The ion channel TRPA1 is required for chronic itch

Abstract

Chronic itch is a debilitating condition that affects one in 10 people. Little is known about the molecules that mediate chronic itch in primary sensory neurons and skin. We demonstrate that the ion channel TRPA1 is required for chronic itch. Using a mouse model of chronic itch, we show that scratching evoked by impaired skin barrier is abolished in TRPA1-deficient animals. This model recapitulates many of the pathophysiological hallmarks of chronic itch that are observed in prevalent human diseases such as atopic dermatitis and psoriasis, including robust scratching, extensive epidermal hyperplasia, and dramatic changes in gene expression in sensory neurons and skin. Remarkably, TRPA1 is required for both transduction of chronic itch signals to the CNS and for the dramatic skin changes triggered by dry-skin-evoked itch and scratching. These data suggest that TRPA1 regulates both itch transduction and pathophysiological changes in the skin that promote chronic itch.

Figure 1.

Cheek model of dry skin pruritus induces scratching and epidermal thickening. A, Time course for the dry skin assay in the mouse cheek. First day: the right cheek of each mouse was shaved. Days one to five: the shaved cheek was treated twice daily with a 1:1 mixture of acetone and ether, followed by water. Scratching behaviors were recorded for 20 min on days three and five after the second AEW treatment. Mouse cheeks were removed for histological analysis following recording on either day three or day five. B, Photo displaying the area of treatment in the cheek model of itch C, Scratching behaviors were observed on day three and day five of cheek treatment. The total time spent scratching was quantified for 20 min. Application of vehicle (VEH, water) failed to elicit scratching or wiping. All error bars represent SEM (n ≥ 12 mice/group, **p < 0.01, one-way ANOVA). D, H&E-stained cheek skin sections from mice treated for 3 d (top) or 5 d (bottom) with AEW on ipislateral (right) and VEH on contralateral cheeks (left). E indicates epidermis; D, dermis. E, Thickness of nucleated epidermal layers was quantified from AEW-treated and VEH-treated skin. Means ± SEM are shown (n = 16–24 sections from two mice per group, ***p < 0.002, Student's two-tail t test).

Figure 2.

AEW caudal back model of dry skin pruritus induces off-site scratching, epidermal thickening, and increased locomotor activity. A, Photo displaying the area of treatment in the caudal back model of itch. This assay produces dry skin, but the inaccessible location prevents scratching, wiping, or biting of the treated area. B, Image displaying location of off-site scratching after treatment to the caudal back. Off-site scratching was observed in response to both acute subcutaneous injection of chloroquine (CQ) and chronic (5 d) AEW treatment to the caudal back. AEW-evoked scratching behaviors were recorded on day five of treatment. After both treatments, the total time spent scratching or biting was quantified for 20 min. Application of vehicle (VEH, water) failed to elicit scratching or biting toward any site. C, Image displaying AEW site-to-wall contact behavior. Site-to-wall behaviors were observed on day five of treatment. The total time spent in a back-to-wall configuration was quantified for 20 min. Application of VEH failed to elicit back-to-wall contact. D, AEW-treated mice display a significant increase in locomotor activity in which they move and frequently attempt to contact the caudal back to the chamber wall. The percentage of time spent moving was quantified for 20 min. E, H&E-stained caudal back skin sections from mice treated for 5 d with AEW or VEH on the caudal back (VEH; water; left). E indicates epidermis; D, dermis. F, Thickness of nucleated epidermal layers was quantified from AEW-treated and VEH-treated skin. All error bars represent SEM (n ≥ 6 mice/group, ***p < 0.001, one-way ANOVA).

Figure 3.

Sensory neurons are required for dry-skin-evoked itch behaviors. A, Mustard oil (MO, 10% in PBS)-evoked wiping was measured in wild-type, vehicle (CONTROL, 0.05% ascorbic acid, and 7% Tween 80, black), or RTX-injected (RTX ABLATED; 1 μg/ml RTX in 0.05% ascorbic acid, and 7% Tween 80, red) mice 3 d after injection. Nocifensive behavior was quantified for 2 min. All error bars represent SEM (n ≥ 4 mice/genotype, *p < 0.05). B, Dry-skin-evoked scratching was measured in wild-type vehicle (CONTROL, 0.05% ascorbic acid, and 7% Tween 80, black) or RTX-injected (RTX ABLATED; 1 μg/ml RTX in 0.05% ascorbic acid, and 7% Tween 80, red) mice on days three and five of treatment. The total time spent scratching was quantified for 20 min. Error bars represent SEM (n ≥ 9 mice/genotype; *p < 0.05) C, Gene expression was measured in whole TG isolated from VEH- and AEW-treated mice normalized to VEH-treated mice. Green indicates AEW-evoked increase in expression; magenta, AEW-evoked decrease in expression. Differentially expressed genes are clustered based on cellular function. Among differentially expressed receptors, the itch receptors MrgprA3 and Bdkr2 (*) were highly expressed in the TG of AEW-treated mice relative to VEH-treated mice.

Figure 4.

TRPA1 is required for dry-skin-evoked itch behaviors and morphological changes in the skin. A, Dry-skin-evoked scratching was measured in wild-type (WT AEW; black), TRPV1^−/− (V1 AEW; gray), and TRPA1^−/− (A1 AEW; red) mice after 3 and 5 d of AEW treatment to the cheek. The total time spent scratching was quantified for 20 min. Treatment with vehicle on wild-type mice (VEH, water) failed to elicit scratching or wiping (VEH; white). All error bars represent SEM (n ≥ mice/genotype, **p < 0.01). B, Dry-skin-evoked scratching was measured in wild-type mice on AEW treatment on days three and five immediately after (≤2 min) subcutaneous injection with control PBS (CON) or the TRPA1 inhibitor HC-030031 (HC). The total time spent scratching was quantified for 20 min postinjection and normalized to CON-treated mice. All error bars represent SEM (n ≥ 9 mice/genotype or treatment, **p < 0.01). C, H&E-stained skin sections from wild-type mice (WT; top) and TRPA1^−/− littermates (A1^−/−; bottom) treated ipsilaterally with AEW (left) and contralaterally with VEH. E indicates epidermis; D, dermis. D, The increase in epidermal thickness induced by AEW was quantified from four to five mice per condition. NS indicates the “no scratching” condition in the caudal back model. Means ± SEM are shown (*p = 0.01, Student's two-tail t test).

Figure 5.

TRPA1 is required for dry-skin-evoked expressional changes in the skin. A, Gene expression was measured in cheek skin biopsies isolated from VEH- or AEW-treated TRPA1^+/+ mice that were free to scratch (WT SCR), and TRPA1^−/− mice. Gene expression was also measured in caudal back skin biopsies in which scratching was prevented (WT NSCR). The AEW gene expression data are normalized to VEH for each genotype and treatment area. Green indicates the AEW-evoked increase in expression; magenta, the AEW-evoked decrease in expression. All genes significantly altered in WT mice are shown (p < 0.05). B, Changes in gene expression with AEW treatment for FLG, IL31RA, AQP3, and IL33 in WT (black) and A1^−/− (white) mice as measured by quantitative PCR. Expression is reported as ΔΔCt. C, Micrographs of WT and A1^−/− skin are stained with KRT6 (brown). In VEH-treated skin, KRT6 is localized to hair follicles (arrows), whereas AEW-treated skin shows induction of KRT6 in the interfollicular epidermis (brackets). Nuclei are stained in blue. Scale bar, 50 μm.

Figure 6.

TRPA1 promotes scratch-dependent and scratch-independent expressional changes in the skin. A, Venn diagram displaying relative numbers of scratch-dependent, TRPA1-dependent, and scratch- and TRPA1-dependent genes significantly modulated in WT AEW mouse cheek skin. B, Analysis of a subset of genes that were previously implicated in processes related to chronic itch to determine dependence on scratching and/or TRPA1 (Fig. 5A). Green indicates AEW-evoked increase in expression; magenta, AEW-evoked decrease in expression. C, Venn diagram displaying relative numbers of scratch-dependent, TRPA1-dependent, and scratch- and TRPA1-dependent itch-related genes significantly modulated in WT AEW mouse cheek skin.