Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper ...microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium‐permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature‐dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high‐end confocal imaging techniques, we show a decrease in actin‐rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.
Main Points
Acute inhibition of TRPV4 abrogates microtubule and actin‐rich filopodia dynamism in microglia in vitro.
Trpv4 KO microglia exhibit less ramification complexity and slower process extension speed in situ.
Background The therapeutic action of capsaicin treatment in patients with idiopathic rhinitis (IR) is based on ablation of the transient receptor potential cation channel subfamily V, receptor 1 ...(TRPV1)–substance P nociceptive signaling pathway. However, the functional consequences of capsaicin treatment on nasal nerve activation and the association between the reduction in nasal hyperreactivity (NHR) and response to capsaicin treatment remain unknown. Objective We sought to study the effects of capsaicin nasal spray on the afferent innervation of the nasal mucosa by monitoring trigeminal nerve activity in patients with IR and healthy control (HC) subjects. Methods A double-blind, placebo-controlled randomized trial with capsaicin nasal spray was performed involving 33 patients with IR and 12 HC subjects. Before and at 4, 12, and 26 weeks after treatment, nasal mucosal potentials (NMPs) were measured while exposing the nasal mucosa of patients with IR and HC subjects to aerosols with increasing doses of the chemical irritants allyl isothiocyanate (AITC; also known as mustard oil) or capsaicin. The threshold for each compound was determined for each subject. The results of the NMP measurements were evaluated in parallel with the therapeutic response, visual analog scale scores for nasal symptoms, self-reported NHR, and mRNA expression of PGP9.5 ; TRPV1 ; transient receptor potential cation channel subfamily A, receptor 1 ( TRPA1 ); TRPV4 ; transient receptor potential cation channel subfamily M, member 8 (TRPM8) ; and nerve growth factor (NGF) in nasal biopsy specimens. Results AITC turned out to be the best stimulus because the coughing induced by capsaicin interfered with measurements. At baseline, the threshold for evoking changes in NMPs based on AITC was significantly lower for patients with IR compared with HC subjects ( P = .0423). Capsaicin treatment of IR patients increased the threshold for the response to AITC at 4 and 12 weeks compared with placebo ( P = .0406 and P = .0325, respectively), which returned to baseline by week 26 ( P = .0611). This increase correlated with changes in visual analog scale major symptom ( P = .0004) and total symptom ( P = .0018) scores. IR patients with self-reported NHR at baseline showed a trend to being better responders to capsaicin treatment compared with patients with IR but without NHR ( P = .10). Conclusion The lower threshold for AITC based on NMPs in patients with IR compared with HC subjects and the increased threshold for AITC after capsaicin treatment in patients with IR demonstrate the crucial role of TRPA1 and TRPV1 in IR pathophysiology. The strong correlation between the increase in AITC threshold in patients with IR and symptom reduction after capsaicin treatment demonstrates the clinical relevance of these findings.
Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., ...2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism.
The increase in cytosolic Ca
is essential in key effector functions of dendritic cells (DCs), including differentiation, maturation, cytokine expression, and phagocytosis. Although several Ca
...-permeable ion channels have been described in DCs, the contribution of transient receptor potential (TRP) channels remains poorly understood. Here, we investigated whether TRPV4 plays a role in the differentiation, maturation, and phagocytosis of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced mouse bone marrow-derived cells (BMDCs). Using intracellular Ca
imaging experiments, we found that TRPV4 was functionally expressed in the plasma membrane of immature CD11c
BMDCs and that its activity and expression were downregulated in CD11c
BMDCs matured with lipopolysaccharide (LPS). Comparative analysis of the GM-CSF-stimulated cells showed that
knockout and wild-type bone marrow cultures had a similar distribution of differentiated cells, generating a heterogenous culture population rich in CD11c
, CD11b
cells, and low levels of F4/80
cells. The lack of TRPV4 did not prevent the LPS-induced nuclear translocation of NF-κB, the upregulation of the proinflammatory cytokines IL-6 and IL-12, or the upregulation of the maturation markers CD40, CD80, and CD86. In contrast, TRPV4-deficient CD11c
BMDCs exhibited a significantly reduced endocytic capacity of IgG-coated beads, but the internalization of uncoated beads in the absence of TRPV4 was not affected. Taken together, our results demonstrate that TRPV4 was dispensable in the differentiation and maturation of mouse CD11c
BMDCs but contributed to the mechanism underlying Fc receptor-mediated phagocytosis. Overall, our results further strengthen the role of TRPV4 in immune-related processes.
TRPA1 is a nonselective cation channel activated by a wide variety of noxious chemicals. Intriguingly, several TRPA1 modulators induce a bimodal effect, activating the channel at micromolar ...concentrations and inhibiting it at higher concentrations. Here we report the bimodal action of cinnamaldehyde (CA) and camphor, which are thus far reported as agonist and antagonist of TRPA1, respectively. Whole-cell patch-clamp experiments in TRPA1-expressing CHO cells revealed that, as previously reported, extracellular application of 100 μM CA strongly stimulates TRPA1 currents. However, subsequent application of 3 mM CA induced fast and reversible current inhibition. Application of 3 mM CA in basal conditions induced a rather small current increase, followed by current inhibition and a dramatic rebound of current amplitude upon washout. These observations are reminiscent of the effects of TRPA1 modulators having bimodal effects, e.g., menthol and nicotine. In line with previous reports, extracellular application of 1 mM camphor induced a decrease of basal TRPA1 currents. However, the current amplitude showed a significant overshoot upon washout. On the other hand, application of 100 μM camphor induced a 3-fold increase of the basal current amplitude measured at −75 mV. The bimodal effects of CA and camphor on TRPA1 were also observed in microfluorimetric measurements of intracellular Ca
2+
in intact TRPA1-expressing CHO cells and in primary cultures of mouse dorsal root ganglion neurons. These findings are essential for the understanding of the complex sensory properties of these compounds, as well as their utility when used to study the pathophysiological relevance of TRPA1.
Abstract Background Acute exposure of part of the skin to cold stimuli can evoke urinary urgency, a phenomenon termed acute cold-induced urgency (ACIU). Despite its high prevalence, particularly in ...patients with overactive bladder, little is known about the mechanisms that induce ACIU. Objective To develop an animal model of ACIU and test the involvement of cold-activated ion channels transient receptor potential (TRP) M8 and TRPA1. Design, setting, and participants Intravesical pressure and micturition were monitored in female mice (wild-type C57BL/6J, Trpa1 −/− , Trpm8 +/+ , and Trpm8 −/− ) and Sprague Dawley rats. Interventions An intravesical catheter was implanted. Localized cooling of the skin was achieved using a stream of air or topical acetone. The TRPM8 antagonist ( N -(3-aminopropyl)-2-{(3-methylphenyl) methyloxy}- N -(2-thienylmethyl)benzamide (AMTB) or vehicle was injected intraperitoneally. Outcome measurements and statistical analysis Frequencies of bladder contractions and voids in response to sensory stimuli were compared using the Mann-Whitney or Kruskal-Wallis test. Results and limitations Brief, innocuously cold stimuli applied to different parts of the skin evoked rapid bladder contractions and voids in anesthetized mice and rats. These responses were strongly attenuated in Trpm8 −/− mice and in rats treated with AMTB. As rodent bladder physiology differs from that of humans, it is difficult to directly extrapolate our findings to human patients. Conclusions Our findings indicate that ACIU is an evolutionarily conserved reflex rather than subconscious conditioning, and provide a useful in vivo model for further investigation of the underlying mechanisms. Pharmacological inhibition of TRPM8 may be useful for treating ACIU symptoms in patients. Patient summary Brief cold stimuli applied to the skin can evoke a sudden desire to urinate, which can be highly bothersome in patients with overactive bladder. We developed an animal model to study this phenomenon, and found that it depends on a specific molecular cold sensor, transient receptor potential M8 (TRPM8). Pharmacological inhibition of TRPM8 may alleviate acute cold-induced urinary urgency in humans.
Asthma may be induced by chemical sensitisers, via mechanisms that are still poorly understood. This type of asthma is characterised by airway hyperreactivity (AHR) and little airway inflammation. ...Since potent chemical sensitisers, such as toluene-2,4-diisocyanate (TDI), are also sensory irritants, it is suggested that chemical-induced asthma relies on neuro-immune mechanisms.We investigated the involvement of transient receptor potential channels (TRP) A1 and V1, major chemosensors in the airways, and mast cells, known for their ability to communicate with sensory nerves, in chemical-induced AHR.In vitro intracellular calcium imaging and patch-clamp recordings in TRPA1- and TRPV1-expressing Chinese hamster ovarian cells showed that TDI activates murine TRPA1, but not TRPV1. Using an in vivo model, in which an airway challenge with TDI induces AHR in TDI-sensitised C57Bl/6 mice, we demonstrated that AHR does not develop, despite successful sensitisation, in Trpa1 and Trpv1 knockout mice, and wild-type mice pretreated with a TRPA1 blocker or a substance P receptor antagonist. TDI-induced AHR was also abolished in mast cell deficient Kit(Wsh) (/Wsh) mice, and in wild-type mice pretreated with the mast cell stabiliser ketotifen, without changes in immunological parameters.These data demonstrate that TRPA1, TRPV1 and mast cells play an indispensable role in the development of TDI-elicited AHR.
Airway hyperreactivity (AHR) is a key feature of bronchial asthma, and inhalation of irritants may facilitate development of nonallergic AHR. Swimmers exposed to hypochlorite (ClO(-))-containing ...water show a higher risk of developing AHR. We developed a mouse model in which instillation of ClO(-) before ovalbumin (OVA) induces AHR without bronchial inflammatory cells.
To investigate the mechanisms of ClO(-)-OVA-induced nonallergic AHR.
The involvement of the transient receptor potential ankyrin (TRPA)1 channel was checked in vivo by the use of TRPA1(-/-) mice and in vitro by Ca(2+) imaging experiments. The role of substance P (SP) was investigated by pretreating animals with the receptor antagonist RP67580, by replacing ClO(-) with SP in vivo, and by immunofluorescent staining of large airways of exposed mice. The role of mast cells was evaluated by exposing mast cell-deficient Kit(Wh)/Kit(Wsh) mice to ClO(-)-OVA with or without mast cell reconstitution.
ClO(-)-OVA did not induce AHR in TRPA1(-/-) mice, and ClO(-) generates a Ca(2+) influx in TRPA1-transfected cells. Pretreatment with RP67580 reduces ClO(-)-OVA-induced AHR, although no increased SP expression was shown in the airways. SP-OVA exposure resulted in the same AHR as induced by ClO(-)-OVA. Kit(Wsh)/Kit(Wsh) mice did not develop AHR in response to ClO(-)-OVA unless they were reconstituted with bone marrow-derived mast cells.
Induction of AHR by exposure to ClO(-)-OVA depends on a neuroimmune interaction that involves TRPA1-dependent stimulation of sensory neurons and mast cell activation.
The powerful plant-derived irritant allyl isothiocyanate (AITC, aka mustard oil) induces hyperalgesia to heat in rodents and humans through mechanisms that are not yet fully understood. It is ...generally believed that AITC activates the broadly tuned chemosensory cation channel transient receptor potential cation channel subfamily A member 1 (TRPA1), triggering an inflammatory response that sensitizes the heat sensor transient receptor potential cation channel subfamily V member 1 (TRPV1). In the view of recent data demonstrating that AITC can directly activate TRPV1, we here explored the possibility that this compound sensitizes TRPV1 to heat stimulation in a TRPA1-independent manner. Patch-clamp recordings and intracellular Ca
2+
imaging experiments in HEK293T cells over-expressing mouse TRPV1 revealed that the increase in channel activation induced by heating is larger in the presence of AITC than in control conditions. The analysis of the effects of AITC and heat on the current–voltage relationship of TRPV1 indicates that the mechanism of sensitization is based on additive shifts of the voltage dependence of activation towards negative voltages. Finally, intracellular Ca
2+
imaging experiments in mouse sensory neurons isolated from
Trpa1
KO mice yielded that AITC enhances the response to heat, specifically in the subpopulation expressing TRPV1. Furthermore, this effect was strongly reduced by the TRPV1 inhibitor capsazepine and virtually absent in neurons isolated from double
Trpa1
/
Trpv1
KO mice. Taken together, these findings demonstrate that TRPV1 is a locus for cross sensitization between AITC and heat in sensory neurons and may help explaining, at least in part, the role of this channel in AITC-induced hyperalgesia to heat.
Urinary tract infections (UTI) affect a large proportion of the population, causing among other symptoms, more frequent and urgent micturition. Previous studies reported that the gram-negative ...bacterial wall component lipopolysaccharides (LPS) trigger acute epithelial and bladder voiding responses, but the underlying mechanisms remain unknown. The cation channel TRPV4 is implicated in the regulation of the bladder voiding. Since TRPV4 is activated by LPS in airway epithelial cells, we sought to determine whether this channel plays a role in LPS-induced responses in urothelial cells (UCs). We found that human-derived UCs display a fast increase in intracellular Ca
concentration upon acute application of
LPS. Such responses were detected also in freshly isolated mouse UCs, and found to be dependent on TRPV4, but not to require the canonical TLR4 signaling pathway of LPS detection. Confocal microscopy experiments revealed that TRPV4 is dispensable for LPS-induced nuclear translocation of NF-κB in mouse UCs. On the other hand, quantitative RT PCR determinations showed an enhanced LPS-induced production of proinflammatory cytokines in TRPV4-deficient UCs. Cystometry experiments in anesthetized wild type mice revealed that acute intravesical instillation of LPS rapidly increases voiding frequency. This effect was not observed in TRPV4-deficient animals, but was largely preserved in
KO and
KO mice. Our results suggest that activation of TRPV4 by LPS in UCs regulates the proinflammatory response and contributes to LPS-induced increase in voiding frequency. These findings further support the concept that TRP channels are sensors of LPS, mediating fast innate immunity mechanisms against gram-negative bacteria.
PDF
