The Journal of Neuroscience, May 29, 2013 • 33(22):9283–9294 • 9283 Cellular/Molecular The Ion Channel TRPA1 Is Required for Chronic Itch Sarah R. Wilson,1,2 Aislyn M. Nelson,3,4 Lyn Batia,1 Takeshi Morita,1,2 Daniel Estandian,1,2 David M. Owens,3,5 Ellen A. Lumpkin,3,6 and Diana M. Bautista1,2 1Department of Molecular and Cell Biology and 2Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720-3200, 3Department of Dermatology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, 4Medical Scientist Training Program, Baylor College of Medicine, Houston, Texas 77030, 5Department of Pathology and Cell Biology and 6Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, New York 10032 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,includingrobustscratching,extensiveepidermalhyperplasia,anddramaticchangesingeneexpressioninsensoryneuronsand 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. Introduction better understand the cellular and molecular mechanisms under- Chronic itch is a widespread and debilitating condition that re- lying chronic itch. sults from a variety of pathological conditions such as eczema, A variety of cell types contribute to chronic itch pathologies and kidney failure, cirrhosis, nervous system disorders, and some sensations. In the skin, tissue-resident cells such as keratinocytes and cancers (Bowcock and Cookson, 2004; Yosipovitch, 2004; Ikoma cells that infiltrate during inflammation, such as lymphocytes, mast et al., 2006). Although a number of factors have been identified cells, and eosinophils, release pruritogens that activate primary affer- that mediate acute itch transduction in primary sensory afferents ent neurons (Ikoma et al., 2006). These pruritogens are detected by and the CNS, several major obstacles persist that cloud our un- the free nerve endings of primary afferent C-fibers located within the derstanding of chronic itch (Dong et al., 2001; Sun and Chen, epidermis (Ikoma et al., 2006). Itch is mediated by both histamine- 2007; Liu et al., 2009; Sun et al., 2009; Lagerstro¨m et al., 2010; sensitive and histamine-insensitive C-fibers, although most forms of Ross et al., 2010; Wilson et al., 2011; Liu et al., 2011; Han et al., chronic itch are insensitive to antihistamine treatment and are me- 2013). First, very little is known about the fundamental basis for diated by histamine-insensitive neurons (Jeffry et al., 2011; Han et chronic itch perception, including the molecules that underlie al., 2013). chronic itch and promote itch sensations, in both primary sen- The ion channel TRPA1 is required for acute histamine- sory neurons and the skin. Second, the signaling pathways in the independent itch (Wilson et al., 2011). However, whether TRPA1 skin and the peripheral nervous system that initiate and sustain ion channels are required for chronic itch has not been examined. chronic itch conditions remain unknown. Finally, the contribu- Using a mouse cheek model of chronic itch adapted from the tion of the itch-scratch cycle, in which repetitive scratching wors- model used by Miyamoto et al. (2002b), we show that scratching ens itch symptoms, to the progression of chronic itch has not evoked by a dry skin model is abolished in TRPA1-deficient ani- been examined thoroughly. Therefore, there is an urgent need to mals. This model recapitulates many of the pathophysiological hallmarks of chronic itch that are observed in prevalent human Received Nov. 15, 2012; revised April 17, 2013; accepted April 19, 2013. diseases such as psoriasis, including robust scratching, extensive Authorcontributions:S.R.W.,A.M.N.,D.M.O.,E.A.L.,andD.M.B.designedresearch;S.R.W.,A.M.N.,L.B.,andD.E. epidermal hyperplasia, and changes in gene expression in the skin performed research; S.R.W., A.M.N., and T.M. analyzed data; S.R.W. and D.M.B. wrote the paper. This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases–National (Bowcock and Cookson, 2004). We provide clear evidence that Institutes of Health (Grant #R01 AR051219 to E.A.L. and Grant #R01 AR059385 to D.M.B.), the McNair Scholars TRPA1-expressing sensory neurons drive all of these dry-skin- Program (fellowship to A.M.N.), and the National Science Foundation (fellowship to S.R.W.). Histology was per- evoked phenotypes. In addition, we show for the first time that a formed with support from the Columbia University Skin Disease Research Center Tissue Culture and Histology Core model of chronic itch leads to significant expressional changes in and Advanced Imaging Core (supported by National Institutes of Health Grant #P30AR044535) and the Herbert Irving Comprehensive Cancer Center Confocal and Specialized Microscopy Shared Resource (supported by National the sensory neurons that innervate the affected skin. Finally, we Institutes of Health Grant #P30CA013696). demonstrate that dry, itchy skin triggers TRPA1-dependent hy- The authors declare no competing financial interests. perplasia in both the presence and absence of scratching. Re- Correspondence should be addressed to Diana Bautista, University of California, Department of Molecular and markably, TRPA1 is required not only for transduction of Cell Biology, 142 Life Sciences Addition #3200, Berkeley, CA 94720-3200. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.5318-12.2013 chronic itch signals to the CNS, but also for the changes in gene Copyright © 2013 the authors 0270-6474/13/339283-12$15.00/0 expression that we observed in both neurons and skin. 9284 • J. Neurosci., May 29, 2013 • 33(22):9283–9294 Wilson et al. • TRPA1 Is Required for Chronic Itch Sigma) was applied topically with a cotton swab to shaved mouse cheeks. Nocifensive behavior was quantified for 2 min after application. Behav- ioral scoring was performed while the observer was blind to the experi- mental condition. All experiments were performed under the policies and recommendations of the International Association for the Study of Pain and were approved by the University of California, Berkeley, Animal Care and Use Committee. Histology. Skin specimens were dissected immediately after animals were killed and were then fixed in 4% paraformaldehyle overnight at 4°C, dehydrated in 70% ethanol, and embedded in paraffin. Sections (8 ␮m) were stained with H&E or with rabbit anti-keratin-6 (KRT6) polyclonal antibodies (Covance), followed by immunoperoxidase labeling. Slides were imaged with a bright-field microscope outfitted with 10ϫ, 0.3 nu- merical aperture (NA) and 20ϫ, 0.4 NA lenses and an Axiocam color CCD camera (AxioObserver.Z1; Zeiss). All specimens were blinded with respect to genotype and treatment before imaging. The thickness of nu- cleated epidermal layers was measured from 20 ϫ bright-field images taken at 2–3 random fields per section using ImageJ software. Real-time quantitative PCR. Total RNA from skin was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s specifica- tions; 500 ng of total RNA was used to generate cDNA using SuperScript III Reverse Transcriptase (Invitrogen). Real-time PCR was performed using SYBR GreenER qPCR SuperMix for ABI PRISM (Invitrogen) on a StepOnePlus ABI machine. To determine gene expression in keratino- cytes, threshold cycles for each transcript (Ct) were normalized to GAPDH (⌬Ct). Calibrations and normalizations were performed using the 2 Ϫ⌬⌬Ct method in which GAPDH was used as the reference gene, samples from vehicle-treated mice were used as the calibrator, and ⌬⌬Ct ϭ [(Ct (target gene) Ϫ Ct (reference gene)] Ϫ [Ct (calibrator) Ϫ Ct (reference gene)]. Real-time PCR measurements were performed in triplicate. Microarray. Trigeminal ganglia (TG) were removed from either wild- type C57BL6 or Trpa1Ϫ/Ϫ C57BL6 6- to 8-week-old mice treated with Figure 1. Cheek model of dry skin pruritus induces scratching and epidermal thickening. A, either AEW or vehicle for 5 d and RNA was isolated with TRIzol (Invit- Time course for the dry skin assay in the mouse cheek. First day: the right cheek of each mouse rogen) according to the manufacturer’s protocol. RNA integrity was con- was shaved. Days one to five: the shaved cheek was treated twice daily with a 1:1 mixture of firmed with an Agilent 2100 BioAnalyzer. RNA (100 ng) was used to acetone and ether, followed by water. Scratching behaviors were recorded for 20 min on days make cRNA and samples were analyzed for gene expression with Af- three and five after the second AEW treatment. Mouse cheeks were removed for histological fymetrix Mouse Genome 430.2 GeneChip arrays, which cover transcripts analysis following recording on either day three or day five. B, Photo displaying the area of and variants from 34,000 well characterized mouse genes using standard treatment in the cheek model of itch C, Scratching behaviors were observed on day three and Affymetrix reagents and protocols. Probe sets on this array are derived day five of cheek treatment. The total time spent scratching was quantified for 20 min. Appli- from sequences from GenBank and dbEST. Samples from three indepen- cation of vehicle (VEH, water) failed to elicit scratching or wiping. All error bars represent SEM dent mice from each group were analyzed using one microarray per (n Ն 12 mice/group, **p Ͻ 0.01, one-way ANOVA).