Keratinocytes control skin immune homeostasis through de novo–synthesized glucocorticoids

INTRODUCTION

The skin epithelial immune microenvironment deploys several mechanisms to regulate internal and environment-derived stress signals. While tolerance is induced toward innocuous factors, invasion of pathogens and contact with damaging substances activate keratinocytes and skin-resident immune cells to initiate proinflammatory processes resulting in skin-associated inflammation. Mechanisms for resolution and tissue recovery are necessary to maintain a balanced homeostasis and prevent the development of cutaneous immunopathologies (1).

The immunosuppressive effect of glucocorticoids (GC) is widely accepted and exploited in the therapy of inflammatory diseases (2). Despite their pleiotropic and adverse side effects, GC represent the mainstay in clinical therapy for more than 70 years, as diverse inflammatory disorders are still efficiently treated using synthetic GC (3). However, the potential impact of endogenous GC in the regulation of local inflammation in several immune-related diseases remains unclear. The skin and other so-called extra-adrenal organs have been reported to produce immunoregulatory GC independent from the adrenal cortex (4–9). Several studies have shown that de novo GC synthesis in the skin is regulated by a local network equivalent to the hypothalamus-pituitary-adrenal (HPA) axis and its regulatory elements (10–13). GC synthesis involves cytochrome P450 enzymes, including the 11β-hydroxylase (CYP11B1) catalyzing the final hydroxylation of 11-deoxycortisol to cortisol, respectively 11-deoxycorticosterone to corticosterone (14). Local de novo GC synthesis in the skin allows the integumentary system to react toward tissue insults and inflammation independent from the central stress response by the HPA axis. In this regard, cutaneous GC were shown to be involved in epithelial development, differentiation, and homeostasis, and recent studies also linked deficient skin GC synthesis to inflammatory skin diseases (15–18). However, the capacity and scope of skin-derived GC regarding their immunoregulatory function under physiological and immunopathological conditions and their clinical relevance remain to be investigated.

Here, we addressed this question and generated an inducible in vivo knockout (KO) model with keratinocyte-specific deletion of the critical enzyme Cyp11b1. Cyp11b1 depletion resulted in a substantial reduction of keratinocyte-produced GC and spontaneously primed skin draining lymph node (dLN) cells through skin-derived antigen-presenting cells (APCs). Abrogation of keratinocyte de novo GC synthesis also facilitated sensitization and inflammation in a contact hypersensitivity (CHS) model, associated with an enhanced interleukin-17A (IL-17A) response and increased myeloid cell infiltration. However, atopic dermatitis (AD)–like skin pathology developed nevertheless in both KO mice and littermate controls, although IL-4–mediated pruritus was reduced in the absence of keratinocyte-derived GC. In contrast, psoriasiform inflammation in skin of KO mice was exacerbated and associated with increased cytotoxicity and tissue damage. High-dimensional flow cytometry coupled with algorithm-guided computational analysis revealed decreased regulatory T (T_reg) cells and the involvement of interferon-γ (IFN-γ)– and IL-17A–expressing innate-like, unconventional, and invariant T cells in sustained psoriasiform skin inflammation in KO mice. Most notably, long-term Cyp11b1 deficiency in the skin alone resulted in the development of a spontaneous skin inflammation involving type 1 and 17 cytokines, accompanied by mononuclear phagocyte infiltration. Our study thus emphasizes the important immunoregulatory role of skin-derived GC in the regulation of the cutaneous immune system and provides mechanistic insight on the critical role of skin GC in maintaining homeostasis under physiological conditions and in different prevalent inflammatory skin diseases.

RESULTS

Corticosteroidogenic key enzymes are expressed in human and mouse skin

The skin is considered as an extra-adrenal source of GC since its expression of enzymes involved in de novo synthesis, such as CYP11B1, allows autonomous and local production of GC. In addition, the de novo synthesis in the skin is subjected to a local regulatory network, which resembles the regulatory elements of the central HPA axis. It is proposed that skin GC synthesis contributes to skin homeostasis and that deficient steroidogenic enzyme expression and GC synthesis may be involved in the pathogenesis of inflammatory skin diseases. We here show that epidermal expression of not only the critical enzyme CYP11B1 but also CYP11A1, responsible for the generation of the steroid precursor pregnenolone, is abolished in human psoriatic skin lesions compared to healthy donor skin (Fig. 1, A and B). Similar results, obtained from laser capture microdissected epidermis of healthy donor and patient skin sections, demonstrate decreased CYP11B1 and HSD11B1 expression in both AD- and psoriasis-involved lesioned skin, while CYP11A1 transcripts were still detected in AD skin (Fig. 1C). These results confirm previous studies indicating that epidermal steroidogenesis is dysregulated in prevalent inflammatory skin disorders, such as AD and psoriasis (17, 18).

We next continued to investigate de novo GC synthesis in mouse skin to experimentally assess its in vivo relevance. To elucidate the capacity of mouse skin to produce GC and to assess its responsiveness toward inflammatory stimuli, we analyzed ex vivo corticosterone production in skin biopsies from untreated and lipopolysaccharide (LPS)–treated mice. Our results show that local ex vivo GC synthesis in mouse skin biopsy and other extra-adrenal organs was reduced using the GC synthesis inhibitor metyrapone, while in vivo LPS challenge further enhanced local GC synthesis indicating the skin's sensitive steroidogenic responsiveness toward bacterial endotoxin–triggered inflammatory stimuli (Fig. 1D). Furthermore, expression of steroidogenic enzymes was validated in mouse skin (fig. S1A) and primary immortalized mouse keratinocytes, demonstrating that the expression of Cyp11b1, Hsd11b1, and the transcriptional regulator Sf-1 (Nr5a1) was inducible upon treatment with adenosine 3′,5′-monophosphate/protein kinase A signaling activators using adrenocorticotropic hormone (ACTH) and forskolin (Fig. 1E). Together, consistent with previous reports, we demonstrate keratinocyte expression of key steroidogenic enzymes and the steroidogenic responsiveness of the skin under homeostatic and inflammatory conditions, raising the question of the physiological impact, resp. benefit of local corticosteroidogenesis in human and mouse skin.

Genetic ablation of keratinocyte-specific Cyp11b1 abrogates de novo GC synthesis in the skin

To specifically address the role of keratinocyte-derived de novo GC synthesis in the regulation of tissue homeostasis and under inflammatory conditions of the skin, we generated a genetic model system that allows us to specifically target Cyp11b1 expression in keratinocytes to compromise skin GC synthesis. To do so, two LoxP (L2) sites, flanking exons 3, 4, and 5 of the Cyp11b1 locus (Cyp11b1^L2/L2), were inserted into the locus, to allow for Cre recombinase–mediated deletion of Cyp11b1 (Fig. 2A). Cyp11b1^L2/L2 mice were backcrossed to mice carrying the tamoxifen-inducible Cre recombinase transgene under the Krt14 promoter (K14-CreER^Tam) to generate K14-CreER^TamCyp11b1^L2/L2 animals (Fig. 2B and fig. S1, B and C). Cre recombinase–negative littermates of the K14-CreER^TamCyp11b1^L2/L2 mouse line were used as controls for subsequent experiments (fig. S1D). For all experimental animals, the dorsal skin hair was gently clipped, and all mice received topical application of tamoxifen to the back and ear skin for five consecutive days (Fig. 2C). Specific deletion product resulting from successful tamoxifen-induced Cre-mediated recombination was exclusively detected in the skin of Cre⁺ mice (referred to as KO) but not in other extra-adrenal tissue or in Cre⁻ littermate control mice (referred to as L2/L2) (Fig. 2D and fig. S1E). Accordingly, Cyp11b1 expression in the skin was significantly decreased in KO mice compared to controls, while Hsd11b1 expression, which mediates the reactivation of inactive, serum-derived GC, was not significantly altered (Fig. 2E). These results indicate that skin of KO mice is deficient for de novo GC synthesis while leaving the capability to provide local GC through reactivation of inactive, serum-derived GC unaffected. Accordingly, serum GC levels were also not altered after the consecutive tamoxifen application (Fig. 2F). In line with keratinocyte-specific Cyp11b1 deletion in the skin, local ex vivo corticosterone production in skin explant cultures from KO mice was significantly reduced compared to controls, while additional ex vivo treatment with the steroid precursor pregnenolone further increased skin de novo corticosterone production in control skin explant cultures in contrast to KO skin explant cultures (Fig. 2G). Furthermore, the bioactivity of ex vivo secreted corticosterone in the supernatant from dorsal and ear skin explant cultures of control and KO mice was analyzed. Therefore, human embryonic kidney (HEK) 293T cells, transfected with the GC response element (GRE) luciferase reporter, were treated with the supernatants to assess their potential in activating the GC receptor (GR) signaling. Bioactive GC secretion was significantly decreased in KO skin explant cultures irrespective of biopsy source compared to controls. Metyrapone-mediated inhibition of GC synthesis further confirmed the ex vivo production of specific, local, and adrenal-independent corticosterone in skin explants (Fig. 2H). Together, this model system represents a unique and valid tool to study the role of skin-derived GC synthesis in the regulation of skin immune homeostasis under physiological and pathological conditions.

Skin GC deficiency facilitates skin APC emigration toward dLNs

We hypothesized that local GC synthesis in the skin may fine-tune the epithelial microenvironment by regulating skin-resident immune cells and shaping barrier immunity and immunopathology. A large proportion of skin-resident immune cells is represented by APCs, which consist of several heterogeneous dendritic cell (DC) and macrophage populations populating the epidermal and dermal areas of the skin (19, 20). Trafficking to skin dLNs to prime peripheral immune cells represents one of their major functions. Since keratinocyte-derived GC may have paracrine modes of action within the skin, we hypothesized that skin APCs are likely to be targeted by locally secreted GC. We thus investigated whether keratinocyte-secreted GC may affect the residency and trafficking of skin APC toward dLN. Tamoxifen-treated K14-CreER^TamCyp11b1^L2/L2 mice and littermate controls were used to analyze APC populations in skin and dLN. Our results indicate that epidermal major histocompatibility complex II (MHCII)⁺ APC (fig. S2A) were less abundant in epidermal sheets of KO skin as observed by immunofluorescence. Flow cytometry analysis of specifically CD11c⁺ MHCII⁺ APC did not show reduced CD11b⁺ skin APC in KO ear skin compared to controls, whereas a significant decrease was observed within the CD11b⁻ subset, which include conventional DC type 1 (cDC1) cells (Fig. 3, A and B, and fig. S2, A and B) (20, 21). In line with altered cDC1 frequencies, increased keratinocyte proliferation in tamoxifen-treated K14-CreER^TamCyp11b1^L2/L2 mice was observed by means of Ki-67 staining indicating epithelial activation (fig. S2C). Furthermore, substantial increase of migratory CD11c⁺ MHCII^hi APC, but not resident CD11c⁺ MHCII^int APC, in skin dLN suggest increased skin APC trafficking toward dLN in KO mice compared to controls (Fig. 3C and fig. S2D). In line with the reduced CD11b⁻ APC subset in the skin, our results suggest that the increase of migratory skin APC in the dLN of KO mice is likely due to the increased abundance of the CD11b⁻ CD11c⁺ MHCII^hi APC subset (Fig. 3D). To specifically confirm the enhanced migration of skin-derived APC toward dLN in KO mice, we performed a previously described in vivo migration assay by painting the skin of mice with fluorescein isothiocyanate (FITC) and the sensitizer dibutyl phthalate (DBP) (22, 23). The dLN of untreated or FITC-treated control and KO mice were then analyzed for skin-emigrated FITC⁺ CD11c⁺ MHCII^hi APC. Our results show that skin-derived FITC⁺ APC in dLN of KO mice were elevated compared to control mice, confirming an increased trafficking of skin-resident APC from skin with deficient de novo GC synthesis (Fig. 3E and fig. S2E). In this regard, the CD11b⁻ subset of skin-derived FITC⁺ CD11c⁺ MHCII^hi APC was increased in dLN, as evidenced by higher frequency and total cell numbers, confirming that abrogation of epidermal GC synthesis facilitates emigration of skin-resident APC (Fig. 3F). In line with increased dLN migration of skin-resident APC cells, type 1 and 17 proinflammatory cytokine expression, but also lineage regulators, was elevated in dLN of KO mice (fig. S2E). We also observed increased T cell numbers with central memory phenotype (CD44⁺ CD62L⁺) in dLN of KO mice, indicating that peripheral T cells were primed upon in vivo Cyp11b1 depletion in the skin (fig. S2F). Collectively, these findings indicate that cutaneous abrogation of keratinocyte-mediated de novo GC synthesis increased skin immune sensitivity and responsiveness and consequently facilitated spontaneous dLN priming by skin-resident APC.

Abrogation of skin de novo GC synthesis aggravates CHS

We next investigated whether abrogation of de novo–synthesized GC in the skin, and consequent skin sensitization and immune priming may also affect inflammatory processes in acute skin disease models. The hapten FITC was used as a contact allergen to induce a classical CHS, an established experimental model for allergic contact dermatitis (23, 24). Skin GC–deficient K14-CreER^TamCyp11b1^L2/L2 mice (KO) and littermate controls (L2/L2) were sensitized with FITC on their back skin or left untreated as nonsensitized controls. Five days later, all animals received topical application of FITC to their ears (Fig. 4A). Only FITC-sensitized mice responded with a FITC-specific CHS reaction. However, KO mice exhibited an aggravated CHS response with increased ear swelling, edema, and cellular infiltration compared to L2/L2 littermate controls (Fig. 4, B to D). The aggravated skin inflammation in KO mice was associated with an increased infiltration of monocytic and granulocytic phagocytes and an inflammation-induced increase in Il17a expression (fig. S3, A and B, and Fig. 4, E to G). Notably, steroidogenic enzyme transcript levels were not induced upon CHS challenge, suggesting that CHS-induced skin inflammation does not substantially alter their transcriptional expression (fig. S3C). Although no skin pathology was observed for nonsensitized KO mice, Il17a transcript levels in the skin were elevated, indicating a potential for enhanced susceptibility to skin inflammation in the absence of de novo–synthesized skin GC (Fig. 4F). Together, we demonstrate that Cyp11b1 deficiency in keratinocytes and subsequent abrogation of de novo–synthesized GC in the skin not only increased the susceptibility to skin sensitization and dLN priming against contact allergens (Fig. 2) but also resulted in the aggravation of skin inflammation during the CHS effector response, associated with enhanced Il17a expression and increased recruitment of inflammatory myeloid monocytes and granulocytes. Our results therefore suggest that keratinocyte-derived GC pose an immunomodulatory potential in regulating skin sensitization but also restrict inflammatory processes during an acute CHS response.

Skin-derived GC promote pruritus but do not alter AD-like skin inflammation

Our results demonstrate that deficient de novo GC synthesis in keratinocytes predisposes to skin inflammation in regard of CHS reactions. We further continued to investigate the importance of skin GC in prevalent inflammatory skin disorders, such as AD and psoriasis. Given alterations in CYP11B1 expression in the context of AD lesional skin (Fig. 1C), we next tested the immunoregulatory impact of skin-derived GC in the pathogenesis of experimental AD. To do so, tamoxifen-treated K14-CreER^TamCyp11b1^L2/L2 mice and Cre⁻ littermate controls were skin-sensitized with ovalbumin (OVA) on a developing AD-like skin lesion induced by the vitamin D analog MC903 or on a vehicle-treated skin as previously reported (Fig. 5A) (25–27). Using this treatment regimen, both KO and control mice exhibited similar clinical and histological features of AD-like skin inflammation, characterized by skin edema and myeloid cell infiltration to sites of topical MC903 treatment (Fig. 5, B to G, and fig. S4, A and B). Furthermore, local GC synthesis in skin of MC903-treated littermate controls was substantially increased compared to vehicle-treated controls, whereas skin GC production in KO mice was still reduced indicating the capability of AD-like skin inflammation to potently trigger local de novo GC synthesis (fig. S4C). Serum GC levels in both vehicle- and MC903-treated KO mice were not significantly different (fig. S4C). Likewise, the increase of activated skin dLN T cells and the OVA-specific IL-2 response in ex vivo restimulated skin dLN cultures from MC903-treated KO and control mice were similar (fig. S4, D and E, and Fig. 5H). However, we found KO mice to exhibit dampened OVA-specific IL-4 responses and significantly reduced IL-4 protein in AD-like skin compared to L2/L2 littermate controls (Fig. 5, H and I). In line with IL-4 as an activator of itch-sensory pathways (28), reduced IL-4 production in the absence of skin de novo GC synthesis was associated with reduced pruritus as measured by episodes of scratching, suggesting that keratinocyte-derived GC signaling is involved in the IL-4–mediated itch-sensory pathway (Fig. 5E). In conclusion, our findings provide evidence that local de novo–synthesized GC in the skin do not restrict the pathogenesis of MC903-induced experimental AD and associated AD-like skin inflammation but do promote the IL-4–mediated itch-sensory pathway.

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.

Loss of keratinocyte de novo GC synthesis exacerbates psoriasiform inflammation

Our results have shown that de novo GC synthesis in keratinocytes is able to regulate skin sensitization and consequent inflammation. Given that ablation of skin GC synthesis resulted in a spontaneous skin dLN priming and enhanced Il17a induction in the CHS response, we next investigated the impact of keratinocyte-derived GC on the pathogenesis of psoriasis, a chronic, immune-mediated inflammatory skin disease driven by IL-17 effector cytokine responses. We therefore used an experimental model of psoriasis that is induced by topic application of Aldara (ALD) creme, which contains the potent Toll-like receptor 7/8 ligand imiquimod to activate cutaneous APC in vivo (29). ALD-induced psoriasiform skin inflammation closely resembles the human plaque psoriasis that is mediated through the IL-23/IL-17 axis (30). We topically applied ALD over 8 days to the back or ear skin of control and KO mice after tamoxifen-induced Cyp11b1 deletion (Fig. 6A). Our results revealed that both pathogenesis and progression of psoriasiform dermatitis was significantly exacerbated in KO mice, with increased abscess and scale formation compared to ALD-treated L2/L2 littermate controls (Fig. 6B). In addition, clinical scoring at days 6 to 7 indicated the onset of remission with decreasing signs of inflammation in ALD-treated L2/L2 control mice, whereas aggravation of skin inflammation in KO mice was sustained without signs of amelioration (Fig. 6, B to E). Histological analysis further revealed abnormal abscess formation in KO mice, which was, however, not associated with epidermal hyperproliferation as an psoriatic hallmark but was rather followed by tissue-damaging processes leading to increased leukocyte infiltration and to the loss of an intact epidermis (Fig. 6F). Compared to control mice, the psoriasiform skin inflammation in ear and dorsal skin of KO mice featured increased tissue damage and cell death (Fig. 6, G and H, and fig. S5, A and B). Decreased frequencies of myeloid phagocytes, which are known to initially infiltrate psoriasiform skin lesions, in line with increased cell death, further demonstrate the advanced progression of psoriasiform skin inflammation in KO mice compared to littermate controls, while untreated KO mice exhibited a developing spontaneous skin inflammation as evidenced by clinical scoring and increased myeloid cell infiltration (Fig. 6, C and D and G to I, and fig. S5A). Besides, KO skin biopsies from ALD-treated mice were still deficient in the local de novo production of GC compared to skin biopsies of ALD-treated littermate controls, confirming the long-lasting targeting in the K14-CreER^TamCyp11b1^L2/L2 mice (fig. S5C). Our results demonstrate that in vivo abrogation of de novo skin GC synthesis exacerbated pathogenesis and progression of ALD-induced psoriasiform skin inflammation and thus highlight the endogenous potential of keratinocyte-derived GC in the regulation of type 17–mediated skin inflammation.

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.

Sustained psoriasiform inflammation in KO skin is associated with reduced T_reg cells and involves invariant and unconventional T cells

IL-17 effector cytokines are key drivers in the pathogenesis of psoriasis and are primarily produced by several innate-like and γδ T cells in the model of ALD-induced experimental psoriasis (31, 32). Since GC activate various immunoregulatory mechanisms and exert pleiotropic effects in different immune cell types, we aimed to uncover those T cell populations, which were associated with the aggravated psoriasiform skin inflammation in mice with deficient de novo GC synthesis in the skin. Therefore, we used computational flow cytometry analysis using t-distributed stochastic neighbor embedding (tSNE) algorithm–based visualization and FlowSOM algorithm–based meta clustering to identify T cell populations in ears of untreated and ALD-treated control and KO mice (Fig. 7A). Our comprehensive analysis showed that CD4⁻CD8⁻ αβ T cells (CD4⁻CD8⁻ T cells), CD8⁺ γδ T cell receptor–positive (TCR⁺) cells (CD8 γδ T cells), and several NK1.1-expressing T cell (NKT) subsets were dominantly recruited in ALD-treated KO ears, whereas CD4⁺ T helper (T_H) and CD4⁺ Foxp3⁺ T_reg cells were most notably decreased (Fig. 7B). These results indicate that the exacerbated psoriasiform inflammation in KO skin was maintained because of the unique recruitment of innate-like, unconventional, and invariant T cells and the reduction of T_reg cells. Analysis of the inflammatory profile of each population additionally revealed the presence of IL-17A⁺ and IFN-γ⁺ T cells (Fig. 7C and fig. S7, A and B). Accordingly, increased dermal γδ TCR^int T cells (γδ T cells), mostly representing Vγ4⁺ γδ T cells, are described to represent the major source of IL-17A in psoriasiform skin inflammation, which was consistently observed in ALD-treated control mice (Fig. 7C) and reported previously (31, 32). However, ALD-treated KO mice showed increased IFN-γ⁺ γδ T cells and CD8⁺ γδ T cells with IL-17A expression in ear skin immune cell infiltrates (Fig. 7C and fig. S7, A to E). Our results therefore suggest that psoriasis-like inflammation in skin lacking keratinocyte-derived de novo–synthesized GC results in reduced T_reg cell numbers and specifically promotes IFN-γ– and IL-17A–expressing innate-like, unconventional γδ T cells and invariant NK1.1⁺ T cells to exacerbate psoriasiform skin inflammation, which is associated with increased cytotoxicity and epithelial damage.

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.

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

Our ALD experiments demonstrated that even untreated KO mice exhibited clinical signs of skin inflammation starting around days 7 to 8 after tamoxifen application (Fig. 6, B to D). Skin sensitization and dLN priming, observed directly after genetic in vivo ablation of Cyp11b1 in keratinocytes, appear to contribute to the development of a spontaneous skin inflammation in KO mice (Fig. 3, A to D, and fig. S2, C, E, and F). Further analysis revealed that 10 to 14 days after tamoxifen application, affected KO mice exhibited ear skin thickening, epidermal hyperproliferation, and myeloid cell infiltration involving Ly-6C⁺ monocyte subsets and Ly-6G⁺ neutrophils (Fig. 8, A and B and fig. S8A). In addition, RNA expression analysis from ears of KO mice demonstrated elevated levels of several type 1 and 17 signature cytokines and the Cxcl9 chemokine, consistent with the myeloid phagocyte infiltration in contrast to ear skin of littermate controls (Fig. 8C). These results indicate that long-term deficiency of GC de novo synthesis in keratinocytes alone is capable to promote the activation of type 1 and 17 immune response, which subsequently can develop into a spontaneous skin inflammation consequently involving the recruitment of inflammatory myeloid cells. Since the given immunophenotype of the spontaneous inflammation in KO skin resembles, to a certain degree, the psoriasiform skin inflammation, we suggest that endogenous and locally derived keratinocyte GC specifically regulate skin immune pathologies and potently counteract IL-17–driven skin inflammation to maintain tissue homeostasis.

DISCUSSION

The concept of local GC synthesis in the regulation of the skin immune microenvironment has gained increasing scientific and clinical attention since the first demonstration of extra-adrenal GC synthesis at this epithelial barrier (4, 5, 17). Keratinocytes were shown to produce GC in the skin, tightly regulated by a local HPA-like axis via steering hormones and neuropeptides. Rodent and human skin were shown to be capable in producing GC, while patients with inflammatory skin diseases were suspected to have a defective steroidogenesis. Yet, a clear demonstration that local GC synthesis in the skin regulates local immune responses h...

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