Figure 9.

Keratinocyte-γδ T cell crosstalk is involved in rHMGB1-induced skin inflammation. (A and B) The Krt14^+/+-Hmgb1^f/f and Krt14^Cre/+-hmgb1^f/f mice were treated with IMQ for 5 d, n = 5/group. (A) Representative and quantitative results from intracellular FACS analysis of IL17A-producing cells, neutrophils, Vγ5⁺ cells and IFNG⁺ cells in the dorsal skin. (B) qRT-PCR analysis of the mRNA levels of the indicated genes in back skin. (C) Epidermal or dermal cell suspensions were isolated from Krt14^+/+-Atg5^f/f and Krt14^Cre/+-atg5^f/f mice that treated with IMQ for 2 d. The coculture system of the epidermal and dermal cells was treated with HMGB1 neutralizing antibody (10 μg/mL) or rHMGB1 (10 μg/mL) for 48 h, and the extracellular IL17 expression was analyzed by ELISA, n = 3/group. (D) Representative H&E-stained sections (left, scale bar: 50 μm) of dorsal skin and quantification of the thickness (right) are shown, n = 5/group. (E) qRT-PCR analysis of each indicated gene in the skin, n = 5/group. (F and G) C57BL/6 WT and tcrd KO mice received daily intradermal injections with 2 μg of rHMGB1 or vehicle control for 7 d, n = 5/group. (F) Representative H&E-stained sections (left) and quantification of the thickness (right) are shown, scale bar: 50 μm. (G) The qRT-PCR analysis was performed for each indicated gene in the skin. (H) The coculture system of the epidermal and dermal cells from wild-type C57BL/6 mice was stimulated with rHMGB1 (10 μg/mL) for 48 h, and the extracellular IL17A expression was analyzed by ELISA, n = 3/group. (I) Dermal cell suspensions from wild-type C57BL/6 mice were stimulated with rHMGB1 (10 μg/mL), rIL1B (10 ng/mL) and rIL23A (50 ng/mL) for 48 h, and the extracellular IL17A expression was analyzed by ELISA, n = 3/group. (J and K) KCs were stimulated with 10 μg/mL rHMGB1 for 24 h or 48 h, n = 3/group. (J) The protein level of CXCL8 in the culture medium was assessed by ELISA at 48 h. (K) qRT-PCR analysis of the indicated genes at 24 h. (L) Dermal cells of tcrd KO mice or WT mice were treated with 10 μg/mL rHMGB1-induced epidermal supernatant for 48 h in the absence or presence of anti-HMGB1 IgY antibody (10 μg/mL) or IgY antibody (10 μg/mL), and the extracellular IL17A secretion was analyzed by ELISA. n = 3/group. Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant. Two-tailed Student’s t-test (A, B, D-H, J and K), One-way ANOVA (C, I and L). All the data are representative of three independent experiments