Fig 1: Corticosteroidogenic key enzymes are expressed in human and mouse skin.(A) Immunohistochemistry with anti-human CYP11B1 or rabbit immunoglobulin G (IgG) of human skin from healthy donors or patients with lesional AD or psoriasis. Representative images from healthy donors (n = 5), patients with lesional AD (n = 8), or lesional psoriasis (n = 10). Scale bar, 100 µm. (B) Immunofluorescence for CYP11A1 (green) and CD45 (red) or rabbit and rat isotype IgG on frozen human skin sections from healthy donors or patients with lesional AD or psoriasis. Cell nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI) (blue). Representative images from three individual donors and patients are shown. White dashed line indicates dermal-epidermal junction. Scale bar, 50 µm. (C) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of CYP11B1, HSD11B1, and CYP11A1 in laser capture microdissected epidermis from frozen human skin sections of healthy donors (HD), patients with lesional AD (AD) or psoriasis (PS). Expression was normalized to GAPDH, and data are depicted as 2(-?Ct). Columns represent means ± SEM (n = 2 to 3 individuals per group) of one experiment. (D) Corticosterone radioimmunoassay from untreated (UT) or metyrapone (MET)–treated ex vivo mouse tissue culture in response to phosphate-buffered saline (PBS) or LPS. Symbols represent individual animals. Columns show means ± SEM (n = 4 to 6 per group), pooled from two independent experiments. (E) RNA expression in immortalized C57BL/6 keratinocytes that were untreated or treated with 1 µM ACTH or 20 µM forskolin (FSK) for overnight. Expression was normalized to Actb, and data are depicted as fold change to untreated samples. Data represent means ± SEM (n = 4 to 8 per group), pooled from two to three independent experiments. Statistical significance for (E) was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons test and the ordinary one-way analysis of variance (ANOVA) with Dunn’s multiple comparisons test for Hsd11b1 expression analysis.
Fig 2: Genetic ablation of keratinocyte-specific Cyp11b1 abrogates de novo GC synthesis in the skin.(A) Scheme of Cre/LoxP strategy for Cyp11b1 exons 3 to 5 excision. (B) Generation of mice with inducible Cyp11b1 deletion in keratinocytes (K14-CreERTamCyp11b1L2/L2) by breeding Cyp11b1L2/L2 mice with K14-CreERTam mice. (C) Experimental protocol for in vivo Cyp11b1 deletion in the skin. (D) Agarose gel electrophoresis of the PCR analysis of genomic Cyp11b1 excision from dorsal skin of control (L2/L2) and KO animals. Deletion fragment (349 base pairs) indicates successful Cyp11b1 in vivo deletion. bp, base pairs. (E) Cyp11b1 and Hsd11b1 expression in ear skin. Expression was normalized to Actb. Data are presented as 2(-?Ct) and shown as fold change over L2/L2 controls. Dots represent individual animals (n = 10 to 13 per group), pooled from three independent experiments. (F and G) Corticosterone radioimmunoassay of blood serum (F) and supernatant of untreated (G, left) or adrenocorticotropin (ACTH), forskolin, or pregnenolone-treated dorsal skin ex vivo cultures (G, right). Dots represent individual animals (n = 6 to 12 per group), pooled from three (F), four (G, left), or two (G, right) independent experiments. (H) GRE luciferase (Luc) reporter assay with HEK 293T cells using supernatant of untreated or metyrapone-treated ex vivo skin culture from L2/L2 or KO animals. Empty luciferase vector–transfected cells served as controls. Normalized relative luciferase activity was depicted as fold change over the mean of untreated L2/L2 controls (dashed line). Paired dots represent skin biopsies from one individual animal (n = 10 to 13 per group). Data are pooled from two to three independent experiments. Box plots (E to G) show the 25th to 75th percentiles with whiskers indicating minimum to maximum values. Statistical significance was determined using unpaired two-tailed t test (F), two-tailed Mann-Whitney test (E and G, left) and ordinary two-way ANOVA with Sidak’s multiple comparisons test (G, right and H). (A) and (B) were created with biorender.com.
Fig 3: Abrogation of skin de novo GC synthesis aggravates CHS.(A) Experimental protocol for FITC skin sensitization and CHS induction following in vivo Cyp11b1 deletion in the skin. (B) Hematoxylin and eosin staining of frozen ear sections from naïve or FITC-sensitized controls (L2/L2) or KO mice 24 hours after CHS induction. Representative images of three independent experiments. Scale bar, 200 µm. (C and D) Ear swelling and ear cell numbers of naïve and sensitized mice. Dots represent individual animals (n = 9 to 16 per group), pooled from three to four independent experiments. (E) Anti–Ly-6G (red) and DAPI (blue) immunofluorescence of frozen ear sections from naïve or sensitized mice. Yellow-stained areas represent FITC treatment–induced fluorescence. Representative images of three independent experiments. Scale bar, 100 µm. (F) RT-qPCR analysis of Il17a expression in ear skin. Expression was normalized to Actb and shown as fold change over naïve L2/L2 mice. Dots represent individual animals (n = 5 to 9 per group), pooled from three independent experiments. (G) Flow cytometry analysis of myeloid granulocytes and monocyte subsets depicted as total cell numbers per ear. Dots represent individual animals (n = 9 to 16 per group), pooled from three independent experiments. Box plots (C, D, F, and G) show the 25th to 75th percentiles with whiskers indicating minimum to maximum values. Statistical differences were determined using ordinary two-way ANOVA with Sidak’s multiple comparisons test.
Supplier Page from MilliporeSigma for Anti-CYP11B1 antibody produced in rabbit