key: cord-0307737-tyu0x0si authors: Graf, M.; von Stuckrad, S. L.; Uruha, A.; Klotsche, J.; Zorn-Pauly, L.; Unterwalder, N.; Buttgereit, T.; Krusche, M.; Meisel, C.; Burmester, G. R.; Hiepe, F.; Biesen, R.; Kallinich, T.; Stenzel, W.; Schneider, U.; Rose, T. title: SIGLEC1 enables straightforward assessment of type I interferon activity in idiopathic inflammatory myopathies date: 2021-09-16 journal: nan DOI: 10.1101/2021.09.13.21263325 sha: b14df2e75c9f48ede4a970188f8d019309cff20a doc_id: 307737 cord_uid: tyu0x0si Objective: To evaluate SIGLEC1 expression on monocytes by flow cytometry as a type I interferon biomarker in idiopathic inflammatory myopathies (IIM). Methods: We performed a retrospective analysis of adult and pediatric patients with the diagnosis of IIM. SIGLEC1 expression was assessed by flow cytometry and was compared with Physician Global Assessment (PGA) or Childhood Myositis Assessment Scale (CMAS) disease activity scores. Mann Whitney-U test and receiver operating characteristic (ROC) curves were used for cross-sectional data analysis (n=96), two-level mixed-effects linear regression model for longitudinal analyses (n=26, 110 visits). Response to treatment was analyzed in 14 patients within 12 months, using Wilcoxon test. SIGLEC1 was compared to ISG15/MxA status by immunohistochemical staining of muscle biopsies (n=17). Results: 96 patients with adult (a) and juvenile (j) dermatomyositis (DM, n=38), antisynthetase syndrome (AS, n=19), immune-mediated necrotizing myopathy (IMNM, n=8), inclusion body myositis (IBM, n=9), and overlap myositis (n=22) were included. SIGLEC1 distinguished significantly between active and inactive disease with an area under the curve (AUC) of 0.92 (95% CI: 0.83-1) in DM and correlated with disease activity longitudinally (aDM: standardized beta=0.54, p<0.001; jDM: standardized beta=-0.70, p<0.001). Response to treatment in DM was associated with a decreasing SIGLEC1 (p<0.01, Wilcoxon test). A positive ISG15/MxA stain was highly associated with a SIGLEC1 upregulation. SIGLEC1 was found upregulated in AS (42.1%) and IBM (22,2%) and not in IMNM. Conclusion: SIGLEC1 is a candidate biomarker to assess type I interferon activity in IIM and proved useful for monitoring disease activity and response to treatment in juvenile and adult DM. Abbreviations: ISG15 = interferon-stimulated gene 15; MxA = myxovirus resistance protein A; MDA5 = melanoma differentiation-associated protein 5; NXP2 = nuclear matrix protein 2; TIF1γ = transcriptional intermediary factor 1-gamma; PL7 = anti-threonyl-tRNA synthetase; PL12 = alanyl-tRNA-synthetase; Jo1 = anti-histidyl tRNA synthetase; SRP = signal recognition particle; HMGCR = 3-hydroxy-3-methylglutaryl-coenzyme A reductase; COVID-19 = coronavirus disease 2019; EULAR/ACR = European League Against Rheumatism/American College of Rheumatology What is already known about this subject? • There is an unmet need for routine clinical biomarkers to assess type I interferon activity in rheumatic musculoskeletal diseases • SIGLEC1 is part of the type I interferon signature and transcripts were found to be upregulated in various autoimmune diseases such as systemic lupus erythematosus (SLE), Sjoegren syndrome and dermatomyositis. • SIGLEC1 is expressed on the surface of monocytes and thus is easily assessable by flow cytometry, which enables straightforward monitoring of type I interferon activity What does this study add? • An activation of the type I interferon system in IIM can be identified by flow cytometry analysing SIGLEC1 expression. SIGLEC1 correlated to disease activity and improvement under therapy in juvenile and adult dermatomyositis. • SIGLEC1 expression is a suitable biomarker for monitoring type I interferon activation in rheumatic musculoskeletal diseases, which has clinical implications for patient stratification and treatment decision making especially in the context of interferon inhibitory therapies. Idiopathic inflammatory myopathies are a group of autoimmune diseases that can affect the muscles, skin, lungs, joints and other organs. The EULAR/ACR classification of 2017 divides them into dermatomyositis, polymyositis, and inclusion body myositis. [1] However, increasing knowledge about sub-entities such as antisynthetase syndrome and immunemediated necrotizing myopathy, and the discovery of new myositis-specific autoantibodies, has led to even more differentiation. Furthermore, the existence of polymyositis has recently been challenged. [2] [3] [4] Serum creatine kinase is used in clinical practice for diagnosis and monitoring of disease activity despite its shortcomings in subtypes of dermatomyositis. [5, 6] Studies have pointed out a crucial role of type I interferons in the etiopathogenesis of connective tissue diseases such as systemic lupus erythematosus and idiopathic inflammatory myopathies, [7, 8] and therefore have become therapeutic targets of interest. [9] [10] [11] [12] An upregulation of type I interferon-inducible transcripts was found in peripheral blood, muscle and skin biopsy specimens, particularly in patients with adult and juvenile dermatomyositis, showing a correlation with disease activity in adult and juvenile dermatomyositis. [7, [13] [14] [15] [16] [17] [18] [19] [20] However, type I interferon activation varies individually and is frequently not present. Thus an easy assessable type I interferon biomarker for patient stratification and disease activity monitoring in routine clinical diagnostics is highly needed. [21] Analyzing the type I interferon signature in whole blood, SIGLEC1 (sialic acid binding Iglike lectin 1, CD169), although monocyte-restricted, was found to be a highly upregulated transcript in patients with adult and juvenile dermatomyositis, systemic lupus erythematosus and various genetic interferonopathies. [13, [22] [23] [24] [25] However, the whole blood type I interferon signature is influenced by potentially disruptive factors (such as changes in immune blood cell distribution) that prevent optimal longitudinal comparative analysis. [ Blood samples for the analysis of SIGLEC1 (CD169) expression on monocytes, creatine kinase and autoantibody profiles were obtained on an outpatient or inpatient basis as part of routine diagnostics at the Charité -Universitätsmedizin Berlin, and they were analyzed at Labor Berlin -Charité Vivantes GmbH. SIGLEC1 expression in whole blood was determined by flow cytometry using a highly standardized quantitative assay (Supplementary eText S1), as described by Stuckrad et al. [28] The lower limit of quantitation of this assay was 1200 monoclonal antibodies bound per cell (mAb/cell). The reference range for the expression of SIGLEC1 in healthy controls was determined to be less than 2400 mAb/cell. Juvenile patients with idiopathic inflammatory myopathies were routinely evaluated with the Childhood Myositis Assessment Scale (CMAS) by a trained physiotherapist at the time of their visit, as described by Rider et al. [36] CMAS is a validated tool to measure muscle strength and endurance in juvenile idiopathic inflammatory myopathies. [36, 37] Physiotherapists were not informed about laboratory parameters, such as creatine kinase or SIGLEC1 expression. The Physician Global Assessment (PGA), with scores ranging from 0 (no disease activity) to 10 (high disease activity), was used to rate overall disease activity in juveniles and adults. If PGA data was missing in the medical records, it was determined retrospectively by two experienced rheumatologists (U.S., A.v.S.) based on all available information except for SIGLEC1 expression. Visit 1 was defined as the date of the patient's first SIGLEC1 expression measurement at our clinic. To assess a clinically meaningful response to treatment in juvenile and adult patients with dermatomyositis we included all patients with (a) active disease (PGA≥5) on the first visit (b) a follow up visit in a time frame of 3 to 12 months and (c) reduction of PGA by at least 20% (as proposed by Rider et al. [38] ). Interferon-stimulated gene 15 (ISG15) and myxovirus resistance protein A (MxA) reflect the type I interferon activity [39] and their protein expression can be assessed by immunohistochemical analysis. Briefly, 7-8 μ m cryosections of skeletal muscle biopsies were All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; stained using MxA (Santa Cruz polyclonal, Mx1/2/3, H-285, sc-50509, 1:100) and ISG15 (Abcam, clone ab14374, 1:50) as the primary antibodies, and immunohistochemical (IHC) analysis was performed using the iVIEW DAB (3,3'-diaminobenzidine) Detection Kit (Ventana Medical Systems, Tucson, Arizona, USA), as previously described. [40, 41] Appropriate biotinylated secondary antibodies were used for signal amplification, and visualization of the reaction product was carried out on a Benchmark XT immunostainer (Ventana) using a standardized procedure. As previously described, MxA and ISG15 staining in the cytoplasm was considered positive except for necrotic or regenerating fibers. Fibers with equivocally faint staining were considered negative. [40, 41] Biopsies were included in this study only if performed within seven days of SIGLEC1 measurement in blood. Quantitative data are presented as mean (range) or median (interquartile range), and qualitative data are presented as absolute numbers (percentage). The Mann Whitney U-test (MWU) was used to compare groups with non-normally distributed data. Spearman's rank test was used to evaluate correlations between SIGLEC1 or creatine kinase and disease activity scores (PGA, CMAS). Receiver operating characteristic curves were plotted to compare the capability of SIGLEC1 and creatine kinase to distinguish between active (PGA≥5) and inactive disease (PGA<5). Longitudinal data analyses were performed using a two-level mixed-effects linear regression model. Standardized beta coefficients (betaST) were reported to compare the strength of association between the following parameters of interest: (a) CMAS score and SIGLEC1 / creatine kinase, respectively, in juvenile dermatomyositis and (b) PGA score and SIGLEC1 / creatine kinase, respectively, in adult dermatomyositis. Clinically meaningful response to treatment was analyzed using Wilcoxon matched-pairs signed rank test. Linear mixed models analyses were performed with STATA 12.1, and Graphpad Prism Version 9.1.2 was used for all other data analyses and graphs. P values of <0.05 were considered statistically significant. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; Seventy-four patients with confirmed diagnoses of idiopathic inflammatory myopathies were included (Table 1) and forty-four patients were excluded due to unclear diagnoses and/or insufficient clinical data. According to the EULAR/ACR criteria for idiopathic inflammatory myopathies, [1] 82.4% to 100% of the patients with adult and juvenile dermatomyositis, antisynthetase syndrome and inclusion body myositis had a probable or definite diagnosis, compared to only 62.5% of the immune-mediated necrotizing myopathy patients. All immune-mediated necrotizing myopathy diagnoses were conclusive and based on clinical and morphological criteria, as discussed at the 224th ENMC International Workshop. [42] The "Overlap" group consisted of twenty-two patients with overlap myositis (n=16), mixed connective tissue disease (n=5) and systemic sclerosis (n=1). No patient with isolated polymyositis as an independent entity could be identified in the time frame of 5 years. Adult and juvenile dermatomyositis patients expressed high levels of SIGLEC1 (median, 5876 and 5272 mAb/cell) ( Figure 1 and Table 1 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; To determine if SIGLEC1 expression is associated with disease activity, each subgroup of idiopathic inflammatory myopathies was divided into two groups by PGA score: PGA<5 (no to moderate disease activity) and PGA≥5 (moderate to severe disease activity) (Figure 2 and Table 2 ). The correlations between disease activity and SIGLEC1 were stronger than those between disease activity and creatine kinase in all analyses. The increase or decrease of SIGLEC1 between two consecutive visits was associated with changes in disease activity scores. Results for creatine kinase were not significant. Longitudinal graphs of biomarkers and disease activity are presented in Supplementary eFigure S4 and S5. 14 patients with dermatomyositis (adult, n=6 and juvenile, n=8) fulfilled the inclusion criteria for our response to treatment analysis. Between the two visits, there was a significant reduction of SIGLEC1 expression (median of differences -10059, IQR -6058 to -12152, p<0.01, Wilcoxon test) (Figure 2 ). PGA scores between the respective visits improved by -All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; 76.5% (median of differences, IQR -63% to -91%). Medication for the treatment were as followed: Prednisolone (n=14), Methotrexate (n=9), Intravenous Immunoglobulin (n=9), Hydroxychloroquine (n=4), Azathioprine (n=3), Cyclophosphamide (n=2). No significant correlation between SIGLEC1 and disease activity was detected in patients with antisynthetase syndrome, inclusion body myositis or immune-mediated necrotizing myopathy (Figure 3 and Supplementary eFigure S3). Of the 17 muscle samples eligible for immunohistochemical staining (inclusion criteria: 7 days or less between SIGLEC1 measurement and muscle biopsy), 7 (41.2%) were positive for ISG15 and/or MxA. The average time between SIGLEC1-measurement and muscle biopsy was 3 days (range: 0-7 days). All muscle biopsy patients that were positive for ISG15/MxA (in staining) had elevated SIGLEC1 expression on monocytes in peripheral blood ( Figure 5 ), but one patient with antisynthetase syndrome had only minimal elevation (2449 mAb/cell). Three patients with upregulation of SIGLEC1 expression had a negative ISG15/MxA status and were diagnosed with dermatomyositis: one had anti-TIF1γ antibodies, one did not have any myositis-specific autoantibodies, and one had anti-MDA5 + amyopathic dermatomyositis. One patient with inclusion body myositis who was positive for SIGLEC1 expression on monocytes and in immunohistochemical staining in muscle biopsy was also positive for anti-Ku, anti-U1RNP and anti-Ro52 antibodies (Supplementary eTable S1). We found that SIGLEC1 expression on monocytes correlated with disease activity in patients with juvenile and adult dermatomyositis. Clinically meaningful improvement under therapy was associated with a significant decrease in SIGLEC1 expression. These results are in line All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; with studies that analyzed type I interferon transcripts, [13] [14] [15] [16] [17] [18] [19] but with the advantage of being easily assessable by flow cytometry. Very recently, Lerkvaleekul et al. [43] published data of SIGLEC1 in 21 patients with newly diagnosed juvenile dermatomyositis. They found, that SIGLEC1 expression correlated to disease activity and was superior to predict treatment response compared to an interferon-stimulated gene score consisting of five genes. Our results validate the findings in juvenile dermatomyositis and by that underline the potential of SIGLEC1 in clinical routine diagnostics. In our study, SIGLEC1 could distinguish between active and inactive disease in adult and juvenile dermatomyositis patients with a large area under the curve. In this context, published results in a preprint article and comment suggest the utility of direct assessment of interferon by using a highly sensitive interferon-alpha single-molecule array (SIMOA) digital enzymelinked immunosorbent assay (ELISA) in adult and juvenile dermatomyositis. [44, 45] A direct comparison of these two type I interferon biomarkers would be highly interesting, as they are both candidates for routine clinical diagnostics. In the present study, expression of SIGLEC1 -which is mostly type I interferonregulated [34, 46] -was lower in antisynthetase syndrome than in dermatomyositis. This finding is in line with preprint published results of interferon-alpha in dermatomyositis and antisynthetase syndrome using SIMOA technology. [44] It has been proposed that type II interferons might play a more prominent role in the etiopathogenesis of antisynthetase syndrome. [39, 47] However, Reed et al. found high interferon scores (including IP-10, I-TAC and MCP-1) in patients with antisynthetase syndrome. [48] We also identified patients with high expression of SIGLEC1 indicating a type I interferon response. This interesting finding warrants further investigation. Two of our nine patients with inclusion body myositis exhibited high expression of SIGLEC1: both were positive for anti-Ro and one was also positive for anti-U1RNP autoantibodies, which are known to induce type I interferons. [49] One of these anti-U1RNP + inclusion body myositis patients was also analyzed for MxA/ISG15 in muscle tissue and showed an unusual positive staining on myofibers (cf. patient AD010). Data on the role of interferons in inclusion body myositis are inconsistent and need to be clarified. [39, 50, 51 ] The detection of an activated type I interferon system might have important treatment implications in this debilitating chronic disease. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; We did not find evidence of an upregulation of SIGLEC1 expression in immune-mediated necrotizing myopathy, which is in line with other current studies analyzing type I interferonregulated transcripts (in muscle) or interferon-alpha by SIMOA technology (in blood). [39, 44] Few studies have explored differences in interferon signatures in blood according to myositisspecific autoantibody status. [48] We found the highest SIGLEC1 expression in anti-MDA5 + dermatomyositis patients, but could not detect a specific association of SIGLEC1 to certain myositis-specific autoantibodies in dermatomyositis otherwise. Our findings underline, that the autoantibody status alone is not predictive for the detection of a type I interferon activation. Thus, the assessment of myositis-specific autoantibodies in conjunction with the assessment of type I interferon activity might be useful for patient stratification. The current study has strengths and limitations. A strength is that we could demonstrate that SIGLEC1, as an implemented routine biomarker, was able to validate findings from other studies investigating type I interferon signature with the advantage of being easily assessable by flow cytometry. A limitation is that muscle strength scores equivalent to the CMAS were not available for adult patients. However, the PGA is a commonly used instrument that reflects disease activity in idiopathic inflammatory myopathies. [36] Secondly, the PGA was performed retrospectively in most cases. Since the missing scores were determined by two experienced rheumatologists with access to all clinical data for each visit but blinded to SIGLEC1 expression, we are confident that we were able to reduce a potential bias. In conclusion, analysis of SIGLEC1 expression by flow cytometry enables the individual assessment of type I interferon activity in idiopathic inflammatory myopathies. Present and published data on SIGLEC1 expression in patients with interferon-associated rheumatic and musculoskeletal diseases, interferonopathies, and viral infections confirm that SIGLEC1 is an easy-to-use type I interferon biomarker and demonstrate its potential for patient stratification, disease activity monitoring and assessment of treatment response in routine clinical practice. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted September 16, 2021. ; European League Against Rheumatism/American College of Rheumatology classification criteria for adult and juvenile idiopathic inflammatory myopathies and their major subgroups Integrated classification of inflammatory myopathies Development of a New Classification System for Idiopathic Inflammatory Myopathies Based on Clinical Manifestations and Myositis-Specific Autoantibodies Where are we moving in the classification of idiopathic inflammatory myopathies? The Utility of Serum Aldolase in Normal Creatine Kinase Dermatomyositis Different phenotypes in dermatomyositis associated with anti-MDA5 antibody: Study of 121 cases Interferon-signature in idiopathic inflammatory myopathies Drivers of the immunopathogenesis in systemic lupus erythematosus JAK inhibitor improves type I interferon induced damage: proof of concept in dermatomyositis Trial of Anifrolumab in Active Systemic Lupus Erythematosus Study of Tofacitinib In Refractory Dermatomyositis (STIR): An open label pilot study of 10 patients Tofacitinib for refractory interstitial lung diseases in anti-melanoma differentiation-associated 5 gene antibody-positive dermatomyositis Correlation of cutaneous disease activity with type 1 interferon gene signature and interferon β in dermatomyositis Interleukin-6 and type I interferon-regulated genes and chemokines mark disease activity in dermatomyositis Relationship between disease activity and type 1 interferon-and other cytokine-inducible gene expression in blood in dermatomyositis and polymyositis An Interferon Signature in the Peripheral Blood of Dermatomyositis Patients is Associated with Disease Activity MxA gene expression in juvenile dermatomyositis peripheral blood mononuclear cells: association with muscle involvement Type I interferon-inducible gene expression in blood is present and reflects disease activity in dermatomyositis and polymyositis Changes in novel biomarkers of disease activity in juvenile and adult dermatomyositis are sensitive biomarkers of disease course Dermatomyositis and Type 1 Interferons Biomarkers in Inflammatory Myopathies-An Expanded Definition Assessment of Type I Interferon Signaling in Pediatric Inflammatory Disease Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-α Monoclonal Antibody Trials in Systemic Lupus Erythematosus Sialic acid-binding Ig-like lectin 1 expression in inflammatory and resident monocytes is a potential biomarker for monitoring disease activity and success of therapy in systemic lupus erythematosus Type I interferon as a biomarker in autoimmunity and viral infection: a leukocyte subset-specific analysis unveils hidden diagnostic options Siglec-1 expression on monocytes is associated with the interferon signature in juvenile dermatomyositis and can predict treatment response SIGLEC1 (CD169) is a sensitive biomarker for the deterioration of the clinical course in childhood systemic lupus erythematosus IFNalpha and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus Elevated STAT1 expression but not phosphorylation in lupus B cells correlates with disease activity and increased plasmablast susceptibility Are interferon-related biomarkers advantageous for monitoring disease activity in systemic lupus erythematosus? A longitudinal benchmark study A macrophage marker, Siglec-1, is increased on circulating monocytes in patients with systemic sclerosis and induced by type I interferons and toll-like receptor agonists SIGLEC1 is a biomarker of disease activity and indicates extraglandular manifestation in primary Sjogren's syndrome SIGLEC1 (CD169) as a potential diagnostical screening marker for monogenic interferonopathies CD169/SIGLEC1 is expressed on circulating monocytes in COVID-19 and expression levels are associated with disease severity Measures of adult and juvenile dermatomyositis, polymyositis, and inclusion body myositis: Physician and Patient/Parent Global Activity, Manual Muscle Testing (MMT), Health Assessment Questionnaire (HAQ)/Childhood Health Assessment Questionnaire (C-HAQ) Validation and clinical significance of the Childhood Myositis Assessment Scale for assessment of muscle function in the juvenile idiopathic inflammatory myopathies Defining Clinical Improvement in Adult and Juvenile Myositis Identification of distinctive interferon gene signatures in different types of myositis Diagnostic potential of sarcoplasmic myxovirus resistance protein A expression in subsets of dermatomyositis Sarcoplasmic MxA expression: A valuable marker of dermatomyositis 224th ENMC International Workshop:: Clinico-sero-pathological classification of immune-mediated necrotizing myopathies Zandvoort Siglec-1 expression on monocytes is associated with the interferon signature in juvenile dermatomyositis and can predict treatment response Ultrasensitive Interferons quantification in idiopathic inflammatory myopathies serve as biomarkers of activity in dermatomyositis and antisynthetase syndrome Circulating Interferon-α Measured With a Highly Sensitive Assay as a Biomarker for Juvenile Inflammatory Myositis Activity: Comment on the Article by Mathian et al. Arthritis Rheumatol Online Role of the interferons in CD64 and CD169 expressions in whole blood: Relevance in the balance between viral-or bacterial-oriented immune responses Distinct interferon signatures stratify inflammatory and dysimmune myopathies Biologic predictors of clinical improvement in rituximab-treated refractory myositis U1 small nuclear ribonucleoprotein immune complexes induce type I interferon in plasmacytoid dendritic cells through TLR7