Keratinocytes control skin immune homeostasis through de novo-synthesized glucocorticoids

Abstract

Glucocorticoids (GC), synthesized by the 11β-hydroxylase (Cyp11b1), control excessive inflammation through immunosuppressive actions. The skin was proposed to regulate homeostasis by autonomous GC production in keratinocytes. However, their immunosuppressive capacity and clinical relevance remain unexplored. Here, we demonstrate the potential of skin-derived GC and their role in the regulation of physiological and prevalent inflammatory skin conditions. In line with 11β-hydroxylase deficiency in human inflammatory skin disorders, genetic in vivo Cyp11b1 ablation and long-term GC deficiency in keratinocytes primed the murine skin immune system resulting in spontaneous skin inflammation. Deficient skin GC in experimental models for inflammatory skin disorders led to exacerbated contact hypersensitivity and psoriasiform skin inflammation accompanied by decreased regulatory T cells and the involvement of unconventional T cells. Our findings provide insights on how skin homeostasis and pathology are critically regulated by keratinocyte-derived GC, emphasizing the immunoregulatory potential of endogenous GC in the regulation of epithelial immune microenvironment.

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-CreER^TamCyp11b1^L2/L2) by breeding Cyp11b1^L2/L2 mice with K14-CreER^Tam 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. Skin GC deficiency facilitates skin APC emigration toward dLNs.

(A and B) Flow cytometry plot (left) and quantification (right) of CD11c⁺ MHCII⁺ skin APC (A) and CD11b⁺/CD11b⁻ subsets of CD11c⁺MHCII⁺ cells (B) as frequency of live, single ear cells. Columns (A and B) show means ± SD with n = 7 mice per group. PE, phycoerythrin; FITC, fluorescein isothiocyanate. (C and D) Flow cytometry plot (left) and quantification (right) of migratory CD11c⁺ MHCII^hi skin APC and resident CD11c⁺ MHCII^int APC (C) and CD11b⁺/CD11b⁻ subsets of CD11c⁺ MHCII^hi cells (D) in skin dLNs, depicted as frequency of live, single dLN cells (left) and total cell numbers (right). Columns (C) and stacked columns (D) show means ± SD with n = 11 to 12 mice per group. (E and F) Flow cytometry plot (left) and quantification (right) of migratory CD11c⁺ MHCII^hi FITC⁺ skin-derived APC in skin dLN (E) and CD11b⁺/CD11b⁻ subsets out of CD11c⁺ MHCII^hi FITC⁺ APC (F), depicted as frequency of single, live cells and total cell numbers. Columns (E) and stacked columns (F) show means ± SD with n = 6 to 10 mice per group. Flow cytometry plots are representative and quantification graphs are pooled from two (A, B, E, and F) or three (C and D) independent experiments. Symbols represent individual animals. Statistical significance was determined using unpaired two-tailed t test (A to F) and Mann-Whitney test [(C) for CD11c⁺ MHCII^int and (D) for CD11b⁺ subset].

Fig. 4. 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.

Fig. 5. AD-like skin inflammation is not restricted by de novo–produced skin GC.

(A) Experimental protocol for OVA skin sensitization on vehicle-treated (EtOH) skin or MC903-induced AD-like skin. (B) Dorsal skin images of four individual control (L2/L2) and KO mice during treatment period. Representative images of three independent experiments. (C) Hematoxylin and eosin staining of frozen ear skin sections. Representative images of three independent experiments; scale bar, 100 μm. (D) Skin thickness change during treatment period as percentage of the base line skin thickness (day 0, untreated). Data show pooled means ± SD of three independent experiments (n = 8 to 10 mice per group). (E) Scratch episodes during treatment period. Data show pooled means ± SD of three independent experiments (n = 6 to 8 mice per group). (F) Total cell numbers per ear after treatment period. Data are pooled from three independent experiments (n = 8 to 10 mice per group). (G) Flow cytometry quantification of monocytes and granulocytes from ear skin single cells. Data are pooled from two to three independent experiments (n = 6 to 10 per group). (H) OVA-specific IL-2 and IL-4 protein levels in cell-free supernatants from dLN restimulated cultures. Data show pooled means ± SD of three independent experiments (n = 6 to 10 mice per group). (I) IL-4 protein levels from individual dorsal skin biopsies. Data are pooled from three independent experiments (n = 8 to 9 per group). Box plots (F, G, and I) show the 25th to 75th percentiles with whiskers indicating minimum to maximum values with dots representing individual animals. Statistical differences were determined using repeated-measures (RM) two-way ANOVA with Tukey’s multiple comparisons test (D and E) and ordinary two-way ANOVA with Sidak’s multiple comparisons test (F to H) and with Tukey’s multiple comparisons test (I). ns, not significant. Photo credit: Truong San Phan, University of Konstanz.

Fig. 6. Loss of keratinocyte de novo GC synthesis exacerbates ALD-induced psoriasiform inflammation.

(A) Experimental protocol for ALD-induced psoriasiform skin inflammation. (B) Dorsal skin images of individual untreated and ALD-treated control (L2/L2) and KO mice. Representative images (n = 6 to 8 mice per group) of two independent experiments. (C) Clinical erythema (top) and desquamation score (bottom) and (D) PASI score (sum of erythema and desquamation score) of untreated and ALD-treated dorsal skin during the treatment period. Data represent means ± SD (n = 6 to 8 mice per group), pooled from two independent experiments. (E) Skin thickness change of untreated and ALD-treated mice as percentage of the respective base line skin thickness (day 0). Data show means ± SD (n = 8 to 11 mice per group), pooled from three independent experiments. (F) Hematoxylin and eosin staining of frozen dorsal skin sections from untreated and ALD-treated mice. Representative images of two independent experiments. Scale bar, 300 μm. (G) Anti–Ly-6G (red) and anti–keratin 14 (green) immunofluorescence of frozen dorsal skin sections. Representative images of two independent experiments. Scale bar, 100 μm. (H and I) Flow cytometry frequencies of single dead cells (H) and single live monocyte subsets and granulocytes (I) from ears of indicated mice. Box plots show the 25th to 75th percentiles with whiskers indicating minimum to maximum values. Dots represent individual animals, pooled from two independent experiments with n = 4 to 6 per group (H) and n = 3 to 6 per group (I). Statistical differences were determined using RM two-way ANOVA with Tukey’s multiple comparisons test (C to E) and ordinary two-way ANOVA with Sidak’s multiple comparisons test (H). Photo credit: Truong San Phan, University of Konstanz.

Fig. 7. Psoriasiform inflammation in KO skin is associated with reduced T_reg cells and increased IL-17A⁺CD8⁺ γδ T cells.

(A and B) Computational flow cytometry analysis of single CD45⁺CD11b⁻CD3⁺ T cells from untreated and ALD-treated ears of control (L2/L2) and KO mice are visualized using tSNE algorithm with overlaid distribution of cell populations defined by FlowSOM algorithm–based clustering (top, A). tSNE map depicts aggregated samples (each downsampled to 1000 cells) with n = 4 to 6 mice per group from two independent experiments. Frequencies of FlowSOM-defined clusters as stacked bar graph (bottom) and as box plots (B) showing the 25th to 75th percentiles with whiskers indicating the range to the smallest and largest data point until the 1.5 × interquartile range (IQR). (C) tSNE maps as in (A) visualizing T cell clusters with overlaid distribution of IL-17A⁺ cells and total IL-17A⁺ cells per ear presented as box plots (bottom) showing the 25th to 75th percentiles with whiskers indicating the range to the smallest and largest data point until the 1.5 × IQR. Stacked bar graph (A) and dots in box plots (B and C) represent individual animals (n = 4 to 6 per group), pooled from two independent experiments. DETC, dendritic epidermal T cell; NKT, NK1.1⁺ T cells.

Fig. 8. Long-term deficiency of skin de novo GC synthesis results in spontaneous type 1 and 17 skin inflammation.

(A) Hematoxylin and eosin staining (top) and anti–Ly-6G (red) with anti–keratin-14 (green) (middle) or rat/rabbit IgG isotype immunofluorescence (bottom) on frozen ear skin sections of control (L2/L2) and KO mice. Representative images of three independent experiments. Scale bar, 100 μm. (B) Flow cytometry plots (top) and quantification (bottom) of myeloid granulocytes and monocyte subsets, isolated from ears 14 days after tamoxifen treatment, depicted as total cell numbers per ear. Flow cytometry plots are representative, and columns show means ± SEM with dots representing individual animals (n = 8 to 9 per group), pooled from three independent experiments. (C) RT-qPCR analysis of indicated target gene expressions in ears of L2/L2 and KO mice 10 days after tamoxifen treatment. Target genes were normalized to Actb and shown as fold change over L2/L2 controls. Box plots show the 25th to 75th percentiles with whiskers indicating minimum to maximum values. Dots represent individual animals (n = 5 to 6 per group), pooled from two independent experiments. Statistical differences were determined using two-tailed unpaired t test for CD11b⁺, Ly-6C^int, and Ly-6C^hi cells (B) and two-tailed Mann-Whitney test for Ly-6G⁺ cells (B). PerCP, peridinin-chlorophyll-protein.