Broad locations of antigenic regions for anti-TRPM1 autoantibodies in paraneoplastic retinopathy with retinal ON bipolar cell dysfunction.

PURPOSE: Cancer-associated retinal ON bipolar cell dysfunction (CARBD), which includes melanoma-associated retinopathy (MAR), has been reported to be caused by autoantibodies against the molecules expressed in ON bipolar cells, including TRPM1. The purpose of this study was to determine the antigenic regions of the autoantibodies against TRPM1 in the sera of CARBD patients, in whom we previously detected anti-TRPM1 autoantibodies. METHODS: The antigenic regions against TRPM1 in the sera of eight CARBD patients were examined by Western blots using HEK293T cells transfected with the plasmids expressing FLAG-tagged TRPM1 fragments. The clinical course of these patients was also documented. RESULTS: The clinical course differed among the patients. The electroretinograms (ERGs) and symptoms were improved in three patients, deteriorated in one patient, remained unchanged for a long time in one patient, and were not followable in three patients. Seven of the eight sera possessed multiple antigenic regions: two sera contained at least four antigen recognition regions, and three sera had at least three regions. The antigen regions were spread over the entire TRPM1 protein: five sera in the N-terminal intracellular domain, six sera in the transmembrane-containing region, and six sera in the C-terminal intracellular domain. No significant relationship was observed between the location of the antigen epitope and the patients' clinical course. CONCLUSIONS: The antigenic regions of anti-TRPM1 autoantibodies in CARBD patients were present not only in the N-terminal intracellular domain, which was reported in an earlier report, but also in the transmembrane-containing region and in the C-terminal intracellular domain. In addition, the antigenic regions for TRPM1 were found to vary among the CARBD patients examined, and most of the sera had multiple antigenic regions.

A putative anti-inflammatory role for TRPM8 in irritable bowel syndrome-An exploratory study.

BACKGROUND: Chronic and recurring pain is a characteristic symptom in irritable bowel syndrome (IBS). Altered signaling between immune cells and sensory neurons within the gut may promote generation of pain symptoms. As transient receptor potential melastatin 8 (TRPM8) agonists, such as L-menthol in peppermint oil, have shown to attenuate IBS pain symptoms, we began investigating potential molecular mechanisms. METHODS: Colonic biopsy tissues were collected from patients with IBS and controls, in two separate cohorts. Immunohistochemistry was performed to identify TRPM8 localization. Quantitative PCR was performed to measure mucosal mRNA levels of TRPM8. In addition, functional experiments with the TRPM8 agonist icilin were performed ex vivo to examine cytokine release from biopsies. Daily diaries were collected to ascertain pain symptoms. RESULTS: In biopsy tissue from IBS patients, we showed that TRPM8 immunoreactivity is colocalized with immune cells predominantly of the dendritic cell lineage, in close approximation to nerve endings, and TRPM8 protein and mRNA expression was increased in IBS patients compared to controls (p < 0.001). TRPM8 mRNA expression showed a significant positive association with abdominal pain scores (p = 0.015). Treatment of IBS patient biopsies with icilin reduced release of inflammatory cytokines IL-1ß, IL-6, and TNF-α (p < 0.05). CONCLUSIONS AND INFERENCES: These data indicate TRPM8 may have important anti-inflammatory properties and by this virtue can impact neuro-immune disease mechanisms in IBS.

TRPM2 channel in oxidative stress-induced mitochondrial dysfunction and apoptotic cell death.

Mitochondria, conserved intracellular organelles best known as the powerhouse of cells for generating ATP, play an important role in apoptosis. Oxidative stress can induce mitochondrial dysfunction and activate mitochondria-mediated apoptotic cell death. TRPM2 is a Ca2+-permeable cation channel that is activated by pathologically relevant concentrations of reactive oxygen species (ROS) and one of its well-recognized roles is to confer susceptibility to ROS-induced cell death. Increasing evidence from recent studies supports TRPM2 channel-mediated cell death as an important cellular mechanism linking miscellaneous oxidative stress-inducing pathological factors to associated diseased conditions. In this chapter, we will discuss the role of the TRPM2 channel in neurons in the brain and pancreatic ß-cells in mediating mitochondrial dysfunction and cell death, focusing mainly on apoptotic cell death, that are induced by pathological stimuli implicated in the pathogenesis of neurodegenerative diseases, ischemic stroke and diabetes.

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling.

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.

TRPM2 Is Not Required for T-Cell Activation and Differentiation.

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling.

The thymic development of regulatory T (Treg) cells, crucial suppressors of the responses of effector T (Teff) cells, is governed by the transcription factor FOXP3. Despite the clinical importance of Treg cells, there is a dearth of druggable molecular targets capable of increasing their numbers in vivo. We found that inhibiting the function of the TRPM7 chanzyme (ion channel and enzyme) potentiated the thymic development of Treg cells in mice and led to a substantially higher frequency of functional Treg cells in the periphery. In addition, TRPM7-deficient mice were resistant to T cell-driven hepatitis. Deletion of Trpm7 and inhibition of TRPM7 channel activity by the FDA-approved drug FTY720 increased the sensitivity of T cells to the cytokine interleukin-2 (IL-2) through a positive feed-forward loop involving increased expression of the IL-2 receptor α-subunit and activation of the transcriptional regulator STAT5. Enhanced IL-2 signaling increased the expression of Foxp3 in thymocytes and promoted thymic Treg (tTreg) cell development. Thus, these data indicate that inhibiting TRPM7 activity increases Treg cell numbers, suggesting that it may be a therapeutic target to promote immune tolerance.

Exposure to formaldehyde at low temperatures aggravates allergic asthma involved in transient receptor potential ion channel.

Studies have indicated that formaldehyde and low temperature are considered to be the factors associated with several respiratory diseases. However, the effect of co-exposure to formaldehyde and low temperature on allergic asthma, and the potential mechanisms, are unknown. In this study, an allergic asthma mouse model was built and mice were exposed to 0.8 mg/m3 formaldehyde and/or subjected to low temperatures of 16 °C. The data showed that exposure to either low temperature or formaldehyde did not induce a significant aggravation on allergic asthma. However, simultaneous exposure to formaldehyde and low temperature was shown to aggravate mucus hypersecretion and inflammation in the lung, lead to an exacerbation of allergic asthma. After blocking the TRPM8 and TRPA1 ion channels, the levels of inflammatory factors reduced. These results demonstrated that co-exposure to formaldehyde and low temperature exacerbate allergic asthma, and that TRPM8 and TRPA1 are involved in this process.

A case of melanoma-associated retinopathy with autoantibodies against TRPM1.

PURPOSE: To report a case of melanoma-associated retinopathy (MAR) with autoantibodies against the transient receptor potential cation channel, subfamily M, member 1 (TRPM1) with asymmetric severe vision loss. METHODS: We evaluated a patient with heel skin melanoma showing progressive vision loss in both eyes confirmed with a baseline ophthalmic examination, fluorescein angiography, spectral domain optical coherence tomography (OCT), visual field test, and full-field electroretinogram (ERG). Immunofluorescence assays and western blot analysis revealed autoantibodies in the patient's serum. RESULTS: The patient's best-corrected visual acuities were 20/50 in the right eye and hand motion in the left eye. Visual field test showed severely depressed visual fields especially in the left eye. Fluorescein angiography and OCT revealed extrafoveal choroidal neovascularization in the left eye. The patient had an electronegative ERG, suggesting MAR, and autoantibodies against TRPM1 and aldolase C were detected in the patient's blood sample. CONCLUSIONS: The clinical features of MAR patients with positive anti-TRPM1 autoantibodies can be manifested as severe vision loss, and the identification of autoantibodies can be helpful for confirming the diagnosis.

Identification and characterization of novel TRPM1 autoantibodies from serum of patients with melanoma-associated retinopathy.

Melanoma-associated retinopathy (MAR) is a rare paraneoplastic retinal disorder usually occurring in the context of metastatic melanoma. Patients present with night blindness, photopsias and a constriction of the visual field. MAR is an auto-immune disorder characterized by the production of autoantibodies targeting retinal proteins, especially autoantibodies reacting to the cation channel TRPM1 produced in melanocytes and ON-bipolar cells. TRPM1 has at least three different isoforms which vary in the N-terminal region of the protein. In this study, we report the case of three new MAR patients presenting different anti-TRPM1 autoantibodies reacting to the three isoforms of TRPM1 with variable binding affinity. Two sera recognized all isoforms of TRPM1, while one recognized only the two longest isoforms upon immunolocalization studies on overexpressing cells. Similarly, the former two sera reacted with all TRPM1 isoforms on western blot, but an immunoprecipitation enrichment step was necessary to detect all isoforms with the latter serum. In contrast, all sera labelled ON-bipolar cells on Tprm1+/+ but not on Trpm1-/- mouse retina as shown by co-immunolocalization. This confirms that the MAR sera specifically detect TRPM1. Most likely, the anti-TRPM1 autoantibodies of different patients vary in affinity and concentration. In addition, the binding of autoantibodies to TRPM1 may be conformation-dependent, with epitopes being inaccessible in some constructs (truncated polypeptides versus full-length TRPM1) or applications (western blotting versus immunohistochemistry). Therefore, we propose that a combination of different methods should be used to test for the presence of anti-TRPM1 autoantibodies in the sera of MAR patients.

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.

TRPM2, linking oxidative stress and Ca2+ permeation to NLRP3 inflammasome activation.

The NLRP3 inflammasome is an innate immune platform that senses various pathogens and sterile insults. NLRP3 stimulation leads to activation of caspase-1, the secretion of pro-inflammatory cytokines and an inflammatory cell death called pyroptosis. Effectors of the NLRP3 inflammasome efficiently drive an immune response, not only providing protection in physiological settings but also promoting pathology when over activated. Generation of reactive oxygen species (ROS) and intracellular calcium mobilization can activate the NLRP3 inflammasome. Recent studies suggest that TRPM2 is a calcium-permeable cation channel mediating ROS-dependent NLRP3 activation. Here, we review the role of TRPM2 in NLRP3 inflammasome activation and provide an update on new functional and structural discoveries. Understanding the molecular mechanism of TRPM2 dependent NLRP3 inflammasome activation will shed lights on this complex pathway and help the developing of therapeutic strategies.

Identification and validation of an immune-related gene signature predictive of overall survival in colon cancer.

The heterogeneity and complexity of tumor-immune microenvironments lead to diverse immunotherapy effects among colon cancer patients. It is crucial to identify immune microenvironment-related biomarkers and construct prognostic risk models. In this study, the immune and stromal scores of 415 cases from TCGA were calculated using the ESTIMATE algorithm. AXIN2, CCL22, CLEC10A, CRIP2, RUNX3, and TRPM5 were screened and established a prognostic immune-related gene (IRG) signature using by univariate, LASSO, and multivariate Cox regression models. The predicted performance of IRG signature was external validated by GSE39582 (n=519). Stratified survival analysis showed IRG signature was an effective predictor of survival in patients with different clinical characteristics. The protein expression level of six genes was validated by immunohistochemistry analysis. Difference analysis indicated the mutation rate, immune cell of resting NK cells and regulatory T cells infiltration and four immune checkpoints of PD-1, PD-L1, LAG3 and VSIR expression levels in the high-risk group were significantly higher than those in the low-risk group. A nomogram incorporating the gene signatures and clinical factors was demonstrated had a good accuracy (1-, 3-, and 5-year AUC= 0.799, 0.791, 0.738). Our study identified a novel IRG signature, which may provide some references for the clinical precision immunotherapy of patients.

Naltrexone Restores Impaired Transient Receptor Potential Melastatin 3 Ion Channel Function in Natural Killer Cells From Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a seriously long-term and debilitating illness of unknown cause hallmarked by chronic pain and fatigue, memory and concentration impairment, and inflammation. ME/CFS hypothesis involves impaired Transient receptor potential melastatin 3 (TRPM3) ion channel function, affecting calcium signaling and Natural killer (NK) cell functions. Currently, substances called opioids, agonists of mu (µ)-opioid receptors (µOR), are the strongest painkillers clinically available for people suffering from strong or long-lasting pain characteristic of ME/CFS. µOR have been reported to specifically inhibit TRPM3 and to be expressed in immune cells where they play an immunomodulatory and immunosuppressive role. Naltrexone hydrochloride (NTX) acts as an antagonist to the µOR thus negating the inhibitory function of this opioid receptor on TRPM3. Therefore, understanding the mechanism of action for NTX in regulating and modulating TRPM3 channel function in NK cells will provide important information for the development of effective therapeutic interventions for ME/CFS. Whole-cell patch-clamp technique was used to measure TRPM3 activity in Interleukin-2 (IL-2) stimulated and NTX-treated NK cells for 24 h on eight ME/CFS patients and 8 age- and sex-matched healthy controls, after modulation with a TRPM3-agonist, pregnenolone sulfate (PregS), NTX and a TRPM3-antagonist, ononetin. We confirmed impaired TRPM3 function in ME/CFS patients through electrophysiological investigations in IL-2 stimulated NK cells after modulation with PregS and ononetin. Importantly, TRPM3 channel activity was restored in IL-2 stimulated NK cells isolated from ME/CFS patients after incubation for 24 h with NTX. Moreover, we demonstrated that NTX does not act as an agonist by directly coupling on the TRPM3 ion channel gating. The opioid antagonist NTX has the potential to negate the inhibitory function of opioid receptors on TRPM3 in NK cells from ME/CFS patients, resulting in calcium signals remodeling, which will in turn affect cell functions, supporting the hypothesis that NTX may have potential for use as a treatment for ME/CFS. Our results demonstrate, for the first time, and based on novel patch clamp electrophysiology, potential pharmaco-therapeutic interventions in ME/CFS.

TRPM2 channel regulates cytokines production in astrocytes and aggravates brain disorder during lipopolysaccharide-induced endotoxin sepsis.

Sepsis is one of the most significant challenges in intensive care units, which is associated with increased morbidity and mortality. Sepsis-associated encephalopathy (SAE) is a severe complication which can cause death and serious disabilities. Calcium signaling in astrocyte is essential for cellular activation and the potential resolution of infection or inflammation in SAE patients. The transient receptor potential melastatin 2 (TRPM2) channel has been identified as a unique fusion of a Ca2+-permeable nonselective cation channel, which plays an important role in inflammation and immune response. Because of its role as an oxidative stress sensor in astrocytes, we investigated the function of TRPM2 in inflammation mediators (interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α) release, Bcl-2/E1B-19 K-interacting protein 3 (BNIP3), apoptosis inducing factor (AIF) and Endonuclease G (Endo G) expression. We showed that TRPM2-KO mice, when intraperitoneally (i.p) injected with LPS, exhibited better neurologic assessment scores and decreased inflammatory injury in hippocampal neurons compared with wild-type (WT) mice. The absence of TRPM2 triggered less production of inflammatory mediators (IL-1ß, IL-6, TNF-α) and decreased apoptosis related proteins (BNIP3, AIF, Endo G) expressions in response to LPS induced sepsis. Furthermore, TRPM2-deficient astrocytes (transfected with TRPM2 siRNA) upon LPS stimulation also induced decreased IL-1ß, IL-6 and TNF-α level. Our data suggested that decreased production of inflammatory cytokines and apoptosis related proteins with TRPM2 deletion could regulate inflammatory stress and decrease inflammatory injury in hippocampal neurons, and consequently, ameliorate brain disorder.

TRPM1 Autoantibodies in Melanoma Patients Without Self-Reported Visual Symptoms.

Purpose: Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma (CMM). Visual symptoms include night blindness, photopsia, and reduced-contrast sensitivity. An abnormal ERG b-wave and the presence of anti-bipolar cell autoantibodies, including autoantibodies reacting with the ON-bipolar cell TRPM1 channel, help to confirm the diagnosis. The goal of this study was to determine if CMM patients without visual symptoms also express anti-TRPM1 autoantibodies. Methods: Serum samples from 15 CMM patients were tested using three assays: immunofluorescent labeling of TRPM1-transfected HEK cells, immunofluorescent labeling of retinal sections from wild-type and TRPM1 knockout mice, and immunoblot detection of a bacterially produced recombinant TRPM1 peptide. Results: Serum specimens from 5 of the 15 CMM patients without declared visual symptoms were positive for anti-TRPM1 autoantibodies in at least one of the three assays. One of 50 control sera from patients not known to have cancer was also weakly reactive with the TRPM1 peptide. Conclusions: Autoantibodies against TRPM1 are present in CMM patient sera without self-reported visual symptoms. Most patients had advanced (stage III and IV) disease and were undergoing aggressive treatments, including immunotherapy. It is unknown if immunotherapy affects the expression of TRPM1 autoantibodies. The presence of TRPM1 autoantibodies may predispose patients for MAR.

CLINICAL COURSE OF PARANEOPLASTIC RETINOPATHY WITH ANTI-TRPM1 AUTOANTIBODY IN JAPANESE COHORT.

PURPOSE: To report the clinical course of eyes with paraneoplastic retinopathy caused by an autoantibody against transient receptor potential cation channel, subfamily M, member 1 (TRPM1). METHODS: Ten paraneoplastic retinopathy patients with retinal ON-bipolar cell dysfunction, including six melanoma-associated retinopathy, from eight institutions in Japan were evaluated for the presence of an anti-TRPM1 antibody. The results of ophthalmic examinations and the presence of anti-TRPM1 antibody were analyzed. RESULTS: Five patients were positive for the anti-TRPM1 antibody. These patients had similar clinical findings in both eyes at the time of diagnosis; relatively preserved best-corrected visual acuity, absence of fundus and optical coherence tomography abnormalities, and specific abnormalities of the electroretinography (ERG); and negative-type ERGs with bright stimulus flashes. One patient whose retinal ON-bipolar cells remained dysfunctional for the entire testing period, although the anti-TRPM1 antibody had disappeared. On the other hand, the ERGs recovered in 2 cases within 2 years after the onset. One case progressed to additional impairment of the photoreceptors with deterioration of ERGs. One case died and the clinical course was unavailable. CONCLUSION: Paraneoplastic retinopathy patients with retinal ON-bipolar cell dysfunction possess autoantibodies against TRPM1 at the onset of the disease process; however, the clinical course of these eyes can be different.

TRPM2 modulates neutrophil attraction to murine tumor cells by regulating CXCL2 expression.

In recent years, immune cells were shown to play critical roles in tumor growth and metastatic progression. In this context, neutrophils were shown to possess both pro- and anti-tumor properties. To exert their anti-tumor effect, neutrophils need to migrate towards, and form physical contact with tumor cells. Neutrophils secrete H2O2 in a contact-dependent mechanism, thereby inducing a lethal Ca2+ influx via the activation of the H2O2-dependent TRPM2 Ca2+ channel. Here, we explored the mechanism regulating neutrophil chemoattraction to tumor cells. Interestingly, we found that TRPM2 plays a role in this context as well, since it regulates the expression of potent neutrophil chemoattractants. Consequently, cells expressing reduced levels of TRPM2 are not approached by neutrophils. Together, these observations demonstrate how tumor cells expressing reduced levels of TRPM2 evade neutrophil cytotoxicity in two interrelated mechanisms-downregulation of neutrophil chemoattractants and blocking of the apoptotic Ca2+-dependent cascade. These observations demonstrate a critical role for TRPM2 in neutrophil-mediated immunosurveillance and identify cells expressing low levels of TRPM2, as a potential target for cancer therapy.

TRPM2 Exacerbates Central Nervous System Inflammation in Experimental Autoimmune Encephalomyelitis by Increasing Production of CXCL2 Chemokines.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the CNS characterized by demyelination and axonal injury. Current therapies that mainly target lymphocytes do not fully meet clinical need due to the risk of severe side effects and lack of efficacy against progressive MS. Evidence suggests that MS is associated with CNS inflammation, although the underlying molecular mechanism is poorly understood. Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable nonselective cation channel, is expressed at high levels in the brain and by immune cells, including monocyte lineage cells. Here, we show that TRPM2 plays a pathological role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Knockout (KO) or pharmacological inhibition of TRPM2 inhibited progression of EAE and TRPM2-KO mice showed lower activation of Iba1-immunopositive monocyte lineage cells and neutrophil infiltration of the CNS than WT mice. Moreover, CXCL2 production in TRPM2-KO mice was significantly reduced at day 14, although the severity of EAE was the same as that in WT mice at that time point. In addition, we used BM chimeric mice to show that TRPM2 expressed by CNS-infiltrating macrophages contributes to progression of EAE. Because CXCL2 induces migration of neutrophils, these results indicate that reduced expression of CXCL2 in the CNS suppresses neutrophil infiltration and slows progression of EAE in TRPM2-KO mice. Together, the results suggest that TRPM2 plays an important role in progression of EAE pathology and shed light on its putative role as a therapeutic target for MS.SIGNIFICANCE STATEMENT Current therapies for multiple sclerosis (MS), which mainly target lymphocytes, carry the risk of severe side effects and lack efficacy against the progressive form of the disease. Here, we found that the transient receptor potential melastatin 2 (TRPM2) channel, which is abundantly expressed in CNS-infiltrating macrophages, plays a crucial role in development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE progression was suppressed by Knockout (KO) or pharmacological inhibition of TRPM2; this was attributed to a reduction in CXCL2 chemokine production by CNS-infiltrating macrophages in TRPM2-KO mice, resulting in suppression of neutrophil infiltration into the CNS. These results reveal an important role of TRPM2 in the pathogenesis of EAE and shed light on its potential as a therapeutic target.

Inhibition of transient receptor potential melastatin 8 alleviates airway inflammation and remodeling in a murine model of asthma with cold air stimulus.

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.