Trpa1 KO Mice Research Articles

An LPAR 5 -antagonist that reduces nociception and increases pruriception.

IntroductionThe G-protein coupled receptor LPAR5 plays a prominent role in LPA-mediated pain and itch signaling. In this study we focus on the LPAR5-antagonist compound 3 (cpd3) and its ability to affect pain and itch signaling, both in vitro and in vivo.MethodsNociceptive behavior in wild type mice was induced by formalin, carrageenan or prostaglandin E2 (PGE2) injection in the hind paw, and the effect of oral cpd3 administration was measured. Scratch activity was measured after oral administration of cpd3, in mice overexpressing phospholipase A2 (), in wild type mice (WT) and in TRPA1-deficient mice (Trpa1 KO). In vitro effects of cpd3 were assessed by measuring intracellular calcium release in HMC-1 and HEK-TRPA1 cells.ResultsAs expected, nociceptive behavior (induced by formalin, carrageenan or PGE2) was reduced after treatment with cpd3. Unexpectedly, cpd3 induced scratch activity in mice. In vitro addition of cpd3 to HEK-TRPA1 cells induced an intracellular calcium wave that could be inhibited by the TRPA1-antagonist A-967079. In Trpa1 KO mice, however, the increase in scratch activity after cpd3 administration was not reduced.ConclusionsCpd3 has in vivo antinociceptive effects but induces scratch activity in mice, probably by activation of multiple pruriceptors, including TRPA1. These results urge screening of antinociceptive candidate drugs for activity with pruriceptors.

Drug Delivery through the Psoriatic Epidermal Barrier-A "Skin-On-A-Chip" Permeability Study and Ex Vivo Optical Imaging.

Psoriasis is a chronic inflammatory disease with unmet medical needs. To clarify potential therapeutic targets, different animal models have been developed. In the current study, imiquimod-induced psoriasiform dermatitis was used for monitoring the changes in skin thickness, transepidermal water loss, body weight, blood perfusion and drug permeability for a topical cream formulation of caffeine, both in wild type and in knock out mice. Morphological characterization of control and diseased tissues was performed by scanning electron microscopy and two-photon microscopy. The chemically induced psoriatic group showed increased skin permeability for the model drug during disease progression. In wild type and TRPA1 KO mice, however, enhanced skin thickness and hyperkeratosis blocked further increase of drug penetration at the late phase (96 h). These results indicate that topical drug therapy can be more effective in early phases of plaque development, when skin thickness is lower. Although paracellular connections (tight junctions) are looser in the advanced phase, hyperkeratosis blocks drug delivery through the transappendageal routes. Novel drug formulations may have the potency for effective drug delivery across the epidermal barrier even in the advanced phase. For development of more effective topical drugs, further research is proposed to explore drug penetration both in healthy and diseased conditions.

TRPV4 Mediates Acute Bladder Responses to Bacterial Lipopolysaccharides.

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 Ca2+ concentration upon acute application of Escherichia coli 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 Tlr4 KO and Trpa1 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.

Behavioral characterization of a CRISPR-generated TRPA1 knockout rat in models of pain, itch, and asthma

The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.

Signs of chronic itch in the mouse imiquimod model of psoriasiform dermatitis: sex differences and roles of TRPV1 and TRPA1.

Plaque psoriasis is a chronic inflammatory skin disease that affects a substantial proportion of the world population. This disorder is characterized by scaly, thick skin, intense ongoing itch, and itch from light touch (such as clothing contacting skin, called “alloknesis”). Imiquimod is a topical treatment for basal cell carcinomas and warts that has been used to create a mouse model of plaque psoriasis. Imiquimod-treated male, but not female, wildtype B6 mice showed significant increases in spontaneous scratching, while both sexes exhibited increased alloknesis, indicative of chronic itch. TRPV1 and TRPA1 knockout (KO) mice all exhibited numeric increases in spontaneous scratching which were significant for TRPV1KO mice and TRPA1KO males. Female TRPV1KO and TRPA1KO mice exhibited imiquimod-induced increases in alloknesis scores that did not significantly differ from wildtypes, while alloknesis scores in imiquimod-treated male TRPV1KO and TRPA1KO mice were significantly lower compared with wildtypes, suggesting that these ion channels are necessary for the development of alloknesis in males but not females in this model. Curiously, none of the groups exhibited any significant overall change in chloroquine-evoked scratching following imiquimod treatment, indicating that hyperknesis does not develop in this mouse model. Overall, the data indicate that there are sex differences in this mouse model of psoriasis, and that TRPV1 and TRPA1 ion channels have a small role in promoting the development of itch sensitization. This contrasts with the far greater role these channels play in the manifestation of skin changes in psoriatic dermatitis.

Attenuated lipopolysaccharide-induced inflammatory bladder hypersensitivity in mice deficient of transient receptor potential ankilin1

Transient receptor potential ankyrin 1 (TRPA1) channel expressed by urothelial cells and bladder sensory nerve fibers might act as a bladder mechanosensor and nociceptive transducer. To disclose the role of TRPA1 in bladder function and inflammation-associated hypersensitivity, we evaluated in vitro and in vivo bladder function and inflammatory mechanosensory and nociceptive responses to intravesical lipopolysaccharide (LPS)-instillation in wild type (WT) and TRPA1-knock out (KO) mice. At baseline before treatment, no significant differences were observed in frequency volume variables, in vitro detrusor contractility, and cystometric parameters between the two groups in either sex. LPS-instillation significantly increased voiding frequency and decreased mean voided volume at 24–48 hours after instillation in WT but not in TRPA1-KO mice. LPS-instillation also significantly increased the number of pain-like behavior at 24 hours after instillation in WT mice, but not in TRPA1-KO mice. Cystometry 24 hours after LPS-instillation revealed shorter inter-contraction intervals in the WT mice compared with TRPA1-KO mice. In contrast, inflammatory cell infiltration in the bladder suburothelial layer was not significantly different between the two groups. These results indicate that TRPA1 channels are involved in bladder mechanosensory and nociceptive hypersensitivity accompanied with inflammation but not in physiological bladder function or development of bladder inflammation.

Transient Receptor Potential Ankyrin 1 Mediates the Afferent Arm of the Inflammatory Reflex

Abstract The vagus nerve plays an important role in maintaining an organism’s immune homeostasis through the inflammatory reflex. Inflammatory molecules including LPS, TNF and IL-1β can mediate electrophysiological changes in vagus nerve signaling. Reasoning that these signals are mediated though specific neuronal fibers that can be identified by molecular markers, we administered endotoxin to mice that received optovin, a molecule that interacts with TRPA1 and is light sensitive. Selective stimulation of vagus nerve TRPA1+ fibers significantly reduce serum TNF levels (sham: 920.9 ± 87.9 pg/ml vs. stimulated: 456.8 ± 69.4 pg/ml). This reduction is ablated by proximal vagotomy, indicating that the signals are afferent in nature. Prior work suggests that the afferent vagus nerve is required for IL-1β induced febrile responses and sickness behavior. Here we administered IL-1β to TRPA1 KO mice and observed that body temperature was maintained as compared to wildtype (peak temperature change; wildtype: −2.35 ± 0.15 °C vs. TRPA1 KO:0.18 ± 0.40 °C). When recording electrical activity from the VN, IL-1β induces a significant increase in VN activity in WT mice. However, no significant increase is observed in TRPA1 KOs. Cecal ligation and puncture in TRPA1 KOs caused significantly higher disease severity scores, percent weight loss, and mortality. These results identify TRPA1 as both a marker for fiber specific inflammatory signaling as well as a necessary component for regulating inflammatory homeostasis.

Transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 contribute to the progression of colonic inflammation in dextran sulfate sodium-induced colitis in mice: Links to calcitonin gene-related peptide and substance P

Transient receptor potential (TRP) vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1), which are non-selective cation channels, play important roles in the sensation of pain. This study investigated the roles of TRPV1 and TRPA1 in dextran sulfate sodium (DSS)-induced murine colitis. DSS (2%) administered for 7 days caused severe colitis that was significantly less severe in TRPV1-deficient (TRPV1KO) and TRPA1-deficient (TRPA1KO) mice than that in wild-type (WT) mice. Similar colitis attenuations were observed in TRPV1KO and TRPA1KO mice but not in WT mice that had been transplanted with bone marrow cells from WT, TRPA1KO, or TRPV1KO mice. DSS treatment upregulated calcitonin gene-relative peptide (CGRP)- and substance P (SP)-positive nerve fibers in the colonic mucosa of WT mice. TRPV1KO and TRPA1KO mice showed significant reductions in the DSS-induced upregulation of SP, but the DSS-induced upregulation of CGRP was not reduced. Sensory deafferentation evoked by pretreatment with high doses of capsaicin markedly exacerbated DSS-induced colitis with reductions in DSS-induced upregulation of SP- and CGRP-positive nerve fibers. These findings suggest that neuronal TRPV1 and TRPA1 contribute to the progression of colonic inflammation. While these responses may be mediated by the upregulation of SP-mediated deleterious mechanisms, CGRP may be associated with protective mechanisms.

Effects of Lipopolysaccharide on Sensory TRP Channels of Dorsal Root Ganglion Sensory Neurons

Neurogenic inflammation and pain associated to bacterial infection have been ascribed to sensitization and activation of sensory nerve afferents. We have recently unveiled a role of Transient Receptor Potential (TRP) A1 channel as sensor of lipopolysaccharide (LPS) in nociceptive neurons. However, here we show that responses to LPS are still detected in 30% of dorsal root ganglion neurons isolated from Trpa1 KO mice. The proportion of cells responding to LPS was dramatically lower in double Trpa1/Trpv1 KO neurons. Using intracellular calcium imaging and patch-clamp in a recombinant expression system, we studied the effects of LPS on TRPV1, TRPV2, TRPM3 and TRPM8 heterologously expressed in HEK cells. In isolated sensory neurons, we compared the activation of TRPV1 and TRPA1 by LPS. In contrast, LPS was ineffective on TRPV2, weakly activated TRPM3 and activated TRPM8 at 25°C but not at 35°C. Our results indicate that, in addition to TRPA1, other TRP channels in sensory neurons can be targets of LPS, raising the possibility that they may also contribute to trigger and regulate innate immune responses.

Upregulation of the transient receptor potential ankyrin 1 ion channel in the inflamed human and mouse colon and its protective roles.

Transient Receptor Potential Ankyrin 1 (TRPA1) channels are localized on sensory nerves and several non-neural cells, but data on their functional significance are contradictory. We analysed the presence and alterations of TRPA1 in comparison with TRP Vanilloid 1 (TRPV1) at mRNA and protein levels in human and mouse intact and inflamed colons. The role of TRPA1 in a colitis model was investigated using gene-deficient mice. TRPA1 and TRPV1 expressions were investigated in human colon biopsies of healthy subjects and patients with inflammatory bowel diseases (IBD: ulcerative colitis, Crohn's disease) with quantitative PCR and immunohistochemistry. Mouse colitis was induced by oral 2% dextran-sulphate (DSS) for 10 days. For investigating the functions of TRPA1, Disease Activity Index (weight loss, stool consistency, blood content) was determined in C57BL/6-based Trpa1-deficient (knockout: KO) and wildtype (WT) mice. Sensory neuropeptides, their receptors, and inflammatory cytokines/chemokines were determined with qPCR or Luminex. In human and mouse colons TRPA1 and TRPV1 are located on epithelial cells, macrophages, enteric ganglia. Significant upregulation of TRPA1 mRNA was detected in inflamed samples. In Trpa1 KO mice, Disease Activity Index was significantly higher compared to WTs. It could be explained by the greater levels of substance P, neurokinins A and B, neurokinin 1 receptor, pituitary adenylate-cyclase activating polypeptide, vasoactive intestinal polypeptide, and also interleukin-1beta, macrophage chemoattractant protein-1, monokine induced by gamma interferon-1, tumor necrosis factor-alpha and B-lymphocyte chemoattractant in the distal colon. TRPA1 is upregulated in colitis and its activation exerts protective roles by decreasing the expressions of several proinflammatory neuropeptides, cytokines and chemokines.

Allyl isothiocyanate sensitizes TRPV1 to heat stimulation

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.

Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4‐induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B₄ (LTB₄) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B₄ receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB₄-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.

TRPV1 Activation by Allyl Isothiocyanate

Allyl isothiocyanate (mustard oil, MO) is a highly reactive electrophilic compound known to cause irritation, pain and inflammation. These effects are thus far thought to be mediated by activation of TRPA1, a Transient Receptor Potential (TRP) cation channel expressed in nociceptive neurons. Recent research has shown that TRPV1, the heat and capsaicin receptor, can be also activated by reactive compounds such as allicin and leek and onion extracts. Here, we show that both human and mouse TRPV1 are activated by MO, at concentrations at which TRPA1 undergoes fast desensitization and block. In Ca2+ imaging experiments of intact HEK293 cells, MO induces an increase of the intracellular Ca2+, which was not present when Ca2+ was omitted in the bath solution. Activation of TRPV1 by MO is dose-dependent and is caused by a shift of the voltage dependence of channel activation to more negative potentials, similar to the activation of TRPV1 by capsaicin. Stimulation of TRPV1 by MO can be observed in inside-out patches, indicating a membrane-delimited mechanism of activation. Furthermore, the heat-induced activation of TRPV1 could be sensitized with sub-activating MO concentrations.Notably, MO was able to stimulate a large population of sensory neurons isolated from Trpa1 KO mice. This population was significantly reduced in Trpa1/Trpv1 double KO mice, indicating the physiological importance of TRPV1 activation by MO. WT, Trpa1 and Trpv1 KO mice displayed significantly stronger aversion to MO than double KO mice in forced drinking and open field exploration assays. The identification of TRPV1 as a novel target of MO is essential for the full understanding of the mechanisms of action of this compound in vivo and prompts to re-evaluate the results of previous research, in which MO was used as specific activator of TRPA1.