biomedicines Article Thalidomide Exerts Anti-Inflammatory Effects in Cutaneous Lupus by Inhibiting the IRF4/NF-ҡB and AMPK1/mTOR Pathways Sandra Domingo 1 , Cristina Solé 1, * , Teresa Moliné 2 , Berta Ferrer 2 and Josefina Cortés-Hernández 1 1 Lupus Unit, Rheumatology Departament, Hospital Universitari Vall d’Hebron, Institut de Recerca (VHIR), Universitat Autonoma de Barcelona, 08035 Barcelona, Spain; sandra.domingo@vhir.org (S.D.); fina.cortes@vhir.org (J.C.-H.) 2 Department of Pathology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; teresa.moline@vhir.org (T.M.); bferrer@vhebron.net (B.F.) * Correspondence: cristina.sole@vhir.org; Tel.: +34-93-489-4045 Abstract: Thalidomide is effective in patients with refractory cutaneous lupus erythematosus (CLE). However, the mechanism of action is not completely understood, and its use is limited by its potential, severe side-effects. Immune cell subset analysis in thalidomide’s CLE responder patients showed a reduction of circulating and tissue cytotoxic T-cells with an increase of iNKT cells and a shift towards a Th2 response. We conducted an RNA-sequencing study using CLE skin biopsies performing a Therapeutic Performance Mapping System (TMPS) analysis in order to generate a predictive  model of its mechanism of action and to identify new potential therapeutic targets. Integrating  RNA-seq data, public databases, and literature, TMPS analysis generated mathematical models Citation: Domingo, S.; Solé, C.; which predicted that thalidomide acts via two CRBN-CRL4A- (CRL4CRBN ) dependent pathways: Moliné, T.; Ferrer, B.; IRF4/NF-ҡB and AMPK1/mTOR. Skin biopsies showed a significant reduction of IRF4 and mTOR Cortés-Hernández, J. Thalidomide in post-treatment samples by immunofluorescence. In vitro experiments confirmed the effect of Exerts Anti-Inflammatory Effects in thalidomide downregulating IRF4 in PBMCs and mTOR in keratinocytes, which converged in an Cutaneous Lupus by Inhibiting the NF-ҡB reduction that led to a resolution of the inflammatory lesion. These results emphasize the IRF4/NF-ҡB and AMPK1/mTOR anti-inflammatory role of thalidomide in CLE treatment, providing novel molecular targets for Pathways. Biomedicines 2021, 9, 1857. https://doi.org/10.3390/ the development of new therapies that could avoid thalidomide’s side effects while maintaining biomedicines9121857 its efficacy. Academic Editor: Anna Campanati Keywords: cutaneous lupus; thalidomide; mechanism of action; new therapy Received: 26 October 2021 Accepted: 6 December 2021 Published: 7 December 2021 1. Introduction Cutaneous Lupus Erythematosus (CLE) is common and encompasses a wide range Publisher’s Note: MDPI stays neutral of dermatologic manifestations. As many as 70–80% of patients can develop skin lesions, with regard to jurisdictional claims in which can be an early sign of systemic involvement [1,2]. CLE can be classified into published maps and institutional affil- specific and non-specific lesions, of which discoid lupus erythematosus (DLE) and subacute iations. cutaneous lupus erythematosus (SCLE) are the most prevalent forms [3]. Early effective treatment may resolve the lesions, but delayed or inadequate treatment can result in permanent scarring, especially in DLE [4]. First-line therapies for CLE are antimalarial agents and/or topical steroids, together Copyright: © 2021 by the authors. with sun protection [5]. Although most patients respond to this regimen, 30 to 40% of Licensee MDPI, Basel, Switzerland. cases will be refractory [6]. For this minority, there is no consensus treatment algorithm, This article is an open access article and several systemic agents have shown a variable response [7]. Thalidomide, a glutamic distributed under the terms and acid derivative with immunomodulatory and anti-inflammatory effects, has been used suc- conditions of the Creative Commons cessfully in several oncological and chronic inflammatory dermatological conditions [8,9]. Attribution (CC BY) license (https:// First prescribed for refractory CLE in 1975 [10], thalidomide use has increased following its creativecommons.org/licenses/by/ reported efficacy, reaching 80–90% [6,10,11]. However, large-scale clinical trials are lacking, 4.0/). Biomedicines 2021, 9, 1857. https://doi.org/10.3390/biomedicines9121857 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 1857 2 of 18 and serious adverse events such as teratogenicity, neurotoxicity, and thrombosis restrict its use [12–15]. Although thalidomide’s mechanism of action (MoA) has been studied, little is known of the molecular basis of its immunomodulatory effect in CLE. In vitro studies have demon- strated that thalidomide inhibits neutrophil chemotaxis, phagocytosis, angiogenesis, and production of tumour necrosis factor alpha (TNF-α). It also interacts with the T-helper response and regulation of transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ҡB) [16–19]. In the absence of an effective and safe treatment, a better understanding of thalidomide’s MoA can help to identify key target molecules for the development of new therapeutic agents. Genome-wide gene expression profiling is increasingly used to investigate patho- genetic mechanisms and identify potential disease biomarkers [20]. RNA sequencing (RNA-seq) is a widely used method to study overall transcriptional activity. RNA-seq is a powerful investigative tool using transcriptome changes as a proxy for drug effect and has led to the discovery of potential biomarkers in several diseases, yet standard library construction is costly [21–23]. Using a mathematical model including the comparative RNA-sequencing data, we identified different molecular signalling signatures that provide novel insights into thalidomide’s MoA and potential therapeutic targets. 2. Materials and Methods 2.1. Patients Six-millimetre punch skin biopsies for RNA-seq and blood samples for peripheral blood mononuclear cells (PBMCs) isolation were taken from 10 patients with active CLE, before and 4 weeks after thalidomide treatment (Table S1; see Supplementary Materials). CLE diagnosis and classification was based on clinical and histological criteria according to the 2004 Dusseldorf classification [24]. Disease activity was assessed by the validated modified CLE Disease Area and Severity Index (CLASI) [25]. The study was approved by the Vall d’Hebrón Ethics Committee and informed consent was obtained from all subjects. 2.2. RNA-Seq and System Biology Analysis Whole skin RNA was extracted following the protein and RNA Isolation kit’s instruc- tions (Thermofisher Scientific, Waltham, MA, USA). For library construction, total RNA (1 µg) was used following the Illumina TruSeq™ RNA Sample Prep Kit (Illumina, San Diego, CA, USA) manufacturer’s instructions. The resulting libraries were subjected to Illu- mina Hiseq 2000 sequencing platform version 3, producing 2 × 75 bp run with >65 M reads (Illumina, San Diego, CA, USA). Sequences were analyzed for quality control (FASTQC) and aligned to the Human genome (GRCh38) with STAR program V2.5.2a [26,27]. Sequenc- ing reads were processed using the RSEM program (version 1.2.28) [28] and differential expression calculated by DESeq2 [29]. Data are available from Gene Expression Omnibus (GSE162424). We generate models to predict thalidomide’s MoA by a Therapeutic Perfor- mance Mapping System (TMPS) approach (Anaxomics Biotech, Barcelona, Spain). TPMS combines RNA-seq data with a complete characterization of CLE/Thalidomide using biological information from KEGG, Binding Database, BioGRID, REACTOME, Pubmed, Drug Bank, Stich and Supertarget [30–37] (See Supplementary Materials). 2.3. Flow Cytometry PBMCs cell phenotype was analyzed by seven-colour flow cytometry (LSR Fortessa, BD Biosciences, Franklin Lakes, NJ, USA). For cell surface staining, conjugated monoclonal antibodies were used (BD Biosciences) (Table S2; see Supplementary Materials). Isotype controls were used for gate setting. Data were analyzed using FCS Express 4 Flow Research software (BD Biosciences, Erembodegem, Belgium). Biomedicines 2021, 9, 1857 3 of 18 2.4. Immunofluorescence and Immunohistochemistry Immunohistochemistry (IHC) and immunofluorescence (IF) were performed as de- scribed on paraffin-embedded and frozen sections, respectively [37,38] using purified monoclonal antibodies listed in Table S3. Stained samples were evaluated by two blinded dermatopathologists and cell counts were quantified using Image J V1.42 (see Supplemen- tary Materials). 2.5. In Vitro Ubiquitination Assay Recombinant Human CRBN + DDB1 + CUL-4A + RBX1 (Abcam, Cambridge, UK) (1 µM), Human AMPK1 Fisher (ThermoFisher, Waltham, MA, USA) (1.5 µM) and Thalido- mide (100 µM) were used with the E3 Ligase Auto-Ubiquitilylation Assay Kit (Abcam, Cambridge, UK) following the manufacturer’s instructions. Reactions were incubated at 37 ◦ C for 2 h before separation by SDS–PAGE followed by western blot analysis. 2.6. Co-Immunoprecipitation for Cell-Based Ubiquitination Assay Epidermal keratinocytes were stimulated with UVB for 6h and then treated with thalidomide. After 24 h, cells were washed twice with PBS and lysed with RIPA buffer (Sigma Aldrich, St. Louis, MI, USA) together with protease inhibitor cocktail (Sigma Aldrich, St. Louis, MI, USA). After centrifugation at 10,000 rpm for 15 min, supernatant was collected. Concurrently, Dynabeads™ Protein G for Immunoprecipitation (Invitrogen, Waltham, MA, USA), were washed and incubated with anti-AMPK antibody (1:250) (Abcam, Cam- bridge, UK) in PBS 0.02% Tween™ 20 for 15 min at room temperature in order to obtain the Antibody-bead complex. Then, the mix was incubated with the obtained supernatant from the cell lysis. After 1 h at room temperature, the antibody-bead-AMPKprotein com- plexes were obtained. Finally, AMPK protein was eluted with elusion buffer (50 mM glycine pH 2.8) for 2 min at room temperature. Supernatants were subjected to western blot analysis for AMPK and ubiquitin protein analysis. 2.7. Protein Extraction and Western Blot Skin protein samples were obtained using the PARIS kit following manufacturer’s instructions (see Supplementary Materials). Protein concentrations were determined using the BCA protein assay kit (Bio-Rad, Hercules, CA, USA). Then, 50 µg of protein was loaded into 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA) by Semi-Dry Electrophoretic Transfer (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% BSA (RT, 1 h) followed by overnight incubation (4 ◦ C) with specific primary antibodies (Abcam, Cambridge, UK, Table S3). Secondary HRP-labelled antibodies were added (1:500) and visualized using ECL Detection System (Santa Cruz Biotechnology, Dallas, TA, USA). 2.8. RNA Extraction and RT-qPCR RNA from cultured lysed cells was obtained with RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was transcribed into cDNA with High-Capacity cDNA Reverse Transcrip- tion Kit (Applied Biosystems, Foster City, CA, USA). Gene expression was assessed by TaqMan assays (Applied Biosystems, Foster City, CA, USA) (Table S4; see Supplementary Materials). 2.9. Proliferation Assays Proliferation assays were performed using CyQUANT NF Cell Proliferation Assay Kit (Invitrogen), following manufacturer’s instructions. 2.10. Cell Culture Human epidermal adult Keratinocytes (HEKa) were cultured in EpiLife serum-free media with Human Keratinocyte Growth Supplement (Life Technologies, Carlsbad, CA, Biomedicines 2021, 9, 1857 4 of 18 USA) and isolated PBMCs from healthy volunteers (Vacutainer CPT, BD Biosciences) in RPMI medium (Life Technologies, Carlsbad, CA, USA; see Supplementary Materials). 2.11. Gene Silencing Third passage cultured cells at 30–50% confluence were transfected with interferon regulatory factor 4 (IRF4) or mechanistic target of rapamycin (mTOR) small interfering RNA (siRNA, ThermoFisher) or a silencer negative control (ThermoFisher, AM4615) using the lipofectamine CRISPRMAX Cas9 Reagent following the manufacturer’s instructions (ThermoFisher, Waltham, MA, USA). After 24 h, cells were treated with TNF-α (10 ng/mL) or U.V for 6 h and analyzed by qPCR-RT or immunofluorescence. 2.12. Co-Culture Experiments Co-cultures were performed in modified 24-well plates with cell culture inserts (0.4-µm pore; BD Biosciences, Franklin Lakes, NJ, USA). HEKa cells were cultured at the bottom overnight. Isolated healthy donor PBMCs, stimulated with TNF-α for 6 h, were placed in the upper part of the insert and treated with thalidomide (100 ng/mL). After 24 h, the insert was removed, and HEKa cells were analyzed by immunofluorescence or RT-qPCR. 2.13. Statistical Analysis Data are represented as mean ± SEM. Comparison between groups and differential gene expression was calculated with paired or unpaired t-tests as applicable using Prism GraphPad (GraphPad Software, v7.0, San Diego, CA, USA). p values ≤ 0.05 were considered statistically significant. RT-qPCR analysis was calculated using Fold Change differences with 2− ∆∆Ct method. 3. Results 3.1. Immunoregulatory Effects in CLE Peripheral Blood and Skin Ten thalidomide-treated CLE patients were included (Table S1) (see Supplementary Materials). Seven (70%) achieved clinical remission (CLASI = 0). Following treatment, responder patients had a reduction in peripheral cytotoxic CD8+ T-cells (p = 0.044) and an increase of iNK T-cells (p = 0.006) (Figure 1a). iNK T-cell related cytokines were not different after treatment; however we observed a tendency to decrease granulate cytokines (perforin A and granzyme B) in post-treatment samples (Figure S1). No significant changes in CD4+ T percentages or in dendritic cells, B-cells, or T regs were observed. Analysis of distinct Th subsets showed a skew towards a Th2 response (p = 0.018) (Figure 1b). The immunohistochemical study results of the skin infiltrating cells mirrored the ones observed in peripheral blood with a significant reduction in the number of CD8+ T-cells (p = 0.013, Figure 1c) and an increase in iNK T-cells post-thalidomide (p = 0.004, Figure 1d). 3.2. RNA-Sequencing with Therapeutic Performance Mapping System (TMPS) Analysis Revealed Thalidomide’s Mechanisms in CLE We first identified relevant proteins in CLE pathogenesis and thalidomide through the analysis of published biological information that allowed us to establish the protein network (Tables S5 and S6; Figures S2 and S3). To obtain further insight into the molecular basis of CLE, we performed an RNA-seq. Comparative analysis of the skin RNA sequenc- ing of thalidomide responder patients revealed 448 differentially expressed transcripts, of which 339 were protein coding genes (|log2(FC)| ≥ 1; Adj. p value < 0.05, data avail- able GSE162424). To construct the Thalidomide’s MoA, the RNA-seq data was used to restrict the models (see Supplementary Materials). Finally, we identified twenty-seven differential molecules of which 14 were CLE effectors (Table S7, Figure 2). In our model, we found thalidomide to act by two CRL4CRBN -dependent mechanisms: (a) downregu- lating IRF4 leading to an inhibition of the NF-ҡB signalling pathway; and (b) regulating AMPK1/mTOR signalling pathway (Figure 2). Biomedicines 2021, 9, 1857 5 of 18 Biomedicines 2021, 9, 1857 5 of 19 Figure1.1.Thalidomide Figure Thalidomideameliorates amelioratesskin skininflammation inflammationby bydecreasing decreasingCD8+ CD8+ TT cells, cells, increasing increasing iNK-Tcells iNK-Tcellsandandpromoting promotingaa Th2response Th2 responseinin CLE. CLE. (a)(a) Flow Flow cytometry cytometry percentages percentages of T of T helper helper (CD3+CD4+), (CD3+CD4+), T cytotoxic T cytotoxic cells (CD3+CD8+) cells (CD3+CD8+) and iNKand iNK Tcells Tcells (CD3+6B11+) (CD3+6B11+) in PBMCsin PBMCs of responder of responder patientspatients (n = 7)(n = 7) before before andthalidomide and after after thalidomide treatment. treatment. (b) Post-thalidomide, (b) Post-thalidomide, CLE CLE patients had lower percentages of Th1 (CCR5+ CXCR3+) T cells and higher percentages of Th2 (CCR4+CCR3+). (c) patients had lower percentages of Th1 (CCR5+ CXCR3+) T cells and higher percentages of Th2 (CCR4+CCR3+). (c) Skin Skin immunohistochemistry to evaluate infiltrating CD4+ and CD8+ in skin biopsies of CLE. Graphs represent the aver- immunohistochemistry to evaluate infiltrating CD4+ and CD8+ in skin biopsies of CLE. Graphs represent the average signal age signal intensity (n = 3). (d) Immunofluorescence of post-treatment skin samples showed a significant increase of iNK intensity (n = 3). Tcells (6B11+ (d) Immunofluorescence cells). Scale bar = 200mm. * of p