HTR7 Mediates Serotonergic Acute and Chronic Itch

Abstract

Chronic itch is a prevalent and debilitating condition for which few effective therapies are available. We harnessed the natural variation across genetically distinct mouse strains to identify transcripts co-regulated with itch behavior. This survey led to the discovery of the serotonin receptor HTR7 as a key mediator of serotonergic itch. Activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors. In addition, acute itch triggered by serotonin or a selective serotonin reuptake inhibitor required both HTR7 and TRPA1. Aberrant serotonin signaling has long been linked to a variety of human chronic itch conditions, including atopic dermatitis. In a mouse model of atopic dermatitis, mice lacking HTR7 or TRPA1 displayed reduced scratching and skin lesion severity. These data highlight a role for HTR7 in acute and chronic itch and suggest that HTR7 antagonists may be useful for treating a variety of pathological itch conditions.

Figure 1. A survey of mouse natural variation reveals Htr7 as a candidate itch transducer in DRG neurons

(A) The mouse strains C57BL/6J and DBA/2J display differential scratching behavior to various pruritogens: TSLP, thymic stromal lymphopoietin (15 nM); 5-HT, 5-hydroxytryptamine (1 mM); BAM, bovine adrenal medulla peptide 8–22 (3.5 mM); CQ, chloroquine (40 mM); 48/80, compound 48/80 (4 mM); HIS, histamine (27 mM). Student’s t-test; *p < 0.05; **p < 0.01; n = 4–8/strain. Error bars represent SEM. At bottom, schematic representation of the BXD recombinant inbred strains from a cross between C57BL/6J and DBA/2J mouse strains. (B) Heatmap visualization of the 20 genes whose expression in dorsal root ganglia (DRG) neurons correlates most highly with itch behavior (time spent scratching, 30 min) across BXD mouse strains; each row reports normalized gene expression from one strain (white, low relative to gene median; dark blue, high) ordered based on CQ itch sensitivity (bottom). Black box highlights Htr7. See also Tables S1, and S2.

Figure 2. HTR7 is expressed in sensory neurons that innervate the skin

(A) Htr7 expression in DRG neurons correlates strongly with CQ itch across BXD strains. Each point represents one BXD strain. (B) PCR amplification of Htr7 transcripts shows expression in mouse and human DRG. (C) Left, in situ hybridization of an Htr7 probe in mouse DRG. Scale bar, 100 μm. Middle, a higher magnification of the region encased by the yellow box in the left panel. Htr7-positive cell bodies are highlighted in yellow, Htr7-negative cell bodies in white. Scale bar, 50 μm. Right, Htr7 is expressed mostly in small diameter cells (n = 5 sections, 642 cells). (D) Immunostaining of hairy skin shows expression of HTR7 in a subset of TRPA1-positive sensory neuronal fibers. Left, Immunostaining of hairy skin with antibodies against HTR7 and TRPA1, counter stained with DAPI. Right, a higher magnification of region encased by the yellow box in the left panel. Arrowheads mark HTR7- and TRPA1-positive neuronal fibers, the arrow marks an HTR7-negative, TRPA1-positive neuronal fiber. Dotted line demarcates dermal-epidermal boundary based on DAPI staining. Scale bar, 10 μm. (E) Left, Retrogradely labeled cutaneous afferents with cholera toxin subunit B (CTB) were cultured and LP44 (100 μM) responses were assayed using calcium imaging. Scale bar, 100 μm. Right, LP44 promotes calcium influx in labeled cutaneous afferent 1, but not 2. (F) LP44 does not activate primary mouse keratinocytes; quantification of peak LP44-evoked Ca²⁺ responses in mouse primary keratinocytes to vehicle (VEH), LP44 and thapsigargin (THAP). One-way ANOVA, Tukey-Kramer post hoc test; ns, p ≥ 0.05; ***p < 0.001. Error bars represent SEM. See also Figure S1.

Figure 3. HTR7 signals through Gβγ, adenylate cyclase, and TRPA1 to promote neuronal excitation

(A) Fura-2 loaded DRG neurons treated with LP44 (100 μM) and KCl (75 mM). Arrowhead depicts an LP44-responsive neuron. Pseudocolor bar represents Fura-2 ratio. (B) Representative trace shows a neuron that responds to LP44 (100 μM), 5-HT (100 μM), and allyl isothiocyanate (AITC; 100 μM). (C) Left, LP44-responsive neurons are all sensitive to 5-HT (100 μM), AITC (100 μM), and capsaicin (1 μM). Right, the majority of these neurons overlap with the population of chloroquine-sensitive neurons (64%), and smaller subsets overlap with chloroquine/BAM8-22-sensitive (6%) or histamine-sensitive neurons (27%). No overlap is observed in the CQ-insensitive, TSLP-positive population. (D) Current-clamp recording showing action potential firing evoked by LP44 (100 μM) in a representative DRG neuron. Inset, single action potential. (E) Percentage of calcium responders to LP44 (100 μM) is reduced in neurons from Htr7^−/− and Trpa1^−/− mice, but not Trpv1^−/− mice, relative to wild-type (WT). (F) Percentage of calcium responders to LP44 (100 μM) is reduced in neurons pretreated with the adenylate cyclase blocker 2′,3′-dideoxyadenosine (DDA, 50 μM) or the Gβγ blocker gallein (GAL, 100 μM), but not the PLC blocker, U73122 (U7, 1 μM), relative to vehicle treatment (VEH). One-way ANOVA, Tukey-Kramer post hoc test; ns, p ≥ 0.05; **p < 0.01; n = 3 mice/genotype or treatment, n ≥ 200 cells/genotype or treatment. (G) An inward current is evoked by LP44 (100 μM) in HEK293 cells transfected with HTR7 and TRPA1 but not with HTR7 alone using voltage clamp recording. (H) Representative current-voltage trace of HEK293 cell transfected with HTR7 and TRPA1 in the absence (background, BKG) or presence of LP44 (100 μM) or AITC (100 μM). (I) HTR7 and TRPA1 are both required for LP44-evoked currents in transfected HEK cells; Quantification of LP44-induced peak currents (normalized to vehicle treatment) in HEK293 cells transfected as in panel (G). Each point represents one cell. Fisher’s exact test; *p < 0.05; n = 16 cells/transfection. (J) HTR7 and TRPA1 are both required for LP44-evoked calcium influx in transfected HEK cells; Quantification of peak LP44-evoked Ca²⁺ response in HEK293 cells transfected with pcDNA3 (CON), HTR7 and/or TRPA1. (K) HTR7 and TRPA1 are both required for 5-HT-evoked calcium influx in transfected HEK cells; Quantification of peak 5-HT-evoked (100 μM) Ca²⁺ response in HEK293 cells transfected with pcDNA3 (CON), HTR7 and/or TRPA1. One-way ANOVA, Tukey-Kramer post hoc test; ns, p ≥ 0.05; **p < 0.01; n = 3–5 transfections. Error bars represent SEM.

Figure 4. HTR7 triggers robust acute itch behaviors, but not pain

(A) LP44 (2 mM), but not vehicle (VEH; 20 μL, 2% DMSO in PBS) injection induces scratching behaviors in the cheek model of itch. This response is blocked by the pre-injection of HTR7 antagonist SB-269970 (SB, 5 mM). (B) Scratching evoked by LP44 injection is attenuated in Htr7^−/− mice relative to Htr7^+/+ wild-type mice (WT). (C) Scratching evoked by LP44 injection is attenuated in Trpa1^−/− mice relative to Trpa1^+/+ wild-type mice (WT). (D) LP44-evoked itch behavior is similar in Trpv1^+/+ (WT) and Trpv1^−/− mice. (E) TRPV1-positive neuronal ablation by resiniferatoxin (RTX) eliminates LP44-evoked scratching. (F) LP44-evoked itch behaviors are similar in mast cell deficient mice (Kit^W-sh) and wild-type control mice (WT). (G) LP44-evoked itch behaviors are similar in K14-Cre;Trpa1^fl/fl mice and Trpa1^fl/fl control mice. (H) LP44 (2 mM) does not evoke wiping behavior, while 1 mM 5-HT causes robust wiping. VEH, vehicle (I) Thermal hypersensitivity in mice injected with 5-HT (10 μM) and LP44 (2 mM). One-way ANOVA, Tukey-Kramer post hoc test; ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; n = 5–9 mice/genotype/behavior. Error bars represent SEM. See also Figure S1.

Figure 5. HTR7 is required for the detection of 5-HT dependent acute itch

(A) 5-HT (100 μM and 1 mM) but not vehicle injection (VEH; 20 μL, PBS) causes robust scratching in the cheek model of itch. (B) Injection of 1 mM, but not 100 μM 5-HT evokes wiping behaviors. (C) Scratching behaviors evoked by 1 mM 5-HT are similar between Htr7^+/+ (WT) and Htr7^−/− mice. (D) Scratching behaviors evoked by 100 μM 5-HT are significantly attenuated in Htr7^−/− versus Htr7^+/+ (WT) mice. VEH; 20 μL, PBS. (E) Scratching behaviors evoked by 100 μM 5-HT are reduced in Trpa1^−/− compared to Trpa1^+/+ (WT) mice. (F) Skin 5-HT levels increase after injection with the selective serotonin reuptake inhibitor (SSRI), sertraline (100 μM). One-way ANOVA, ***p < 0.001 (G) Time course of cumulative scratching behavior to sertraline (100 μM) follows the time-dependent increase in skin 5-HT levels following sertraline injection, as shown in (F). (H) Htr7^−/− and Trpa1^−/− mice show significant decreases in scratching behaviors compared to wild-type (WT) mice following sertraline injection (100 μM). (I) Chloroquine (CQ, 40 mM), compound 48/80 (48/80, 4 mM), and histamine (HIS, 27 mM) injection evokes similar itch behaviors in both Htr7^+/+ (WT) and Htr7^−/− mice. (J) There is no difference in acute heat sensitivity between Htr7^+/+ (WT) and Htr7^−/− mice. (K) AITC application (10% in mineral oil) evokes similar nocifensive behaviors in Htr7^+/+ (WT) and Htr7^−/− mice. One-way ANOVA, Tukey-Kramer post hoc test; ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; n = 5–9 mice/genotype/behavior. Error bars represent SEM.

Figure 6. HTR7 is required for the development of chronic itch in a model of atopic dermatitis

(A) Representative images of skin isolated from wild-type (WT), Htr7^−/−, and Trpa1^−/− mice treated topically with vehicle control (CON; 20 μl 100% ethanol) or the vitamin D analog, MC903 for the induction of atopic dermatitis (AD; 200 μM). (B) Atopic dermatitis lesion severity scores in mice treated with MC903 or vehicle (as described in panel A). (C) The AD model triggers and increases in the concentration of 5-HT treated skin isolated from WT, Htr7^−/−, and Trpa1^−/− mice. One-way ANOVA, Tukey-Kramer post hoc test; ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; n = 5/genotype/treatment. (D) Scratching behavior during and after VITD treatment is attenuated in Htr7^−/− and Trpa1^−/− mice. ANOVA for multivariate linear models; ***p < 0.001; n = 10/genotype. Error bars represent SEM.

Figure 7. Schematic diagram depicting the putative roles of HTR7 and TRPA1 signaling in acute and chronic itch

Model by which increased 5-HT levels in the skin, triggered by direct injection of 5-HT, SSRI administration or atopic dermatitis activates the 5-HT receptor, HTR7. HTR7 in turn signals to adenylate cyclase via Gα and Gβγ, to open TRPA1 ion channels, neuronal depolarization, action potential firing and ultimately trigger itch-evoked scratching.