IL-27 signaling mediates skin inflammation in experimental psoriasis and atopic dermatitis

Abstract

Background

Psoriasis and atopic dermatitis (AD) are two prevalent inflammatory skin disorders, each characterized by distinct adaptive immune responses. However, recent evidence suggests that these diseases may share overlapping immune mechanisms, especially concerning keratinocyte function. The specific cytokines that coordinate these inflammatory pathways remain largely undefined.

Methods

The expression of IL-27 and its receptor was analyzed using data derived from GEO datasets. Imiquimod-induced psoriasis-like and MC903-induced AD-like skin inflammation models were established in wild-type and Il27ra knockout littermates. Skin inflammation was evaluated using clinical scoring, histology, and immunostaining. Flow cytometry was employed to characterize immune cell populations in skin. Expression of relevant cytokines and signaling molecules was assessed using quantitative PCR, bulk RNA sequencing, and Western blotting.

Results

We found significantly elevated expression of the IL-27 receptor in the lesional skin of patients with psoriasis or AD. IL-27 receptor-deficient mice exhibited markedly reduced skin inflammation in both psoriasis-like and AD-like murine models. Mechanistic investigations revealed that IL-27 induces tumor necrosis factor-α production via signal transducer and activator of transcription 1 activation in keratinocytes, thereby potentiating inflammatory responses.

Conclusions

Our findings identify IL-27 signaling in keratinocytes as a pivotal regulator of skin inflammation in both psoriasis and AD. This highlights IL-27 as a promising therapeutic target for inflammatory skin diseases.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13578-025-01527-2.

Introduction

Psoriasis and atopic dermatitis (AD) are inflammatory skin disorders that significantly impact patients’ quality of life [1–4]. Both conditions likely arise from a combination of genetic factors and environmental influences [1–4]. Common features of psoriasis and AD include immune cell infiltration, changes in the dermis, and alterations in the epidermis characterized by acanthosis, hyperkeratosis, and parakeratosis [5]. Keratinocytes play crucial roles in the pathogenesis of both diseases by reacting to various danger signals and producing proinflammatory cytokines and chemokines, which recruit cells from both the innate and adaptive immune systems [1–5]. Despite these similarities, psoriasis and AD are thought to develop through different immunological pathways, with type 17 immune responses prevalent in psoriasis and type 2 immune responses dominating in AD [1–5]. However, the identification of cytokines that may serve as common drivers of skin inflammation, relevant to both psoriasis and AD, is still in its early stages of exploration.

Interleukin (IL)-27 is a heterodimeric cytokine composed of subunits p28 and Epstein-Barr virus-induced 3 (EBI3). It interacts with a heterodimeric receptor complex, which consists of IL-27RA and GP130 [6]. It is an immune-regulatory cytokine with diverse functions, particularly in barrier tissues like the skin. For instance, IL-27 facilitates keratinocyte proliferation [7, 8] and CD301b⁺ cells-derived IL-27 promotes skin wound healing by enhancing keratinocyte proliferation and antiviral defenses [9]. Additionally, IL-27 protects against Zika virus infection by inducing the expression of antiviral proteins such as OAS1, OAS2, OASL, and MX1 in skin keratinocytes, through a signal transducer and activator of transcription (STAT) 1- and interferon regulatory factor 3-dependent but STAT2-independent mechanism [10]. However, IL-27 derived from macrophages exacerbates allergic hypersensitivity responses by inducing keratinocytes to produce IL-15 in a STAT1-dependent manner, which subsequently promotes the survival of skin T cells [11].

However, the role of IL-27 in psoriasis remains contentious. Studies conducted in Japanese psoriasis patients reveal increased IL-27 expression that correlates with the severity of the disease [12]. In an imiquimod (IMQ)-induced psoriasis mouse model, antibody-mediated blockade of IL-27 signaling mitigates the severity of dermatitis by downregulating the Th1 immune response [13]. Furthermore, macrophage-derived IL-27 aggravates IMQ-induced psoriasis-like skin inflammation by enhancing the Th1 immune response [14], whereas inhibition of GP130 signaling with the monomeric IL-27 subunit p28, also known as IL-30, alleviates skin inflammation in two murine models of psoriasis [15, 16]. In contrast, studies in Chinese psoriasis patients demonstrate decreased IL-27 expression, and antibody-mediated blockade of IL-27 signaling exacerbates IMQ-induced psoriasis-like skin inflammation by enhancing the Th17 responses [17]. Supporting this finding, conditional knockout of Il27ra in T cells worsens IMQ-induced psoriasis-like inflammation by promoting the generation of IL-17 A⁺ γδT cell [18], and oral administration of Lactococcus lactis expressing IL-27 suppresses IMQ-induced psoriasis-like inflammation [19]. These discrepancies suggest that the role of IL-27 in psoriasis and possibly other inflammatory skin diseases, such as AD, warrants further investigation.

In our study, utilizing Il27ra germline knockout (KO) mice, we demonstrate that IL-27 signaling is critical for the full induction of both IMQ-induced psoriasis-like and MC903-induced AD-like skin inflammation in mice. Furthermore, we provide evidence that IL-27 signaling enhances the expression of tumor necrosis factor (TNF)-α in keratinocytes through activation of STAT1. These findings highlight the pro-inflammatory role of IL-27 in both psoriasis and AD, suggesting that targeting IL-27 could be a promising approach for treating these common inflammatory skin conditions.

Results

Deficiency of IL-27 signaling ameliorates IMQ-induced psoriasis-like skin inflammation

At first, we investigated the expression of IL-27 and its receptor components in psoriatic lesions using the GEO datasets. Our analysis revealed a significant upregulation of both IL-27 subunits in psoriatic skin lesions compared to normal skin from healthy controls and non-lesional skin from psoriasis patients (Fig. S1A-B). Importantly, while IL-27RA expression was notably elevated in psoriatic lesions (Fig. S1C), the expression of GP130 did not show a comparable increase (Fig. S1D).

Next, we investigated the role of IL-27 in psoriasis by utilizing Il27ra KO mice. Il27ra KO mice were generated using CRISPR/Cas9 technology. Consistent with previous reports [20], Il27ra KO mice are viable, fertile, and show no obvious abnormalities at baseline (data not shown). IL-27R is known to be highly expressed by T cells [6]. Thus, we collected skin-draining lymph nodes from IMQ-treated wild-type (WT) and Il27ra KO mice and conducted flow cytometry to assess its expression on T cells (Fig. 1A). Consistent with expectations, IL-27RA was undetectable on T cells from the skin-draining lymph nodes of Il27ra KO mice (Fig. 1B-C, Fig. S1E-F). Remarkably, IMQ-induced skin inflammation was significantly reduced in Il27ra KO mice compared to their control littermates, as evidenced by lower mPASI scores on both day 3 and day 5 (Fig. 1D-E). Histological analysis corroborated these findings, revealing reduced epidermal hyperplasia in Il27ra KO mice following IMQ treatment (Fig. 1F-G). Additionally, the number of Ki67⁺ epidermal cells was lower (Fig. 1H-I), while the expression of differentiation-associated proteins, such as keratin 1 and filaggrin, was markedly higher in the skin lesions of Il27ra KO mice compared to controls (Fig. 1J-K). These results suggest that disrupted IL-27 signaling may affect keratinocyte proliferation and differentiation.

Fig. 1.

IL-27RA deficiency ameliorates IMQ-induced psoriasis-like skin inflammation in mice. A Experimental design for IMQ treatment in B-K. B-C Flow cytometry results of IL-27RA expression on T cells of skin-draining lymph nodes from IMQ-treated WT (n = 4) and KO (n = 3) mice. Representative flow cytometry plots (B) and ratio of IL-27RA⁺ T cells to live cells (C) are shown. D Representative photographs of IMQ-treated WT and KO mice on day 5. E Quantification of the mPASI score. n = 5–7 mice per group. F Representative skin histology (H&E staining) on day 5. Scale bar = 100 μm. G Quantification of epidermal thickness. n = 5–7 mice per group. H Representative immunohistochemistry images of Ki67 protein in the lesional skin of IMQ-treated WT and KO mice on day 5. n = 4 mice per group. Scale bar = 50 μm. I Quantification of Ki67⁺ epidermal cells in the lesional skin of IMQ-treated WT and KO mice on day 5. n = 4 mice per group. J-K Representative immunofluorescence images of keratin 1 (J) and filaggrin (K) in the lesional skin of IMQ-treated WT and KO mice on day 5. n = 4 mice per group. Scale bar = 50 μm. DAPI stains the nuclei. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p values were calculated via 2-tailed unpaired Student’s t test (C, G and I) or two-way ANOVA (E)

Given that psoriasis is characterized by aberrant immune responses [1], we further explored whether IL-27 signaling deficiency influences immune cell infiltration. Notably, neutrophil counts were significantly reduced in the lesional skin of Il27ra KO mice, while the infiltration of other leukocyte types remained unchanged (Fig. 2A-B, Fig. S1G). Real-Time quantitative PCR (RT-qPCR) analyses revealed a significant downregulation of mRNA levels for Mmp9, which is critical for neutrophil migration [21], Cxcl2, a well-known neutrophil chemoattractant, and Tnf, a common inflammatory mediator, in the lesional skin of Il27ra KO mice compared to controls (Fig. 2C). Collectively, these findings underscore a pro-inflammatory role for IL-27 in psoriasis.

Fig. 2.

IL-27RA deficiency abrogates neutrophil accumulation in psoriasis-like skin inflammation. A-B Flow cytometry analysis of the indicated immune cells in the lesional skin of IMQ-treated WT and KO mice on day 5. Representative flow cytometry plots (A) and quantification of the relative number of the indicated immune cells (B) are shown. n = 7 mice per group. C RT‒qPCR results showing the expression of the indicated genes in the whole skin of IMQ-treated WT (n = 5) and KO (n = 4) mice on day 5. Data are presented as the means + SEMs. *p < 0.05, **p < 0.01, ***p < 0.001. p values were calculated via 2-tailed unpaired Student’s t test

Deficiency of IL-27 signaling dampens MC903-induced AD-like skin inflammation

We then explored whether this cytokine also contributes to other inflammatory skin disorders, such as allergic contact dermatitis (ACD) and AD.

First, we analyzed IL-27 and its receptor components in ACD patients using GEO datasets. Our data showed that while EBI3 and IL27RA were significantly upregulated in ACD skin lesions (Fig. S2A-B), IL27A and GP130 did not exhibit notable changes (Fig. S2C-D). To gain further insights, we induced ACD in mice by applying 1-fluoro-2,4-dinitrobenzene (DNFB) to their ears, and observed that Il27ra deficiency did not influence disease severity (Fig. S2E). These data indicate that IL-27 signaling might be not required for the development of ACD.

We then assessed the role of IL-27 in AD. GEO dataset analysis revealed a substantial increase in IL27RA expression in AD lesional skin compared to both healthy skin and non-lesional AD skin, whereas GP130 expression remained unchanged (Fig. S3A-B). IL27A and EBI3 were not detected in this dataset (data not shown). To evaluate the functional role of IL-27 in AD, we used the MC903-induced mouse model of AD [22]. In this model, Il27ra deficiency significantly reduced the severity of AD-like dermatitis, as evidenced by decreased ear thickness and reduced epidermal hyperplasia (Fig. 3A-D). Furthermore, Ki67⁺ epidermal cells’ number was reduced (Fig. 3E-F), while the expression of keratin 1 and filaggrin, was drastically elevated in the AD-like skin lesions of Il27ra KO mice compared to controls (Fig. 3G-H).

Fig. 3.

IL-27RA deficiency reduced MC903-induced AD-like skin inflammation in mice. A Representative photographs of MC903-treated WT and KO mice on day 11. B Ear thickness (mean ± SEM) of MC903-treated WT (n = 10) and KO (n = 8) mice. C Representative skin histology (H&E staining) on day 11. Scale bar = 100 μm. D Quantification of epidermal thickness. n = 8–9 mice per group. E Representative immunohistochemistry images of Ki67 in the lesional skin of MC903-treated WT and KO mice on day 11. n = 4 mice per group. Scale bar = 50 μm. F Quantification of Ki67⁺ epidermal cells in the lesional skin of MC903-treated WT and KO mice on day 11. n = 4 mice per group. G-H Representative immunofluorescence images of keratin 1 (G) and filaggrin (H) in the lesional skin of MC903-treated WT and KO mice on day 11. n = 4 mice per group. Scale bar = 50 μm. DAPI stains the nuclei. **p < 0.01, ****p < 0.0001. p values were calculated via two-way ANOVA (B) or 2-tailed unpaired Student’s t test (D and F)

To gain further insights into the molecular and cellular changes in MC903-treated Il27ra KO mice, we performed RT-qPCR and flow cytometry analyses on mouse skin lesions. We found that Tslp expression was notably decreased in the skin lesions of Il27ra KO mice, while other inflammatory mediators were not significantly altered (Fig. 4A). Additionally, immune cell infiltration analysis revealed that CD4⁺ T cell and neutrophil numbers were reduced in the AD-like lesions of Il27ra KO mice, without significant changes in other immune cell types (Fig. 4B-E, Fig. S3C-D).

Fig. 4.

IL-27RA deficiency partially decreased MC903-induced inflammatory responses in mice. A RT‒qPCR results showing the expression of the indicated genes in the whole skin of MC903-treated WT (n = 4) and KO (n = 3) mice on day 11. Data are presented as the means + SEMs. B-E Flow cytometry analysis of the indicated immune cells in the lesional skin of MC903-treated WT and KO mice on day 11. Representative flow cytometry plots (B and D) and quantification of the relative number of the indicated immune cells (C and E) are shown. n = 3–4 mice per group. *p < 0.05. p values were calculated via 2-tailed unpaired Student’s t test

Collectively, these findings indicate that while IL-27 does not play a significant role in ACD, it is implicated in promoting AD-like dermatitis.

Neutralization of IL-27 dampens disease severity in both psoriasis-like and AD-like dermatitis

Given that IL‑27RA deficiency ameliorates skin inflammation in murine models of psoriasis and AD, we investigated whether blocking IL‑27 signaling with a neutralizing antibody could be therapeutic. Administration of an IL‑27-neutralizing antibody significantly attenuated skin inflammation and epidermal hyperplasia in IMQ‑induced psoriasis‑like lesions (Fig. 5A-D), and similarly suppressed MC903‑induced ear swelling and epidermal hyperplasia (Fig. 5E-I). These findings indicate that IL‑27 blockade may represent a promising therapeutic strategy for both psoriasis and AD.

Fig. 5.

IL-27 blockade alleviates IMQ-induced and MC903-induced skin inflammation in mice. A Experimental design for IL-27 antibody and IMQ treatment in B-D. B Representative photographs of IL-27 antibody- or IgG-treated WT mice on day 5. C Representative skin histology (H&E staining) on day 5. Scale bar = 50 μm. D Quantification of epidermal thickness. n = 5 mice per group. E Experimental design for IL-27 antibody and MC903 treatment in F-H. F Ear thickness (mean ± SEM) of IL-27 antibody- or IgG-treated WT mice. n = 4–5 mice per group. G Representative photographs of IL-27 antibody- or IgG-treated WT mice on day 11. H Representative skin histology (H&E staining) on day 11. Scale bar = 200 μm. I Quantification of epidermal thickness. n = 4–5 mice per group. *p < 0.05, ***p < 0.001, ****p < 0.0001. p values were calculated via 2-tailed unpaired Student’s t test (D and I) or two-way ANOVA (F)

Transcriptomic analysis of IL-27-mediated downstream signaling in psoriasis-like dermatitis

IL-27 is known to repress IL-17 A expression of T cells in various disease models including psoriasis [17, 18, 23], which cannot explain the attenuated skin inflammation observed in IMQ-treated Il27ra KO mice (Figs. 2 and 3). We hypothesized that IL-27 might promote psoriasis-like skin inflammation via its interaction with epidermal cells, particularly keratinocytes. To minimize secondary changes resulting from alleviated skin inflammation, we collected epidermal samples from IMQ-treated Il27ra KO mice and their control littermates at an early time point (day 3), followed by bulk RNA sequencing (RNA-seq) (Fig. 6A). Deficiency of IL-27 signaling resulted in the upregulation of 207 genes and the downregulation of 213 genes (Fig. 6B-C). Metascape pathway analysis revealed that the downregulated genes were significantly enriched in terms such as “positive regulation of cytokine production,” “cytokine-cytokine receptor interaction,” “EGFR interacts with phospholipase C-gamma,” “positive regulation of cell fate commitment,” and “epidermal cell differentiation (Fig. 6D).” Conversely, the upregulated genes were enriched in pathways including “myeloid cell homeostasis,” “hair follicle morphogenesis,” “neutrophil homeostasis,” “pattern specification process,” and “supramolecular fiber organization (Fig. 6E).” Differentially expressed genes (DEGs) related to “positive regulation of cytokine production” and “myeloid cell homeostasis” are displayed (Fig. 6F). RT-qPCR analysis validated the DEGs identified in the “positive regulation of cytokine production” pathway (e.g., Il1a, Il17a, Il23a, Il12b), as well as from other pathways (e.g., Slc1a1, Lrrc61, Zbtb16) (Fig. 6G). Taken together, these data suggest that IL-27 might coordinately regulate inflammatory responses and keratinocyte proliferation and differentiation during the development of psoriasis-like skin inflammation.

Fig. 6.

IL-27RA signaling primarily regulates inflammatory responses in the epidermis of IMQ-treated mice. A Experimental design for IMQ treatment in B-G. B-C Volcano plots (B) and heatmap (C) showing the DEGs of the epidermis of IMQ-treated KO mice on day 3. n = 4 mice per group. D-E Metascape analysis results of the downregulated (D) and upregulated (E) genes. F Heatmap showing the genes associated with the indicated pathways. G RT-qPCR results showing the expression of the indicated genes in the epidermis of IMQ-treated WT and KO mice on day 3. n = 4 mice per group. Data are presented as the means + SEMs. *p < 0.05, **p < 0.01, ***p < 0.001. p values were calculated via 2-tailed unpaired Student’s t test

IL-27 induces TNF expression of keratinocytes in a STAT1-dependent manner

To further investigate how IL-27 promotes skin inflammation, we integrated bulk RNA-seq data from IL-27-treated normal human epidermal keratinocytes (NHEKs) [24]. IL-27 treatment results in 269 upregulated genes and 24 downregulated genes in NHEKs (Fig. 7A). Among the 269 upregulated genes, 6 genes are overlapped with the downregulated genes in IMQ-treated Il27ra KO mice (Fig. 7B). These genes are ATF3, TNF, CSF2, PTGS2, CD274 and IFI44. ATF3 encodes a transcription factor involved in cellular stress response [25]. TNF encodes TNF-α, a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor superfamily [26]. CSF2 encodes GM-CSF, a cytokine that controls the production, differentiation, and function of granulocytes and macrophages [27]. PTGS2 encoding prostaglandin-endoperoxide synthase, also known as cyclooxygenase, is the key enzyme in prostaglandin biosynthesis, and acts both as a dioxygenase and as a peroxidase [28]. CD274 encodes PD-L1, an immune inhibitory receptor ligand that binds to PD-1 and acts to block T-cell activation [29]. IFI44 encodes a protein that is involved in immune responses [30]. TNF-α is known to promote inflammatory responses in both psoriasis and AD [31, 32]. We therefore investigated whether IL‑27 aggravates psoriasis‑ and AD‑like pathology by upregulating TNF‑α expression. Both TNF‑α mRNA and protein levels were markedly reduced in the skin lesions of Il27ra KO mice in the murine psoriasis model (Fig. 7C-D). Importantly, intradermal administration of recombinant murine TNF‑α was sufficient to restore psoriasis‑like skin inflammation and epidermal hyperplasia in IMQ‑treated Il27ra KO mice (Fig. 7E-H). Likewise, TNF‑α mRNA and protein were significantly decreased in lesions from Il27ra KO mice in the AD model (Fig. 7I-J), and intradermal TNF‑α exacerbates MC903‑induced ear swelling and epidermal hyperplasia in these animals (Fig. 7K-O). Together, these data indicate that IL‑27 drives both psoriasis‑ and AD‑like skin inflammation at least in part by upregulating TNF‑α.

Fig. 7.

Activation of IL-27RA signaling enhances TNF-α expression via STAT1 in keratinocytes. A Volcano plots showing the differentially expressed genes (DEGs) of IL-27-treated NHEKs. The data were derived from published RNA-seq results (GSE188242). B Venn diagrams of DEGs showing overlap between the IL-27-induced genes and downregulated genes in IMQ-treated KO mice. C RT-qPCR results showing the expression of Tnf in the epidermis of IMQ-treated WT and KO mice on day 3. n = 4 mice per group. D Representative immunohistochemistry images of TNF-α in the lesional skin of IMQ-treated WT and KO mice on day 3. n = 4 mice per group. Scale bar = 50 μm. E Experimental design for TNF-α and IMQ treatment in F-H. F Representative photographs of TNF-α- or PBS-treated KO mice on day 5. G Representative skin histology (H&E staining) on day 5. Scale bar = 50 μm. H Quantification of epidermal thickness. n = 4 mice per group. I RT-qPCR results showing the expression of Tnf in the whole skin of MC903-treated WT and KO mice on day 11. n = 4 mice per group. J Representative immunohistochemistry images of TNF-α in the lesional skin of MC903-treated WT and KO mice on day 11. n = 4 mice per group. Scale bar = 50 μm. K Experimental design for TNF-α and MC903 treatment in L-O. L Ear thickness (mean ± SEM) of TNF-α- or PBS-treated KO mice. n = 3–4 mice per group. M Representative photographs of TNF-α- or PBS-treated KO mice on day 7. N Representative skin histology (H&E staining) on day 7. Scale bar = 200 μm. O Quantification of epidermal thickness. n = 3–4 mice per group. P RT-qPCR results showing the expression of TNF in NHEKs treated with IL-27 for 24 h. Q RT-qPCR results showing the expression of TNF in DMSO- or Fludarabine-treated HaCaT with or without IL-27 treatment for 24 h. R Western blotting results showing p-STAT1 expression in DMSO-, Fludarabine-, or Stattic-treated HaCaT with or without IL-27 treatment for 15 min. n = 3 independent experiments (P-R). S-T Representative immunofluorescence images of p-STAT1 in the lesional skin of IMQ-treated WT and KO mice on day 3 (S) or MC903-treated WT and KO mice on day 11 (T). n = 4 mice per group. Scale bar = 50 μm. The dashed lines in S and T indicate the basement membrane, and DAPI was used to stain the nuclei. Data are presented as the means + SEMs (C, I, P and Q). **p < 0.01, ***p < 0.001, ****p < 0.0001. p values were calculated via 2-tailed unpaired Student’s t test (C, H, I, O and P) or two-way ANOVA (L and Q)

Next, we investigated whether and how IL-27 regulates TNF expression in keratinocytes. We found that IL-27 treatment increased TNF expression in both NHEKs and HaCaT cells (Fig. 7P-Q). Furthermore, inhibition of STAT1 with its specific inhibitor, fludarabine, attenuated IL-27-induced TNF-α expression (Fig. 7Q-R). Additionally, STAT1 phosphorylation was markedly impaired in the epidermis of Il27ra KO mice in both murine models of psoriasis and AD (Fig. 7S-T). Taken together, these findings indicate that IL-27 may promote TNF-α expression through STAT1 activation during skin inflammation.

Discussion

Psoriasis is mainly driven by Th17 immune responses while AD is primarily induced by Th2 immune responses, thus they were considered as different types of skin diseases. However, recent studies have shown that these two inflammatory skin diseases might share common inflammatory mediators, especially those affecting keratinocyte, such as LIGHT, TWEAK and IL-17D [33–35]. Discovering these common mediators holds notable clinical significance since blocking Th17 immune response with antibodies in psoriasis patients sometimes leads to a skewing Th2 immune response that causes AD in the same patient, and vice versa [36, 37]. Targeting key inflammatory factors of psoriasis or AD along with blocking these common mediators might prevent the immune shift and avoid the development of the other skin disease. Our study identifies IL‑27 as a previously unrecognized common mediator in both psoriasis and AD, acting through regulation of the epithelial immune microenvironment.

Our research also sheds light on previously unrecognized players of IL-27 signaling in promoting neutrophil accumulation during inflammation. Specifically, IL-27 signaling deficiency abrogates neutrophil infiltration in both experimental psoriasis and AD. These results are correlated with decreased Mmp9 and Cxcl2 expression in Il27ra KO mice, suggesting IL-27 might promote neutrophil infiltration through upregulating these factors. However, the precise mechanism warrants further investigation.

In this study, Th17-associated inflammatory cytokine expression (e.g., IL-17, IL-23) is notably reduced in the epidermis of Il27ra deficiency mice 2 days after IMQ treatment. However, their expression is not significantly altered in the skin lesions of Il27ra deficiency mice 4 days after IMQ treatment. One explanation is that IL-27 might exert different functions through different cell types at different disease stages during the development of psoriasis. It is possible that IL-27 acts as a prime signal for other inflammatory cytokines [e.g., TNF-α [38] on keratinocytes to initiate the inflammatory cascades early in psoriasis. Thus, significant reduction of Th17-associated inflammatory cytokine expression is observed 2 days after IMQ treatment. However, when disease progresses, abundant immune cells (e.g., dermal γδT cells) are accumulated in the skin lesions and IL-27 might directly act on these cells to suppress IL-17 expression.

IL‑27 can promote inflammation in keratinocytes by inducing TNF‑α and other pro‑inflammatory mediators, as shown in our study and in previous reports [7–13, 38]. At the same time, IL‑27 has well‑documented anti‑inflammatory effects on T cells, including the suppression of IL‑17 [6]. These opposing functions suggest that systemic and non‑selective IL‑27 blockade could attenuate keratinocyte‑driven inflammation, but might also interfere with beneficial regulatory pathways in T cells. Keratinocyte‑directed delivery of IL‑27 pathway inhibitors, such as topical and nanoparticle‑based small interfering RNAs against IL-27RA, could allow selective blockade of the pro‑inflammatory IL‑27 response in the epidermis while preserving the regulatory functions of IL‑27 in circulating immune cells. Furthermore, antibody-drug conjugates [39] that selectively target keratinocytes represent another potential strategy. For example, antibodies recognizing keratinocyte‑enriched surface molecules could be conjugated to inhibitors of IL‑27 signaling or to small interfering RNAs. This approach would allow precise delivery of the inhibitory cargo directly to epidermal keratinocytes, reducing the likelihood of unwanted effects on T cells or other immune populations. Conversely, systemic IL‑27 inhibitors might be appropriate only when keratinocyte‑driven inflammation clearly outweighs potential immune regulatory benefits.

A limitation of this study is the use of germline Il27ra KO mice. This approach does not allow us to distinguish cell autonomy from indirect effects mediated by other compartments. For example, loss of IL-27 signaling in T cells or myeloid cells could alter recruitment, activation or cytokine production [18, 40] and thereby change epidermal responses secondarily. To reduce this concern, we focused analyses on epidermis collected at an early disease stage, validated key findings in primary human keratinocytes and in HaCaT cells, and performed intradermal TNF-α rescue experiments. These complementary approaches strengthen the argument for a keratinocyte-centered mechanism. However, definitive assignment of the relevant cellular targets will require keratinocyte- and T cell-specific deletion of Il27ra and the use of inducible systems to avoid developmental compensation in the future.

Conclusions

In conclusion, our study has unveiled IL-27 as a common mediator of both psoriasis and AD by coordinately regulating epidermal hyperplasia and immune responses, and may ultimately lead to the development of alternative therapeutic approaches in psoriasis and AD.

Materials and methods

Mice

Il27ra gene KO mice were generated using CRISPR/Cas9 technology. Target sites ending with NGG were designed near the shared exon 1–2 region to facilitate precise cleavage by the CRISPR/Cas9 system. Cas9‑mediated cleavage at these sites resulted in a frameshift deletion that disrupts Il27ra expression. The specific guide RNA sequences used were: Il27ra‑sgRNA1: TGG GAG CCA TGA ACC GGC TCC GG, Il27ra‑sgRNA2: GAG TGG TCT AGG GGA AAC TGA GG. Il27ra KO mice are viable, fertile, and show no obvious abnormalities at baseline. Heterozygous mice were used for breeding, and 7-week-old WT and KO mice from the same litter were utilized for experiments. Both female and male mice were used in the experiments. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Shanghai Skin Disease Hospital.

IMQ-induced psoriasis mouse model and MC903-induced AD mouse model

In the IMQ-induced psoriasis mouse model, 7-week-old mice were treated topically with a daily dose of 62.5 mg of 5% IMQ cream (Shichuan MedShine Pharmaceuticals Co.), applied to their shaved backs for the designated period. Mouse skin lesions were evaluated using the modified Psoriasis Area and Severity Index (mPASI) scores, which included assessments of scale (scores ranging from 0 to 4), erythema (scores ranging from 0 to 4), and infiltration (scores ranging from 0 to 4). For the MC903-induced AD mouse model, 1 nmol of MC903 (MedChemExpress, Cat# HY-10001) or an equivalent volume of ethanol was applied to the mouse ears once daily for 10 consecutive days. The mice were sacrificed on day 11, and ear thickness was measured daily using a caliper.

Skin cell preparation and flow cytometry

To prepare a single-cell suspension from skin-draining lymph nodes, inguinal lymph nodes were harvested from mice and placed in cold Phosphate Buffered Saline (PBS). Using the rubber end of a syringe plunger, the lymph nodes were gently ground to break down the tissue and release individual cells. The resulting suspension was subsequently filtered through a 40 μm filter to remove debris.

Single cell suspension of skin tissues was prepared as described previously [41, 42]. In brief, the skin of mice was minced with fine scissors after the removal of subcutaneous fat. The minced tissue was digested in 10 ml of RPMI 1640 medium (Gibco, Cat# 11875093) containing 0.25% collagenase (Sigma-Aldrich, Cat# C9091), 0.01 M HEPES (Thermo Fisher Scientific, Cat# BP310), 0.001 M sodium pyruvate (Thermo Fisher Scientific, Cat# BP356), and 0.1 mg/ml DNase (Sigma, Cat# DN25) for 1 h at 37 °C with rotation. The resulting cell suspension was filtered through a 40-µm filter, then centrifuged and resuspended in staining buffer (2% fetal bovine serum in PBS).

For cell surface staining, cells were incubated with TruStain FcX™ Antibody (Biolegend, Cat# 101319) to block Fc receptors for 10 min at room temperature and incubated with antibodies for 30 min at 4 ℃. Following staining, the cells were incubated with SYTOX Blue Dead Cell Stain (Invitrogen, Cat# S34857) for 5 min, and 10,000 counting beads (catalog no. C36950, Invitrogen) were added per sample before analysis. Skin cells were analyzed using the FACSAria Fusion Sorter (BD Biosciences), and data were processed using FlowJo v.10 software (TreeStar). Antibodies used were: Brilliant Violet 510 anti-mouse CD45 (Biolegend, Cat# 103137, Clone 30-F11), allophycocyanin (APC) anti-mouse CD45 (Biolegend, Cat# 103112, Clone 30-F11), Alexa Fluor 488 anti-mouse CD11b (Biolegend, Cat# 101219, Clone M1/70), APC/Cyanine7 anti-mouse Ly6g (Tonbo Biosciences, Cat# 25-1276, Clone 1A8), APC anti-mouse CD11c (Biolegend, Cat# 117310, Clone N418), phycoerythrin (PE) anti-mouse F4/80 (Biolegend, Cat# 101219, Clone BM8), PE/Cyanine7 anti-mouse EpCAM (Biolegend, Cat# 118216, Clone G8.8), PerCP/Cyanine5.5 anti-mouse CD3 (Biolegend, Cat# 100217, Clone 17A2), PE anti-mouse γδTCR (Biolegend, Cat# 118108, Clone GL3), Brilliant Violet 605 anti-mouse CD4 (Biolegend, Cat# 100548, Clone RM4-5), Fluorescein isothiocyanate (FITC) anti-mouse CD8 (Biolegend, Cat# 100706, Clone 53 − 6.7), PerCP/Cyanine5.5 anti-mouse CD90.2 (Biolegend, Cat# 105338, Clone 30-H12), and PE anti-mouse IL-27Rα Antibody (Biolegend, W16125D).

Histology and immumohistochemistry staining

Mouse skin tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. For histology, the sections were stained with hematoxylin and eosin (H&E), and images were obtained using a Zeiss AxioScope microscope.

For immunohistochemical analysis of mouse TNF-α and Ki67 expression, paraffin-embedded sections were deparaffinized in xylene and rehydrated before undergoing antigen retrieval by boiling in a 10 mM Citrate buffer (pH 6.0), and treated with 3% H₂O₂ to inactivate intrinsic peroxidases. After blocking with 3% bovine serum albumin (BSA) for one hour at room temperature (RT), the sections were incubated overnight at 4 °C with a rabbit anti-mouse TNF-α polyclonal antibody (1:500, Servicebio, Cat# GB115702-100) or a mouse anti-Ki67 mono clonal antibody (1:500, Servicebio, Cat# GB121141-100). Following PBS washes, the sections were incubated for one hour at room temperature with rabbit anti-mouse IgG. The secondary antibody was visualized using DAB, and nuclei were counterstained with hematoxylin. For immunohistochemical analysis of mouse Filaggrin, Keratin 1 and p-STAT1, paraffin-embedded sections were deparaffinized in xylene and rehydrated before undergoing antigen retrieval by boiling in an EDTA buffer (1 mM EDTA containing 0.05% Tween 20, pH 9.0). The sections were then treated with H₂O₂ for 10 min, followed by permeabilization with 0.5% Triton X-100 for 15 min, and washed with PBS. Blocking was performed in 3% BSA in PBS at RT for 1 h, after which the sections were incubated overnight at 4 °C with primary antibodies. The following day, a two-color fluorescence kit (Recordbio Biological Technology, Shanghai, China) was used to stain the sections based on tyramide signal amplification technology, according to the manufacturer’s instructions, then nuclei were stained with DAPI (1:1000, Cell Signaling Technology, Cat# 4083). Finally, the fluorescence-stained sections were mounted with ProLong Gold Antifade reagent and visualized under a Leica microscope. The primary antibodies used were Rabbit anti-Filaggrin (1:1000, Abcam, Cat# ab81468), Rabbit anti-Cytokeratin 1 (1:1000, Abcam, Cat# ab185628) and Rabbit anti-p-STAT1 (1:200, Cell Signaling Technology, Cat# 9167).

RNA extraction and real-time quantitative PCR (RT-qPCR)

RNA was isolated by using GeneJET RNA Purification Kit (Thermo Scientific, Cat# K0732) according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Cat# R212-01) per manufacturer’s instructions. RNA expression levels were detected by using HiScript II Q RT SuperMix for qPCR (Vazyme, Cat# R222-01) per manufacturer’s instructions. Primer sequences were listed in Table S1.

Epidermis separation

Dorsal skin samples were taken from adult mice and incubated with 3 mg/ml Dispase II (Thermo Fisher Scientific, Cat# 17105041) for 16 h at 4 °C. Following this incubation, the epidermis was separated from the dermis. RNA was then extracted from the epidermis for subsequent analysis.

In vivo IL-27 antibody and TNF-α treatment

For IL‑27 blockade, mice were injected intraperitoneally with 500 µg of IL‑27‑neutralizing antibody (BioXcell, Cat# BE0326, Clone MM27.7B1) or control IgG (BioXcell, Cat# BE0085) on day 0, followed by 200 µg on the indicated days [43]. For TNF‑α experiments, mice received daily intradermal injections of 200 ng recombinant murine TNF‑α (GenScript, Cat# Z03333) in 20 µl PBS for ear treatments or 100 µl PBS for dorsal skin treatments, with PBS alone administered as a volume‑matched control [44].

RNA sequencing and data analysis

For RNA sequencing (RNA-seq), Ribo-Zero Gold (Illumina) was utilized for cDNA library construction. The resulting cDNAs were amplified via PCR to construct the library. The libraries were validated using the Agilent 2100 bioanalyzer and sequenced on the NovaSeq 6000 (Illumina). In our RNA-seq analysis, differentially expressed genes (DEGs) were identified using the following criteria: an absolute fold change of ≥ 1.5 and a significance level of p < 0.05. Pathway enrichment analysis was performed using Metascape, accessible at https://metascape.org [45]. The raw sequence data have been deposited in the Genome Sequence Archive [46] in National Genomics Data Center [47], China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA033948) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Public RNA-seq datasets related to psoriasis [GDS4602 [48] and GDS5392 [49], allergic contact dermatitis [GDS2935 [50] and AD [GDS4491 [51] and GDS4444 [52] were analyzed with the online tool GEO2R. Additionally, public RNA-seq data of IL-27-treated NHEKs [GSE188242 [24] were analyzed using an online platform for data analysis and visualization, accessible at https://www.bioinformatics.com.cn [53].

Human keratinocyte culture

In this study, NHEKs were obtained from ATCC (Cat# PCS-200-010) and cultured in DermaCult™ Keratinocyte Expansion Medium (STEMCELL Technologies, Cat# 100–0500). Cells at passages 3 to 5 were used for subsequent experiments. The immortalized human keratinocyte cell line HaCaT was maintained in Dulbecco’s Modified Eagle Medium (Gibco, Cat# 10567014) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml), and streptomycin (100 µg/ml) under standard culture conditions at 37 °C with 5% CO2. The cells were treated with recombinant human IL-27 (100 ng/ml, PeproTech, Cat# 200 − 38) for the specified duration. To inhibit STAT1 activity, the cells were pre-treated with 5 µM Fludarabine (MedChemExpress, Cat# HY-B0069) for 1 h prior to the addition of IL-27.

Western blotting

To assess the effects of Fludarabine and Stattic on the inhibition of IL-27-induced STAT1 phosphorylation, HaCaT cells were treated with either 5 µM Fludarabine (MedChemExpress, Cat# HY-B0069), 10 µM Stattic (MedChemExpress, Cat# HY-13818), or DMSO for 24 h. IL-27 (100 ng/ml, PeproTech, Cat# 200 − 38) was added during the last 15 min of the treatment period. Protein extraction and Western blotting were performed as described previously [41]. Phospho-STAT1 antibody (Cell Signaling Technology, Cat# 9167, 1:1000) was used.

Statistical analysis

The statistical significance was assessed by two-tailed unpaired Student’s t-test, one-way ANOVA or two-way ANOVA as required. All analysis was performed with GraphPad Prism software. Significant differences were considered when p < 0.05.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AD

Atopic dermatitis

IL

Interleukin

EBI3

Epstein-Barr virus-induced 3

STAT

Signal Transducer and Activator of Transcription

IMQ

Imiquimod

KO

Knockout

WT

Wild-type

PASI

Psoriasis Area and Severity Index

RT-qPCR

Real-Time quantitative PCR

ACD

Allergic contact dermatitis

DNFB

1-fluoro-2,4-dinitrobenzene

TNF

Tumor necrosis factor

PBS

Phosphate buffered saline

H&E

Hematoxylin and eosin

BSA

Bovine serum albumin

RNA-seq

RNA sequencing

Author contributions

Y.S., Q.Y. and J.G. designed and supervised the study. Z.C., L.C., Z.L., S.L., N.Y., S.L., Z.Z., J.C., Y.W., T.L., Y.Y., J.L., X.Z. and C.G. performed the experiments. Z.C., L.C. and Z.L. drafted the manuscript. Z.C., L.C., Z.L., Y.S., Q.Y. and J.G. analyzed the data and corrected the manuscript. All authors approved the final manuscript.

Funding

This work was supported by National Key Research and Development Program of China (2023YFC2508106), National Natural Science Foundation of China (No. 82430101, 82273510, 82173405, 82473517), Innovation Program of Shanghai Municipal Education Commission (2025GDZKZD06), Shanghai Dermatology Research Center (2023ZZ02017), Clinical Research Plan of SHDC (No. 22022302), Shanghai Science and Technology Development Funds (24YF2738100), and Shanghai Pujiang Program (24PJA113).

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. Request for materials and correspondence should be addressed to Y.S. (shiyuling1973@tongji.edu.cn), Q.Y. (yuervictory@163.com) and J.G. (gujun79@163.com).

Declarations

Ethics approval and Consent for participate

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Skin Disease Hospital.

Competing interests

The authors declare no competing interests.