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Immunocytochemical detection of TRPM8 protein in BEAS-2B cells. Immunocytochemical staining using a polyclonal antibody of TRPM8 demonstrated protein expression in BEAS-2B cells. The nuclei were counterstained blue using DAPI. (a) 1 mM of TDI intensified green staining in the plasma membranes in a time-dependent manner. (b) Pretreatment with TDI (0.25–1 mM) or (c) menthol (2–4 mM) for 3 h led to increased green staining in a dose-dependent manner. DAPI, 4′, 6-diamidino-2-phenylindole; TDI, toluene diisocyanate.
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Toluene diisocyanate (TDI) is the most important cause of occupational asthma (OA), and various pathogenic mechanisms have been suggested. Of these mechanisms, neurogenic inflammation is an important inducer of airway inflammation. Transient receptor potential melastatin 8 (TRPM8) is a well-established cold-sensing cation channel that is expressed...
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... Respiratory cells exposed to TDI undergo an inflammatory response and airway remodeling, resulting in reduced pulmonary function and respiratory disease [6,8,34]. Despite these risks and increasing usage, studies on the molecular mechanisms of chronic TDI exposure are still insufficient [35]. Therefore, we aim to identify TDI-induced pathological characteristics and mechanisms through our chronic TDI exposure model. ...
Toluene diisocyanate (TDI) is commonly used in manufacturing, and it is highly reactive and causes respiratory damage. This study aims to identify the mechanism of tumorigenesis in bronchial epithelial cells induced by chronic TDI exposure. In addition, transcriptome analysis results confirmed that TDI increases transforming growth factor-beta 1 (TGF-β1) expression and regulates genes associated with cancerous characteristics in bronchial cells. Our chronically TDI-exposed model exhibited elongated spindle-like morphology, a mesenchymal characteristic. Epithelial-mesenchymal transition (EMT) was evaluated following chronic TDI exposure, and EMT biomarkers increased concentration-dependently. Furthermore, our results indicated diminished cell adhesion molecules and intensified cell migration and invasion. In order to investigate the cellular regulatory mechanisms resulting from chronic TDI exposure, we focused on TGF-β1, a key factor regulated by TDI exposure. As predicted, TGF-β1 was significantly up-regulated and secreted in chronically TDI-exposed cells. In addition, SMAD2/3 was also activated considerably as it is the direct target of TGF-β1 and TGF-β1 receptors. Inhibiting TGF-β1 signaling through blocking of the TGF-β receptor attenuated EMT and cell migration in chronically TDI-exposed cells. Our results corroborate that chronic TDI exposure upregulates TGF-β1 secretion, activates TGF-β1 signal transduction, and leads to EMT and other cancer properties.
... Airway epithelial cells provide an important barrier function in the superficial layer of the airways, preventing the entry of harmful substances from the environment. In addition to this barrier function, the airway epithelium also plays a role in controlling inflammatory conditions by producing and secreting cytokines and chemokines that regulate various inflammatory responses [5]. ...
... Toluene diisocyanate (TDI) is a leading cause of occupational asthma (OA) and is also known to induce allergic rhinitis [5]. Regarding TDI-induced immune responses, induction of interleukins (ILs), including IL-4, 13, 25, and 33, in human bronchial epithelial cells has been reported [5]. ...
... Toluene diisocyanate (TDI) is a leading cause of occupational asthma (OA) and is also known to induce allergic rhinitis [5]. Regarding TDI-induced immune responses, induction of interleukins (ILs), including IL-4, 13, 25, and 33, in human bronchial epithelial cells has been reported [5]. ...
Toluene diisocyanate (TDI) is a major cause of occupational asthma and rhinitis. Shoseiryuto (SST) is one of the traditional herbal medicines (Kampo medicine) and has long been used as a natural medicine for allergic diseases such as allergic rhinitis (AR) and asthma. Recent studies have shown that the expression and release of IL-33, which regulates the TH2 cytokine response in epithelial cells, is an important step in developing the inflammatory response of the nasal mucosa. In this study, we investigated whether SST may ameliorate the TDI-induced AR-related symptoms in rats and inhibit IL-33 release from nasal epithelial cells. An AR rat model was generated by sensitization and induction with TDI. SST was administered during the sensitization period. AR-related symptoms in rats were evaluated, and IL-33 release was measured both in vivo and in vitro. SST suppressed symptoms appearing in TDI-induced AR model rats, such as elevated serum histamine and IL-33 levels in nasal lavage fluid (NLF)/serum, which were suppressed by SST administration. TDI-induced IL-33 release from the nasal epithelial cell nuclei was also observed and suppressed in SST-treated rats and cultured nasal epithelial cells. These results suggest that SST ameliorates the symptoms of TDI-induced AR at least partially by inhibiting IL-33 release from nasal epithelial cells.
... These contrasting findings could be due to the difference in the mechanism of action of HDM and toluene diisocyanate. HDM activates pathogen recognition receptors (PRRs) like dectin-1 and Toll-like receptor (TLR)-4 resulting in the release of pro-inflammatory cytokines like CCL17, CCL20, IL-5, and IL-13 (Gregory and Lloyd, 2011;Post et al., 2014), whereas toluene diisocyanate acts on transient receptor potential melastatin 8 (TRPM8) resulting in the release of IL-25, IL-4, and IL-13 (Kim et al., 2017a). This suggests that TRPM8, but not PRR activation is regulated by β-catenin/CBP signaling. ...
Excessive mucus production is a major feature of allergic asthma. Disruption of epithelial junctions by allergens such as house dust mite (HDM) results in the activation of β-catenin signaling, which has been reported to stimulate goblet cell differentiation. β-catenin interacts with various co-activators including CREB binding protein (CBP) and p300, thereby regulating the expression of genes involved in cell proliferation and differentiation, respectively. We specifically investigated the role of the β-catenin/CBP signaling pathway in goblet cell metaplasia in a HDM-induced allergic airway disease model in mice using ICG-001, a small molecule inhibitor that blocks the binding of CBP to β-catenin. Female 6- 8-week-old BALB/c mice were sensitized to HDM/saline on days 0, 1, and 2, followed by intranasal challenge with HDM/saline with or without subcutaneous ICG-001/vehicle treatment from days 14 to 17, and samples harvested 24 h after the last challenge/treatment. Differential inflammatory cells in bronchoalveolar lavage (BAL) fluid were enumerated. Alcian blue (AB)/Periodic acid–Schiff (PAS) staining was used to identify goblet cells/mucus production, and airway hyperresponsiveness (AHR) was assessed using invasive plethysmography. Exposure to HDM induced airway inflammation, goblet cell metaplasia and increased AHR, with increased airway resistance in response to the non-specific spasmogen methacholine. Inhibition of the β-catenin/CBP pathway using treatment with ICG-001 significantly attenuated the HDM-induced goblet cell metaplasia and infiltration of macrophages, but had no effect on eosinophils, neutrophils, lymphocytes or AHR. Increased β-catenin/CBP signaling may promote HDM-induced goblet cell metaplasia in mice.
... Another study revealed that the phenotype of toluene diisocyanate (TDI)-induced occupational asthma (OA) was significantly associated with TRPM8 gene (Kim et al., 2009). TDI led to a robust increase in the protein level of TRPM8 in human bronchial epithelial cells (cell line BEAS-2B), coupled with significantly enhanced expression of inflammatory cytokines mRNA (IL-4, IL-13 and IL-25) (Kim et al., 2017). Treatment with the TRPM8 antagonist BCTC partially attenuated the increased cytokine expression, which also supports the view that TRPM8 is involved in the pathogenesis of TDI-induced OA. ...
... Concerning treatment, TRPM8 agonists ameliorate pruritus in clinical studies (Misery et al., 2019;Stander et al., 2017). TRPM8 antagonists have been reported as potential drugs for bladder hypersensitive disorders (Aizawa et al., 2019;Ito et al., 2016;Lashinger et al., 2008;Mistretta et al., 2016;Ohmi et al., 2014), cancer (Bai et al., 2010;Liu et al., 2014Liu et al., , 2018aOkamoto et al., 2012), migraine (Horne et al., 2018), COPD (Li et al., 2011) and asthma (Kim et al., 2017). ...
Phosphatidylinositol 4-kinase (PI4K) is a lipid kinase that can catalyze the transfer of phosphate group from ATP to the inositol ring of phosphatidylinositol (PtdIns) resulting in the phosphorylation of PtdIns at 4-OH sites, to generate phosphatidylinositol 4-phosphate (PI4P). Studies on biological functions reveal that PI4K is closely related to the occurrence and development of common clinical diseases such as virus replication, cancer and inflammations. PI4K-related inhibitors have been found to have the effects of inhibiting virus replication, anti-cancer, treating malaria and reducing rejection in organ transplants, among which MMV390048, an anti-malaria drug, has entered phase II clinical trial. This review discusses the classification, structure, distribution and related inhibitors of PI4K and their role in the progression of cancers, viral replication, and other inflammation induced diseases to explore their potential as therapeutic targets.
... Surprisingly, IL-33 which is a known damage-associated pattern used to evaluate epithelial damage was not changed after dermal and/or airway challenge with TDI. We expected increased levels of IL-33 caused by irritant exposure to TDI on the airway epithelium, which was already proven in vitro by Kim et al. 41 It is also known that IL-33 initiates the production of Th2 cytokines, such as IL-4, IL-5 and IL-13, which were increased in the TDI/TDI-treated group. 41 It needs to be mentioned that IL-33 is known to be released from the epithelium immediately after exposure and can already be cleared from the body at the time of collection, which is 24 hours after the last exposure. ...
... We expected increased levels of IL-33 caused by irritant exposure to TDI on the airway epithelium, which was already proven in vitro by Kim et al. 41 It is also known that IL-33 initiates the production of Th2 cytokines, such as IL-4, IL-5 and IL-13, which were increased in the TDI/TDI-treated group. 41 It needs to be mentioned that IL-33 is known to be released from the epithelium immediately after exposure and can already be cleared from the body at the time of collection, which is 24 hours after the last exposure. Additionally, these findings also indicate that other damage-associated patterns (IL-25 or TSLP) are possibly responsible for the induction of cytokine production and play a more important role in our model for chemical-induced asthma. ...
Purpose:
Exposure to low concentrations of toluene diisocyanate (TDI) leads to immune-mediated chemical-induced asthma. The role of the adaptive immune system has already been thoroughly investigated; nevertheless, the involvement of innate immune cells in the pathophysiology of chemical-induced asthma is still unresolved. The aim of the study is to investigate the role of innate lymphoid cells (ILCs) and dendritic cells (DCs) in a mouse model for chemical-induced asthma.
Methods:
On days 1 and 8, BALB/c mice were dermally treated (20 μL/ear) with 0.5% TDI or the vehicle acetone olive oil (AOO; 2:3). On days 15, 17, 19, 22 and 24, the mice received an oropharyngeal challenge with 0.01% TDI or AOO (1:4). One day after the last challenge, airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and immune-related parameters, including the cytokine pattern in bronchoalveolar lavage fluid, lymphocyte subpopulations of the lymph nodes and their ex vivo cytokine production profile, blood immunoglobulins and DC and ILC subpopulations in the lungs.
Results:
Both DC and ILC2 were recruited to the lungs after multiple airway exposures to TDI, regardless of the prior dermal sensitization. However, prior dermal sensitization with TDI alone results in AHR and predominant eosinophilic airway inflammation, accompanied by a typical type 2 helper T (Th2) cytokine profile.
Conclusions:
TDI-induced asthma is mediated by a predominant type 2 immune response, with the involvement of adaptive Th2 cells. However, from our study we suggest that the innate ILC2 cells are important additional players in the development of TDI-induced asthma.
... Another study revealed that the phenotype of toluene diisocyanate (TDI)-induced occupational asthma (OA) was significantly associated with TRPM8 gene (Kim et al., 2009). TDI led to a robust increase in the protein level of TRPM8 in human bronchial epithelial cells (cell line BEAS-2B), coupled with significantly enhanced expression of inflammatory cytokines mRNA (IL-4, IL-13 and IL-25) (Kim et al., 2017). Treatment with the TRPM8 antagonist BCTC partially attenuated the increased cytokine expression, which also supports the view that TRPM8 is involved in the pathogenesis of TDI-induced OA. ...
... Concerning treatment, TRPM8 agonists ameliorate pruritus in clinical studies (Misery et al., 2019;Stander et al., 2017). TRPM8 antagonists have been reported as potential drugs for bladder hypersensitive disorders (Aizawa et al., 2019;Ito et al., 2016;Lashinger et al., 2008;Mistretta et al., 2016;Ohmi et al., 2014), cancer (Bai et al., 2010;Liu et al., 2014Liu et al., , 2018aOkamoto et al., 2012), migraine (Horne et al., 2018), COPD (Li et al., 2011) and asthma (Kim et al., 2017). ...
Ion channels are important therapeutic targets due to their plethoric involvement in physiological and pathological consequences. The transient receptor potential cation channel subfamily M member 8 (TRPM8) is a nonselective cation channel that controls Ca²⁺ homeostasis. It has been proposed to be the predominant thermoreceptor for cellular and behavioral responses to cold stimuli in the TRP channel subfamilies and exploited so far to reach the clinical-stage of drug development. TRPM8 channels can be found in multiple organs and tissues, regulating several important processes such as cell proliferation, migration and apoptosis, inflammatory reactions, immunomodulatory effects, pain, and vascular muscle tension. The related disorders have been expanded to new fields ranging from cancer and migraine to dry eye disease, pruritus, irritable bowel syndrome (IBS), and chronic cough. This review is aimed to summarize the distribution of TRPM8 and disorders related to it from a clinical perspective, so as to broaden the scope of knowledge of researchers to conduct more studies on this subject.
... BEAS2B cells were used because they were found to express TRPM8 in our previous study. 22 The cells were treated with various concentrations of menthol (0-2.0 mM), and both the mRNA and protein expression of TRPM8 robustly increased in a dose-dependent manner (Fig. 1). The results of ELISA to assess cell supernatants and cell lysates were similar to those of Western blotting (Supplementary Fig. S1). ...
... However, literature reviews show that various stimuli can induce the activation of this receptor and subsequently produce proinflammatory cytokines. Calcium-rich particulate matter and smoking can activate TRPM8, 27 and our previous study reported that toluene diisocyanate, 22 which is a major cause of occupational asthma, also induces TRPM8 activation in airway epithelial cells. Moreover, human rhinovirus infection can cause upregulation of TRPM8, which is dependent on viral replication. ...
Purpose:
Cold air is a major environmental factor that exacerbates asthma. Transient receptor potential melastatin family member 8 (TRPM8) is a cold-sensing channel expressed in the airway epithelium. However, its role in airway inflammation remains unknown. We investigated the role of TRPM8 in innate immune responses in bronchial epithelial cells and asthmatic subjects.
Methods:
The TRPM8 mRNA and protein expression on BEAS2B human bronchial epithelial cells was examined by real-time polymerase chain reaction (PCR), immunofluorescence staining and western blotting. Additionally, interleukin (IL)-4, IL-6, IL-8, IL-13, IL-25 and thymic stromal lymphopoietin (TSLP) levels before and after menthol, dexamethasone and N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl) piperazine-1-carboxamide (BCTC) treatments were measured via real-time PCR. TRPM8 protein levels in the supernatants of induced sputum from asthmatic subjects and normal control subjects were measured using enzyme-linked immunosorbent assay, and mRNA levels in sputum cell lysates were measured using real-time PCR.
Results:
Treatment with up to 2 mM menthol dose-dependently increased TRPM8 mRNA and protein in BEAS2B cells compared to untreated cells (P < 0.001) and concomitantly increased IL-25 and TSLP mRNA (P < 0.05), but not IL-33 mRNA. BCTC (10 μM) significantly abolished menthol-induced up-regulation of TRPM8 mRNA and protein and IL-25 and TSLP mRNA (P < 0.01). TRPM8 protein levels were higher in the supernatants of induced sputum from asthmatic subjects (n = 107) than in those from healthy controls (n = 19) (P < 0.001), and IL-25, TSLP and IL-33 mRNA levels were concomitantly increased (P < 0.001). Additionally, TRPM8 mRNA levels correlated strongly with those of IL-25 and TSLP (P < 0.001), and TRPM8 protein levels were significantly higher in bronchodilator-responsive asthmatic subjects than in nonresponders.
Conclusions:
TRPM8 may be involved in the airway epithelial cell innate immune response and a molecular target for the treatment of asthma.
... Pulmonary Pharmacology & Therapeutics 53 (2018) [39][40][41][42][43][44][45][46][47][48][49][50][51] generation, BALF cells were harvested 24 h after last challenge, the cells were rinsed with Hank's balanced salt solution (HBSS) and incubated with 20 μM 2′,7′-dichlorofluorescein diacetate (DCFDA) reagent for 60 min. DCFDA forms a fluorescent product upon oxidation of dichlorofluorescein by ROS. ...
... Apart from the heterogeneity of the inflammatory responses, TDI sensitisation and challenge also triggered desquamation of airway epithelium, collagen deposition and intracellular ROS surge. These suggest the presence of airway remodelling and oxidative damage in TDI-exposed animals [3,8,[47][48][49]. ...
Toluene diisocyanate (TDI) is a major cause of chemical-induced occupational asthma, which contributes about 15% of global asthma burden. Resistance and compounded side effects associated with the use of corticosteroid in asthma necessitate the search for alternative drugs. Andrographolide (AGP), a naturally occurring diterpene lactone is known to exhibit various bioactivities. Its ability to ameliorate cardinal features of allergic asthma was previously suggested in an eosinophilic asthma endotype. However, its potential antiasthma activity and mechanism of action in a neutrophilic occupational asthma model, as well as its effect on epithelial dysfunction remain unknown. BALB/c mice were dermally sensitised with 0.3% TDI or acetone olive oil (AOO) vehicle on day 1 and 8, followed by 0.1% TDI intranasal challenge on days 15, 18 and 21. Endpoints were evaluated via bronchoalveolar lavage fluid (BALF) cell analysis, 2',7'-dichlorofluorescein diacetate (DCFDA) assays, immunoblotting, immunohistochemistry and methacholine challenge test. Decreases in total and differential leukocyte counts of BALF were recorded in AGP-treated animals. The compound dose-dependently reduced intracellular de-esterification of DCFDA, thus suggesting AGP's potential to inhibit intracellular reactive oxygen species (ROS). Mechanistically, the treatment prevented TDI-induced aberrant E-cadherin distribution and restored airway epithelial β-catenin at cell to cell contact site. Furthermore, AGP ameliorated TDI induced pulmonary collagen deposition. In addition, the treatment significantly upregulated pulmonary HO-1, Nrf2 and phospho-p38 levels. Airway hyperresponsiveness was markedly suppressed among AGP-treated animals. Collectively, these findings suggest AGP's protective function against TDI-induced airway epithelial barrier dysfunction and oxidative lung damage possibly through the upregulation of adherence junction proteins and the activation of p38/Nrf2 signalling. This study elucidates the therapeutic potential of AGP in the control and management of chemical-induced allergic asthma. To the best of our knowledge, the potential anti-asthma activity of AGP in TDI-induced occupational asthma has not been reported previously.
... N-(4-Tertiarybutylphenyl)-4-(3chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC), thio-BCTC, and capsazepine are some listed potent inhibitors of TRPM8 responses [5]. Recent studies have shown that activation of TRPM8 in various cells (bronchial epithelial cells, mast cells, and vagal ganglion neurons) leads to certain respiratory responses including mucus hypersecretion, enhanced inflammatory cytokine expressions, histamine release, and autonomic nerve reflex, which suggests the potential involvement of TRPM8 in the pathogenesis of asthma [6][7][8][9]. ...
... The present study showed that TRPM8 could enhance the expression of mRNAs of various cytokines significantly (including IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13, GM-CSF, and TNF-α) in the primary AEC of asthmatic mice cultured at a low temperature (18°C). Overexpression or downregulation of TRPM8 could accordingly increase or decrease [8,21]. However, there is also paradoxical finding. ...
Bronchial asthma is a chronic inflammatory airway disease that can be aggravated by cold air. However, its mechanism remains largely unknown. As a thermo-sensing cation channel, transient receptor potential melastatin 8 (TRPM8) can be activated by cold stimuli (8–22 °C) and cooling agents. Whereas TRPM8 activation leads to enhanced expression of inflammatory cytokines and mucus hypersecretion in human bronchial epithelial cell lines, no previous study has examined its role in regulating the cold-induced inflammatory responses and its mechanism in asthmatic airway epithelium. Airway epithelial cells were isolated from asthma model mice and exposed to low temperature (18 °C). The TRPM8 overexpression plasmid and siRNA lentivirus were transfected to up- or downregulate the TRPM8 level. The expression of mRNAs of inflammatory cytokines was tested using real-time reverse transcription–polymerase chain reaction (RT-PCR). The activities of phosphorylated protein kinase C (PKC) and phosphorylated inhibitor of nuclear factor kappa B (IκB) were measured using the immunofluorescence assay. The expression of mRNAs of inflammatory cytokines [interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-13, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-α] increased significantly under cold conditions, which was boosted after TRPM8 overexpression and augmented further in the presence of PKC inhibitor, calphostin C. However, the downregulation of TRPM8 and nuclear factor kappa B (NF-κB) impaired the transcription of these cytokine genes. In addition, the phosphorylated PKC and phosphorylated IκB were activated by cold stimuli. Moreover, the expression of phosphorylated IκB protein improved in the presence of TRPM8, while disruption with the TRPM8 gene or TRPM8 antagonist prohibited the activation of IκB. Cold air could induce inflammatory responses through the TRPM8-mediated PKC/NF-κB signal pathway in primary airway epithelial cells of asthmatic mice.
... Moreover, we demonstrated that inhibition of TRPM8 suppressed MAPK and NF-κB pathways. These data supported previous findings and indicated that TRPM8 may be involved in the pathogenesis of asthma, including airway inflammation and remodeling [19,20]. ...
... Activation of cold-sensitive channels TRPM8 and TRPA1 inhibits the proliferative airway smooth muscle cell phenotype [20]. Toluene diisocyanate exposure induces airway inflammation of bronchial epithelial cells via the activation of TRPM8 [19]. Activation of the TRPM8 variant in lung epithelial cells by cold air leads to increased expression of several cytokine and chemokine genes [12]. ...
Cold air stimulus is an important environmental factor that exacerbates asthma. At the molecular level, the transient receptor potential melastatin 8 (TRPM8) plays a crucial part in cold detection. The roles of TRPM8 in airway inflammation and remodeling in a murine model of asthma with cold stimulus and the related molecular mechanism are largely unknown. In this study, C57BL/6 mice were randomly divided into four groups: phosphate-buffered saline control group (control), ovalbumin (OVA)-induced asthma group (OVA), OVA with cold air stimulus group (OVA+cold), and OVA+cold+shTRPM8 (TRPM8 short hairpin RNA) group. We showed that cold air stimulus-induced TRPM8 upregulation in the OVA+cold group. Moreover, TRPM8 knockdown significantly attenuated cold-induced inflammation and infiltration, decreased levels of immunoglobulin E, restored the Th1/Th2 balance, and reduced inflammatory cell accumulation and airway remodeling. Furthermore, we demonstrated that TRPM8 knockdown dramatically inhibited mitogen-activated protein kinase and nuclear factor-κB pathways. Collectively, these results revealed that cold air stimulus induced an airway inflammatory response and remodeling by increasing TRPM8 expression and that downregulation of TRPM8 alleviated these responses.
