Exploring neuronal mechanisms involved in the scratching behavior of a mouse model of allergic contact dermatitis by transcriptomics

Abstract

Background: Allergic contact dermatitis (ACD) is a common skin condition characterized by contact hypersensitivity to allergens, accompanied with skin inflammation and a mixed itch and pain sensation. The itch and pain dramatically affects patients' quality of life. However, still little is known about the mechanisms triggering pain and itch sensations in ACD.

Methods: We established a mouse model of ACD by sensitization and repetitive challenge with the hapten oxazolone. Skin pathological analysis, transcriptome RNA sequencing (RNA-seq), qPCR, Ca²⁺ imaging, immunostaining, and behavioral assay were used for identifying gene expression changes in dorsal root ganglion innervating the inflamed skin of ACD model mice and for further functional validations.

Results: The model mice developed typical ACD symptoms, including skin dryness, erythema, excoriation, edema, epidermal hyperplasia, inflammatory cell infiltration, and scratching behavior, accompanied with development of eczematous lesions. Transcriptome RNA-seq revealed a number of differentially expressed genes (DEGs), including 1436-DEG mRNAs and 374-DEG-long noncoding RNAs (lncRNAs). We identified a number of DEGs specifically related to sensory neuron signal transduction, pain, itch, and neuroinflammation. Comparison of our dataset with another published dataset of atopic dermatitis mouse model identified a core set of genes in peripheral sensory neurons that are exclusively affected by local skin inflammation. We further found that the expression of the pain and itch receptor MrgprD was functionally upregulated in dorsal root ganglia (DRG) neurons innervating the inflamed skin of ACD model mice. MrgprD activation induced by its agonist β-alanine resulted in exaggerated scratching responses in ACD model mice compared with naïve mice.

Conclusions: We identified the molecular changes and cellular pathways in peripheral sensory ganglia during ACD that might participate in neurogenic inflammation, pain, and itch. We further revealed that the pain and itch receptor MrgprD is functionally upregulated in DRG neurons, which might contribute to peripheral pain and itch sensitization during ACD. Thus, targeting MrgprD may be an effective method for alleviating itch and pain in ACD.

Keywords: Allergic contact dermatitis; Itch; Pain; RNA-seq; Sensory neurons.

Fig. 1

The establishment of the mouse model of ACD by oxazolone sensitization and challenge and evaluations. A Protocol for the establishment of the oxazolone (Oxa)-induced ACD model in mice. Control group receive vehicle (acetone) treatment only. B Typical pictures showing the neck skin condition of mice in vehicle- or oxazolone-treated group. C Pathological dermatitis scores of control and oxazolone group mice. D Bi-fold skin thickness evaluation of the neck skin of control and oxazolone group of mice. E Scratching behavior of mice treated with oxazolone or vehicle. F Representative pictures of hematoxylin and eosin (H&E) staining of neck skin of control and oxazolone group of mice. G Summary of epidermal thickness of control and oxazolone group mice. H Representative pictures of immunohistochemistry staining of CD3⁺ cells in neck skin of control and oxazolone group mice. I Summary of the number of CD3⁺ cells per observation field. n = 6 mice per group. **p < 0.01 versus control group. Scale bar, 100 μm

Fig. 2

Bioinformatics analysis of DRG from oxazolone-induced mouse ACD model. A Volcano graph of mRNA expression in DRG of oxazolone (Oxa) group versus control (Con) group. Dots with different colors indicate corresponding expression changes. B Heatmap illustration of the hierarchical clustering of DEmRNAs of oxazolone group versus control group. C, E, and G The top ten most significantly enriched biological processes, molecular functions, and cellular components of upregulated DEmRNAs. D, F, and H The top ten most significantly enriched biological processes, molecular functions, and cellular components of downregulated DEmRNAs. The dotted line indicates q-value of 0.05

Fig. 3

Heatmap summaries of representative DEmNRAs related to sensory neuron signal transduction, pain, itch, and inflammation. Red genes indicate upregulated DEmRNAs, whereas blue genes indicate downregulated DEmRNAs

Fig. 4

Validation of DEmNRAs and DElncRNAs by qPCR testing. A Five randomly picked upregulated DEmRNAs were examined via qPCR testing. B qPCR examination of the expression of three randomly picked downregulated DEmRNAs. C, D qPCR examination of the expression of certain representative DEmRNAs relevant with itch, pain, or inflammation process. E qPCR examination of the expression of four randomly selected upregulated DElncRNAs. F qPCR examination of the expression of two randomly selected upregulated DElncRNAs. *p < 0.05, **p < 0.01 versus control group. n = 6 mice per group

Fig. 5

The expression and functional activity of pain- and itch-related receptor MrgprD are upregulated in DRG neurons from oxazolone-induced ACD model mice. A Heatmap showing the expression of Mrgpr family genes identified in DRG of oxazolone versus control group mice. n = 4 mice per group. B qPCR validations of the expression of three itch- or pain-related Mrgpr genes, namely, Mrgprx1, Mrgpra3, and Mrgprd. n = 6–8 mice per group. *p < 0.05, **p < 0.01 versus control group. C Representative immunofluorescence images indicating MrgprD antibody staining of DRGs from control and oxazolone group of mice. Scale bar is 50 μm. Areas staining positive for MrgprD are shown in green. NeuN staining (red) was used to illustrate DRG neurons. D Summarized percentages of MrgprD⁺ neurons per field. The total neuronal cell numbers were deduced from NeuN⁺ staining. n = 6 mice per group. **p < 0.01 versus control group. E Pseudo-color images of Fura-2-based ratiometric Ca²⁺ imaging of C1–T1 DRG neurons from oxazolone and control group of mice in response to endogenous MrgprD-specific agonist β-alanine (8 mM). At the end of all recordings, 40 mM KCl was perfused to trace all alive neurons. F Summary of the percentages of neurons responding to β-alanine challenge. G Summary of Δ increase in peak 340/380 ratio upon β-alanine challenge. n = 7 tests per group. Each group included 200–300 neurons. *p < 0.05, **p < 0.01 versus control group. H Representative Ca²⁺ traces of DRG neurons in response to β-alanine and KCl from oxazolone and control group of mice. I Protocol for behavioral studies. J The effect of different dosages of β-alanine (10, 30, and 100 mM in 50 μl) on scratching behaviors in naïve mice. Beta-alanine was subcutaneously injected into the neck of naïve mice. Vehicle group received PBS injection. **p < 0.01 as indicated. NS, no significance. K Summary of scratching bouts upon vehicle/β-alanine (30 mM) injection in control or oxazolone group of mice. n = 6–7 mice per group