All punch biopsies of patients with different inflammatory skin disorders (N = 20) were taken for diagnostic purposes from active skin lesions (CDLE, n=5; SCLE, n=5; atopic dermatitis, n=5; psoriasis, n=5). Biopsies of safety margin skin tumor surgery remnants were used as healthy controls (n=5). Skin samples were fixed with 4% formalin overnight and proceeded for histology and immunohistochemistry. The study was performed in accordance to the principles of the Declaration of Helsinki and approved by the local ethics committee in Bonn (BN 09004). The patients provided written informed consent to participate in this study.

In addition, we analyzed data from Gene Expression Omnibus (GEO GSE280220) including gene-expression profiles of 19 lesional CLE skin biopsies (CDLE and SCLE) and 8 controls (healthy skin) (9).

The biopsy samples were subjected to hematoxylin and eosin (H&E) staining to facilitate diagnostic evaluation, which was conducted by a board-certified dermatopathologist (JW). The three-dimensional epidermis models (MatTek) were stained with H&E as well. Immunohistochemistry was conducted with DAKO-Omnis using the Envision staining system (Agilent, Glostrup, Denmark). The staining intensity was assessed on a scale (0=zero, 1=low, 2=moderate, 3=strong) as described before (10). For the evaluation of these staining results, Group differences were assessed using the nonparametric Kruskal–Wallis test, followed by Dunn’s post hoc test with correction for multiple comparisons. One-tailed p-values < 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism (version 9.5). The specific antibodies used were those directed towards IFNAR1 (AA 28-227, antikoerper-online.de), CXCL10 (Abcam, ab9807, Cambridge, United Kingdom), and MxA (M143, Prof. Haller, Freiburg).

In vitro experiments utilized immortalized keratinocytes HaCaT (CLS Cell Line Service GmbH, Eppelheim, Germany), N/TERT (provided by collaborators at Biomedical Center II, Bonn), and a 3D epidermis model, constructed from normal epidermal human keratinocytes (=NHEK: EPI-200/EPI-212) from MatTek Life Sciences Biotechnology Company, Bratislava, Slovak Republic. Cell cultivation followed the protocols provided by the respective companies. Cultivation conditions were maintained at 36.6°C, 5% CO₂ concentration in the air, 95% relative humidity, and 21% oxygen content.

For the HaCaT cell line, DMEM medium supplemented with 10% FCS and 5% PBS was used. N/TERT keratinocytes were cultured in Keratinocyte-SFM medium, which contained all necessary supplements. Cells were split at 80% confluence using Trypsin-EDTA for HaCaT cells and Accutase or N/TERT cells, following the manufacturer’s protocol (all reagents purchased from Thermo Fisher, Waltham, USA).

During the experiments, cells were first treated with the IFN-α receptor blocker anifrolumab (AstraZeneca, Cambridge, UK) at a target concentration of 10 µg/ml and then incubated for 1 h. Subsequently, the cells were treated with various stimuli: 1µg/ml IFN-α (PeproTech, Hamburg, Germany), 10 µg/ml PolyIC (InvivoGen, San Diego, USA), 1 µg/ml PolydAdT (InvivoGen, San Diego, USA), and 12.5 µg/ml eNA extracted from the N/TERT cell line using the NucleoSpin-Kit (Macherey-Nagel, Dueren, Germany). For transfection, a volume of 12.5 µl/ml Lipofectamine 2000 (Invitrogen, Carlsbad, USA) was used to ensure intracellular delivery of PolydAdT and eNA. Cells were then incubated for 24 hours at 37°C. All experiments were implemented in biological triplicates.

After 24 hours, an enzyme-linked immunosorbent assay for human CXCL10 was performed using a CXCL10 DuoSet ELISA (DY266, R&D Systems, Minneapolis, USA), following the company’s protocol. Measurements were taken with the Synergy HT Multi-Detection Microplate Reader (BioTek, Winooski, VT, United States) and analyzed using Gen5 software (Version 1.11.5).

RNA was processed by the Next Generation Sequencing (NGS) Core Facility of the Medical Faculty of the University of Bonn using the QuantSeq 3’-mRNA Library Prep Kit by Lexogen. Illumina HiSeq 2500 was used for RNA sequencing (Standard 3’RNA seq with 50 cycles). NGS-gene expression was analyzed with Subio™ using Welch’s t-test. Statistical analysis of ELISA-analyses were performed with GraphPad prism software (version 9.5) using one-sided Kruskal-Wallis test and Mann-Whitney U-test. Analysis of GEO-based gene-expression data was performed using the nSolver platform from Nanostring/Bruker™ and Welch’s t-test (https://nanostring.app.box.com). Confidence intervals were determined at 95%. P < 0.05 was considered as “significant” (*), p < 0.01 as “highly significant” (**), p < 0.1 as “tendency (TD)”. KEGG and Reactome pathways were mapped to differentially expressed genes using DAVID v2024q2 (Database for Annotation, Visualization and Integrated Discovery) and EnrichR (https://maayanlab.cloud/Enrichr) based on Fisher’s exact test. Z-score was calculated using the following formula: (X=data point - μ=mean of the data set)/σ=standard deviation of the data set.

In the first step, immunohistochemistry was used to investigate the extent to which IFNaR1, the target of anifrolumab, is expressed by lesional keratinocytes in patients with CLE. Our analyses showed that both in CDLE and SCLE this receptor is not only expressed by infiltrating cells, but also strongly by epidermal keratinocytes. This mode of expression was significantly stronger than in patients with atopic dermatitis and healthy controls, while a moderate expression was also found in patients with psoriasis. High expression of the IFNaR1 was closely associated with a stronger lesional expression of the IFN-inducible proteins MxA and CXCL10 ( Figures 1B, C ).

In parallel we analyzed gene expression profiles of lesional CLE skin biopsies focusing on the IFN-pathway. The analyses revealed a strong activation of the IFN-associated signaling pathways. Importantly, these analyses supported our immunohistological data with CXCL10 (239-fold) and MxA (also known as Mx1: 35-fold) being strongly upregulated in CLE versus healthy control ( Figure 1D ). The top 10 most upregulated genes also included other ligands of CXCR3 (CXCL9 and CXCL11) and other proinflammatory cytokines (CXCL13, CCL8) as well as typical IFN-regulated genes (ISG15, IDO1, OASL) and GZMB, an IFN-regulated cytotoxic marker. Figure 1E details the expression of typical IFN-associated markers, including upregulation of the IFNαβ-receptors in individual patients. In reactome analyses “Interferon-αβ-Signaling” was among the top 20 activated pathways. These analyses reflected the strong activation of immune pathways of the innate (“Innate Immune System”, Cytokine Signaling”, “Toll-like Receptor Cascades”, “Interferon Signaling”, “Interleukin-1 Family Signaling “, “Neutrophil Degranulation”) and the adaptive immune system (“Adaptive Immune System”, “Immunoregulatory Interactions Between a Lymphoid and a non-Lymphoid Cell”) in parallel ( Table 1 ).

The functional analyses of the efficacy of anifrolumab were initially carried out in a HaCaT cell culture. Stimulation was performed with synthetic immunostimulatory nucleic acids (DNA: polydAdT, RNA: polyIC), which we had already established for an in vitro model of CLE. Ruxolitinib, a selective JAK1/2 inhibitor which was established in our in vitro system in earlier studies (11), served as a positive control for the inhibition. The studies showed that anifrolumab significantly inhibited the expression of the proinflammatory cytokine CXCL10 after stimulation to a level almost comparable to the inhibitory effect of ruxolitinib ( Figures 2A, B ).

In the next step, we carried out analyses in the 3D keratinocyte model with human cells. Stimulation was performed with the stimulatory RNA polyIC and with recombinant interferon alpha. Our immunohistochemical analyses showed that in this model, anifrolumab was able to significantly reduce both the intraepidermal expression of type I/III-interferon-inducible MxA and the expression of the IFN-regulated proinflammatory cytokine CXCL10 ( Figures 2C–E ). A similar picture was seen in the cell supernatants: Here, treatment with anifrolumab resulted in a highly significant reduction in CXCL10 expression ( Figure 2F ).

In the final step, the efficacy of anti-IFNaR1 therapy on keratinocytes was confirmed in a further approach. For this purpose, N/TERT cells were stimulated with extracted endogenous nucleic acids, which are an established model for the in vivo activation of keratinocytes in CLE skin lesions (12), and then treated with anifrolumab. Stimulation with IFNα and polyIC was carried out in parallel. Here, a significant inhibition of CXCL10 by anifrolumab was shown in all three investigated systems ( Figures 3A–C ).

Finally, next-generation sequencing (NGS) analyses were performed to confirm our earlier results. These analyses showed that anifrolumab significantly inhibits the activation of IFN-associated proinflammatory genes and their pathways in stimulated keratinocytes. In the Reactome analysis, the top regulated pathways (depicted in Figure 3D ) were “Interferon Signaling” (R-HSA-913531), “Cytokine Signaling in Immune system” (R-HSA-1280215), “Interferon Gamma Signaling” (R-HSA-877300), “Interferon Alpha/Beta Signaling” (R-HSA-909733), and “Immune System” (R-HSA-168256). These pathways include several CLE-typical proinflammatory factors, including IRF7, IFITM1, IFITM2, IFNL2/IL28a and CXCL10, which were downregulated by anifrolumab treatment. Interestingly, when comparing these data with the previously identified top 20 CLE pathways, there was a high degree of consistency in anifrolumab inhibiting these CLE-typical pathways, even though only the effect of the drug on keratinocytes was investigated here ( Table 1 ).

Subsequently a network-analysis supported the central role of CXCL10 within the proinflammatory pathways downregulated by anifrolumab ( Figure 3E ).