Analysis of cellular and behavioral responses to imiquimod reveals a unique itch pathway in transient receptor potential vanilloid 1 (TRPV1)-expressing neurons

Abstract

Despite its clinical importance, the mechanisms that mediate or generate itch are poorly defined. The identification of pruritic compounds offers insight into understanding the molecular and cellular basis of itch. Imiquimod (IQ) is an agonist of Toll-like receptor 7 (TLR7) used to treat various infectious skin diseases such as genital warts, keratosis, and basal cell carcinoma. Itch is reportedly one of the major side effects developed during IQ treatments. We found that IQ acts as a potent itch-evoking compound (pruritogen) in mice via direct excitation of sensory neurons. Combined studies of scratching behavior, patch-clamp recording, and Ca(2+) response revealed the existence of a unique intracellular mechanism, which is independent of TLR7 as well as different from the mechanisms exploited by other well-characterized pruritogens. Nevertheless, as for other pruritogens, IQ requires the presence of transient receptor potential vanilloid 1 (TRPV1)-expressing neurons for itch-associated responses. Our data provide evidence supporting the hypothesis that there is a specific subset of TRPV1-expressing neurons that is equipped with diverse intracellular mechanisms that respond to histamine, chloroquine, and IQ.

Fig. 1.

Pain and scratching behavioral analysis following injection of IQ. (A) Dose–response curve for IQ-induced scratching behavior in C57BL/6 mice (n = 4–8/each dose). Inset indicates structure of IQ. (B) Time course of scratching following s.c. injection of IQ (100 μg) (n = 12). The number of scratches is indicated at each 5-min interval during a 30-min test period. (C) Time to onset of scratching after injection of eight different pruritic compounds. Each symbol indicates an individual mouse tested for each compound. The following substances were tested: IQ (100 μg), histamine (100 μg), CQ (200 μg), compound 48/80 (100 μg), 5-HT (100 μg), ET-1 (10 pmol), SLIGRL-NH₂ (100 nmol), and U-46619 (3.5 μg). Seven to eight mice were used for each pruritic compound. (D) Scratching and wiping directed toward the site of injection of each compound into the cheek. The tested compounds are as follows: capsaicin (10 μg), histamine (20 μg), IQ (20 μg), and CQ (20 μg). At least 4–10 mice were used for each substance. (E) Latency to paw withdrawal from radiant heat before (BL) and after IQ injection into a hind paw (n = 10). (F) Paw-withdrawal threshold (g) to von Frey filaments before (BL) and after IQ injection into a hind paw (n = 10). No significant differences at any point were found in both thermal sensitivity and mechanical sensitivity between saline treated and IQ treated groups (P >0.05; two-way ANOVA with Bonferroni correction). The paw-withdrawal theshold or latency time obtained from IQ treated and saline treated groups was compared using two-way ANOVA with Bonferroni correction to test the significance of the difference. All data are presented as means ± SEM.

Fig. 2.

The characterization of IQ-induced responses in cultured DRG neurons. (A) Application of IQ (20 μg/mL) increased intracellular Ca²⁺ in DRG neurons. Each trace indicates responses of representative DRG neurons in calcium imaging assay. (B) Extracellular calcium was not required for IQ-induced Ca²⁺ responses in DRG neurons. (C) Effects of inhibitors in blocking IQ-induced Ca²⁺ responses. The cells were pretreated with U73122 (10 μM) or 2-APB (20 μM) for 3–5 min before the addition of IQ. For PTX (200 ng/ml), at least 4 h of preincubation preceded application of IQ. Note that pretreatment of 2-APB profoundly impaired IQ-induced Ca²⁺ responses [only 2.2% (14/633) cells responded]. The percentages are given as the number of IQ-responding neurons of total DRG neurons counted. Approximately 400–800 cells were studied in at least four separate experiments. Asterisks mark significant differences compared with control (P < 0.05, Student's t test, unpaired). Error bars represent SEM. (D) Patch-clamp recording: the treatment of IQ (20 μg/mL) generated a train of action potentials in DRG neurons.

Fig. 3.

TLR7 does not affect scratching, calcium, and electrophysiological responses to IQ. (A) IQ-induced scratches in TLR7^+/+ and TLR7^−/− mice. There was no significant difference between TLR7-deficient mice (n = 12) and their control animals (n = 5). (B) Compared with wild-type neurons, IQ-induced Ca²⁺ responses were not impaired in TLR7^−/− DRG neurons. (C) Representative IQ-elicited action potentials from WT (n = 4) and TLR7-deficient DRG neurons (n = 7). Three of four WT and six of seven mutant DRG neurons responded to IQ (20 μg/mL), causing depolarization. Among depolarized neurons, one WT and three TLR7^−/− DRG neurons generated action potentials in the presence of IQ. IQ induced comparable magnitudes of membrane depolarization in WT and TLR7 deficient neurons. (bar graph). (D) Compared with IQ, a scratching behavioral response to loxoribine (100 μg) was not significant in wild-type mice (n = 8). (E) Loxoribine (20 μg/mL) did not evoke Ca²⁺ responses without affecting IQ-induced Ca²⁺ responses.

Fig. 4.

TRPV1⁺ neurons are responsible for mediating behavioral responses to IQ. (A) Scratching behavioral responses to IQ in various mutant mice (n = 12 for PLCβ3^+/+ and PLCβ3^−/− mice, n = 11 for TRPV1^+/+ and TRPV1^−/− mice, and n = 4 for mast-cell–deficient mice and their control animals). (B) Capsaicin-treated mice exhibit a profound loss of heat pain sensitivity. Withdrawal latency to radiant heat was measured 1 d after i.t. injection of capsaicin or vehicle. (C) TRPV1 immunostaining in lumbar dorsal horn after behavioral experiments. Shown are 1-μm confocal optical sections of adult mouse DRG neurons. (Scale bar, 100 μm.) (D) Ablation of central terminals of TRPV1-expressing neurons led to profound loss of behavioral responses to IQ (**P < 0.01, Student's t test, unpaired). n = 10 for capsaicin-treated or vehicle-treated mice.

Fig. 5.

A small subset of IQ-sensitive neurons responded to histamine, CQ, and capsaicin. (A) Fluo-3 images of DRG neurons before and after applying IQ (20 μg/mL). The calcium responses of the individual neurons from the visual field were monitored by fluorescence microscopy. The white fluorescence in individual neurons reflects an increase in intracellular calcium in response to IQ. Arrows indicate large-diameter neurons (>30 μm) and arrowheads indicate medium-diameter neurons (19–28 μm). (Scale bar, 40 μm.) (B) Twenty-six percent (100/385) of IQ-responding neurons were sensitive to capsaicin (10 μM). (C and D) A large fraction of histamine- or CQ-responding neurons was sensitive to IQ. (E) A small subset of capsaicin-responding neurons also responded to histamine, CQ, and IQ.