Figure S3.
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 or NRF2-KO mice (n = 4–10 mice per group; three to four independent experiments) were either shaved only or tape-stripped (TAPE). Blood and skin biopsies were collected on day 5 (D5) after tape stripping. (A) pDCs infiltration in inflamed skin was analyzed by flow cytometry. (B and C) Neutrophil infiltration in inflamed skin of (B) TAPE C57BL/6 mice on day 1 and day 5 and (C) WT or NRF2-KO TAPE mice on day 5 were analyzed by flow cytometry. (D and E) Gene expression levels of (D) MX1 and ISG15 and (E) non-ISGs inflammatory genes such as IL1B were analyzed by Q-PCR. (F–I) 8–10-wk-old C57BL/6 mice (n = 4–15 mice per group; three independent experiments) 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. (F) CD45+ cell infiltration in inflamed skin was analyzed by flow cytometry. (G) pDCs infiltration in skin was analyzed by flow cytometry. (H) pDCs (CD45+CD11b−Ly6G−CD317+) were sorted from the skin of TAPE mice treated with either vehicle or KI696 and gene expression levels of HMOX1 were analyzed by Q-PCR. (I) Gene expression levels of IRF7, MX1, ISG20, ISG15, and OAS2 were analyzed in the skin by Q-PCR. (J) pDC depletion was achieved in 9–16-wk-old BDCA2-DTR mice (pDC-dep) (n = 3 mice per group; two independent experiments) by i.p. injection of diphtheria toxin on day −3 and day −1 before tape stripping. Spleens of BDCA2-DTR and C57BL/6 WT mice were harvested one day after tape stripping and the presence of pDCs (CD45+B220+SiglecH+CD317+) cells was analyzed by flow cytometry. (K) 8–12-wk-old C57BL/6 or NRF2-KO mice (n = 3–13 mice per group; three independent experiments) 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 ISG20, ISG15, IRF7, and MX1 were analyzed in the skin by Q-PCR. (L and M) 8–10-wk-old C57BL/6 mice (n = 6–10 mice per group; three independent experiments) 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. (L) Neutrophils infiltration in inflamed skin of TAPE mice injected with either vehicle or KI696 was analyzed by flow cytometry. (M) Gene expression levels of IL1B, TNFA, and IL6 were analyzed in the skin by Q-PCR. 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 or NRF2-KO mice (n = 4–10 mice per group; three to four independent experiments) were either shaved only or tape-stripped (TAPE). Blood and skin biopsies were collected on day 5 (D5) after tape stripping. (A) pDCs infiltration in inflamed skin was analyzed by flow cytometry. (B and C) Neutrophil infiltration in inflamed skin of (B) TAPE C57BL/6 mice on day 1 and day 5 and (C) WT or NRF2-KO TAPE mice on day 5 were analyzed by flow cytometry. (D and E) Gene expression levels of (D) MX1 and ISG15 and (E) non-ISGs inflammatory genes such as IL1B were analyzed by Q-PCR. (F–I) 8–10-wk-old C57BL/6 mice (n = 4–15 mice per group; three independent experiments) 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. (F) CD45+ cell infiltration in inflamed skin was analyzed by flow cytometry. (G) pDCs infiltration in skin was analyzed by flow cytometry. (H) pDCs (CD45+CD11b−Ly6G−CD317+) were sorted from the skin of TAPE mice treated with either vehicle or KI696 and gene expression levels of HMOX1 were analyzed by Q-PCR. (I) Gene expression levels of IRF7, MX1, ISG20, ISG15, and OAS2 were analyzed in the skin by Q-PCR. (J) pDC depletion was achieved in 9–16-wk-old BDCA2-DTR mice (pDC-dep) (n = 3 mice per group; two independent experiments) by i.p. injection of diphtheria toxin on day −3 and day −1 before tape stripping. Spleens of BDCA2-DTR and C57BL/6 WT mice were harvested one day after tape stripping and the presence of pDCs (CD45+B220+SiglecH+CD317+) cells was analyzed by flow cytometry. (K) 8–12-wk-old C57BL/6 or NRF2-KO mice (n = 3–13 mice per group; three independent experiments) 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 ISG20, ISG15, IRF7, and MX1 were analyzed in the skin by Q-PCR. (L and M) 8–10-wk-old C57BL/6 mice (n = 6–10 mice per group; three independent experiments) 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. (L) Neutrophils infiltration in inflamed skin of TAPE mice injected with either vehicle or KI696 was analyzed by flow cytometry. (M) Gene expression levels of IL1B, TNFA, and IL6 were analyzed in the skin by Q-PCR. 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.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal