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. 2020 Aug 28;5(50):eaba5683.
doi: 10.1126/sciimmunol.aba5683.

Activation of TRPA1 nociceptor promotes systemic adult mammalian skin regeneration

Jenny J Wei  1 , Hali S Kim  1 , Casey A Spencer  1 , Donna Brennan-Crispi  1 , Ying Zheng  1 , Nicolette M Johnson  1 , Misha Rosenbach  1 , Christopher Miller  1 , Denis H Leung  2 , George Cotsarelis  1   3 , Thomas H Leung  4   3   5
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Free PMC article

Activation of TRPA1 nociceptor promotes systemic adult mammalian skin regeneration

Jenny J Wei et al. Sci Immunol. .
Free PMC article

Abstract

Adult mammalian wounds, with rare exception, heal with fibrotic scars that severely disrupt tissue architecture and function. Regenerative medicine seeks methods to avoid scar formation and restore the original tissue structures. We show in three adult mouse models that pharmacologic activation of the nociceptor TRPA1 on cutaneous sensory neurons reduces scar formation and can also promote tissue regeneration. Local activation of TRPA1 induces tissue regeneration on distant untreated areas of injury, demonstrating a systemic effect. Activated TRPA1 stimulates local production of interleukin-23 (IL-23) by dermal dendritic cells, leading to activation of circulating dermal IL-17-producing γδ T cells. Genetic ablation of TRPA1, IL-23, dermal dendritic cells, or γδ T cells prevents TRPA1-mediated tissue regeneration. These results reveal a cutaneous neuroimmune-regeneration cascade triggered by topical TRPA1 activators that promotes adult mammalian tissue regeneration, presenting a new avenue for research and development of therapies for wounds and scars.

Conflict of interest statement

Competing interests: G.C. is a founder and member of the scientific advisory board of Follica. A provisional patent has been filed with the US Patent and Trademark Office regarding TRPA1 activation on reducing scar formation and promoting tissue regeneration.

Figures

Figure 1:
Figure 1:. Imiquimod induces a circulating factor that promotes mammalian tissue regeneration.
(A) Representative photographs of control and imiquimod (IMQ)-treated WT ears. Dotted circle represents original 2mm hole-punch size. (B) Percentage of wound closure in Cetaphil control- (solid black line) and IMQ-treated (dashed red line) WT mouse ears. N=10. (C) Schematic of histology orientation. (D) Representative Hematoxylin & Eosin stained tissue sections from control- and IMQ-treated WT mouse ears. Distance between opposing cartilage endplates are marked. Original wound size represented by blue box; Regenerated tissue represented by green box; Uninjured skin represented by yellow box. Higher magnification images from boxed areas. Arrows denote new hair follicles and sebaceous glands, which are further highlighted in inset images. N=10. Scale bars, 100μM. (E) Percentage of wound closure in control- (solid black line) and IMQ- treated (dashed red line) WT mice, where one ear was treated and the contralateral ear was injured. N=7 (control); N=9 (IMQ). (F) Percentage of wound closure in WT mice, where time of IMQ treatment initiation was varied. N=7 (control); N=8 (IMQ). (G) Wound induced hair neogenesis (WIHN): representative images from control- and IMQ-treated dorsal back skin. Area of WIHN shown in red box. Quantification of regenerated follicle numbers. N=14. Scale bars, 1mm. N=biological replicates per group. Data are pooled from at least two independent experiments. (B, E, and F) Two-way analysis of variance (ANOVA) and Friedman rank sum test with replicated blocks. (G) 2-tailed unpaired t test. Data presented are means ± SEM. ** P<0.01 and *** P<0.001.
Figure 2:
Figure 2:. Activated TRPA1 nociceptor promotes tissue regeneration.
(A) Schematic of imiquimod (IMQ) signaling pathway. (B-E) Representative photographs of ear holes 4 weeks after injury and percentage of wound closure in control- (solid black line) and IMQ-treated (dashed red line) mice. (b) Tlr7–/– mice. N=4 (control); N=9 (IMQ). (c) Trpv1–/– mice. N=7. (d) Trpa1+/– mice. N=3 (control); N=6 (IMQ). (e) Trpa1–/– mice. N=9 (control); N=15 (IMQ). (F) Representative H&E; stained sections from IMQ-treated Trpa1–/– and Trpa1+/– mice. Distance between cartilage endplates are marked. (G) Percentage of wound closure in mineral oil control- (solid black line) and allyl isothiocyanate (AITC)-treated (dashed red line) WT (C57/BL6) and Trpa1–/– mice. C57/BL6 mice: N=4. Trpa1–/– mice: N=7 (control); N=6 (AITC). (H) Relative value units of mouse TRPA1 transcript in different mouse tissues by qPCR. Dorsal root ganglion (DRG). N=3 (DRG); N=5 (Immune cells and skin). Dotted circle represents original 2mm-diameter hole size. N=biological replicates per group. Data are pooled from at least two independent experiments. (B, C, D, E, and G) Two-way analysis of variance (ANOVA) and Friedman rank sum test with replicated blocks. Data presented are means ± SEM. ** P<0.01 and *** P<0.001.
Figure 3:
Figure 3:. Activated TRPA1 induces the type 17 innate immune axis to promote tissue regeneration.
(A) IL-17, IL-22, and IL-23 protein levels in wound edge tissue isolated from control- (black bars) and imiquimod (IMQ)-treated (red bars) WT and Trpa1–/– mice. IL-17: N=3 (WT D3); N=8 (WT D7); N=4 (Trpa1–/– D7). IL-22: N=3 (WT D3); N=9 (WT D7); N=7 (Trpa1–/– D7). IL-23: N=3 (WT D3); N=4 (WT D7); N=6 (Trpa1–/– D7). (*) P<0.05; (**) P<0.01. (B) Serum IL-17 protein levels from control- (solid black line) and IMQ-treated (dashed red line) WT and Trpa1–/– mice. Left panel, N=5 (WT), N=4 (Trpa1–/–); Right panel, N=8 (WT), N=7 (Trpa1–/–). (**) P<0.01. (C) CD11c-DTR mice were treated with diphtheria toxin to deplete dendritic cells. Percentage of wound closure in control- (solid black line) and IMQ-treated mice (dashed red line). N=6 (control); N=4 (IMQ). (D) Percentage of wound closure in control- (solid black line) and IMQ-treated (dashed red line) Il-12p40–/– (functionally IL-23 deficient) mice. N=7 (control); N=10 (IMQ). (E) Trpa1–/– mice received daily subcutaneous injections of recombinant IL-23 (dashed blue line) or PBS (solid black line) near the ear. Percentage of wound closure in control- and IMQ-treated mice. N=5 (PBS); N=13 (recombinant IL-23). (F) Percentage of wound closure in control- (solid black line) and IMQ-treated (dashed red line) Scid–/– mice. N=5. (G) Percentage of wound closure in control- (solid black line) and IMQ-treated (dashed red line) Foxn1–/– mice. N=5. N=biological replicates per group. Data are pooled from at least two independent experiments. (A and B) 2-tailed unpaired t test. (B-G) Two-way analysis of variance (ANOVA) and Friedman rank sum test with replicated blocks. Data presented are means ± SEM. * P<0.05, ** P<0.01, and *** P<0.001. For (C), p=.16 for ANOVA and p=0.03 for Friedman rank sum.
Figure 4:
Figure 4:. γδ T cells are required for TRPA1-mediated tissue regeneration.
(A) Representative immunofluorescence sections for CD3 (red), keratin-14 (green), and nuclei (blue) in control- and imiquimod (IMQ)-treated wound edge tissue from WT mice at day 7 after injury. Scale bars, 25μM. (B) Quantification dermal and epidermal CD3 staining in control- and IMQ-treated ears. N=3. (C) Percentage of CD3+ T cells, αβ T cells, and γδ T cells relative to total live cells in wound edge tissue from control- and IMQ-treated WT and Trpa1–/– mice. N=5 (baseline); N=6 (control and IMQ). (D) Percentage of γδ T cells relative to total live cells in wound edge tissue taken at day 7 after injury from control- and IMQ-treated WT and Trpa1–/– mice. N=5. (E) Percentage of wound closure in control- (solid black line) and IMQ-treated (dashed red line) Tcrd–/– mice. N=6. (F) TCRD transcript assessed in injured wound edge tissue from non-regenerating Mus musculus mice and regenerating Acomys rodents. Data obtained from previously published analysis (33). (G) Schematized model of neuroimmune-regeneration. N=biological replicates per group. Data are pooled from at least two independent experiments. (B-D) 2-tailed unpaired t test. (E) Two-way analysis of variance (ANOVA) and Friedman rank sum test with replicated blocks. Data presented are means ± SEM. * P<0.05, ** P<0.01 and *** P<0.001.
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