TRPC3 Antagonizes Pruritus in a Mouse Contact Dermatitis Model

Abstract

Contact dermatitis (CD), including allergic and irritant CD, are common dermatological diseases and are characterized by an erythematous rash and severe itch. In this study, we investigated the function of TRPC3, a canonical transient receptor potential channel highly expressed in type 1 nonpeptidergic (NP1) nociceptive primary afferents and other cell types, in a mouse CD model. Although TrpC3 null mice had little deficits in acute somatosensation, they showed significantly increased scratching with CD. In addition, TrpC3 null mice displayed no differences in mechanical and thermal hypersensitivity in an inflammatory pain model, suggesting that this channel preferentially functions to antagonize CD-induced itch. Using dorsal root ganglia and panimmune-specific TrpC3 conditional knockout mice, we determined that TrpC3 in dorsal root ganglia neurons but not in immune cells is required for this phenotype. Furthermore, the number of MRGPRD⁺ NP1 afferents in CD-affected dorsal root ganglia is significantly reduced in TrpC3-mutant mice. Taken together, our results suggest that TrpC3 plays a critical role in NP1 afferents to cope with CD-induced excitotoxicity and that the degeneration of NP1 fibers may lead to an increased itch of CD. Our study identified a role of TrpC3 and NP1 afferents in CD pathology.

Figure 1.. TrpC3 antagonizes scratching in the SADBE-CD model.

a. Protocol and timeline of SADBE-induced CD model. b. Diagram of the TrpC3 gene, depicting excision of exons 7–8 in null mice. c. Schematic representation of relative expression levels of TrpC3 (based on mouse single neuron RNAseq data) in systems relevant to CD induced itch sensation. d. H & E staining of skin from SADBE-treated mice with dermal layers labeled: epidermis (E), dermis (D), ulcer (U). e-f. Quantification of ulcer thickness and cellular infiltration of treated neck skin (≥10 measurements/mouse, n=3). g-h. Quantification of spontaneous scratch behavior of TrpC3 null and WT mice on day 16 of the SADBE model (Cohort W, n=8 (males); Cohort P, WT n=10 (5 males, 5 females), TrpC3 null n=8 (3 males, 5 females). i. Quantification of scratch duration per bout (s). j. Frequency distribution of scratch bout numbers in 5-minute bins. k. Bar graph comparing the percentage of low (<60 bouts) and high (≥60 bouts) scratching bout bins. l. Quantification of wiping bouts. Scale bars = 50 μm. Student’s two-tailed t test (e-i, l). Chi-squared test (k). ns, not significant. Asterisks indicate statistical significance. *p<0.05, **p<0.01; error bar, SEM.

Figure 2.. Loss of TRPC3 does not alter mechanical allodynia, thermal hyperalgesia, or gait.

Behavioral assays with WT and TrpC3 null mice after CFA (n=7/genotype; 2 males, 5 females) or saline (n=6/genotype; 1 male, 5 females) injection. a. 50% Paw withdrawal threshold (PWT) in response to von Frey filaments. b. Paw withdrawal score (PWS) in response to dynamic brush stimulation. c. Paw withdrawal latency (PWL) in Hargreaves test. d. Representative footprints of TrpC3 null and WT mice in gait assay. e-f. Quantification of gait width and the alternative coefficient (indicating step alternation uniformity) (n=5/genotype, females). Scale bar = 100 μm. Two-way ANOVA (a-c); Student’s one-tail t test (e-f); error bar, SEM.

Figure 3.. TrpC3 is required in DRG neurons to antagonize CD-induced scratching.

a. Diagram of floxed TrpC3 gene illustrating the loxP sites surrounding exons 7 and 8 of TrpC3 gene and the location of the forward (FP) and reverse RT-PCR primers (RP). b. Schematic representation of knocking out TrpC3 expression in DRG neurons. c. RT-PCR performed on RNA isolated from floxed control and TrpV1 CKO DRG, cerebellum, and spleen tissue. d. Quantification of scratch behavior of TrpV1 CKO (n =9; 4 males, 5 females) and control mice on Day 16 (n =9; 5 males, 4 females). e. Frequency distribution of bout numbers in 5-minute bins. f. Bar graph comparing the total percentage of low (<60 bouts) and high (≥60 bouts) scratching bout bins. g. Quantification of scratch duration per bout (s). h. Quantification of wiping bouts. Student’s two-tailed t test (b, e, f); Chi-squared test (d). Asterisks indicate statistical significance. **p<0.01; error bar, SEM.

Figure 4.. TrpC3 in immune cells is not required to modulate CD-induced scratching.

a. Schematic illustration showing CKO of TrpC3 from immune cells. b. Quantification of scratch bouts of Vav1-CKO and control mice on Day 16 (n =9; 4 males, 5 females/genotype). c. Quantification of scratch duration per bout (s). d. Frequency distribution of bout numbers in a 5-minute bins. e. Bar graph comparing the percentage of low (<60 bouts) and high (≥60 bouts) scratching bout bins. f. Quantification of wiping bouts. Student’s two-tailed t test (c, d, g); Chi-squared test (f); error bar, SEM.

Figure 5.. Degeneration of MRGPRD⁺ neurons in the CD affected region of TrpC3 null mice.

a. Immunostaining of adult Mrgprd^EGFP(+/−) and TrpC3 null; Mrgprd^EGFP(+/−) mouse cervical DRG, spinal cord, and treated neck skin sections using IB4 and antibodies against CGRP and GFP. Skin layers: ulcer (U); dermis (D); subcutaneous layer (S). b. Quantification of marker positive DRG neuron numbers per section. c. Quantification of the innervation thickness of MRGPRD⁺ central terminals. d. Quantification of dermal innervation of MRGPRD⁺ fibers in 1000μm² area (≥4 sections/mouse; n=3). Scale bars = 50 μm. Student’s two-tailed t test. Asterisks indicate statistical significance. *p<0.05, ***p<0.001, ****p<0.0001; error bar, SEM.

Figure 6.. MRGPRD⁺ neurons are not affected in the control region of TrpC3 null mice.

a. Immunostaining of adult Mrgprd^EGFP(+/−) and TrpC3 null; Mrgprd^EGFP(+/−) mouse lumbar DRG, sections using IB4 and antibodies against CGRP and GFP. b. Quantification of marker positive DRG neuron numbers per section (≥4 sections/mouse, n=3). c. Schematic of the hypothetical model of how TRPC3 functions in MRGPRD⁺ neurons to antagonize CD-induced itch. Scale bars = 50 μm. Student’s two-tailed t test.; error bar, SEM.