pivotal roles in dry skin-induced pruritus.


Assuntos
Citocinas/imunologia , Neuroimunomodulação , Prurido/imunologia , Pele/imunologia , Canais de Cátion TRPV/imunologia , Animais , Células Cultivadas , Gânglios Espinais , Humanos , Queratinócitos/imunologia , Masculino , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Morfolinas/farmacologia , Neurônios/imunologia , Pirróis/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Linfopoietina do Estroma do Timo
2.
Front Immunol ; 12: 767319, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34795674

RESUMO

The importance of innate immune cells to sense and respond to their physical environment is becoming increasingly recognized. Innate immune cells (e.g. macrophages and neutrophils) are able to receive mechanical signals through several mechanisms. In this review, we discuss the role of mechanosensitive ion channels, such as Piezo1 and transient receptor potential vanilloid 4 (TRPV4), and cell adhesion molecules, such as integrins, selectins, and cadherins in biology and human disease. Furthermore, we explain that these mechanical stimuli activate intracellular signaling pathways, such as MAPK (p38, JNK), YAP/TAZ, EDN1, NF-kB, and HIF-1α, to induce protein conformation changes and modulate gene expression to drive cellular function. Understanding the mechanisms by which immune cells interpret mechanosensitive information presents potential targets to treat human disease. Important areas of future study in this area include autoimmune, allergic, infectious, and malignant conditions.


Assuntos
Imunidade Inata/imunologia , Macrófagos/imunologia , Mecanotransdução Celular/imunologia , Neutrófilos/imunologia , Transdução de Sinais/imunologia , Animais , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Canais Iônicos/imunologia , Canais Iônicos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Canais de Cátion TRPV/imunologia , Canais de Cátion TRPV/metabolismo
3.
Life Sci ; 287: 120115, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34740578

RESUMO

AIMS: Activation of transient receptor potential vanilloid 1 (TRPV1) ion channels inhibits inflammation, enhance cytotoxic immune response, and may have therapeutic potential in treatment of cancer characterized by increased systemic inflammation. We here determined how activation of TRPV1 alters immune response of tumor-bearing mice. MAIN METHODS: Three different metastatic subset of 4 T1 breast carcinoma cells were used to induce tumors in Balb-c mice. Mix leukocyte cultures (MLCs) using spleens and draining lymph nodes were prepared and stimulated with various challenges. Effects TRPV1 agonists including capsaicin, antagonist (AMG9810) and Gambogic Amide (GA), a TrkA agonist that sensitizes TRPV1, on secreted levels of cytokines were determined. KEY FINDINGS: MLCs of tumor-bearing mice secreted markedly higher levels of IL-6 and lower levels of IFN-γ compared to control mice. We observed differential effects of TRPV1 agonists in control and mice bearing different subset of metastatic cells. TRPV1 increased IFN-γ and IL-17 secretion in control mice while they markedly increased IL-6 secretion and suppressed IFN--γ secretion in tumor-bearing mice. Unexpectedly, AMG9810 acted as an inverse agonist and did not antagonize the effects of TRPV1 agonists. SIGNIFICANCE: Our results demonstrate constitutive activity of TRPV1 in immune cells, suggesting cross activation. To prevent excessive chronic activation of TRPV1 in immune cells in the presence of metastatic breast carcinoma, lower doses of TRPV1 agonist should be considered. Unexpected findings further document that a drug can have multiple intrinsic activities depending on surrounding factors can act on the same receptor as an agonist, antagonist or inverse agonist.


Assuntos
Neoplasias da Mama/imunologia , Imunidade Celular/imunologia , Mediadores da Inflamação/imunologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/imunologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Diterpenos/farmacologia , Diterpenos/uso terapêutico , Feminino , Imunidade Celular/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Canais de Cátion TRPV/metabolismo
4.
Nat Commun ; 12(1): 926, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568652

RESUMO

The endocannabinoid system is a promising target to mitigate pain as the endocannabinoids are endogenous ligands of the pain-mediating receptors-cannabinoid receptors 1 and 2 (CB1 and CB2) and TRPV1. Herein, we report on a class of lipids formed by the epoxidation of N-arachidonoyl-dopamine (NADA) and N-arachidonoyl-serotonin (NA5HT) by epoxygenases. EpoNADA and epoNA5HT are dual-functional rheostat modulators of the endocannabinoid-TRPV1 axis. EpoNADA and epoNA5HT are stronger modulators of TRPV1 than either NADA or NA5HT, and epoNA5HT displays a significantly stronger inhibition on TRPV1-mediated responses in primary afferent neurons. Moreover, epoNA5HT is a full CB1 agonist. These epoxides reduce the pro-inflammatory biomarkers IL-6, IL-1ß, TNF-α and nitrous oxide and raise anti-inflammatory IL-10 cytokine in activated microglial cells. The epoxides are spontaneously generated by activated microglia cells and their formation is potentiated in the presence of anandamide. Detailed kinetics and molecular dynamics simulation studies provide evidence for this potentiation using the epoxygenase human CYP2J2. Taken together, inflammation leads to an increase in the metabolism of NADA, NA5HT and other eCBs by epoxygenases to form the corresponding epoxides. The epoxide metabolites are bioactive lipids that are potent, multi-faceted molecules, capable of influencing the activity of CB1, CB2 and TRPV1 receptors.


Assuntos
Anti-Inflamatórios/administração & dosagem , Dopamina/administração & dosagem , Dor/tratamento farmacológico , Receptor CB1 de Canabinoide/imunologia , Receptor CB2 de Canabinoide/imunologia , Serotonina/administração & dosagem , Animais , Anti-Inflamatórios/química , Dopamina/química , Endocanabinoides/administração & dosagem , Endocanabinoides/química , Compostos de Epóxi/química , Feminino , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nitroso/imunologia , Dor/genética , Dor/imunologia , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genética , Serotonina/química , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologia
5.
Int Arch Allergy Immunol ; 182(4): 324-338, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33567446

RESUMO

In this article, we propose that differences in COVID-19 morbidity may be associated with transient receptor potential ankyrin 1 (TRPA1) and/or transient receptor potential vanilloid 1 (TRPV1) activation as well as desensitization. TRPA1 and TRPV1 induce inflammation and play a key role in the physiology of almost all organs. They may augment sensory or vagal nerve discharges to evoke pain and several symptoms of COVID-19, including cough, nasal obstruction, vomiting, diarrhea, and, at least partly, sudden and severe loss of smell and taste. TRPA1 can be activated by reactive oxygen species and may therefore be up-regulated in COVID-19. TRPA1 and TRPV1 channels can be activated by pungent compounds including many nuclear factor (erythroid-derived 2) (Nrf2)-interacting foods leading to channel desensitization. Interactions between Nrf2-associated nutrients and TRPA1/TRPV1 may be partly responsible for the severity of some of the COVID-19 symptoms. The regulation by Nrf2 of TRPA1/TRPV1 is still unclear, but suggested from very limited clinical evidence. In COVID-19, it is proposed that rapid desensitization of TRAP1/TRPV1 by some ingredients in foods could reduce symptom severity and provide new therapeutic strategies.


Assuntos
COVID-19/dietoterapia , COVID-19/imunologia , Fator 2 Relacionado a NF-E2/imunologia , Nutrientes/imunologia , SARS-CoV-2/imunologia , Canal de Cátion TRPA1/imunologia , Canais de Cátion TRPV/imunologia , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Brassica , COVID-19/complicações , COVID-19/diagnóstico , Teste para COVID-19 , Dessensibilização Imunológica/métodos , Regulação para Baixo , Humanos , Estresse Oxidativo/imunologia , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença , Regulação para Cima
6.
PLoS Pathog ; 16(5): e1008605, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453763

RESUMO

As obligate parasites, viruses highjack, modify and repurpose the cellular machinery for their own replication. Viral proteins have, therefore, evolved biological functions, such as signalling potential, that alter host cell physiology in ways that are still incompletely understood. Retroviral envelope glycoproteins interact with several host proteins, extracellularly with their cellular receptor and anti-envelope antibodies, and intracellularly with proteins of the cytoskeleton or sorting, endocytosis and recirculation pathways. Here, we examined the impact of endogenous retroviral envelope glycoprotein expression and interaction with host proteins, particularly antibodies, on the cell, independently of retroviral infection. We found that in the commonly used C57BL/6 substrains of mice, where murine leukaemia virus (MLV) envelope glycoproteins are expressed by several endogenous MLV proviruses, the highest expressed MLV envelope glycoprotein is under the control of an immune-responsive cellular promoter, thus linking MLV envelope glycoprotein expression with immune activation. We further showed that antibody ligation induces extensive internalisation from the plasma membrane into endocytic compartments of MLV envelope glycoproteins, which are not normally subject to constitutive endocytosis. Importantly, antibody binding and internalisation of MLV envelope glycoproteins initiates signalling cascades in envelope-expressing murine lymphocytic cell lines, leading to cellular activation. Similar effects were observed by MLV envelope glycoprotein ligation by its cellular receptor mCAT-1, and by overexpression in human lymphocytic cells, where it required an intact tyrosine-based YXXΦ motif in the envelope glycoprotein cytoplasmic tail. Together, these results suggest that signalling potential is a general property of retroviral envelope glycoproteins and, therefore, a target for intervention.


Assuntos
Anticorpos Antivirais/imunologia , Canais de Cálcio/imunologia , Membrana Celular/imunologia , Endocitose/imunologia , Vírus da Leucemia Murina/imunologia , Canais de Cátion TRPV/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB C
7.
Infect Immun ; 88(9)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32341116

RESUMO

The orchestration of host immune responses to enteric bacterial pathogens is a complex process involving the integration of numerous signals, including from the nervous system. Despite the recent progress in understanding the contribution of neuroimmune interactions in the regulation of inflammation, the mechanisms and effects of this communication during enteric bacterial infection are only beginning to be characterized. As part of this neuroimmune communication, neurons specialized to detect painful or otherwise noxious stimuli can respond to bacterial pathogens. Highlighting the complexity of these systems, the immunological consequences of sensory neuron activation can be either host adaptive or maladaptive, depending on the pathogen and organ system. These are but one of many types of neuroimmune circuits, with the vagus nerve and sympathetic innervation of numerous organs now known to modulate immune cell function and therefore dictate immunological outcomes during health and disease. Here, we review the evidence for neuroimmune communication in response to bacterial pathogens, and then discuss the consequences to host morbidity and mortality during infection of the gastrointestinal tract.


Assuntos
Sistema Nervoso Entérico/imunologia , Infecções por Enterobacteriaceae/imunologia , Microbioma Gastrointestinal/imunologia , Trato Gastrointestinal/imunologia , Neuroimunomodulação/genética , Células Receptoras Sensoriais/imunologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/imunologia , Citrobacter/crescimento & desenvolvimento , Citrobacter/imunologia , Sistema Nervoso Entérico/microbiologia , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/patologia , Trato Gastrointestinal/inervação , Trato Gastrointestinal/microbiologia , Regulação da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , Células Receptoras Sensoriais/microbiologia , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/imunologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
8.
Food Funct ; 11(4): 3516-3526, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32253400

RESUMO

Chronic obstructive pulmonary disease (COPD) is a chronic, progressive lung disease with few successful treatments, and is strongly associated with cigarette smoking (CS). Since the novel coronavirus has spread worldwide seriously, there is growing concern that patients who have chronic respiratory conditions like COPD can easily be infected and are more prone to having severe illness and even mortality because of lung dysfunction. Loquat leaves have long been used as an important material for both pharmaceutical and functional applications in the treatment of lung disease in Asia, especially in China and Japan. Total flavonoids (TF), the main active components derived from loquat leaves, showed remarkable anti-inflammatory and antioxidant activities. However, their protective activity against CS-induced COPD airway inflammation and oxidative stress and its underlying mechanism still remain not well-understood. The present study uses a CS-induced mouse model to estimate the morphological changes in lung tissue. The results demonstrated that TF suppressed the histological changes in the lungs of CS-challenged mice, as evidenced by reduced generation of pro-inflammatory cytokines including interleukin 6 (IL-6), IL-1ß, tumor necrosis factor α (TNF-α), nitric oxide (NO), and inducible nitric oxide synthase (iNOS) and diminished the protein expression of transient receptor potential vanilloid 1 (TRPV1). Moreover, TF also inhibited phosphorylation of IKK, IκB and NFκB and increased p-Akt. Interestingly, TF could inhibit CS-induced oxidative stress in the lungs of COPD mice. TF treatment significantly inhibited the level of malondialdehyde (MDA) and increased the activity of superoxide dismutase (SOD). In addition, TF markedly downregulated TRPV1 and cytochrome P450 2E1 (CYP2E1) and upregulated the expression of SOD-2, while the p-JNK level was observed to be inhibited in COPD mice. Taken together, our findings showed that the protective effect and putative mechanism of the action of TF resulted in the inhibition of inflammation and oxidative stress through the regulation of TRPV1 and the related signal pathway in lung tissues. It suggested that TF derived from loquat leaves could be considered to be an alternative or a new functional material and used for the treatment of CS-induced COPD.


Assuntos
Fumar Cigarros/efeitos adversos , Medicamentos de Ervas Chinesas/administração & dosagem , Eriobotrya/química , Flavonoides/administração & dosagem , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Canais de Cátion TRPV/imunologia , Animais , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Folhas de Planta/química , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Transdução de Sinais/efeitos dos fármacos , Fumaça/efeitos adversos , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia , Canais de Cátion TRPV/genética
9.
Am J Respir Cell Mol Biol ; 63(2): 198-208, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32182090

RESUMO

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.


Assuntos
Alérgenos/imunologia , Células Epiteliais/imunologia , Imunidade Inata/imunologia , Peptídeo Hidrolases/imunologia , Canais de Cátion TRPV/imunologia , Animais , Asma/imunologia , Brônquios/imunologia , Células Cultivadas , Epitélio/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pyroglyphidae/imunologia , Receptor PAR-2/imunologia , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia
10.
J Immunol ; 204(5): 1310-1321, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969384

RESUMO

Mechanical cell-matrix interactions can drive the innate immune responses to infection; however, the molecular underpinnings of these responses remain elusive. This study was undertaken to understand the molecular mechanism by which the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4), alters the in vivo response to lung infection. For the first time, to our knowledge, we show that TRPV4 protects the lung from injury upon intratracheal Pseudomonas aeruginosa in mice. TRPV4 functions to enhance macrophage bacterial clearance and downregulate proinflammatory cytokine secretion. TRPV4 mediates these effects through a novel mechanism of molecular switching of LPS signaling from predominant activation of the MAPK, JNK, to that of p38. This is accomplished through the activation of the master regulator of inflammation, dual-specificity phosphatase 1. Further, TRPV4's modulation of the LPS signal is mechanosensitive in that both upstream activation of p38 and its downstream biological consequences depend on pathophysiological range extracellular matrix stiffness. We further show the importance of TRPV4 on LPS-induced activation of macrophages from healthy human controls. These data are the first, to our knowledge, to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner through the modulation of dual-specificity phosphatase 1 expression to mediate MAPK activation switching.


Assuntos
Pulmão , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos , Macrófagos/imunologia , Pneumonia Bacteriana , Infecções por Pseudomonas , Pseudomonas aeruginosa/imunologia , Canais de Cátion TRPV/imunologia , Animais , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Lipopolissacarídeos/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/prevenção & controle , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/prevenção & controle , Canais de Cátion TRPV/genética
11.
Channels (Austin) ; 13(1): 264-286, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31237176

RESUMO

Calcium entry is central to the functional processes in mast cells and basophils that contribute to the induction and maintenance of inflammatory responses. Mast cells and basophils express an array of calcium channels, which mediate responses to diverse stimuli triggered by small bioactive molecules, physicochemical stimuli and immunological inputs including antigens and direct immune cell interactions. These cells are also highly responsive to certain venoms (such as Hymenoptera envenomations), which cause histamine secretion, cytokine release and an array of pro-inflammatory functional responses. There are gaps in our understanding of the coupling of venom exposure to specific signaling pathways such as activation of calcium channels. In the present study, we performed a current survey of a model mast cell line selected for its pleiotropic responsiveness to multiple pro-inflammatory inputs. As a heterogenous stimulus, Hymenoptera venom activates multiple classes of conductance at the population level but tend to lead to the measurement of only one type of conductance per cell, despite the cell co-expressing multiple channel types. The data show that ICRAC, IARC, and TRPV-like currents are present in the model mast cell populations and respond to venom exposure. We further assessed individual venom components, specifically secretagogues and arachidonic acid, and identified the conductances associated with these stimuli in mast cells. Single-cell calcium assays and immunofluorescence analysis show that there is heterogeneity of channel expression across the cell population, but this heterogeneity does not explain the apparent selectivity for specific channels in response to exposure to venom as a composite stimulus.


Assuntos
Venenos de Artrópodes/farmacologia , Mordeduras e Picadas/imunologia , Himenópteros/fisiologia , Mastócitos/imunologia , Animais , Venenos de Artrópodes/imunologia , Venenos de Artrópodes/toxicidade , Histamina/imunologia , Humanos , Himenópteros/imunologia , Mastócitos/efeitos dos fármacos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologia
12.
Methods Mol Biol ; 1987: 111-124, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028677

RESUMO

Studying TRP channel expressing nociceptors requires the identification of the respective subpopulations as well as the quantification of dynamic cellular events. However, the heterogeneity of sensory neurons and associated nonneuronal cells demands the analysis of large numbers of cells to reflect the distribution of entire populations. Here we report a detailed workflow how to apply high-content screening (HCS) microscopy to signaling events in TRPV1-positive neurons as well as an approach to use the selective elimination of TRPV1 positive cells from dissociated rat sensory ganglia as base for transcriptomic analysis of TRPV1-positive cells and/or as control for TRPV1 antibody specificity.


Assuntos
Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Células Cultivadas , Imunofluorescência/métodos , Masculino , Camundongos Endogâmicos C57BL , Microscopia/métodos , Microscopia de Fluorescência , Nociceptores/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/imunologia
13.
Korean J Parasitol ; 57(1): 33-38, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30840797

RESUMO

Trichomoniasis is a common sexually transmitted infection caused by Trichomonas vaginalis, which actually does not exist a vaccine for control or prevention. Thus, the identification of new and potent immunogens in T. vaginalis, which can contribute to the development of a vaccine against this parasite, is necessary. Therefore, the aim of this work was to evaluate the potential of a recombinant Transient Receptor Potential-like channel of T. vaginalis (TvTRPV), as a promising immunogen in BALB/c mice. First, TvTRPV was cloned and expressed as a recombinant protein in Escherichia coli BL21 cells and purified by nickel affinity. Next, BALB/c mice were immunized and the antibody levels in mice serum and cytokines from the supernatant of macrophages and from co-culture systems were evaluated. Recombinant TvTRPV triggered high levels of specific total IgG in sera from the immunized mice. Also, a statistically significant increase of cytokines: IL-1ß, IL-6, and TNF-α after stimulation with the corresponding antigens in vitro, was identified. Moreover, co-cultures using CD4+ T cells from immunized mice were able to identify higher levels of IL-10 and IFN-γ. These results were useful to validate the immunogenicity of TvTRPV in BALB/c mice, where IL-10-IFN-γ-secreting cells could play a role in infection control, supporting the potential of TvTRPV as a promising target for vaccine against T. vaginalis.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Citocinas/metabolismo , Macrófagos/imunologia , Vacinas Protozoárias/imunologia , Canais de Cátion TRPV/imunologia , Trichomonas vaginalis/enzimologia , Animais , Antígenos de Protozoários/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPV/genética , Tricomoníase/prevenção & controle , Trichomonas vaginalis/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
14.
Anal Chim Acta ; 1041: 25-32, 2018 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-30340687

RESUMO

A sensitive and highly efficient approach to monitor the expression of proteins on live cells was urgently needed to demonstrate its factor and mechanism and most important for clinical diagnostics and molecular biology. Herein, we developed a simple and highly efficient strategy, nonlinear hybridization chain reaction (nonlinear HCR), for the sensitive determination of proteins on live cells with transient receptor potential vanilloid 4 (TRPV4) and RAW264.7 cells as a model. Unlike the normal hybridization chain reaction (HCR) with multiplicative amplification, an exponential amplified fluorescent response could be obtained in theory based on the proposed nonlinear HCR. As a result, the nonlinear HCR generated a significant enhancement about 3 times compared with the normal HCR and 10 times compared with the directly immunofluorescence assay. Based on the proposed nonlinear HCR, the fluorescent signals increased with the concentration of TRPV4 in the range from 10 pg/mL to 100 ng/mL with a detection limit of 2.8 pg/mL, which would be useful for the sensitive detection of proteins in cell lysis or on cell surface. At the same time, the significant improvements via nonlinear HCR were achieved in the fluorescent imaging system compared with traditional immunofluorescence staining and normal HCR, proving the significant value of nonlinear HCR-based amplification strategy. Success in the establishment of the highly efficient nonlinear HCR strategy offered a simple and sensitive approach to demonstrate the concentration of special proteins on cell and other proteins and nucleotide potentially, revealing a simple and efficient technology for research fields of clinical diagnostics and molecular biology.


Assuntos
Microscopia de Fluorescência , Canais de Cátion TRPV/análise , Animais , Anticorpos/química , Anticorpos/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Camundongos , Oligonucleotídeos/química , Células RAW 264.7 , Canais de Cátion TRPV/imunologia
15.
Mucosal Immunol ; 11(5): 1441-1453, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29867077

RESUMO

Immunological interdependence between the two eyes has been reported for the cornea and the retina but not for the ocular mucosal surface. Intriguingly, patients frequently report ocular surface-related symptoms in the other eye after unilateral ocular surgery. Here we show how unilateral eye injuries in mice affect the mucosal immune response of the opposite ocular surface. We report that, despite the lack of lymphatic cross-drainage, a neurogenic inflammatory reflex in the contralateral conjunctiva is sufficient to increase, first, epithelial nuclear factor kappa B signaling, then, dendritic cell maturation, and finally, expansion of effector, instead of regulatory, T cells in the draining lymph node, leading to disrupted ocular mucosal tolerance. We also show that damage to ocular surface nerves is required. Using pharmacological inhibitors and agonists, we identified transient receptor potential vanilloid 1 (TRPV1) channel as the receptor sensing tissue damage in the injured eye and substance P released in the opposite ocular surface as the effector of the sympathetic response. Finally, blocking either step prevented subsequent ocular allergic reactions in the opposite eye in a unilateral corneal alkali burn model. This study demonstrates that both ocular surfaces are immunologically linked and suggests potential therapeutic targets for intervention.


Assuntos
Olho/imunologia , Inflamação/imunologia , Mucosa/imunologia , Substância P/imunologia , Canais de Cátion TRPV/imunologia , Animais , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Hipersensibilidade/imunologia , Linfonodos/imunologia , Melanoma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia
16.
J Invest Dermatol ; 138(8): 1774-1784, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29550417

RESUMO

This study revealed the modulatory role of transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) cation channels in the Aldara-induced (5% imiquimod) murine psoriasis model using selective antagonists and genetically altered animals. We have also developed a refined localized model to enable internal controls and reduce systemic effects. Skin pathology was quantified by measuring skin thickness, scaling, blood flow, and analyzing dermal cellular infiltrate, whereas nocifensive behaviors were also observed. Cytokine gene expression profiles were measured ex vivo. Psoriasiform dermatitis was significantly enhanced in TRPA1 knockout mice and with TRPA1 antagonist (A967079) treatment. By comparison, symptoms were decreased when TRPV1 function was inhibited. Imiquimod induced Ca2+ influx in TRPA1-, but not in TRPV1-expressing cell lines. Immunohistochemical studies revealed that CD4+ T helper cells express TRPA1 but not TRPV1 ion channels in mice skin. Compared with the TRPV1 knockout animals, additional elimination of the TRPA1 channels in the TRPV1/TRPA1 double knockout mice did not modify the outcome of the imiquimod-induced reaction, further supporting the dominant role of TRPV1 in the process. Our results suggest that the protective effects in psoriasiform dermatitis can be mediated by the activation of neuronal and nonneuronal TRPA1 receptors.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Psoríase/imunologia , Canal de Cátion TRPA1/imunologia , Canais de Cátion TRPV/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Imiquimode/toxicidade , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Oximas/farmacologia , Psoríase/induzido quimicamente , Psoríase/patologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/inervação , Pele/patologia , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/metabolismo
17.
J Invest Dermatol ; 138(7): 1564-1572, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29458120

RESUMO

PAR2 activation in basal keratinocytes stimulates inflammation via the Ca2+-dependent production of mediators such as IL-1ß, TNF-α, and TSLP. In this study, we investigated PAR2 calcium signaling and the consequent production of inflammatory mediators in differentiated human primary keratinocytes (DhPKs). Stimulation with the PAR2-activating peptide SLIGKV promoted Ca2+ store depletion in both undifferentiated human primary keratinocytes and DhPKs. SLIGKV-evoked Ca2+ store depletion did not trigger the store-operated Ca2+ entry (i.e., SOCE) through ORAI1 in DhPKs compared with undifferentiated human primary keratinocytes. The inhibition of phospholipase C and the concomitant inhibition of TRPV1 and inositol triphosphate receptor in DhPKs abrogated the SLIGKV-evoked Ca2+ store depletion; NF-κB activity; and the production of inflammatory mediators such as IL-1ß, TNF-α, and TSLP. Taken together, these results indicate a key role for both InsP3R and TRPV1 in Ca2+ internal stores in the PAR2-evoked Ca2+ release and consequent skin inflammation in DhPKs. These findings may provide clues to understanding the pathological role of DhPKs in skin disorders in which PAR2 is known to be involved, such as atopic dermatitis, Netherton syndrome, and psoriasis.


Assuntos
Mediadores da Inflamação/imunologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Queratinócitos/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Canais de Cátion TRPV/metabolismo , Sinalização do Cálcio/imunologia , Diferenciação Celular , Dermatite/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/imunologia , Queratinócitos/efeitos dos fármacos , Proteína ORAI1/genética , Proteína ORAI1/imunologia , Proteína ORAI1/metabolismo , Oligopeptídeos/farmacologia , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Receptor PAR-2 , Receptores Acoplados a Proteínas G/imunologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologia
18.
Nat Commun ; 9(1): 231, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335450

RESUMO

Atherosclerosis is characterized by the accumulation of lipids within the arterial wall. Although activation of TRPV1 cation channels by capsaicin may reduce lipid storage and the formation of atherosclerotic lesions, a clinical use for capsaicin has been limited by its chronic toxicity. Here we show that coupling of copper sulfide (CuS) nanoparticles to antibodies targeting TRPV1 act as a photothermal switch for TRPV1 signaling in vascular smooth muscle cells (VSMCs) using near-infrared light. Upon irradiation, local increases of temperature open thermo-sensitive TRPV1 channels and cause Ca2+ influx. The increase in intracellular Ca2+ activates autophagy and impedes foam cell formation in VSMCs treated with oxidized low-density lipoprotein. In vivo, CuS-TRPV1 allows photoacoustic imaging of the cardiac vasculature and reduces lipid storage and plaque formation in ApoE-/- mice fed a high-fat diet, with no obvious long-term toxicity. Together, this suggests CuS-TRPV1 may represent a therapeutic tool to locally and temporally attenuate atherosclerosis.


Assuntos
Aterosclerose/prevenção & controle , Cobre/química , Nanopartículas Metálicas , Músculo Liso Vascular/metabolismo , Transdução de Sinais , Sulfetos/química , Canais de Cátion TRPV/metabolismo , Animais , Anticorpos/imunologia , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Autofagia , Cálcio/metabolismo , Dieta Hiperlipídica , Progressão da Doença , Lipoproteínas LDL/farmacologia , Masculino , Camundongos Knockout , Músculo Liso Vascular/citologia , Canais de Cátion TRPV/imunologia
19.
J Invest Dermatol ; 138(2): 365-374, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28964718

RESUMO

Transient receptor potential (TRP) ion channels were first characterized on neurons, where they are classically implicated in sensory functions; however, research in recent decades has shown that many of these channels are also expressed on nonneuronal cell types. Emerging findings have highlighted the role of TRP channels in the skin, where they have been shown to be important in numerous cutaneous functions. Of particular interest is TRPV3, which was first described on keratinocytes. Its functional importance was supported when its gain-of-function mutation was linked to Olmsted syndrome, which is characterized by palmoplantar keratoderma, periorifacial hyperkeratosis, diffuse hypotrichosis and alopecia, and itch. Despite these exciting results, we have no information about the role and functionality of TRPV3 on keratinocytes at the cellular level. In this study, we identified TRPV3 expression both on human skin and cultured epidermal keratinocytes. TRPV3 stimulation was found to function as a Ca2+-permeable ion channel that suppresses proliferation of epidermal keratinocytes and induces cell death. Stimulation of the channel also triggers a strong proinflammatory response via the NF-κB pathway. Collectively, our data show that TRPV3 is functionally expressed on human epidermal keratinocytes and that it plays a role in cutaneous inflammatory processes.


Assuntos
Apoptose/imunologia , Dermatite/imunologia , Epiderme/imunologia , Queratinócitos/imunologia , Canais de Cátion TRPV/imunologia , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Proliferação de Células , Dermatite/patologia , Epiderme/metabolismo , Células HEK293 , Voluntários Saudáveis , Humanos , Queratinócitos/metabolismo , Canais de Cátion TRPV/metabolismo
20.
J Allergy Clin Immunol ; 141(2): 608-619.e7, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28807414

RESUMO

BACKGROUND: Chronic itch is a highly debilitating symptom that underlies many medical disorders with no universally effective treatments. Although unique neuronal signaling cascades in the sensory ganglia and spinal cord have been shown to critically promote the pathogenesis of chronic itch, the role of skin-associated cells remains poorly understood. OBJECTIVE: We sought to examine the cutaneous mechanisms underlying transient receptor potential vanilloid 4 (TRPV4)-mediated allergic and nonallergic chronic itch. METHODS: Expression of TRPV4 in chronic itch and healthy control skin preparations was examined by using real-time RT-PCR. Trpv4eGFP mice were used to study the expression and function of TRPV4 in the skin by means of immunofluorescence staining, flow cytometry, calcium imaging, and patch-clamp recordings. Genetic and pharmacologic approaches were used to examine the role and underlying mechanisms of TRPV4 in mouse models of dry skin-associated chronic itch and spontaneous scratching associated with squaric acid dibutylester-induced allergic contact dermatitis. RESULTS: TRPV4 is selectively expressed by dermal macrophages and epidermal keratinocytes in mice. Lineage-specific deletion of TRPV4 in macrophages and keratinocytes reduces allergic and nonallergic chronic itch in mice, respectively. Importantly, TRPV4 expression is significantly increased in skin biopsy specimens from patients with chronic idiopathic pruritus in comparison with skin from healthy control subjects. Moreover, TRPV4-dependent chronic itch requires 5-hydroxytryptamine (5-HT) signaling secondary to activation of distinct 5-HT receptors in mice with allergic and those with nonallergic chronic itch conditions. CONCLUSION: Our study reveals previously unrecognized mechanisms by which TRPV4-expressing epithelial and immune cells in the skin critically and dynamically mediate chronic itch and unravels novel targets for therapeutics in the setting of chronic itch.


Assuntos
Dermatite Alérgica de Contato/imunologia , Derme/imunologia , Regulação da Expressão Gênica/imunologia , Queratinócitos/imunologia , Macrófagos/imunologia , Prurido/imunologia , Canais de Cátion TRPV/imunologia , Animais , Doença Crônica , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/patologia , Derme/patologia , Feminino , Regulação da Expressão Gênica/genética , Humanos , Queratinócitos/patologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Prurido/genética , Prurido/patologia , Canais de Cátion TRPV/genética
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