Figure 4.
Skin stiffness activates NRF2 to inhibit IFN responses and promotes inflammation resolution following mild skin injury using the tape-stripping model. (A–E) 8–10-wk-old C57BL/6 mice (n = 4–14 mice per group; three independent experiments) were either shaved only or tape stripped (TAPE). Blood and skin biopsies were collected on day 1 (D1) and day 5 after tape stripping. (A) CD45+ cell infiltration in inflamed skin was analyzed by flow cytometry. (B) ISGs such as IRF7, MX1, ISG20, and ISG15 gene expressions were analyzed by Q-PCR. (C) pDCs infiltration in inflamed skin was analyzed by flow cytometry. (D and E) pDCs (CD45+CD11b−Ly6G−CD317+) were sorted from the blood and inflamed skin of TAPE mice. (D) MX1 and ISG15 and (E) NRF2 gene expression were analyzed by Q-PCR. (F–H) 8–10-wk-old C57BL/6 mice or NRF2-KO mice (n = 4–7 mice per group; three independent experiments) were either shaved only or tape stripped (TAPE). Blood and skin biopsies were collected on day 5 after tape stripping. pDCs were sorted from the blood and the inflamed skin of TAPE mice and gene expression level of (F) HMOX1 (NRF2 regulated genes), (G) ISG15 and MX1, and (H) G6PD was quantified by Q-PCR. (I) 8–10 wk old C57BL/6 mice (n = 5 per group) were either shaved only or tape stripped (TAPE) and injected i.p. with either vehicle or KI696. Blood and skin biopsies were collected on day 1 after tape stripping. pDCs (CD45+CD11b−Ly6G−CD317+) were sorted from the skin of TAPE mice treated with either vehicle or KI696 and gene expression levels of ISGs such as MX1, ISG15, and ADAR were analyzed by Q-PCR. (J) pDC depletion was achieved in 9–16-wk-old BDCA2-DTR mice (pDC-dep) (n = 3–5 mice per group; two independent experiments) by i.p. injection of diphtheria toxin at day −3 and day −1 before tape stripping. pDC-dep mice and their WT C57BL/6 littermate mice were either shaved only or tape stripped (TAPE) and injected i.p. with either vehicle or KI696. Skin biopsies were collected on day 1 after tape stripping. Gene expression levels of IFNB1, IFNA2, IFNA4, and IFNA9 were analyzed in the skin by Q-PCR. (K and L) 8–10-wk-old C57BL/6 mice (n = 6–11 mice per group) were either shaved only or tape-stripped (TAPE) and injected i.p. with either vehicle or KI696. Skin biopsies were collected on day 1 after tape stripping. Representative images of H&E-stained skin sections at original magnification 10×. The presence of inflammation spreading in the dermis or adipose tissue is indicated by blue arrows or by the black dotted line for a large band of infiltrating cells. Ulcerations are represented by a square bracket for large areas of disepithelization or by a black arrow for microregions of epidermal loss. The length of the scale bars corresponds to 100 µm. (L) Average histology parameters scored from zero to three in 4–5 skin sections of naive (black), tape + vehicle (green), or tape + KI696 (red) for ulcer score (middle panel), for the degree of inflammation (right panel) and for total score (represented as the average sum of the two histology parameters). Individual mice from three different experiments are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann–Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. Refer to the image caption for details.

Skin stiffness activates NRF2 to inhibit IFN responses and promotes inflammation resolution following mild skin injury using the tape-stripping model. (A–E) 8–10-wk-old C57BL/6 mice (n = 4–14 mice per group; three independent experiments) were either shaved only or tape stripped (TAPE). Blood and skin biopsies were collected on day 1 (D1) and day 5 after tape stripping. (A) CD45+ cell infiltration in inflamed skin was analyzed by flow cytometry. (B) ISGs such as IRF7, MX1, ISG20, and ISG15 gene expressions were analyzed by Q-PCR. (C) pDCs infiltration in inflamed skin was analyzed by flow cytometry. (D and E) pDCs (CD45+CD11b−Ly6G−CD317+) were sorted from the blood and inflamed skin of TAPE mice. (D) MX1 and ISG15 and (E) NRF2 gene expression were analyzed by Q-PCR. (F–H) 8–10-wk-old C57BL/6 mice or NRF2-KO mice (n = 4–7 mice per group; three independent experiments) were either shaved only or tape stripped (TAPE). Blood and skin biopsies were collected on day 5 after tape stripping. pDCs were sorted from the blood and the inflamed skin of TAPE mice and gene expression level of (F) HMOX1 (NRF2 regulated genes), (G) ISG15 and MX1, and (H) G6PD was quantified by Q-PCR. (I) 8–10 wk old C57BL/6 mice (n = 5 per group) were either shaved only or tape stripped (TAPE) and injected i.p. with either vehicle or KI696. Blood and skin biopsies were collected on day 1 after tape stripping. pDCs (CD45+CD11b−Ly6G−CD317+) were sorted from the skin of TAPE mice treated with either vehicle or KI696 and gene expression levels of ISGs such as MX1, ISG15, and ADAR were analyzed by Q-PCR. (J) pDC depletion was achieved in 9–16-wk-old BDCA2-DTR mice (pDC-dep) (n = 3–5 mice per group; two independent experiments) by i.p. injection of diphtheria toxin at day −3 and day −1 before tape stripping. pDC-dep mice and their WT C57BL/6 littermate mice were either shaved only or tape stripped (TAPE) and injected i.p. with either vehicle or KI696. Skin biopsies were collected on day 1 after tape stripping. Gene expression levels of IFNB1, IFNA2, IFNA4, and IFNA9 were analyzed in the skin by Q-PCR. (K and L) 8–10-wk-old C57BL/6 mice (n = 6–11 mice per group) were either shaved only or tape-stripped (TAPE) and injected i.p. with either vehicle or KI696. Skin biopsies were collected on day 1 after tape stripping. Representative images of H&E-stained skin sections at original magnification 10×. The presence of inflammation spreading in the dermis or adipose tissue is indicated by blue arrows or by the black dotted line for a large band of infiltrating cells. Ulcerations are represented by a square bracket for large areas of disepithelization or by a black arrow for microregions of epidermal loss. The length of the scale bars corresponds to 100 µm. (L) Average histology parameters scored from zero to three in 4–5 skin sections of naive (black), tape + vehicle (green), or tape + KI696 (red) for ulcer score (middle panel), for the degree of inflammation (right panel) and for total score (represented as the average sum of the two histology parameters). Individual mice from three different experiments are indicated; all results are represented as mean ± SEM; and statistical significance was evaluated using Mann–Whitney U test or one-way ANOVA, Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

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