MDA5+ Dermatomyositis Is Associated with Stronger Skin Type I Interferon Transcriptomic Signature with Upregulation of IFN-κ Transcript

Dermatomyositis (DM) is a chronic inflammatory and autoimmune disorder belonging to the idiopathic inflammatory myopathies affecting primarily skin and muscle with variable extent. Disease mechanism comprehension is growing, with a pivotal role of both the innate and adaptive immune system and involvement of both cellular and humoral actors (Nagaraju and Lundberg, 2011Nagaraju K. Lundberg I.E. Polymyositis and dermatomyositis : pathophysiology.Rheum Dis Clin North Am. 2011; 37: 159-171Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The immune transcriptomic signature of DM is characterized by a type I IFN signature in peripheral blood mononuclear cells, muscle, and skin, which has been shown to correlate with disease activity (Baechler et al., 2011Baechler E.C. Bilgic H. Reed A.M. Type I interferon pathway in adult and juvenile dermatomyositis.Arthritis Res Ther. 2011; 13: 249Crossref PubMed Scopus (88) Google Scholar, Walsh et al., 2007Walsh R.J. Kong S.W. Yao Y. Jallal B. Kiener P.A. Pinkus J.L. et al.Type I interferon-inducible gene expression in blood is present and reflects disease activity in dermatomyositis and polymyositis.Arthritis Rheum. 2007; 56: 3784-3792Crossref PubMed Scopus (222) Google Scholar, Wong et al., 2012Wong D. Kea B. Pesich R. Higgs B.W. Zhu W. Brown P. et al.Interferon and biologic signatures in dermatomyositis skin: specificity and heterogeneity across diseases.PLOS ONE. 2012; 7e29161Crossref PubMed Scopus (116) Google Scholar); consequently, targeting type I IFN seems to represent a novel therapeutic approach in DM (Higgs et al., 2014Higgs B.W. Zhu W. Morehouse C. White W.I. Brohawn P. Guo X. et al.A phase 1b clinical trial evaluating sifalimumab, an anti-IFN-α monoclonal antibody, shows target neutralisation of a type I IFN signature in blood of dermatomyositis and polymyositis patients.Ann Rheum Dis. 2014; 73: 256-262Crossref PubMed Scopus (121) Google Scholar). Among autoantibodies, MDA5 antibodies are associated with a unique and severe mucocutaneous presentation and a peculiar systemic phenotype with lower incidence of myositis, a higher ferritin level, an elevated risk of interstitial lung disease, and higher mortality (Kurtzman and Vleugels, 2018Kurtzman D.J.B. Vleugels R.A. Anti-melanoma differentiation–associated gene 5 (MDA5) dermatomyositis: A concise review with an emphasis on distinctive clinical features.J Am Acad Dermatol. 2018; 78: 776-785Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Herein, we assessed the global gene expression, and more peculiarly the type I IFN signature, in the skin of three individuals with MDA5+ DM and compared it with five individuals with MDA5− DM and five healthy individuals (Supplementary Table S1). (Supplementary Materials and Methods). The study has been approved by institutional review boards and was conducted in accordance with the ethical and legal frameworks of the Declaration of Helsinki. Informed written consent was received from participants before inclusion in this study, according to our local ethic rules (CPP Paris 12). First, principal component analysis (unsupervised analysis) clustering using all 24,063 genes segregated healthy patients and patients with DM (Figure 1a and b) (Supplementary Figure S1). Using significance analysis of microarray (Tusher et al., 2001Tusher V.G. Tibshirani R. Chu G. Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci U S A. 2001; 98: 5116-5121Crossref PubMed Scopus (9648) Google Scholar), we identified 575 differentially expressed genes in DM compared with healthy skin. As expected and previously described (Wong et al., 2012Wong D. Kea B. Pesich R. Higgs B.W. Zhu W. Brown P. et al.Interferon and biologic signatures in dermatomyositis skin: specificity and heterogeneity across diseases.PLOS ONE. 2012; 7e29161Crossref PubMed Scopus (116) Google Scholar), upregulated genes and enrichment analysis pathway supported increased cellular infiltration of T cells (CD3D) and cytotoxic cells (GZMA and GZMH); activation of immune cells (HLA-DR); and upregulation of type I IFN pathway, RIG-I, IL2-STAT5, complement pathway, and natural killer cytotoxicity pathway in DM compared with healthy skin (Table 1). Gene expression of immune biomarkers previously described in the serum (Cassius et al., 2019Cassius C. Le Buanec H. Bouaziz J.D. Amode R. Biomarkers in adult dermatomyositis: tools to help the diagnosis and predict the clinical outcome.J Immunol Res. 2019; 2019: 9141420Crossref PubMed Scopus (11) Google Scholar) were also upregulated in the skin (IL15 [fold change (FC) = 2.27; q = 0.002], CXCL10 [FC = 12.55; q = 0.004], and CD163 [FC = 4.2; q = 0.002]), confirming their potential pathogenic role. Concerning skin physiology, apoptosis and epithelial mesenchymal transition pathways were upregulated, supporting the cytotoxicity of inflammatory infiltrate toward keratinocytes (Table 1).Table 1GSEA Analysis of Upregulated and Downregulated Genes in DMNameSizeESNESP-valueFDRGSEA analysis of upregulated genes in DM compared with healthy controlsH IFN-α response800.862.970.0000.000H complement1730.652.500.0000.000KEGG natural killer cell–mediated cytotoxicity1190.501.830.0000.005H inflammatory response1690.632.430.0000.000H epithelial mesenchymal transition1650.642.510.0000.000H apoptosis1330.471.750.0000.002GSEA analysis of upregulated genes in MDA5+ compared with MDA5− DMH IFN-α response800.742.710.0000.000H IFN-γ response1680.652.630.0000.000H TNF-α signaling via NF-κB1720.622.520.0000.000H inflammatory response1690.572.340.0000.000Abbreviations: DM, dermatomyositis; ES, enrichment score; FDR, false discovery rate; GSEA, gene set enrichment analysis; H, hallmark; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; TNF-α, tumor necrosis factor-α. Open table in a new tab Abbreviations: DM, dermatomyositis; ES, enrichment score; FDR, false discovery rate; GSEA, gene set enrichment analysis; H, hallmark; KEGG, Kyoto Encyclopedia of Genes and Genomes; NES, normalized enrichment score; TNF-α, tumor necrosis factor-α. Hierarchical clustering segregated MDA5+ from MDA5− DM, highlighting pathogeny differences and potential mechanisms of the distinctive clinical skin findings in this subgroup (Figure 1b). We therefore compared differentially expressed genes and pathways across MDA5+ versus MDA5− DM to identify contrasting elements. Considering genes that had a minimum FC of 2, the principal component analysis clustered independently according to the serology, indicating distinct molecular profiles (Figure 1a). By comparing MDA5+ versus normal skin and MDA5− versus normal skin, we found 305 genes differentially regulated in MDA5+ skin and 100 genes differentially regulated in MDA5− skin (Figure 1c). Among the most highly upregulated genes in MDA5+ skin were IFIT2, CXCL10, and IFIH1; the type I IFN pathway was also significantly upregulated (Table 1). To confirm the type I IFN signature in MDA5+ DM, we showed using MxA immunohistochemistry that MDA5+ DM skin samples expressed more MxA in the epidermis and in the inflammatory infiltrate than MDA5− skin samples (Figure 1d) (Supplementary Table S2). E-selectin gene expression was significantly upregulated in MDA5+ skin compared with MDA5− skin (FC = 11.3, q = 0.02), highlighting the intense vasculitis phenomenon, clinically and histologically observed in practice. Finally, to decipher the origin of the type I IFN signature in DM skin, we focused on both the cell origin of interferons and the type of interferons. Type I IFN signature can originate either from type I IFN (α, β, κ, ε, or ω), among which IFN-α is more specifically secreted by plasmacytoid dendritic cells and IFN-κ is more specifically secreted by keratinocytes, or from type III IFN (λ) (Li et al., 2018Li S.F. Gong M.J. Zhao F.R. Shao J.J. Xie Y.L. Zhang Y.G. et al.Type I interferons: distinct biological activities and current applications for viral infection.Cell Physiol Biochem. 2018; 51: 2377-2396Crossref PubMed Scopus (82) Google Scholar). The IFNK gene was the only interferon significantly elevated in MDA5+ skin (FC = 2.27, q = 0.03) compared with healthy skin (Figure 1e). In MDA5− compared with healthy skin, IFNK was elevated but not significantly (FC = 1.9, q = 0.25). Plasmacytoid dendritic cells express CD123 (or IL3Rα); CD123 gene expression was significantly elevated in DM samples and we observed the same tendency at the protein level by immunohistochemistry (Supplementary Table S2). To confirm the mRNA overexpression of the type I IFN signature and IFNK genes in patients with MDA5+ compared with patients with MDA5−, we analyzed 19 more formalin-fixed paraffin-embedded skin samples from a validation cohort (10 patients with MDA5+ and nine patients with MDA5−) using reverse transcriptase–PCR (Supplementary Materials and Methods). IFNK expression was significantly higher in the skin of patients with MDA5+ than patients with MDA5− (median: 57.6 vs. 0.7, P = 0.03); type I IFN signature was also higher in patients with MDA5+ than patients with MDA5− DM (median: 556 vs. 87, P = 0.02) (Supplementary Figure S2). We confirm here that DM skin involvement is associated with a specific transcriptomic signature comprising activation and infiltration of immune cells and a type I IFN signature. Previous work showed that, when studied in the muscle, IFN signature was higher in MDA5− compared with MDA5+ DM (Allenbach et al., 2016Allenbach Y. Leroux G. Suárez-Calvet X. Preusse C. Gallardo E. Hervier B. et al.Dermatomyositis with or without anti-melanoma differentiation-associated gene 5 antibodies: common interferon signature but distinct NOS2 expression.Am J Pathol. 2016; 186: 691-700Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Our data suggests that the type I IFN signature is expressed more strongly in the skin of MDA5+ than MDA5− DM. This discrepancy could be explained by the organ analyzed, as skin is the main organ affected in patients with MDA5+ DM. IFN-κ, a member of the type I IFNs, is induced following viral infection or treatment of cells with double-stranded RNA or other IFNs, and IFN-κ signaling induces a set of genes via the type I IFN receptor. Unlike other members of the type I IFNs, IFN-κ is highly expressed in keratinocytes (LaFleur et al., 2001LaFleur D.W. Nardelli B. Tsareva T. Mather D. Feng P. Semenuk M. et al.Interferon-κ, a novel Type I interferon expressed in human keratinocytes.J Biol Chem. 2001; 276: 39765-39771Crossref PubMed Scopus (206) Google Scholar, Scarponi et al., 2006Scarponi C. Nardelli B. Lafleur D.W. Moore P.A. Madonna S. De Pità O. et al.Analysis of IFN-kappa expression in pathologic skin conditions: downregulation in psoriasis and atopic dermatitis.J Interferon Cytokine Res. 2006; 26: 133-140Crossref PubMed Scopus (22) Google Scholar) and has been shown to play a pivotal role in the pathophysiology of skin involvement in systemic lupus erythematosus (Sarkar et al., 2018Sarkar M.K. Hile G.A. Tsoi L.C. Xing X. Liu J. Liang Y. et al.Photosensitivity and type i IFN responses in cutaneous lupus are driven by epidermal-derived interferon kappa.Ann Rheum Dis. 2018; 77: 1653-1664Crossref PubMed Scopus (97) Google Scholar, Stannard et al., 2017Stannard J.N. Reed T.J. Myers E. Lowe L. Sarkar M.K. Xing X. et al.Lupus skin is primed for IL-6 inflammatory responses through a keratinocyte-mediated autocrine Type I interferon loop.J Invest Dermatol. 2017; 137: 115-122Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Functional studies on the role of MDA5 antibodies that may modulate inflammatory signaling pathways in patients affected by MDA5+ syndrome in the skin or in the lung may be interesting. Our study corroborates a recent publication (Zhang et al., 2019Zhang S.H. Zhao Y. Xie Q.B. Jiang Y. Wu Y.K. Yan B. Aberrant activation of type I interferon system may contribute to the pathogenesis of anti-MDA5 dermatomyositis.Br J Dermatol. 2019; 180: 1090-1098Crossref PubMed Scopus (29) Google Scholar) and brings insights on the role of IFN-κ in the skin of patients with MDA5+. Our data support the potential role of keratinocytes and IFN-κ over IFN-α in the skin pathophysiology of MDA5+ DM. Datasets related to this article can be found at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128314 Charles Cassius: http://orcid.org/0000-0002-5087-4414 Reyhan Amode: http://orcid.org/0000-0003-1407-2606 Marc Delord: http://orcid.org/0000-0002-0455-6749 Maxime Battistella: http://orcid.org/0000-0002-7053-7431 Justine Poirot: http://orcid.org/0000-0002-6694-7796 Alexandre How-Kit: http://orcid.org/0000-0002-1584-9336 Clémence Lepelletier: http://orcid.org/0000-0003-3204-9194 Marie Jachiet: http://orcid.org/0000-0001-5286-9689 Adèle de Masson: http://orcid.org/0000-0001-7828-6211 Laure Frumholtz: http://orcid.org/0000-0002-3782-3890 Florence Cordoliani: http://orcid.org/0000-0002-0182-9262 David Boccara: http://orcid.org/0000-0002-7365-5943 Jacqueline Lehmann-Che: http://orcid.org/0000-0001-6685-3354 Jennifer Wong: http://orcid.org/0000-0002-1898-6539 Sylvie Dubanchet: http://orcid.org/0000-0003-1290-9708 Antonio José Alberdi: http://orcid.org/0000-0002-9371-1033 Marine Merandet: http://orcid.org/0000-0001-8397-8677 Martine Bagot: http://orcid.org/0000-0002-0400-1954 Armand Bensussan: http://orcid.org/0000-0002-0409-2497 Jean-David Bouaziz: http://orcid.org/0000-0002-4993-2461 Hélène Le Buanec: http://orcid.org/0000-0002-2139-0357 The authors state no conflict of interest. The authors thank Mylène Branchtein for her continuous help and support. The authors thank all medical staff from the dermatology department of Saint-Louis hospital and particularly Estelle Hau, Camille Salle de Chou, and Pauline Laly for their help in including patients. The authors are also grateful to Daniel Zagury for his wise advice and continuous support in their lab. The authors would like to thank Antoine Daunay (CEPH) and Valérie Schiavon (INSERM) for excellent technical assistance. Thanks also go to Cynthia Diziere, Gabrielle Larcheron, and Elisabeth Treillard for their valuable assistance. The authors thank all patients who accepted to be part of this study. Finally, the authors thank the Technological Core Facility of the Saint-Louis Research Institute (Institut de Recherche Saint-Louis), Université Paris-Diderot for continuous support. The Technological Core Facility is supported by grants from Conseil Régional d’Ile-de-France , Canceropôle Ile-de-France , Université Paris-Diderot , Association Saint-Louis , Association Jean-Bernard , Fondation pour la Recherche Médicale , French National Institute for Cancer Research (InCa), and Ministère de la Recherche . Conceptualization: CC, RA, J-DB, HLB; Data Curation: CC, RA, MD, JP, AH-K, AJA; Formal Analysis: CC, RA, MD, MBat, JP, AH-K; Funding Acquisition: MBag, AB, J-DB, HLB; Investigation: CC, RA, MBat, JL-C, JW, SD, AJA, MM; Project Administration: CC, RA, J-DB, HLB; Resources: MBat, CL, MJ, AdM, LF, FC, DB, MBag, J-DB, JL-C, JW; Software: MD, AH-K; Supervision: MBag, AB, J-DB, HLB; Validation: CC, RA; Visualization: CC, RA, MBat; Writing - Original Draft Preparation: CC, RA; Writing - Review and Editing: CC, RA, MD, MBat, JP, AH-K, CL, MJ, AdM, LF, FC, DB, JL-C, JW, SD, AJA, MM, MBag, AB, J-DB, HLB. This study enrolled 27 patients with dermatomyositis (Supplementary Table S1) recruited from the dermatology department of Saint-Louis Hospital, Assistance-Publique Hôpitaux de Paris, Paris, France. Diagnosis was made according to the European Neuromuscular Center criteria (Hoogendijk et al., 2004Hoogendijk J.E. Amato A.A. Lecky B.R. Choy E.H. Lundberg I.E. Rose M.R. et al.119thENMC international workshop: trial design in adult idiopathic inflammatory myopathies, with the exception of inclusion body myositis, 10-12 October 2003, Naarden, The Netherlands. Neuromuscul.Disord. 2004; 14: 337-345Scopus (618) Google Scholar, Sontheimer, 2002Sontheimer R.D. Would a new name hasten the acceptance of amyopathic dermatomyositis (dermatomyositis siné myositis) as a distinctive subset within the idiopathic inflammatory dermatomyopathies spectrum of clinical illness?.J Am Acad Dermatol. 2002; 46: 626-636Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). No patient received immunosuppressive drugs before sample collection. Frozen sections of lesional skin were obtained from liquid frozen stored diagnostic samples. Punch biopsies of normal (n = 5) skin were obtained from healthy subjects through fresh plastic surgery waste and stored in RNA later until RNA extraction. From a lesional dermal-epidermal skin biopsy, 20 frozen sections, each 10-μm-thick, were taken (samples included optimal cutting temperature compound preserved biopsies at −80 °C). Total RNA was extracted from frozen tissue samples using the RNeasy Mini Kit Plus protocol (Qiagen, Hilden, Germany). Healthy skin samples were prepared previously according to the following method: (i) single-use scalpel dissection into 2 × 2 × 2 mm fragments; (ii) immersion of the sample in lysis Buffer RLT (Qiagen) and betamercaptoethanol (10 μl/ml); and (iii) mechanical dissociation by oscillations (tissue lyser, Qiagen), parameterized at 50 Hz, on three sequences of 5 minutes. RNA quality was assessed by microfluidic capillary electrophoretic migration via the Caliper Labchip GX II analyzer (PerkinElmer, Waltham, MA). RNA quality score was 4.3 ± 1.1 for dermatomyositis and 8.84 ± 0.2 for healthy donors. Human genome U 2.0 gene chips (Affymetrix, Santa Clara, CA) were used for this study. After standardization according to the robust multiarray average method, quality control was performed by comparison between individuals of the average raw signal and the median absolute deviations. We excluded from this analysis noncoding RNAs. The bioinformatic used LIMMA for R (The R Foundation, Vienna, Austria). A first approach consisted of unsupervised hierarchical clustering. A second step performed a differential expression study between dermatomyositis and controls, according to the moderate Student test. The gene list and transcripts revealed were confronted with enrichment software in ontological and signaling functions, Molecular Signatures Database hallmark gene set collection/gene set enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathways. The pathways for which the false discovery rate q-value was < 0.05 were considered to be differentially enriched in a statistically significant manner. Gene nomenclature was apprehended via GeneBase. The results interpretation used the Transcriptome Analysis Console software suite (Affymetrix). The pathogenic pathways enrichment analysis used the Molecular Signatures Database platform. The analysis was ordered according to statistical significance (false discovery rate q-value < 0.05). Qlucore Omics Explorer was used to identify most differentially expressed genes and for the Venn diagram. From a lesional dermal-epidermal skin biopsy, 10 formalin-fixed paraffin-embedded sections, each 10-μm-thick, were taken. Total RNA was automatically extracted from tissue samples using the Maxwell RSC RNA FFPE kit protocol (Promega, Charbonnières-les-bains, France). cDNA was synthesized from total RNA using a reverse transcription kit PrimerScript RT reagent Kit with gDNA eraser (Takara Bio Europe SAS, Saint-Germain-en-Laye, France). RNA quantity and quality assessment were performed with a Nanodrop instrument (Thermo Fisher Scientific, Waltham, MA). The expression levels of six genes of the type I IFN signature and of the IFNK gene were tested by real-time quantitative PCR. Real-time quantitative PCR was carried out using the LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland). Primers used are the following: ISG15 (ISG15_F: CGTGCTGGTGGTGGACAA; ISG15_R: CTCGTAGGTGCTGCTGCG), IFIH1 (IFIH1_F: CCTCCTTCAGCCCACTCT; IFIH1_R: CAGCAGCAATCCGGTTTCT), RSAD2 (RSAD2_F: CCTGGATGTTGGTGTAGAA; RSAD2_R:GCAGACAATGGCAGTTAC), and CXCL10 (CXCL10_F: AGGTGCTATGTTCTTAGTGGATGT; CXCL10_R: GGAGGATGGCAGTGGAAGT). MX1, IFIT1, and IFNK were designed and obtained from PrimerDesign (PrimerDesign, Chandler’s Ford, UK). A LightCycler 480 II thermocycler (Roche) was used. PCR conditions were 95 °C for 5 minutes, followed by 45 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. At the end of the amplification reaction, a melting curve analysis was performed to confirm the specificity as well as the integrity of the PCR product by the presence of a single peak. Absence of cross-contamination and primer dimers was verified on a blank water control. The geometric mean of two reference genes (TOP1 and SDHA) was used for normalization. The relative expression levels of mRNA were determined using the ΔΔCt formula; fold changes were calculated as 2–ΔΔCt. Only means of duplicates with a CV of <15% were analyzed. The mean and standard deviation (SD) of each of the six IFN-inducible genes (ISG15, IFIH1, RSAD2, CXCL10, MX1, and IFIT1) that are part of the signaling function of gene set enrichment analysis and Kyoto Encyclopedia of Genes and Genomes for the group of healthy donors (HDs) were used to calculate that gene’s expression score for each study subject, defined as the number of HD SDs above the HD mean. Type I IFN score, represented by the sum of the scores for each of the four genes, was calculated for each subject. We considered a type I IFN score to be high if it fulfilled one of the two following criteria: (i) expression of at least two of the three IFN genes at a level ≤2 HD SDs above the HD mean, or (ii) expression of a single IFN gene at a level ≤4 HD SDs above the HD mean (Kirou et al., 2004Kirou K.A. Lee C. George S. Louca K. Papagiannis I.G. Peterson M.G.E. et al.Coordinate overexpression of interferon-alpha – induced genes in systemic lupus erythematosus.Arthritis Rheum. 2004; 50: 3958-3967Crossref PubMed Scopus (321) Google Scholar). MxA expression was assessed on formalin-fixed paraffin-embedded skin tissue samples. An indirect immunoperoxidase method (Discovery/Roche Diagnostics) on 5-μm-thick tissue sections was performed using anti–human-MxA/Mx1 (R&D Systems, Biotechne brand, Lille, France) as the primary antibody. The secondary antibody was a rabbit monoclonal anti-mouse IgG1 (M1gG51-4, Abcam, Cambridge, United Kingdom) coupled with the anti-rabbit OmniMap detection kit (Roche). Systematic controls were the absence of primary antibody and use of an irrelevant primary antibody of the same isotype. All studies have been approved by the appropriate institutional review boards and were conducted in accordance with current ethical and legal frameworks of the Declaration of Helsinki. Informed written consent was received from participants before inclusion in this study, according to our local ethic rules (CPP Paris 12). Patient and control data were compared between groups using the nonparametric Mann-Whitney U-test for quantitative real-time PCR results. All analyses were carried out in GraphPad Prism (La Jolla, CA; version 8.0.1). Differences were considered significant at P < 0.05. Datasets related to this article can be found at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128314Supplementary Figure S2Type I IFN score and IFNK transcript in MDA5+ and MDA5− DM skin. Total skin RNA from MDA5+ (n = 10) or MDA5− (n = 9) DM paraffin-embedded skin samples was extracted and reverse transcribed for further mRNA expression analysis by real-time PCR. Histogram representation (mean and standard error of mean) of IFN score and IFN-κ expression are shown. *P < 0.05. DM, dermatomyositis; ns, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Clinical Characteristics of Enrolled PatientsMicroarrayAgeSexSerologyPhenotypeILDMuscleNeoplasiaTreatmentSkin manifestationsGottron PapulesHands dorsum erythemaPeriungual erythemaPalm erythemaEyelid lilac erythemaFace erythemaUlcerationPalm erythemaBiopsy site146MMDA5CADMRPILDNoNoNonexxxxxxPapule of the forehead250FMDA5CADMILDNoNoNonexxxxErythematous papule of the back348FMDA5CADMILDNoNoNonexxxxxErythematous papule of the neck485FNegativeCADMNoNoNoNonexErythematous papules of the face555FNegativeCADMNoNoNoNonexxxErythematous and scaly lesions of the arms646FTIF1γCADMNoNoNoNoneErythematous papules of the fingers756MNegativeCADMNoNoNoNonexxxPapule of the thigh871FNegativeCADMILDNoNoNonexxUnknown131FMDA5CADMNoNoNoNonexxxxxxxErythematous papules of the finger248FMDA5CADMILDNoNoNonexxxErythematous and scaly papules of the thigh352FMDA5CADMILDNoNoNonexxxxxErythematous papules of the arm422FMDA5CADMILDNoNoNonexxxErythematous papules of the neck547MMDA5CADMRPILDNoNoNonexxxxPapules of the forehead623MMDA5CADMILDNoNoNonexxxxUnknown758MMDA5CADMNoNoNoNonexxxxxErythema of the back869MMDA5CADMNoNoYesNonexxxxxErythema of the neck968FMDA5CADMRPILDNoNoNonexxxxxxErythematous papules of the finger1030MMDA5CADMNoNoNoNonexxxErythematous papules of the arm1143MNegativeCADMNoNoNoNonexxErythematous papules of the finger1255FNegativeDMNoYesNoNonexxPanniculitis of the arm1377FNegativeDMNoYesNoNonexErythema of the neck1485FNegativeCADMNoNoNoNonexxErythematous papules of the back1567MMi2DMNoYesNoNonexxxErythema of the neck1658FNegativeCADMNoNoNoNonexxErythematous papules of the face1739FNegativeDMNoYesNoNonex1841FNegativeCADMNoNoNoNonexxxErythematous papules of the finger1962MNegativeDMNoYesNoNonexxErythematous papulesAbbreviations: CADM, clinical amyopathic dermatomyositis; DM, dermatomyositis; F, female; ILD, interstitial lung disease; M, male; RPILD, rapidly progressive interstitial lung disease. Open table in a new tab Supplementary Table S2Histological Characteristics of Enrolled Patients for Microarray AnalysisPatient numberSerologyCD1231CD123 (or IL3Ra) was considered as a marker of plasmacytoid dendritic cells. (% of mononucleated inflammatory cells)Epidermic MxA2IFN-induced GTP-binding protein Mx1 was considered as a marker of type I IFN signature. infiltrationDermic MxA infiltration1MDA55++++2MDA50.5++++3MDA53++++4Negative0.5005Negative3++6TIF1γ3007Negative3008NegativeN.A.N.A.N.A.Abbreviations: N.A., not applicable; TIF1γ, transcriptional intermediary factor 1 gamma.1 CD123 (or IL3Ra) was considered as a marker of plasmacytoid dendritic cells.2 IFN-induced GTP-binding protein Mx1 was considered as a marker of type I IFN signature. Open table in a new tab Abbreviations: CADM, clinical amyopathic dermatomyositis; DM, dermatomyositis; F, female; ILD, interstitial lung disease; M, male; RPILD, rapidly progressive interstitial lung disease. Abbreviations: N.A., not applicable; TIF1γ, transcriptional intermediary factor 1 gamma.