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Cluster of highly expressed interferon-stimulated genes associate more with African ancestry than disease activity in patients with systemic lupus erythematosus. A systematic review of cross-sectional studies

Open AccessPublished:August 02, 2021DOI:https://doi.org/10.1016/j.trsl.2021.07.006
      Type I interferons (IFN) are central players in the pathogenesis of systemic lupus erythematosus (SLE) and the up-regulation of interferon-stimulated genes (ISGs) in SLE patients is subjected to increasing scrutiny as for its use in diagnosis, stratification and monitoring of SLE patients. Determinants of this immunological phenomenon are yet to be fully charted. The purpose of this systematic review was to characterize expressions of ISGs in blood of SLE patients and to analyze if they associated with core demographic and clinical features of SLE. Twenty cross-sectional, case-control studies comprising 1033 SLE patients and 602 study controls could be included. ISG fold-change expression values (SLE vs controls), demographic and clinical data were extracted from the published material and analyzed by hierarchical cluster analysis and generalized linear modelling. ISG expression varied substantially within each study with IFI27, IFI44, IFI44L, IFIT4 and RSAD2, being the top-five upregulated ISGs. Analysis of inter-study variation showed that IFI27, IFI44, IFI44L, IFIT1, PRKR and RSAD2 expression clustered with the fraction of SLE cases having African ancestry or lupus nephritis. Generalized linear models adjusted for prevalence of lupus nephritis and usage of hydroxychloroquine confirmed the observed association between African ancestry and IFI27, IFI44L, IFIT1, PRKR and RSAD2, whereas disease activity was associated with expression of IFI27 and RNASE2. In conclusion, this systematic review revealed that expression of ISGs often used for deriving an IFN signature in SLE patients were influenced by African ancestry rather than disease activity. This underscores the necessity of taking ancestry into account when employing the IFN signature for clinical research in SLE.

      INTRODUCTION

      Systemic lupus erythematosus (SLE) is a clinically and immunologically heterogeneous autoimmune disease that is characterized by chronic activation of the interferon (IFN) system. Due to the systemic nature of SLE, any organ may be involved with varying severity. Renal involvement (lupus nephritis) is a common and severe disease phenotype that may progress to renal end-stage disease with non-Caucasian patients, especially those of African ancestry, having the highest risk hereof.
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      The study of interactions between genome and exposome in the development of systemic lupus erythematosus.
       Sustained production of various types of IFNs plays a key role in this process. IFN acts as a potent immune adjuvant and induces the transcription of a plethora of genes,
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      Type I IFNs, especially IFN-α, have attracted attention as they among others are produced by plasmacytoid dendritic cells after stimulation by endogenous nucleic acids via Toll-like-receptor (TLR) 7 and TLR9, but also type II and III IFNs may be centrally involved in the pathogenesis of SLE.
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      Type I and type II IFN-stimulated genes (ISG) are up-regulated in up to 87 % of adult SLE patients.
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      Their pattern of upregulation is often termed as an IFN signature. Most studies of the IFN signature in SLE are available from either analysis of whole blood or peripheral blood mononuclear cells (PBMCs). Yet, researchers are still using pre-selected groups of genes when assaying the IFN signature, although there is no established international consensus on specific definitions of the IFN signature. Previous studies also display great variation as to which genes are found to be activated and to what degree. In some cross-sectional SLE studies, the IFN signature has been shown to correlate with disease activity and severe disease with involvement of the renal, hematopoietic and nervous systems.
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      Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
      On the other hand, longitudinal studies based on 3–5 ISGs have failed to demonstrate such correlations.
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      In recent years, IFN scores have been applied to stratify patients in large-scale randomized clinical trials of anti-IFN treatments; most recently the phase III trial of Anifrolumab, in which a 4-gene score was used. As in other clinical trials using similar approaches, stratification by IFN signature was not useful for an a priori delineation of patients that benefitted from the IFN inhibiting therapy.
      • Morand EF
      • Furie R
      • Tanaka Y
      • et al.
      Trial of anifrolumab in active systemic lupus erythematosus.
      These observations draw attention to other factors that may influence the IFN system including ancestry.
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      • et al.
      Transancestral mapping and genetic load in systemic lupus erythematosus.
      Only few studies have addressed this, but higher serum IFN-α activity has been observed in African and Hispanic American SLE patients compared to European American SLE patients.
      • Weckerle CE
      • Franek BS
      • Kelly JA
      • et al.
      Network analysis of associations between serum interferon-alpha activity, autoantibodies, and clinical features in systemic lupus erythematosus.
      In line with this finding, African American SLE patients displayed differentiated levels of hypomethylation and expression of certain ISGs compared to European American SLE patients.
      • Joseph S
      • George NI
      • Green-Knox B
      • et al.
      Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI.
      ,
      • Sharma S
      • Jin Z
      • Rosenzweig E
      • Rao S
      • Ko K
      • Niewold TB
      Widely divergent transcriptional patterns between SLE patients of different ancestral backgrounds in sorted immune cell populations.
      These findings beg the question to which extent IFN signatures differ among SLE patients with respect to disease activity as well as ancestry.
      Hence, in the context of a systematic review, we wanted (1) to summarize the available data on the expression of ISGs in peripheral blood of patients with SLE compared to healthy controls, (2) to determine which ISGs are most up-regulated in SLE, and (3) to analyze to what extent such ISGs are associated with SLE disease activity and ancestry.

      METHODS

       Eligibility criteria

      This systematic review was developed in accordance with the guidelines and recommendations of the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statements.
      • Moher D
      • Liberati A
      • Tetzlaff J
      • Altman DG
      • Group P.
      Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement.
      A predefined protocol was registered with PROSPERO (an international database of prospectively registered systematic reviews), University of York, York, UK (Registration No. CRD42019132381). Case-control studies assessing the mRNA expression of IFN-stimulated genes in peripheral blood cells from patients with SLE of any age, sex or ancestry were considered eligible. Study controls without any documented systemic autoimmune disorders and of any age, sex or ancestry were considered eligible for inclusion.

       Literature search

      Search strategies for different databases were developed and searches combining key words “systemic lupus erythematosus,” “interferon signature,” “interferon stimulated genes,” “gene expression,” and “mRNA transcription” with Boolean logical operators (“AND” & “OR”) using “Advanced” search options were conducted. Electronic databases including PubMed and EMBASE were searched and screened. The final systematic search was conducted on January 13, 2020. There was no restriction on date of publication. However, only English language articles were considered eligible. Non-human subjects, review articles, editorials, letters, comments and duplicate articles among different databases were excluded. Additionally, articles involving in vitro stimulation of cells prior to gene expression analysis, sorted cells or articles with lack of quantitative data were excluded. There were no restrictions on use of medication among SLE patients. References in the primary selected studies were also examined to further identify potentially relevant articles.

       Quality assessment

      The quality of each of the included studies was assessed by 2 of the authors (KZS and TRW) and finally confirmed by a third author (SJ). Quality was scored by the Newcastle-Ottawa Scale (NOS) modified for cross-sectional studies.
      • Modesti PA
      • Reboldi G
      • Cappuccio FP
      • et al.
      Panethnic differences in blood pressure in europe: a systematic review and meta-analysis.
      The NOS is a scoring system that evaluates features relating to methodological quality, including risk of bias. It assigns a maximum of five points for case selection, 2 points for study comparability and 3 points for handling of outcomes for a total of 1 points.

       Data extraction

      Studies were included based on the inclusion criteria and selection methodology as illustrated in Fig 1. The types of data extracted from the selected articles were as follows: country of study origin, number of subjects with SLE, lupus nephritis, number of study controls, mean study population age, distribution of sex and ancestry of the study population, mean SLE disease activity, number of analyzed ISGs and their expression values or fold-change values as appropriate. Moreover, the number of patients using antimalarial medication (hydroxychloroquine) was also extracted from the selected articles. However, data on other medication usage was not extracted due to insufficient details on dosage. Descriptions of disease activity were based on the Systemic Lupus Disease Activity Index (SLEDAI),
      • Bombardier C
      • Gladman DD
      • Urowitz MB
      • Caron D
      • Chang CH.
      Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE.
      ,
      • Gladman DD
      • Ibanez D
      • Urowitz MB.
      Systemic lupus erythematosus disease activity index 2000.
      the British Isles Lupus Assessment Group (BILAG),
      • Symmons DP
      • Coppock JS
      • Bacon PA
      • et al.
      Development and assessment of a computerized index of clinical disease activity in systemic lupus erythematosus. Members of the British Isles Lupus Assessment Group (BILAG).
      ,
      • Isenberg DA
      • Rahman A
      • Allen E
      • et al.
      BILAG 2004. Development and initial validation of an updated version of the British Isles Lupus Assessment Group's disease activity index for patients with systemic lupus erythematosus.
      the Systemic Lupus Activity Measure (SLAM)
      • Liang MH
      • Socher SA
      • Larson MG
      • Schur PH.
      Reliability and validity of six systems for the clinical assessment of disease activity in systemic lupus erythematosus.
      or in 3 studies by clinical descriptions.
      • Baechler EC
      • Batliwalla FM
      • Karypis G
      • et al.
      Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.
      ,
      • Bouquet J
      • Gardy JL
      • Brown S
      • et al.
      RNA-seq analysis of gene expression, viral pathogen, and B-cell/T-cell receptor signatures in complex chronic disease.
      ,
      • Yao Y
      • Higgs BW
      • Morehouse C
      • et al.
      Development of potential pharmacodynamic and diagnostic markers for anti-IFN-alpha monoclonal antibody trials in systemic lupus erythematosus.
      Based on this information, we imputed missing data on SLEDAI score using previously reported comparative analyses of disease activity indices.
      • Abrahamowicz M
      • Fortin PR
      • du Berger R
      • Nayak V
      • Neville C
      • Liang MH.
      The relationship between disease activity and expert physician's decision to start major treatment in active systemic lupus erythematosus: a decision aid for development of entry criteria for clinical trials.
      ,
      • Cresswell L
      • Yee CS
      • Farewell V
      • et al.
      Numerical scoring for the classic BILAG index.
      Fig 1
      Fig 1Flow diagram of selection process for the included articles according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statements from 2009.
      • Moher D
      • Liberati A
      • Tetzlaff J
      • Altman DG
      • Group P.
      Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement.
      Three of the authors (KZS, TRW and SJ) took part in the discussion to resolve any unclear or missing data. Mean fold change (FC) was used to describe the upregulation of ISGs in SLE patients compared to study controls. If FC values were not given, FC for each gene was calculated by dividing the mean expression in SLE patients by the mean expression in the study controls of the study. Mean FC values were plotted gene-wise for each study in a heatmap format with an explanatory color scale; red indicates high gene expression and green indicates low gene expression. Only genes that were analyzed in at least five studies were presented.

       Statistical analyses

      After finalizing data retrieval, all data was registered in a common database accessible to all authors. Statistical analyses using SPSS statistics V.20.0 software (IBM) included descriptive statistics, coefficient of variance (CoV), hierarchical cluster analysis (HCA) and generalized linear models (GLMs). HCA included FC expression of selected ISGs, disease activity (mean SLEDAI score), ancestry (proportion of African, Asian, Caucasian and Hispanic ancestry), sex (proportion of females), mean age of study populations and antimalarial medication (proportion using hydroxychloroquine). ISGs included for this analysis comprised six genes that previously have been used to define IFN signatures:
      • Morand EF
      • Furie R
      • Tanaka Y
      • et al.
      Trial of anifrolumab in active systemic lupus erythematosus.
      ,
      • Kirou KA
      • Lee C
      • George S
      • et al.
      Coordinate overexpression of interferon-alpha-induced genes in systemic lupus erythematosus.
      IFI27, IFI44, IFI44L, IFIT1, PRKR and RSAD2; any imputations were based on variable means. GLMs were constructed for individual ISG expression by general disease activity (mean SLEDAI score), occurrence of lupus nephritis (percent of study population), occurrence of African ancestry (percent of study population) and occurrence of hydroxychloroquine use (percent of study population). GLMs were weighted for SLE population size and number of NOS quality points and only constructed for ISGs that were represented in a sufficient number of studies to render the models valid.
      For multiple testing correction, the false discovery rate approach according to Benjamini–Hochberg
      • Solari A
      • Goeman JJ.
      Minimally adaptive BH: A tiny but uniform improvement of the procedure of Benjamini and Hochberg.
      was used applying an online calculator (https://tools.carbocation.com/FDR, accessed on June 17, 2021). Results are given with q-values and q<0.05 was considered statistically significant.

      RESULTS

       Selection and inclusion of studies

      Upon searching the databases, a total of 1429 articles were retrieved, of which 154 met the inclusion criteria. Based on the above-stated exclusion criteria, 134 studies were removed. The remaining 20 studies
      • Baechler EC
      • Batliwalla FM
      • Karypis G
      • et al.
      Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.
      ,
      • Joseph S
      • George NI
      • Green-Knox B
      • et al.
      Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI.
      ,
      • Bouquet J
      • Gardy JL
      • Brown S
      • et al.
      RNA-seq analysis of gene expression, viral pathogen, and B-cell/T-cell receptor signatures in complex chronic disease.
      ,
      • Yao Y
      • Higgs BW
      • Morehouse C
      • et al.
      Development of potential pharmacodynamic and diagnostic markers for anti-IFN-alpha monoclonal antibody trials in systemic lupus erythematosus.
      ,
      • Kirou KA
      • Lee C
      • George S
      • et al.
      Coordinate overexpression of interferon-alpha-induced genes in systemic lupus erythematosus.
      ,
      • Panousis NI
      • Bertsias GK
      • Ongen H
      • et al.
      Combined genetic and transcriptome analysis of patients with SLE: distinct, targetable signatures for susceptibility and severity.
      • Alcorta DA
      • Barnes DA
      • Dooley MA
      • et al.
      Leukocyte gene expression signatures in antineutrophil cytoplasmic autoantibody and lupus glomerulonephritis.
      • Zhu H
      • Mi W
      • Luo H
      • et al.
      Whole-genome transcription and DNA methylation analysis of peripheral blood mononuclear cells identified aberrant gene regulation pathways in systemic lupus erythematosus.
      • Assassi S
      • Mayes MD
      • Arnett FC
      • et al.
      Systemic sclerosis and lupus: points in an interferon-mediated continuum.
      • Li Q
      • Z J Z
      • et al.
      Interferon signature gene expression is correlated with autoantibody profiles in patients with incomplete lupus syndromes.
      • Chaussabel D
      • Quinn C
      • Shen J
      • et al.
      A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus.
      • Wither JE
      • Prokopec SD
      • Noamani B
      • et al.
      Identification of a neutrophil-related gene expression signature that is enriched in adult systemic lupus erythematosus patients with active nephritis: clinical/pathologic associations and etiologic mechanisms.
      • Mackay M
      • Oswald M
      • Sanchez-Guerrero J
      • et al.
      Molecular signatures in systemic lupus erythematosus: distinction between disease flare and infection.
      • Lyons PA
      • McKinney EF
      • Rayner TF
      • et al.
      Novel expression signatures identified by transcriptional analysis of separated leucocyte subsets in systemic lupus erythematosus and vasculitis.
      • Lambers WM
      • de Leeuw K
      • Doornbos-van der Meer B
      • Diercks GFH
      • Bootsma H
      • Westra J.
      Interferon score is increased in incomplete systemic lupus erythematosus and correlates with myxovirus-resistance protein A in blood and skin.
      • Han G
      • M S C
      • et al.
      Analysis of gene expression profiles in human systemic lupus erythematosus using oligonucleotide microarray.
      • Ishii T
      • Onda H
      • Tanigawa A
      • et al.
      Isolation and expression profiling of genes upregulated in the peripheral blood cells of systemic lupus erythematosus patients.
      • Bennett L
      • Palucka AK
      • Arce E
      • et al.
      Interferon and granulopoiesis signatures in systemic lupus erythematosus blood.
      • Tang J
      • Gu Y
      • Zhang M
      • et al.
      Increased expression of the type I interferon-inducible gene, lymphocyte antigen 6 complex locus E, in peripheral blood cells is predictive of lupus activity in a large cohort of Chinese lupus patients.
      • Ye S
      • Pang H
      • Gu YY
      • et al.
      Protein interaction for an interferon-inducible systemic lupus associated gene, IFIT1.
      published between 2003 and 2019 were included in the systematic review, not counting 1 study that was based on previously reported patients.
      • Chaussabel D
      • Quinn C
      • Shen J
      • et al.
      A modular analysis framework for blood genomics studies: application to systemic lupus erythematosus.
      The PRISMA flow diagram of the study selection process is illustrated in Fig 1.

       Quality assessment

      Using the NOS to systematically describe basic features of the quality of the included studies, we found that the median NOS score was 6, ranging from 4 to 8, Table Ⅰ. All studies used internationally accepted classification criteria for the case definition
      • Tan EM
      • Cohen AS
      • Fries JF
      • et al.
      The 1982 revised criteria for the classification of systemic lupus erythematosus.
      • Hochberg MC.
      Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.
      • Petri M
      • Orbai AM
      • Alarcon GS
      • et al.
      Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus.
      or adequate clinical descriptions.
      • Joseph S
      • George NI
      • Green-Knox B
      • et al.
      Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI.
      ,
      • Alcorta DA
      • Barnes DA
      • Dooley MA
      • et al.
      Leukocyte gene expression signatures in antineutrophil cytoplasmic autoantibody and lupus glomerulonephritis.
      None of the studies offered sample size calculations and the NOS item on nonresponse rates was not applicable in any of the studies. Fourteen studies offered demographic and clinical data in such detail that comparability analyses were feasible. All studies had clear definitions and description of the statistical methods and gene expression outcomes.
      Table ⅠRisk of bias assessment of the included studies using the Newcastle-Ottawa scale for case-control studies
      • Modesti PA
      • Reboldi G
      • Cappuccio FP
      • et al.
      Panethnic differences in blood pressure in europe: a systematic review and meta-analysis.
      SelectionComparabilityOutcomeTotal score
      1 (*)2 (*)3 (*)4 (**)5 (**)6 (**)7 (*)(Out of 10)
      Alcorta 2007---*-***4
      Assassi 2010---**-***5
      Baechler 2003---**-***5
      Bennet 2003---**-***5
      Bouquet 2017---******6
      Chaussabel 2008---******6
      Han 2003*--*******7
      Ishii 2005*--**-***6
      Joseph 2019*--******7
      Kirou 2004*--*******8
      Lambers 2019---*******7
      Li 2010*--******6
      Lyons 2010---******6
      Mackay 2016---**-***5
      Panousis 2019*--*******8
      Tang 2008*--******7
      Wither 2018*--******7
      Yao 2009---**-***5
      Ye 2009---*******7
      Zhu 2016*--*******8
      1: Representativeness of the cases. 2: Was the sample size justified and satisfactory? 3: Non-response rate. 4: Is the case definition adequate? 5: Study controls for the most important factors. 6: Was the measurement method of the interferon signature described? 7: Statistical testing. Parenthesis indicates maximum number points (*/**) that could be awarded to the criteria if the respective information was available.

       Study characteristics

      All included studies were original peer-reviewed articles originating from North America (60%), Asia (25%), and Europe (15%). The studies comprised 1,033 SLE patients classified by ACR criteria or SLICC criteria and 602 study controls.
      • Tan EM
      • Cohen AS
      • Fries JF
      • et al.
      The 1982 revised criteria for the classification of systemic lupus erythematosus.
      • Hochberg MC.
      Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.
      • Petri M
      • Orbai AM
      • Alarcon GS
      • et al.
      Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus.
      The SLE populations consisted of 924 (89%) women. The population mean age ranged from 13 to 52 years with an average of 37 years. The distribution of ancestry was as follows; Caucasian (39%), Asian (30%), African (16%), Hispanic (11%), and others (4%), Table Ⅱ. The median disease activity expressed as SLEDAI score was 8.7 ranging from <4 to 19.
      Table ⅡStudy characteristics, demographic and clinical information on participants in the 20 included controlled studies of interferon-stimulated genes in patients with systemic lupus erythematosus
      StudyCountryISG (n)SC (n)SLE (n)SLE females (n)SLE age (mean)African
      ancestry of SLE patients.
      (n)
      Caucasian
      ancestry of SLE patients.
      (n)
      Hispanic
      ancestry of SLE patients.
      (n)
      Asian
      ancestry of SLE patients.
      (n)
      Disease activity score (mean)
      given as SLE Disease Activity Index, unless otherwise indicated.
      Nephritis (n)Hydroxy-chloroquine (n)
      Alcorta, 2007USA72284040352018006.7
      British Isles Lupus Assessment Group score.
      4040
      Assassi, 2010USA7121171639268011
      Systemic Lupus Activity Measure score.
      dns
      data for imputation could be deduced from clinical information provided in the article.
      17
      Baechler, 2003USA114248dns45dnsdnsdnsdnsdns
      data for imputation could be deduced from clinical information provided in the article.
      1525
      Bennet, 2003USA49301813831367.518dns
      Bouquet, 2017CAN42251111513503dns
      data for imputation could be deduced from clinical information provided in the article.
      dnsdns
      Chaussabel, 2008USA47122220146311012.1dns10
      Han, 2003CHN111810929000106.132
      Ishii, 2005JPN830313034000313.8dns0
      Joseph, 2019a
      this study was divided into 2 separate datasets by African/Caucasian ancestry, respectively.
      USA558635752630009.8
      data for imputation could be deduced from clinical information provided in the article.
      dnsdns
      Joseph, 2019b
      this study was divided into 2 separate datasets by African/Caucasian ancestry, respectively.
      USA525175752017008.7
      data for imputation could be deduced from clinical information provided in the article.
      dnsdns
      Kirou, 2004USA72877744716411285dns54
      Lambers, 2019NLD112239324303501<41333
      Li, 2010USA591127253912dns15dnsdnsdnsdns
      Lyons, 2010GBR19251313470120119
      British Isles Lupus Assessment Group score.
      713
      Mackay, 2016USA332011113401041370
      Panousis, 2019GRC10058142120400138005.26695
      Tang, 2008CHN760144132330001446.47246
      Wither, 2018CAN34221701493435800278.5130112
      Yao, 2009USA282441394004100dns
      data for imputation could be deduced from clinical information provided in the article.
      dns
      data for imputation could be deduced from clinical information provided in the article.
      10
      Ye, 2003CHN739504529000506.8
      Systemic Lupus Activity Measure score.
      50
      Zhu, 2016CHN6025302629000301215dns
      Abbreviations: dns: data not shown in publication; ISG, interferon stimulated genes investigated; SC, study controls.
      ; * ancestry of SLE patients.
      ; † given as SLE Disease Activity Index, unless otherwise indicated.
      ; ‡ British Isles Lupus Assessment Group score.
      ; § Systemic Lupus Activity Measure score.
      ; || data for imputation could be deduced from clinical information provided in the article.
      this study was divided into 2 separate datasets by African/Caucasian ancestry, respectively.

       The interferon signature in SLE

      The number of ISGs investigated in the included studies ranged from 5 to 100; since only genes represented in at least five studies were included for analysis, the median number of ISGs reviewed was 17 ranging from 5 to 55.
      SLE patients had a markedly higher mRNA expression of a large range of ISGs as compared to their study controls, displaying a large variation in expression within the studies as expressed by mean fold-change expressions ranging from 1.7 for IFITM1 to 13.2 for IFI27. Moreover, large variation was noted for the expression of individual genes across the studies. Genes that showed large variation (CoV > 1) were PLSCR1, LGP2, LY6E, ADAR, IFIT5 and USP18. Genes that showed low variation (CoV < 0.5) were PARP12, IFITM1, IFITM3, DDX60, RTP4, RNASE2, CMPK2, SAMD9L, DHRS9, PRKR, and IFI27, Fig 2.
      Fig 2
      Fig 2Heatmap of expression of interferon stimulated genes in peripheral blood cells from patients with systemic lupus erythematosus vs matched controls in the 20 included studies. Genes presented have been studied in at least 5 works. Gene expression is presented as fold-change according to the depicted color scale. Variation in gene expression across the studies was calculated as the coefficient of variation.

       Association of ISG expressions with demographic and clinical study characteristics

      Six pre-selected ISGs often used to construct an IFN signature were analyzed by HCA together with demographic and clinical data available in 14 studies
      • Baechler EC
      • Batliwalla FM
      • Karypis G
      • et al.
      Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.
      ,
      • Yao Y
      • Higgs BW
      • Morehouse C
      • et al.
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      Leukocyte gene expression signatures in antineutrophil cytoplasmic autoantibody and lupus glomerulonephritis.
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      comprising 785 SLE patients and 413 controls. SLE populations consisted of 650 (83%) women with population ages ranging from 13 to 47 years with a mean of 35 years. The distribution of ancestry was as follows; Caucasian (43%), Asian (35%), African (8%), Hispanic (3%), and others (11%). The median disease activity score (SLEDAI) was 7,2 ranging from <4 to 19.
      The FC expressions of IFI44, IFI44L, IFIT1, PRKR and RSAD2 in SLE patients relative to controls were highly correlated (r: 0.86–0.98), Fig 3. The expression of IFI27 belonged to the same cluster as the previously mentioned genes, but with less pronounced co-expression (r: 0.17–0.44). The selected ISGs also clustered with the population prevalence of African ancestry and lupus nephritis; the latter also being highly inter-correlated (r = 0.82). None of the ISGs clustered with other population characteristics in the 14 included studies, that is, distribution of sex, other ancestries, age, SLE disease activity and hydroxychloroquine usage.
      Fig 3
      Fig 3Heatmap presenting hierarchical clustering and associations between peripheral blood cell expression of six interferon stimulated genes (fold-change of patients with systemic lupus erythematosus vs matched controls), disease activity (transformed to SLEDAI), prevalence of lupus nephritis, prevalence of hydroxychloroquine usage, ancestry (African, Asian, Caucasian, Hispanic), sex, and age. Included in the analysis are 12 studies comprising 727 SLE patients and 368 matched controls.
      These associations prompted us to determine if the prevalence of African ancestry and mean disease activity score associated with ISG expressions using generalized linear modelling adjusted for the prevalence of subjects with a history of LN and prevalence of hydroxychloroquine usage, Table Ⅲ. In these models, the following genes were associated with African ancestry; IFI27, IFI44L, RSAD2, IFIT1, HERC5, USP18, LY6E, OASL, IFIT3, IFI6, OAS1, OAS2, and PRKR. Disease activity was associated with expression of IFI27 and RNASE2 expression. In these analyses, disease activity did not associate with expression of IFI44, IFI44L, IFIT1, PRKR or RSAD2 either.
      Table ⅢPeripheral blood cell expression of interferon stimulated genes by general disease activity (mean SLEDAI score) and African ancestry (%) in systemic lupus erythematosus study populations
      Disease activityAfrican ancestry
      Fold change
      mean fold change expression of the specified gene in the included studies.
      N
      number of studies included with data for the specified gene.
      betaq≤betaq≤
      IFI2710.860.690.0134.080.004
      IFI44L8.710-0.050.971.250.035
      RSAD27.57-0.490.182.820.0008
      IFI445.99-0.101.001.570.050
      IFIT15.410-0.100.941.640.0006
      HERC54.37-0.380.182.160.0005
      ISG154.38-0.140.991.060.37
      USP184.070.340.714.50.022
      LY6E4.014-0.070.931.750.010
      MX13.911-0.010.971.060.10
      OASL3.99-0.380.201.860.002
      IFIT33.99-0.270.361.50.001
      IFI63.88-0.620.172.330.024
      OAS13.8120.080.991.680.011
      PLSCR13.67-0.091.000.940.17
      OAS33.69-0.1011.000.710.38
      SERPING13.58-0.260.530.980.14
      OAS23.310-0.040.892.340.001
      IFIT22.980.070.930.780.45
      RNASE22.760.520.0020.630.32
      PRKR2.77-0.080.580.590.010
      FCGR1A2.160.080.960.880.23
      STAT11.970.180.580.90.07
      Abbreviations: beta, standardized regression coefficients based on normalized variables; q, adjusted P values.
      Models are weighted for number of patients in each study and study quality.
      Generalized linear models are adjusted for history of lupus nephritis (%) and for prevalence of hydroxychloroquine usage (%) in the study populations.
      Bold indicates beta-values that differ from 0 and q-values ≤ 0.05.
      low asterisk mean fold change expression of the specified gene in the included studies.
      number of studies included with data for the specified gene.

      DISCUSSION

      This systematic review provides an overview of ISG expressions in full blood and PBMCs from SLE study populations and shows how these ISGs are associated with clinical and demographic characteristics in these populations. We found considerable variation in the degree of up-regulation of ISGs within and between the included studies and demonstrated that a cluster of highly expressed ISGs associated better with African ancestry than with disease activity in these cross-sectional studies.
      We found that prominent genes of the IFN signature in SLE, that is, IFI27, IFI44L, PRKR, RSAD2 and IFIT1, clustered with African ancestry, but not with other ancestries. In the current literature, there are only few studies specifically investigating ancestry associated ISG expression in SLE. Reynolds et al
      • Reynolds JA
      • Briggs TA
      • Rice GI
      • et al.
      Type I interferon in patients with systemic autoimmune rheumatic disease is associated with haematological abnormalities and specific autoantibody profiles.
      studied patients with systemic autoimmune rheumatic diseases (SLE, Sjögren's syndrome, undifferentiated connective tissue disease, mixed connective tissue disease, systemic sclerosis and idiopathic inflammatory myopathies) and noted an inverse association between the applied 6-ISG score and Caucasian ancestry. Weckerle et al
      • Weckerle CE
      • Franek BS
      • Kelly JA
      • et al.
      Network analysis of associations between serum interferon-alpha activity, autoantibodies, and clinical features in systemic lupus erythematosus.
      observed significantly higher IFN-α activity in African and Hispanic American SLE patients compared to European American SLE patients, which they partially attributed to the more frequent occurrence of SLE-associated autoantibodies in these patients, that in turn may trigger immune-complex induced IFN-production. Nevertheless, a positive association between non-European ancestry and IFN activity remained after controlling for all variables in the study. Banchereau et al
      • Banchereau R
      • Hong S
      • Cantarel B
      • et al.
      Personalized immunomonitoring uncovers molecular networks that stratify lupus patients.
      assessed SLE patients of different ancestral backgrounds for transcriptional differences and identified enrichment of plasmablast, cell cycle and erythropoiesis modules in African Americans, whereas Hispanics and Caucasians had other expression profiles, including enrichment of neutrophil, myeloid lineage and inflammation-related modules. Expression of IFN-associated genes thus seems to be significantly influenced by continental ancestry. This is further supported by results from McDade
      • McDade TW
      • M. Ross K
      • L. Fried R
      • et al.
      Genome-wide profiling of RNA from dried blood spots: convergence with bioinformatic results derived from whole venous blood and peripheral blood mononuclear cells.
      in which healthy African Americans had increased gene expression related to type I interferon signaling compared to healthy Caucasian Americans. However, an increasing degree of ancestral admixture in populations may challenge the precision of self-reported ancestry and it is therefore of great interest that genetic markers of ancestry may have the potential to address this problem.
      • Sharma S
      • Jin Z
      • Rosenzweig E
      • Rao S
      • Ko K
      • Niewold TB
      Widely divergent transcriptional patterns between SLE patients of different ancestral backgrounds in sorted immune cell populations.
      ,
      • Ghodke-Puranik Y
      • Imgruet M
      • Dorschner JM
      • et al.
      Novel genetic associations with interferon in systemic lupus erythematosus identified by replication and fine-mapping of trait-stratified genome-wide screen.
      The basis for ancestral differences in IFN expression is not well-explained but may be influenced by genetic and epigenetic factors. Population genetic studies suggest that pathogen-driven natural selection exerts a strong selective pressure on immune-related genes.
      • Fumagalli M
      • Sironi M.
      Human genome variability, natural selection and infectious diseases.
      In particular, 1 study found that African populations displayed greater genetic IFN diversity compared to Caucasian and Asian populations.
      • Manry J
      • Laval G
      • Patin E
      • et al.
      Evolutionary genetic dissection of human interferons.
      Several studies investigating DNA methylation variation, as the most common regulator of gene expression, indicated pronounced epigenetic differences in immune-related genes in individuals of African ancestry. Using primary monocytes as a model of innate immunity, 1 study found that genes associated with immune response regulation and responses to external stimulation were depleted in healthy individuals of African ancestry compared to those of European ancestry.
      • Husquin LT
      • Rotival M
      • Fagny M
      • et al.
      Exploring the genetic basis of human population differences in DNA methylation and their causal impact on immune gene regulation.
      In an epigenome-wide study of naïve CD4+ T-cells, healthy African Americans were found to have hypomethylation of genes related to positive regulation of apoptosis compared to European Americans, associated with enrichment of autoimmunity-related genes
      • Coit P
      • Ognenovski M
      • Gensterblum E
      • Maksimowicz-McKinnon K
      • Wren JD
      • Sawalha AH.
      Ethnicity-specific epigenetic variation in naive CD4+ T cells and the susceptibility to autoimmunity.
      . Furthermore, the same genes were hypomethylated in African American compared to European American SLE patients. Similar results were found in PBMCs.
      • Joseph S
      • George NI
      • Green-Knox B
      • et al.
      Epigenome-wide association study of peripheral blood mononuclear cells in systemic lupus erythematosus: Identifying DNA methylation signatures associated with interferon-related genes based on ethnicity and SLEDAI.
      Interestingly, 1 study has shown that the pattern of hypomethylation in IFN-regulated genes occurs early in B-cell development in African Americans, and could distinguish African American SLE patients from healthy controls in several B-cell subsets, whereas DNA methylation changes in European American SLE patients occurred in mature B-cell stages.
      • Breitbach ME
      • Ramaker RC
      • Roberts K
      • Kimberly RP
      • Absher D.
      Population-specific patterns of epigenetic defects in the B cell lineage in patients with systemic lupus erythematosus.
      Altogether, evidence from epigenetic studies suggests the existence of DNA methylation differences between populations that might point to etiological differences between African American SLE patients compared to those from other populations.
      The well-known association between African ancestry and lupus nephritis is visualized in the heatmap in Fig 3. An association of lupus nephritis with typical ISGs of the IFN signature is, however, less clear. Most of the ISGs analyzed in this article were not associated with lupus nephritis. IFI6 and OAS2 expressions were inversely associated with lupus nephritis; since also African ancestry was highly correlated with these genes this finding may not be robust. Moreover, earlier studies have provided evidence for a positive association between the IFN signature and active lupus nephritis,
      • Feng X
      • Wu H
      • Grossman JM
      • et al.
      Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
      as well as a history of lupus nephritis,
      • Baechler EC
      • Batliwalla FM
      • Karypis G
      • et al.
      Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.
      ,
      • Kirou KA
      • Lee C
      • George S
      • Louca K
      • Peterson MG
      • Crow MK.
      Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease.
      although the authors did not control for ancestry. More recently, a IFN signature was found to be predictive of a shorter time between SLE diagnosis and development of lupus nephritis, regardless of ancestry.
      • Arriens C
      • Raja Q
      • Husain SA
      • et al.
      Increased risk of progression to lupus nephritis for lupus patients with elevated interferon signature.
      Based on our current understanding of SLE pathogenesis with IFN as a major driver of inflammation, numerous previous studies have investigated a possible association between a blood IFN signature in SLE patients and disease activity. As of yet, no consensus has been reached. Various cross-sectional studies have found the IFN signature to be associated with disease activity.
      • Baechler EC
      • Batliwalla FM
      • Karypis G
      • et al.
      Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.
      • Kirou KA
      • Lee C
      • George S
      • Louca K
      • Peterson MG
      • Crow MK.
      Activation of the interferon-alpha pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease.
      • Feng X
      • Wu H
      • Grossman JM
      • et al.
      Association of increased interferon-inducible gene expression with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
      ,
      • Nikpour M
      • Dempsey AA
      • Urowitz MB
      • Gladman DD
      • Barnes DA.
      Association of a gene expression profile from whole blood with disease activity in systemic lupus erythematosus.
      ,
      • Higgs BW
      • Liu Z
      • White B
      • et al.
      Patients with systemic lupus erythematosus, myositis, rheumatoid arthritis and scleroderma share activation of a common type I interferon pathway.
      In these studies, composite scores of 3–31 ISG expressions were generated to assay the IFN signature. However, these findings are not supported by longitudinal studies on the association between the IFN signature and disease activity. Two studies using composite IFN scores of five and 3 genes, respectively, found the IFN score to remain relatively stable over time, not reflecting changes in disease activity.
      • Landolt-Marticorena C
      • Bonventi G
      • Lubovich A
      • et al.
      Lack of association between the interferon-alpha signature and longitudinal changes in disease activity in systemic lupus erythematosus.
      ,
      • Petri M
      • Singh S
      • Tesfasyone H
      • et al.
      Longitudinal expression of type I interferon responsive genes in systemic lupus erythematosus.
      A modular analysis of the IFN signature
      • Chiche L
      • Jourde-Chiche N
      • Whalen E
      • et al.
      Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures.
      revealed both stable and variable components. However, the same modular approach used by Petri et al
      • Petri M
      • Fu W
      • Ranger A
      • et al.
      Association between changes in gene signatures expression and disease activity among patients with systemic lupus erythematosus.
      showed stability of the IFN signature over time. A more recent and very comprehensive meta-analysis by Catalina et al
      • Catalina MD
      • Bachali P
      • Geraci NS
      • Grammer AC
      • Lipsky PE.
      Gene expression analysis delineates the potential roles of multiple interferons in systemic lupus erythematosus.
      found no association between the IFN signature and the SLEDAI in 2000 patients and did not find changes in the IFN signature to be predictive of disease flares. The authors link this finding to the predominance of monocytes in the IFN signature and show that these cells retain up-regulation of ISGs also in inactive SLE patients. El-Sherbindy et al
      • El-Sherbiny YM
      • Psarras A
      • Md Yusof MY
      • et al.
      A novel two-score system for interferon status segregates autoimmune diseases and correlates with clinical features.
      propose that the discordant findings on the association between the IFN signature and disease activity could be related to a variable association between the IFN signature and different disease manifestations. To this end, it is of interest that kidney biopsies from SLE patients show increased ISG expression, however, more prominent IFN signatures are found in SLE skin and synovium.
      • Catalina MD
      • Bachali P
      • Geraci NS
      • Grammer AC
      • Lipsky PE.
      Gene expression analysis delineates the potential roles of multiple interferons in systemic lupus erythematosus.
      Genes that we found to be associated with African ancestry had generally higher fold-change expression values than genes associated with disease activity. Furthermore, genes associated with African ancestry were all predominantly sensitive to type I IFN stimulation; genes only associated with disease activity were either, mainly type II IFN sensitive (RNASE2) or mainly type I IFN sensitive with type II IFN stimulation synergy (IFI27).
      • Rusinova I
      • Forster S
      • Yu S
      • et al.
      Interferome v2.0: an updated database of annotated interferon-regulated genes.
      ,
      • Sanda C
      • Weitzel P
      • Tsukahara T
      • et al.
      Differential gene induction by type I and type II interferons and their combination.
      ,
      • Liu M
      • Liu J
      • Hao S
      • et al.
      Higher activation of the interferon-gamma signaling pathway in systemic lupus erythematosus patients with a high type I IFN score: relation to disease activity.
      This finding is in line with a previous modular analysis of the IFN signature that identified stable and variable components.
      • Chiche L
      • Jourde-Chiche N
      • Whalen E
      • et al.
      Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures.
      RNASE2 encodes an enzyme with immune cell attractant and anti-viral properties,
      • Rosenberg HF.
      Eosinophil-derived neurotoxin /RNase 2: connecting the past, the present and the future.
      and has previously been associated with disease activity in SLE PBMCs.
      • Baechler EC
      • Batliwalla FM
      • Karypis G
      • et al.
      Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus.
      IFI27 was in this review found to be the ISG with the highest average fold-change expression in SLE patients and like other common ISGs, IFI27 has growth inhibitory and anti-viral functions including the ability to sensitize cells to apoptosis.
      • Gytz H
      • Hansen MF
      • Skovbjerg S
      • et al.
      Apoptotic properties of the type 1 interferon induced family of human mitochondrial membrane ISG12 proteins.
      ,
      • Patro SC
      • Pal S
      • Bi Y
      • et al.
      Shift in monocyte apoptosis with increasing viral load and change in apoptosis-related ISG/Bcl2 family gene expression in chronically HIV-1-infected subjects.
      Thus, in the context of SLE high IFI27 expression could be speculated to exacerbate aberrant apoptosis characteristic of SLE pathogenesis fueling inflammatory activity.
      • Colonna L
      • Lood C
      • Elkon KB.
      Beyond apoptosis in lupus.
      However, this review did not aim for and was not designed for identifying new expression markers of SLE disease activity and other investigations are needed to reveal the genuine robustness of these findings.
      The strength of this review lies in the systematic analysis of data cumulated from study populations with diverse ancestral backgrounds and clinical contexts. However, we recognize several possible limitations.  Firstly, the lack of consensus on the IFN signature was reflected in the diverse selection of ISG for each study. Furthermore, some ISGs were studied in only a few works, making them unsuitable for our analyses. Secondly, some studies lacked data on demographic and clinical characteristics of the included patients. A quantitative comparison of ISG fold-change values from several studies is challenging. Each study has selected a different cohort of controls serving as baseline ISG expression, thereby introducing a possible source of variation in assessing ISG expression across studies. As the variation in ISG expression in healthy populations has not been adequately studied, its impact on the investigated study populations is difficult to predict. However, we think that this drawback is overruled by the comparability between case and control population with respect to ancestry. Additionally, the methods of expression data normalization and correction for multiple testing also vary between studies. These factors are a general concern in comparison with data from microarray studies between laboratories. Lastly, the sparse reporting of medication usage in the selected studies did limit a more extensive correction for the possible influence of immunosuppressive/immunoregulating medicines on the IFN signature; with the data that was available in this systematic review it was only possible to correct for antimalarial medication usage in the HCA and the GLMs.
      In this review, we chose to focus on the available transcriptomic data on the IFN signature in peripheral blood, as this is the method used in most studies and which would be most readily available in a clinical setting. However, the cellular composition of peripheral blood varies greatly among SLE patients, with different cell populations contributing to the IFN signature, making it difficult to interpret the transcriptomic data functionally.
      • Becker AM
      • Dao KH
      • Han BK
      • et al.
      SLE peripheral blood B cell, T cell and myeloid cell transcriptomes display unique profiles and each subset contributes to the interferon signature.
      There is evidence for monocytes to be the strongest contributors to the IFN signature observed in blood, expressing about 3 times as many ISG transcripts as B and T cells,
      • Catalina MD
      • Bachali P
      • Geraci NS
      • Grammer AC
      • Lipsky PE.
      Gene expression analysis delineates the potential roles of multiple interferons in systemic lupus erythematosus.
      although there is considerable overlap between hypomethylation of IFN-regulated genes in different immune cell populations in SLE patients.
      • Ulff-Moller CJ
      • Asmar F
      • Liu Y
      • et al.
      Twin DNA methylation profiling reveals flare-dependent interferon signature and b cell promoter hypermethylation in systemic lupus erythematosus.
      Cell-type specific IFN signatures in blood may, thus, be another way to further dissect the IFN signature.
      In SLE, there is an unmet need for biomarkers for early diagnosis, to help differentiate flares from infection, to identify clinically relevant phenotypes, to predict treatment response of various immunological interventions and thus to enable more tailored treatment strategies. The IFN signature might be promising for several of these purposes; but as pointed out by Paredes et al the current definition and usage of the IFN signature to predict response of IFN antagonists in SLE may not to a sufficient degree discriminate between constitutively high expression of ISGs and IFN signaling that reflects pathway activity.
      • Paredes JL
      • Niewold TB.
      Type I interferon antagonists in clinical development for lupus.
      This view is supported by our findings and suggests that future studies are needed to dissect the IFN signature with a view on the intended purpose of use. El-Sherbindy et al have, for example, established 2 interferon scores,
      • El-Sherbiny YM
      • Psarras A
      • Md Yusof MY
      • et al.
      A novel two-score system for interferon status segregates autoimmune diseases and correlates with clinical features.
      where score A predicted flares of SLE and score B predicted development of SLE in at-risk individuals.
      • Md Yusof MY
      • Psarras A
      • El-Sherbiny YM
      • et al.
      Prediction of autoimmune connective tissue disease in an at-risk cohort: prognostic value of a novel two-score system for interferon status.
      Alase et al showed that a high IFN-score B was associated with a better clinical response to rituximab treatment.
      • Alase A
      • Wigston Z
      • Burska A
      • et al.
      Prediction of response to Rituximab in SLE using a validated two-score system for interferon status.
      Further studies are needed to deepen our systematic understanding of the IFN signature and its determinants not only in patients with SLE but also other systemic autoimmune diseases characterized by IFN mediated immune activation such as primary Sjögren's syndrome
      • Nezos A
      • Gravani F
      • Tassidou A
      • et al.
      Type I and II interferon signatures in Sjogren's syndrome pathogenesis: contributions in distinct clinical phenotypes and Sjogren's related lymphomagenesis.
      and antiphospholipid antibody syndrome.
      • Ugolini-Lopes MR
      • Torrezan GT
      • Gandara APR
      • et al.
      Enhanced type I interferon gene signature in primary antiphospholipid syndrome: association with earlier disease onset and preeclampsia.
      ,
      • Iwamoto T
      • Dorschner J
      • Jolly M
      • Huang X
      • Niewold TB.
      Associations between type I interferon and antiphospholipid antibody status differ between ancestral backgrounds.
      Meta-analyses on existing datasets of the IFN signature are of great value, as they allow for dealing with the great heterogeneity of included patients, treatment and microarray techniques. However, such datasets are mostly cross-sectional and longitudinal studies of large well-characterized clinical cohorts with participants of diverse ancestral backgrounds are highly desirable to move this field forward.
      In conclusion, we were able to show that a cluster of highly expressed ISGs often used to define IFN signatures was more associated with African ancestry than disease activity in cross-sectional studies of SLE patients. This underscores the importance of taking ancestry into account when employing the IFN signature for clinical research in SLE.

      ACKNOWLEDGMENTS

      Conflicts of Interest: All authors have read the journal's authorship statement and policy on disclosure of potential conflicts of interest and state that they have no disclosures as to any financial or personal relationship with organizations that could potentially be perceived as influencing the described research. This manuscript has been reviewed and approved by all named authors.
      Supported by grants from the Danish Rheumatism Association (non-commercial, A3865 ) and the Independent Research Fund Denmark (non-commercial, 0134-00473B ).

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