TRPA1-dependent pruritus in IL-13-induced chronic atopic dermatitis

Abstract

Chronic debilitating pruritus is a cardinal feature of atopic dermatitis (AD). Little is known about the underlying mechanisms. Antihistamines lack efficacy in treating itch in AD, suggesting the existence of histamine-independent itch pathways in AD. Transient receptor potential ankyrin 1 (TRPA1) is essential in the signaling pathways that promote histamine-independent itch. In this study, we tested the hypothesis that TRPA1-dependent neural pathways play a key role in chronic itch in AD using an IL-13-transgenic mouse model of AD. In these mice, IL-13 causes chronic AD characterized by intensive chronic itch associated with markedly enhanced growth of dermal neuropeptide-secreting afferent nerve fibers and enhanced expression of TRPA1 in dermal sensory nerve fibers, their dorsal root ganglia, and mast cells. Inhibition of TRPA1 with a specific antagonist in these mice selectively attenuated itch-evoked scratching. Genetic deletion of mast cells in these mice led to significantly diminished itch-scratching behaviors and reduced TRPA1 expression in dermal neuropeptide containing afferents in the AD skin. Interestingly, IL-13 strongly stimulates TRPA1 expression, which is functional in calcium mobilization in mast cells. In accordance with these observations in the AD mice, TRPA1 expression was highly enhanced in the dermal afferent nerves, mast cells, and the epidermis in the lesional skin biopsies from patients with AD, but not in the skin from healthy subjects. These studies demonstrate a novel neural mechanism underlying chronic itch in AD and highlight the complex interactions among TRPA1(+) dermal afferent nerves and TRPA1(+) mast cells in a Th2-dominated inflammatory environment.

Figures

Figure 1. Itch-evoked scratching correlates with the amount of cutaneous sensory afferent nerves, the number of dermal mast cells, and the severity of the disease in chronic AD of K5-tTA-IL-13 mice

(a) Histology (H&E) of skin samples (20×), and (b) Fluorescent IHC staining with rabbit anti-PGP9.5 IgG (PGP9.5, Green), rabbit anti-CGRP IgG (CGRP, Green), DAPI (Nuclei, Blue), or control IgG (rabbit IgG) in Tg(−) or Tg(+) skin from the neck of mice with chronic AD. (c) Quantification of changes in dermal PGP9.5+ nerves (left) and CGRP+ nerves (right).(d) IHC with rabbit anti-c-Kit (c-Kit, White arrows) for mast cells. (e) Quantification of c-Kit+ mast cells per HPF in the skin (10 fields per skin sample per mouse; n=4–5 each group). Correlation of itch-evoked scratching with the dermal nerve growth CGRP+(f), PGP9.5+(g), c-Kit+ mast cells(h) and the severity of AD (i). In (a, b, and d), the lesional AD samples from the neck of Tg(+) mice were used and the data are pooled from three independent experiments, which gave similar results (Tg(−): n=4; Tg(+): n=5). In (c) and (e), ***P<0.001 versus values from Tg(−) skin. Original magnifications were 20× for(a), 40× for (b) and (d).

Figure 2. Enhanced expression of TRPA1 in the AD skin from K5-tTA-IL-13 mice

(a) Expression of TRPA1 in the epidermis and dermis of the lesional AD skin of the neck (arrows to the epidermis) was determined by fluorescent IHC with rabbit monoclonal anti-TRPA1 (TRPA1− Green, DAPI for nuclei-Blue) (10×). (b) Increased co-localization of TRPA1+ (Green)/CGRP+ afferent nerves (Red) in the AD skin, particularly in the dermis (40×), and (c) Quantification of TRPA1+/CGRP+ sensory nerves. (d) Enhanced co-expression of the dermal TRPA1+(Green)on c-Kit+(Red)mast cells in the AD skin (100×), and (e) increased TRPA1+/c-Kit+ mast cells correlated with itch-evoked scratching. (f) MMCP6+ mast cells (Red) by fluorescent IHC with rabbit polyclonal anti-MMCP6 are in close proximity to CGRP+ afferents (Green), DAPI (Blue for nuclei) in the dermal area of Tg(+) AD skin (100×). (g) Fluorescent IHC for TRPA1 of cryo-sections of cervical sensory DRG isolated from Tg(−) and Tg(+) mice showing markedly augmented expression of TRPA1 (Green) (DAPI for nuclei, Blue) in the sensory neurons, (10×). (g) Photos are representatives of DRG were from individual mice. Tg(−) and Tg(+) mice: n=5 for each group. (c) and (h) Data were pooled from three independent experiments with similar results (Tg(−) mice: n=5 and Tg(+) mice: n=8). (c) and (h), ***P<0.001 vs. corresponding values of Tg(−) skin. (a), (b), (d), (f) and (g), results shown are from one of 3 experiments with identical findings.

Figure 3. Blockade of TRPA1 attenuated itch-evoked scratching

Age- and AD score-matched IL-13 Tg(+) mice were administered with TRPA1 specific antagonist HC-030031 (100 mg/kg, i.p., one dose) or vehicle control. Itch-evoked scratching activities were video recorded, counted, and compared between the two groups. (a) HC-030031 significantly inhibited itch-scratching behaviors in Tg(+) mice with AD. (b) Histamine receptor blocker (Cetirizine, 15 mg/kg, i.p.) failed to reduce itch-induced scratching. (a) and (b) Data were pooled from 2 independent experiments with similar results. (*p<0.05; **p<0.01; n=5 for each group).

Figure 4. Role of mast cells in itch-evoked scratching and TRPA1 expression in the epidermis, dermal cells and dermal sensory afferents and sensory neurons

(a, b) Mast cell-deficient IL-13 Tg(+) mice exhibited slow onset, low incidence, and less severe AD and(c) markedly reduced itch-evoked scratching. Skin samples from upper portion of the back and sensory neurons from DRG collected from cervical region of IL-13 Tg(+) mice on c-Kit+/+ and KitW-sh/W-sh genetic background were fluorescent IHC stained for TRPA1 (Green), neuropeptide-releasing nerves-CGRP (Red), and DAPI (Blue for nuclei). (d) Attenuation and (f) Quantification of TRPA1 in the epidermis and dermis (20×). (e) Diminished dermal co-expression of TRPA1+/CGRP+ afferent sensory nerves (40×). (h, i) Harvested cervical ganglia (6–8 ganglia/mouse) from Tg(−)/c-Kit+/+, Tg(−)/KitW-sh/W-sh, Tg(+)/c-Kit+/+ and Tg(+)/KitW-sh/W-sh mice were stained for TRPA1 (Green), showing reduction of TRPA1 expression in the sensory neurons of DRG from Tg(+)/KitW-sh/W-sh mice as compared to Tg(+)/c-Kit+/+ mice (10×). Tg(+)/c-Kit+/+ mice: n=5, Tg(+)/KitW-sh/W-sh mice: n=5, Tg(−) groups n=4 for each. In (a), (b), and (c) Data are pooled from 5 independent experiments with similar results. Tg(−) mice: n=12 and Tg(+) mice: n=18. In (d), (e), and (h)Each is a representative of 3 independent experiments. Tg(+)/c-Kit+/+ mice: n=8 and Tg(+)/KitW-sh/W-sh mice: n=9. In (f), (g), and (i) Data are pooled from 3 experiments, n=4–7 for each group. (*p<0.05; **p<0.01; ***p<0.001).

Figure 5. Inactivation of the IL-13 transgene in Tg(+) mice with AD over time led to diminished TRPA1 in lesional AD skin associated with diminished itch-induced scratching

After withdrawal of Dox in the drinking water (the transgene was turned on) for 8 weeks, IL-13 was induced and Tg(+) mice began to develop AD (clinical score, 2). These mice were then randomly assigned to receive either Dox to inactivate the IL-13 transgene (IL-13 Tg(+) On-Off group) or normal water to keep the transgene on (IL-13 Tg(+) On-On group). Mice were sacrificed, and skin samples were obtained at the age of 18 weeks. (a) Significantly attenuated TRPA1+/PGP9.5+ afferent nerves in AD skin after the IL-13 transgene was turned off in the Tg(+) On-Off group (dotted line) compared to On-On group (solid line) in which the disease continued to worsen. (b) The number of itch-evoked scratching behaviors was diminished in Tg(+) On-Off group (gray line) compared to the Tg(+) On-On group (black line), (n=5 for each group).

Figure 6. Cytokine regulation of TRPA1 expression in mast cells and functional calcium imaging of mast cells stimulated with TRPA1 agonist

Bone marrow-derived mast cells (BMMC) were obtained by culturing bone marrow cells of wild type C57BL/6 mice in IL-3-containing medium for 4 to 6 weeks, when 98% of the cells were identified as mature mast cells by Toluidine blue staining and by flow cytometry for c-Kit and FcεRI as previously described (39). (a, b) BMMC were stimulated for 24 hours with H2O2 (10 nM), IL-13 (10 ng/ml),IL-4 (10 ng/ml), or IFN-γ (10 ng/ml). (a) Cultured BMMC were stained for TRPA1 using rabbit anti-TRPA1 antibody and Alexa Flour 488 dye labeled-donkey anti-rabbit IgG (H+L), and analyzed by flow cytometry. (b, c) Detection and quantification of TRPA1+ mast cells (Green) byfluore scent IHC. The data represent 4 independent experiments. (*P<0.05, **p<0.01 vs. Medium).Cultured mast cells (C57.1 cell line) were harvested, washed and loaded with Calcium Sensor Dye eFluor® 514 for 30 minutes at 37°C and t he cells were washed and analyzed by flow cytometry as unstimulated (d) and (e) stimulated with media (Red) and with 100 µM allylisothiocyanate (Blue) for 30 minutes. The maximum intracellular calcium mobilization in cells exposed to the agonist was measured, and histogram overlays are displayed as percentage of Max. Shown are representative of three independent experiments.

Figure 7. Increased dermal sensory nerve growth, TRPA1+ sensory afferent nerves, TRPA1+ epidermal and dermal cells and TRPA1+/Tryptase+ mast cells in chronic lesional skin biopsies from patients with AD

Fluorescent IHC with rabbit monoclonal anti-TRPA1, anti-PGP9.5, and anti-human Tryptase was performed in skin biopsysamples from normal subjects (NS) and AD patients. The comparison was made between the skin biopsy samples from the forearms of normal subjects and chronic lesional AD biopsy samples from the forearms of AD patients.(a) Quantification of dermal PGP9.5+afferent nerves, and (b) TRPA1+ afferent nerves and (c) markedly increased TRPA+(Green)/PGP9.5+(Red) afferent nerves in AD skin. (d) Expression of TRPA1 (Green), DAPI (Blue) for nuclei, in the epidermis (keratinocytes) and dermal cells. (e) Increased Tryptase+ (Red) mast cells and (f) Co-expression of TRPA1+ (Green) and Tryptase+ (Red) mast cells and these TRPA1+/Tryptase+mast cells in proximity with TRPA1+ afferent nerves (Arrows). (g) Quantification of TRPA1+/Tryptase+mast cells. In (c), (d), (e), and (f), Each is a representative of 3–4 different sections of the skin from each patient. (g)Cells in 10–12 high power fields per slide were counted (Normal subject n=4 and AD patients n=3). (**p<0.01; ***p<0.001).