Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. TRANSLATIONAL SCIENCE Methylome and transcriptome profiling of giant cell arteritis monocytes reveals novel pathways involved in disease pathogenesis and molecular response to glucocorticoids Elkyn Estupiñán-­Moreno ‍ ‍,1 Lourdes Ortiz-­Fernández ‍ ‍,1 Tianlu Li,2 Jose Hernández-­Rodríguez,3 Laura Ciudad,2 Eduardo Andrés-­León,1 Laura Carmen Terron-­Camero,1 Sergio Prieto-­González,3 Georgina Espígol-­Frigolé,3 Maria Cinta Cid ‍ ‍,3 Ana Márquez ‍ ‍,1,4 Esteban Ballestar ‍ ‍,2 Javier Martín ‍ ‍1 Handling editor Josef S ABSTRACT Smolen Objectives Giant cell arteritis (GCA) is a complex WHAT IS ALREADY KNOWN ON THIS TOPIC systemic vasculitis mediated by the interplay between ⇒ Giant cell arteritis (GCA) is a complex disease ► Additional supplemental material is published online both genetic and epigenetic factors. Monocytes are mediated by multiple genetic and epigenetic only. To view, please visit the crucial players of the inflammation occurring in GCA. factors, in which CD14+ monocytes play journal online (http://d​ x.​doi.​ Therefore, characterisation of the monocyte methylome an important role driving the inflammatory org/1​ 0.​1136/​annrheumdis-​ processes occurring in this vasculitis. and transcriptome in GCA would be helpful to better 2022-2​ 22156). understand disease pathogenesis. ⇒ The study of the DNA methylation and gene For numbered affiliations see Methods We performed an integrated epigenome-­ expression profiles of disease-­relevant cell end of article. and transcriptome-­wide association study in CD14+ types, as well as the integration of omics- monocytes from 82 patients with GCA, cross-­sectionally datasets, has emerged as a successful approach Protected by copyright. Correspondence to classified into three different clinical statuses (active, in to better understand the pathogenesis of Dr Lourdes Ortiz-­Fernández and complex diseases. remission with or without glucocorticoid (GC) treatment), Dr Javier Martín, Institute of Parasitology and Biomedicine and 31 healthy controls. López-­Neyra, IPBLN, Consejo Results We identified a global methylation and WHAT THIS STUDY ADDS Superior de Investigaciones gene expression dysregulation in GCA monocytes. ⇒ We evaluate for the first time the DNA Cientificas, CSIC, 18016 Specifically, monocytes from active patients showed Granada, Spain; methylome and transcriptome landscapes of l​ ourdes@​ipb.​csic.​es, ​ a more proinflammatory phenotype compared with CD14+ monocytes from patients with GCA in javiermartin@​ipb.​csic.​es and healthy controls and patients in remission. In addition to three different states of the disease (patients Dr Esteban Ballestar, Epigenetics inflammatory pathways known to be involved in active with active disease, patients in remission with and Immune Disease Group, GCA, such as response to IL-­6 and IL-­1, we identified or without treatment), identifying profound Josep Carreras Research response to IL-­11 as a new pathway potentially alterations that provide evidence of novel genes Institute (IJC), 08916 Badalona, Barcelona, Spain; implicated in GCA. Furthermore, monocytes from patients and pathways potentially involved in GCA e​ ballestar@​carrerasresearch.​org in remission with treatment showed downregulation pathogenesis. of genes involved in inflammatory processes as well as ⇒ The results of this integrative approach allowed EE-­M and LO-­F contributed overexpression of GC receptor-­target genes. Finally, we equally. the identification of a significant number of identified changes in DNA methylation correlating with CpG-­gene expression interactions, including EE-­M and LO-­F are joint first alterations in expression levels of genes with a potential important genes potentially involved in the authors. role in GCA pathogenesis, such as ITGA7 and CD63, as molecular mechanisms implicated in the active AM, EB and JM are joint senior well as genes mediating the molecular response to GC, state of the disease, such as ITGA7 and CD63, authors. including FKBP5, ETS2, ZBTB16 and ADAMTS2. as well as genes mediating the molecular Received 13 January 2022 Conclusion Our results revealed profound alterations response to glucocorticoids, including FKBP5, Accepted 17 May 2022 in the methylation and transcriptomic profiles of ETS2, ZBTB16 and ADAMTS2. Published Online First monocytes from GCA patients, uncovering novel genes 15 June 2022 and pathways involved in GCA pathogenesis and in the HOW THIS STUDY MIGHT AFFECT RESEARCH, molecular response to GC treatment. PRACTICE AND/OR POLICY ⇒ A better understanding of the pathogenesis of GCA might result in the identification of INTRODUCTION potential biomarkers that allow advances in © Author(s) (or their early diagnosis, classification and therapy for employer(s)) 2022. Re-­use Giant cell arteritis (GCA) is a systemic vasculitis permitted under CC BY. with complex aetiology, presenting a wide range of GCA. Published by BMJ. clinical manifestations.1 The most serious compli- To cite: Estupiñán-­Moreno E, cations such as irreversible blindness and stroke can Ortiz-­Fernández L, Li T, be significantly reduced if patients receive prompt its clinical heterogeneity, including the presence of et al. Ann Rheum Dis treatment with glucocorticoids (GC).2 However, non-­specific symptoms, along with the absence of 2022;81:1290–1300. early recognition of GCA can be challenging due to specific biomarkers.3 1290   Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. The disease results from dysregulated interactions between MATERIAL AND METHODS the vessel wall and the immune system that lead to inflammation Details of the population included in this study, description of and vascular remodelling of medium and large arteries.4 Most of the experimental methods, including DNA methylation and our current knowledge has been obtained from examination of RNA sequencing as well as the statistical analyses applied, are temporal artery biopsies, where granulomatous infiltrates consist provided in online supplemental material. of both innate and adaptive immune cells.5 Besides tissue lesions, GCA is characterised by intense systemic inflammation that is RESULTS driven by IL-­6.6 Although studies focusing on circulating immune Dysregulated DNA methylation and gene expression profiles cells in GCA are scarce, monocytes are considered major players in in CD14+ monocytes of patients with GCA the inflammatory process.7 Indeed, circulating monocytes of GCA CD14+ monocytes from patients with GCA showed a global patients interact with activated endothelial cells in vasa vasorum and hypermethylation pattern neovessels,8 9 and develop tissue invasive capabilities by an aberrant First, the comparison of the DNA methylation landscape of production of matrix metalloproteinase (MMP)-­9, thus allowing CD14+ monocytes between patients with GCA and controls immune cells to access the vascular wall.10 unveiled the existence of 1371 differentially methylated posi- It is well established that epigenetic modifications may exert a tions (DMPs), annotated to 1190 unique genes, across the whole profound influence on cell function by their capacity to modulate genome (figure 1A). Most DMPs were located in intergenic gene expression without altering the DNA sequence.11 Despite the regions (31.8% hypermethylated and 26.6% hypomethylated) high relevance of monocytes in GCA, no study has investigated the and gene bodies (44.3% hypermethylated and 41.4% hypometh- DNA methylation landscape of this cell type to date. Here, with the ylated) and less frequently in promoters, consistent with the aim of shedding light into GCA pathogenesis and identifying molec- findings of studies in other inflammatory conditions,12 13 which ular mechanisms that might serve as novel biomarkers or potential suggests that a substantial part of the methylation aberrations drug targets, we analysed for the first time the methylome and tran- might be located in distal regulatory regions. In addition, DMPs, scriptome of GCA monocytes as well as the correlation between mainly hypermethylated DMPs, were mostly located in open sea DNA methylation and gene expression levels. regions, outside CpG island and surrounding areas (figure 1B). Protected by copyright. Figure 1 Results from the comparison of both DNA methylation and gene expression patterns of CD14+ monocytes between patients with giant cell arteritis and controls. (A) Volcano plot of the epigenome-­wide association study results. False discovery rate (FDR) values are represented on the –log10 scale in the y-­axis. Significant threshold (FDR<0.05) is marked by a dashed line. The effect size and direction obtained for each CpG site is depicted in the x-­axis. Pink and blue dots represent hypermethylated and hypomethylated differentially methylated positions (DMPs), respectively. (B) Bar plots representing the annotation of the significant hypermethylated and hypomethylated DMPs in relation to CpG island (right panel) and gene location (left panel). (C) Representation of selected gene ontology categories obtained from the DMPs enrichment analysis using the GREAT online tool. (D) Volcano plot of the transcriptome-­wide association study results. FDR values are represented on the –log10 scale in the y-­axis. Significant threshold (FDR<0.05) is marked by a dashed line. The effect size and direction obtained for each gene is depicted in the x-­axis. Purple and orange dots represent upregulated and downregulated differentially expressed genes, respectively. GCA, giant cell arteritis. Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 1291 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. Over 85% of the DMPs showed higher DNA methylation levels in online supplemental material). To better characterise the in patients than in controls. Of note, we identified hypermethyl- methylation alterations driving the molecular mechanisms ated DMPs located within or close to genes previously associated responsible of the active state of GCA, we compared the meth- with immune-­mediated diseases, including P4HA2, a suscepti- ylation landscape of patients with GCA with active disease bility genetic factor for GCA 14 (figure 1A and online supple- with those showing no sign or symptoms of this vasculitis mental table 1). Through gene ontology analysis, we observed (healthy controls and patients in remission with and without enrichment in functional pathways of the immune response, GC treatment). In addition, we also aimed to evaluate the such as regulation of interferon-­ gamma (IFN-γ) production, influence of GC on the DNA methylation patterns of CD14+ leucocyte chemotaxis and integrin biosynthesis processes. In monocytes in disease remission by comparing treated and non-­ addition, we detected a significant enrichment in monocyte treated patients. cell biology pathways, such as the colony stimulating factor 1 We observed a large number of significant DNA methyla- (CSF1)-­CSF1 receptor complex, differentiation and prolifera- tion alterations between patients with active disease compared tion of macrophages and cytokine production like macrophage with controls and those patients in remission. In contrast, only colony-­stimulating factor (figure 1C and online supplemental several CpGs were differentially methylated between patients in table 2). The hypomethylated DMPs were also mapped to rele- remission with and without treatment. A summary of the results vant genes in the context of the immune response, like TRIM24, obtained in each analysis is shown in figure 2A. PRDM16, PARP9, ADARB1, CD38 or MICB (figure 1A and online supplemental table 3), and were enriched in significant biological processes like cellular response to IL-­ 4, oxidative Global DNA methylation alterations reflect the active state of the stress response, positive regulation of chemotaxis, complement disease component C3a binding and negative regulation of the platelet-­ We first assessed the differences between the methylation derived growth factor-­beta receptor (PDGF) signalling pathway status of CD14+ monocytes from patients with active disease (figure 1C, online supplemental table 4). and healthy controls, identifying a total of 1444 DMPs (507 hypomethylated and 937 hypermethylated in active patients) Global analysis of patients with GCA shows slight alterations of the (figure 3A and online supplemental tables 7-­8). Interestingly, gene expression profile some of these DMPs mapped to genes encoding chemokine Afterwards, we carried out gene expression analysis between receptors such as CX3CR1, CXCR2P1 and CXCR4, members CD14+ monocytes from patients with GCA and controls. These of the interferon regulatory transcription factor (IRF) family, Protected by copyright. results only revealed 54 differentially expressed genes (DEGs), of including IRF2 and IRF8, as well as genes previously described which 41 were upregulated in GCA patients (figure 1D, online as susceptibility factors for systemic vasculitides, including supplemental tables 5-­ 6). In this regard, ADAMTS2, CD163, TYK2 and KDM4C15 16. In addition, hypermethylated DMPs AMPH, FLT3 and IL1R2 were observed to be among the most were enriched in multiple pathways and biological processes, significantly upregulated DEGs. including positive regulation of cell activation and cell–cell adhe- sion and response to tumour necrosis factor (TNF) (figure 3E Stratified analysis of patients based on clinical status and and online supplemental table 9). Regarding hypomethylated treatment display specific DNA methylation patterns in DMPs, a significant enrichment in interesting pathways in the CD14+ monocytes context of GCA pathogenesis was detected, such as regulation We further stratified patients with GCA according to the of macrophage activation involved in immune response, positive state of the disease at the time of sample collection: patients regulation of lymphocyte migration and regulation of inflamma- with active disease, patients in remission with treatment and tory response, among others (figure 3E and online supplemental patients in remission without treatment (detailed description table 10). Figure 2 Summary of the results obtained from the stratified analysis of patients according to the state of the disease. (A) Number of significant differentially methylated positions (DMPs) and differentially expressed genes (DEGs) obtained in each comparison. (B) Venn diagrams showing the overlap of significant DMPs (left panel) and DEGs (right panel) among the different comparisons performed. ACT, active disease; CRTL, controls; RNT, remission without treatment; RT, remission with treatment. 1292 Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. Protected by copyright. Figure 3 Results of the epigenome-­wide association study obtained from the stratified analysis of patients according to the state of the disease. (A–D) Volcano plots showing the results of the epigenome-­wide association study for each comparison performed. False discovery rate (FDR) values are represented on the –log10 scale in the y-­axis. Significant threshold (FDR<0.05) is marked by a dashed line. The effect size and direction obtained for each CpG site is depicted in the x-­axis. Pink and blue dots represent hypermethylated and hypomethylated differentially methylated positions (DMPs), respectively. (E) Scheme summarising the results from the gene ontology enrichment analysis performed using the GREAT online tool. Columns show the different comparisons carried out in the stratified analysis and rows represent selected gene ontology categories. Pink colour denotes statistical significant enrichment of hypermethylated DMPs and blue colour indicates statistical significant enrichment of hypomethylated DMPs. ACT, active disease; CRTL, controls; RT, remission with treatment; RNT, remission without treatment. Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 1293 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. Subsequently, we identified substantial alterations in the meth- such as response to corticosterone and cellular response to GC ylation patterns of monocytes from active disease compared with stimulus was significantly detected (figure 3E and online supple- patients in remission without treatment. Specifically, we identi- mental table 20). Furthermore, the analysis of the TF binding fied 820 DMPs (668 hypomethylated and 152 hypermethylated motifs revealed that the cluster of hypomethylated DMPs in in active patients) (figure 3B and online supplemental tables 11 patients in remission with GC treatment was enriched in GC and 12). Noteworthy, a considerable part of these findings was response elements (online supplemental figure 1), a family of similar to those obtained in the previous comparison (figure 2B). TF reported to repress the activity of the nuclear factor-­kappa B Aside from the common features, hypomethylated DMPs iden- (NF-­kB) pathway.24 25 tified when compared active patients with patients in remis- sion without treatment were enriched in additional biological GCA remission leads to reversal of methylation changes pathways, such as regulation of reactive oxygen species meta- It should be noted that a significant part of the DMPs identi- bolic processes, positive regulation of IL-­2 production, nega- fied in the comparison between active patients and controls is tive regulation of TNF biosynthetic process and regulation of common to those detected between active patients and patients angiogenesis. Moreover, hypermethylated DMPs were enriched in remission without treatment (figure 2B), suggesting that the in regulatory processes of complement activation, positive regu- DNA methylation landscapes of these two groups of individuals lation of erythrocyte aggregation, regulation of the metabolic are similar. Consistently, no significant DMPs were observed process of reactive oxygen species (figure 3E and online supple- between patients in remission without treatment and healthy mental tables 13 and 14). controls. This evidence indicates that the DNA methylation As expected, CD14+ monocytes from patients with active alterations occurring in monocytes from individuals with active disease showed large differences in the methylation landscape disease are reverted when the disease subsides. compared with those from patients in remission with treatment. We identified a total of 688 DMPs, of which 85% presented low Identification of aberrant gene expression profiles in CD14+ levels of methylation in patients with active disease (figure 3C monocytes through stratified analysis of patients based on and online supplemental tables 15 and 16). These hypomethyl- ated DMPs were enriched in pathways implicated in the immu- clinical status and treatment Following the same reasoning aforementioned, and consid- nopathogenic processes of GCA, including the cellular response ering the few differences observed in the comparison between to IL-­6 as well as response to other members of the IL-­6 family, global GCA patients and healthy controls, we also performed a specifically IL-­11 (figure 3E and online supplemental table Protected by copyright. stratified analysis of the gene expression landscape in patients 17).17 18 In contrast, hypermethylated DMPs were enriched in with GCA according to the state of the disease: patients with pathways of drug metabolism, cellular response to GC stimulus active disease, patients in remission with treatment and patients and regulation of blood vessel remodelling (figure 3E and online in remission without treatment (detailed description in online supplemental table 18). supplemental material). Analysis of the transcription factor (TF) binding motifs Figure 2A summarises the results obtained through each anal- showed that the hypomethylated DMPs identified in the active ysis. Our results revealed that the gene expression profile of patients compared with controls and patients in remission with CD14+ monocytes largely varies according to the clinical status and without treatment were enriched in the basic region-­leucine and treatment. The validity of these results was supported by the zipper (bZIP) family, suggesting that factors in this family might high correlation observed for eight deregulated genes (ITGA7, play a key role in the regulation of the molecular mechanisms CD63, CCRL2, CD300E, CD163, ETS2, FKBP5 and SBNO2) implicated in the active state of the disease (online supplemental between ∆Ct values obtained by qPCR and their normalised figure 1). Particularly, the bZIP TF family has been reported intensities from the RNA-­seq (Spearman rank, |R|=0.79) to regulate the expression of genes involved in angiogenesis, (online supplemental figure 2). Furthermore, as expected, we fibrosis and Th17 cells plasticity control.19–22 Furthermore, IRF have confirmed the significant deregulation of these eight genes family, known to be involved in monocyte/macrophage polari- observed among subgroups (online supplemental table 21). sation,23 was significantly enriched in the cluster of hypermeth- ylated DMPs in active patients in comparison with controls and patients in remission without treatment. Gene expression deregulation in monocytes affects relevant Remarkably, 65% from the total of DMPs observed in the molecular mechanisms during the active state of the disease comparisons between active patients and patients in remission The comparison between the expression patterns of CD14+ with and without treatment were common (figure 2B). This monocytes from patients with GCA with active disease and similarity might be reflecting the success of the GC treatment. controls revealed 292 DEGs (figure 4A and online supplemental tables 22 and 23). The majority (72%) of DEGs presented higher levels of expression in the subgroup of patients with active GC treatment greatly affects methylation levels of glucocorticoid disease. Interestingly, overexpressed genes were found to be receptor target genes enriched in related biological processes such as cell adhesion, We then compared patients in remission with and without treat- extracellular matrix disassembly, cell–matrix adhesion, integrin ment in order to assess the impact of the GC treatment in the DNA complex, among others. The enrichment in negative regulation methylation landscape of CD14+ monocytes, which revealed of angiogenesis and the positive regulation of TNF production 27 CpG sites showing different methylation levels between should also be mentioned (figure 4E and online supplemental the two groups (figure 3D and online supplemental table 19). table 24). Remarkably, the downregulated DEGs were enriched Remarkably, these DMPs, most of which were hypomethylated in the major histocompatibility complex (MHC) class II protein in patients in remission with treatment, showed high differences complex binding including genes like HLA-­DMB and HLA-­DOA in their beta values, for example, ETS2 (ΔBeta=−0.39) and (figure 4E and online supplemental table 25). ZBTB16 (ΔBeta=−0.38). It should be mentioned that, despite Subsequently, we compared the gene expression patterns the low number of DMPs, enrichment of biological processes of CD14+ monocytes from active disease and patients in 1294 Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. Protected by copyright. Figure 4 Results of the transcriptome-­wide association study obtained from the stratified analysis of patients according to the state of the disease. (A–D) Volcano plots showing the results of the transcriptome-­wide association study for each comparison. False discovery rate (FDR) values are represented on the –log10 scale in the y-­axis. Significant threshold (FDR<0.05) is marked by a dashed line. The effect size and direction obtained for each gene is depicted in the x-­axis. Purple and orange dots represent upregulated and downregulated differentially expressed genes (DEGs), respectively. (E) Scheme summarising the results from the gene ontology enrichment analysis performed using the DAVID online tool. Columns show the different comparisons carried out in the stratified analysis and rows represent selected gene ontology categories. Purple colour denotes statistical significant enrichment of upregulated DEGs and orange colour indicates statistical significant enrichment of downregulated DEGs. ACT, active disease; CRTL, controls; RT, remission with treatment; RNT, remission without treatment. Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 1295 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. remission without treatment, identifying 551 DEGs (327 GIMAP2, GIMAP4, GIMAP6, GIMAP7 and GIMAP8) (online upregulated and 224 downregulated in active patients) supplemental table 31). (figure 4B and online supplemental tables 26 and 27). The cluster of 327 upregulated DEGs was enriched in path- ways including platelet degranulation, cell–matrix adhesion, GC treatment reshapes the gene expression landscape of circulating integrin-­mediated signalling pathway, lipid metabolic process, monocytes rheumatoid arthritis and cytokine–cytokine receptor interac- The gene expression landscapes of patients in remission with tion (figure 4E and online supplemental table 28). Of special and without treatment were largely different, with 3550 DEGs mention, HLA-­DMB, HLA-­DOA and HLA-­DRA were under- when comparing both subgroups (1818 upregulated and 1732 expressed in active patients (online supplemental table 27). downregulated in treated patients) (figure 4D and online supple- In this context, these results are in accordance with previous mental tables 34 and 35). As reflected in gene ontology analysis, studies reporting a lower expression of HLA-­DRA in mono- GC treatment might be altering a large number of molecular cytes in inflammatory conditions like sepsis and the immune mechanisms, especially those related with mitochondrial func- dysregulation caused by SARS-­ CoV-­2.26 27 Indeed, among tion. Interestingly, as similar to the previous comparison, we also the most significant results of the gene ontology analysis, we observed enrichment in acetylation of several histones, which observed antigen processing and presentation and MHC class could indicate that other epigenetic mechanisms, besides DNA II protein complex binding. In addition, it is also worth high- methylation, might be affected (figure 4E and online supple- lighting the enrichment of important inflammatory pathways mental table 36). On another side, treated patients showed such as TNF-­mediated and response to IFN-γ (figure 4E and downregulation in multiple inflammatory-­ related pathways, online supplemental table 29). apoptotic processes and, notably, defence response to viruses, Afterwards, we observed greater significant differences including pathways such as Herpes simplex infection, Epstein-­ between the expression profiles of CD14+ monocytes from Barr virus infection, Hepatitis B and Influenza A, among others patients with GCA with active disease and patients in remis- (figure 4E and online supplemental table 37). Unlike the simi- sion with treatment. 5433 DEGs were identified, of which larity observed in DNA methylation patterns, the large differ- 2701 and 2732 DEGs were upregulated and downregulated ences identified in the gene expression patterns between treated in the active disease group, respectively (figure 4C and online and untreated patients are supported by previous evidence supplemental tables 30 and 31). These results were consistent describing, in other immune contexts, that the GC treatment has with the previous knowledge of GCA pathogenesis. In this an important effect in reshaping the gene expression landscape but relatively low impact in the DNA methylation profile.13 Protected by copyright. sense, IL-­6 and MMP9, as well as other members of the MMP family (MMP2, MMP24, MMP14, MMP19 and MMP25), were upregulated in active patients. We also detected over- Aberrant gene expression profile of monocytes in the active state of expression of several genes of the integrin family, such as the disease is lost after remission ITGA2B, ITGA5, ITGA6, ITGA7, ITGAX, ITGAV, ITGB1, Consistent to what we observed in the methylation analyses, ITGB3, ITGB5, ITGB7 and ITGB8, as well as other remark- the gene expression patterns of patients in remission without able genes that are important in monocyte cell biology like treatment and healthy controls were similar. Indeed, 208 DEGs CCR2, CCL2, CCL7, CXCL5, CXCL2 and CXCL3 (online identified in the comparisons of active patients with patients supplemental table 30). 28 Of note, important biological in remission without treatment and controls were common processes and pathways involved in GCA pathogenesis such (figure 2B), and only one gene showed significantly different as angiogenesis, TNF signalling pathway, vascular endothelial expression levels between patients in remission without treat- growth factor (VEGF) receptor pathway, chemokine signal- ment and healthy controls. Specifically, higher expression levels ling, mitogen-­activated protein kinase (MAPK) cascade, Toll-­ of MTCO3P12 were observed in patients in remission without like receptor signalling (TLR) pathway and cellular response treatment (FDR=0.04, logFC=1.51). to IL-­6, were enriched among the set of upregulated genes in patients with active disease (figure 4E and online supple- mental table 32). Among the large number of downregulated Integrative analysis revealed the existence of relationships DEGs, it is notable the presence of genes related with GC between DNA methylation changes and gene expression such as FKBP5, a cochaperone that modulates GC receptor alterations (GR) activity, ZBTB16, transcriptional factor contributing Finally, by performing an integrative analysis, we aimed to to energy balance after GR activation and HPGD which investigate the potential relationship between DNA methyl- encondes a dehydrogenase expressed on dexamethasone ation alterations and gene expression in GCA. We found 10 (online supplemental table 31).29–32 Accordingly, mitochon- 191 significant CpG-­gene expression interactions (FDR<0.05). drial metabolic process characteristic of drug metabolism, To focus on the interactions that might be potentially relevant specifically GC, were significantly enriched (figure 4E and for GCA pathogenesis, we selected 470 CpG-­gene expression online supplemental table 33). In addition, we also found interactions showing both methylation and gene expression enrichment in negative regulation of the type I interferon levels significantly associated in at least one of the comparisons production and the innate response as well as metabolic path- performed. Of these, 34 CpG–gene expression interactions were ways like oxidative phosphorylation or glucose metabolic significant in more than one comparison. Among the 436 unique process. Of special interest is the enrichment of the apop- interactions, we detected 409 DEGs (254 upregulated and totic process that included underexpression of crucial genes 155 downregulated) and 195 DMPs (176 hypomethylated and in the context of immune-­mediated disorders like TNFAIP3, 19 hypermethylated). In addition, we found that 65.53% and DNASE1, AIM2 and PTK2B, among others. We also detected 34.47% of the total interactions associated with disease or clin- downregulation of known autoimmunity-­ related genes, ical status were negative and positive correlations, respectively. as examples: PLD4, FLT3, ERAP2, BTK, MEFV, DNASE1, Finally, when studying the distribution of CpGs in relation to the PADI14, JAZF1 and GIMAP family members (GIMAP1, genes that they interact with, we identified that 10.39% of the 1296 Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. negative and 1.23% of the positive correlations were located at vascular remodelling in GCA.37 38 Therefore, further studies will the gene promoter (figure 5A and online supplemental table 38). be needed in order to determine the impact of this cytokine in We detected interactions that involved relevant genes in the GCA. context of the pathophysiology of GCA. One example was Interestingly, functional categories enriched among the set ITGA7, upregulated in GCA patients with active disease, in of differential methylated genes and DEGs in active patients which it showed three interactions with different CpGs located suggest a relevant role of monocytes in GCA by recruiting in intergenic regions, involving one positive correlation with immune cells and through their interaction with lymphocytes, cg24773560 (r=0.44, FDR=3.03E-­02) and two negative mainly by promoting their differentiation and activation. In this correlations with cg08420353 (r=−0.51, FDR=1.85E-­03) sense, a significant number of genes encoding chemokines, such and cg17016513 (r=−0.41, FDR=4.31E-­02). Interestingly, as CCL2 and CCL7, involved in the recruitment of monocytes, these last two CpGs also showed negative correlation with were overexpressed in active patients.39 Furthermore, genes the CD63 gene (r=−0.41, FDR=4.35E-­02 and r=−0.41, encoding several integrins were also overexpressed. Specifically, FDR=4.92E-­02, respectively), which was also upregulated in the expression level of ITGA7 showed the greatest differences active disease (figure 5B). between active patients and healthy controls. Integrins are essen- Correlation between DNA methylation and gene expression tial in a wide variety of biological processes, including migration, levels was also evident for several known GR target genes, all of proliferation, cytokine production and activation, apoptosis and which were upregulated in the subgroup of patients in remission angiogenesis,40 all of which appear to be dysregulated in active with treatment. These genes include FKBP5, which correlated patients. Additionally, hypomethylation of both ITGA7 and negatively with cg03546163 (r=−0.51, FDR=1.24E-­03); CCRL2, a chemokine receptor involved in both innate and adap- ETS2, which showed a negative interaction with cg06804705 tive immune responses and known to be upregulated in activated that was located in the promoter region of this gene (r=−0.68, cells,41 correlated with higher gene expression levels in active FDR=8.12E-­09); ADAMTS2, which correlated negatively patients. Interestingly, two of the CpGs affecting ITGA7 expres- with cg14727962 (r=0.57, FDR=7.52E-­06) and cg09068128 sion also correlated with overexpression of CD63. Notably, (r=−0.60, FDR=1.54E-­10), positively with cg00854503 CD63 encodes a tetraspanin family member that interacts with (r=0.55, FDR=3.07E-­04) and cg02052156 (r=0.51, integrins, being crucial for the fusion of monocytes to form FDR=1.63E-­03); and ZBTB16, which correlated negatively multinucleated giant cells, which is the hallmark cell type of with cg14388315 (r=−0.60, FDR=8.35E-­05) and cg25345365 GCA.42–45 Finally, CD300E, an immune-­activating receptor that (r=−0.73, FDR=1.23E-­11) (figure 5B). promotes the expression of activation markers and the produc- Protected by copyright. tion of proinflammatory cytokines and reactive oxygen species in monocytes as well as the survival of this cell type,46 47 was DISCUSSION also among the most significantly upregulated genes in active The results of the first methylome and transcriptome profiling patients. Interestingly, expression of this gene was proposed of GCA monocytes have yielded evidence supporting that the to be restricted to CD115+Ly-­ 6Clow/int peripheral blood observed widespread alterations are implicated in the molecular monocytes in mice, which corresponds to human non-­classical mechanisms underlying this disorder. We also found a signifi- (CD14dimCD16+) and intermediate (CD14brightCD16+) cant number of genes whose dysregulation in GCA was mediated monocytes.48 Accordingly, increased levels of CD16+ mono- by an aberrant DNA methylation. In addition, the stratification cytes have been found in temporal artery biopsies of patients of patients according to disease activity allowed us to obtain a with GCA. This subset of monocytes is characterised to be more clearer picture of the changes in both methylation and expres- proinflammatory than the classical one (CD14brightCD16neg) sion driving the molecular processes involved in disease activity and shows a higher capacity to adhere to endothelial cells via and molecular response to GC treatment. CX3CR1,7 which, notably, also appeared to be hypermethylated Monocytes from active patients seemed to have a more proin- in active patients with respect to controls. Taken together, these flammatory phenotype than controls and patients in remission. results could indicate that, similarly to what has been described Supporting the reliability of our results, we observed a dysreg- in GCA biopsies, increased levels of CD16+ monocytes could be ulation of pathways involving cytokines and growth factors present in peripheral blood of active patients. already known to have a key role in GCA, such as IL-­6, TNF, It should be noted that similar results were found when active IL-­1, IL-­4, IL-­2, PDGF and VEGF.6 Interestingly, the response to patients were compared with both healthy controls and patients IL-­11 pathway was enriched among the DMPs hypomethylated in remission, with and without treatment, which suggests that the in active patients with respect to controls and patients in remis- proinflammatory methylation and expression profiles observed sion with and without treatment. Although additional evidence in the active disease are lost during remission. In fact, no differ- is needed to establish the response to IL-­11 as a new molecular ences were found when DNA methylation and gene expression mechanism involved in GCA, the potential role that might play levels were compared between patients in remission without in the active state of the disease is intriguing. IL11, a member of treatment and healthy controls. the IL-­6 family, has been implicated in a range of disease pathol- Furthermore, our results suggest that GC therapy remodel ogies by exerting diverse immunological roles.33 On the one the epigenome and, more robustly, the transcriptome, resulting hand, IL11 inhibits activated macrophages by blocking NF-κB in downregulation of genes involved in pathways with a rele- translocation,34 however, several studies have described a proin- vant role in GCA pathogenesis, including cell migration and flammatory function. For example, it has been reported that proliferation, apoptosis, angiogenesis, NF-κB, TNF, IFNγ and IL-­11 promotes the differentiation of CD4+ T cells into Th17 TLR signalling pathways and positive regulation of cytokines, cells, an important cell type in GCA pathogenesis,35 in multiple such as IL-­6 and IL-­2. Accordingly, several known target genes sclerosis, and it has been also implicated in angiogenesis in rheu- for GR that are involved in controlling inflammation, such as matoid arthritis patients.36 Additionally, it has been reported ETS2, ZBTB16, FKBP5, and ADAMTS2,49–51 appeared among that IL11 is involved in vascular smooth muscle cell phenotype the most upregulated genes in patients in remission with treat- switching, a mechanism that has been proposed to contribute to ment. Notably, expression levels of these four genes negatively Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 1297 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. Protected by copyright. Figure 5 Integrative analysis of DNA methylation and gene expression. (A) General description of the significant CpG-­gene expression interactions identified in our analysis. (B) Selected examples of specific CpG-­gene expression interactions. Box plots representing both the differentially methylated positions (DMPs) and the differentially expressed genes (DEGs). Significant differences are marked (*FDR<0.05). Graphical representation of the correlation of DNA methylation and gene expression of DMP-­DEG pairs is also shown. DNA methylation and gene expression levels are illustrated in each subset of individuals. ACT, active disease; CRTL, controls; RT, remission with treatment; RNT, remission without treatment. 1298 Estupiñán-­Moreno E, et al. Ann Rheum Dis 2022;81:1290–1300. doi:10.1136/annrheumdis-2022-222156 Vasculitis Ann Rheum Dis: first published as 10.1136/annrheumdis-2022-222156 on 15 June 2022. Downloaded from http://ard.bmj.com/ on May 29, 2023 at Univesity Autonomous of Barcelona. 4 correlated with methylation levels, suggesting that GCs modify Systemic Autoimmune Diseases Unit, Hospital Clinico San Cecilio, Instituto de gene expression levels through DNA demethylation of target Investigación Biosanitaria de Granada ibs.GRANADA, Granada, Spain genes. Correction notice This article has been corrected since it was first published. The Interestingly, two scavenger receptors, CD163 and CD163L1, open access licence has been updated to CC BY. 25th May 2023. were the most significant overexpressed genes in the subgroup Acknowledgements We thank all the patients and control donors who of patients in remission with treatment compared with non-­ participated in this study and Sofia Vargas and Gema Robledo for her excellent technical assistance. This research is part of the doctoral degree awarded by treated patients. CD163 is considered a phenotypic marker of E.E.M, within the Biomedicine programme from the University de Granada entitled monocytes with anti-­ inflammatory potential. Specifically, this ’Estudio de las causas moleculares de la arteritis de células gigantes mediante una receptor binds haemoglobin–haptoglobin complexes triggering aproximación sistémica’. endocitosis and activating a signalling cascade that results in the Contributors EE-­M and LO-­F: data analysis, interpretation of the results and production of anti-­ inflammatory molecules, such as IL-­ 10,52 manuscript drafting. EA-­L and LCT-­C: data analysis. TL, LC, GE-­F and SP-­G.: sample another gene upregulated in patients in remission with treat- and data collection. JH-­R and MCC: study design, sample and data collection. AM, ment. Additionally, CD163L1 has been described to have a role EB and JM: study design, interpretation of the results and manuscript drafting. LO-­F and JM: guarantors of the study. All authors read and approved the manuscript. in resolution of inflammation.53 It should be also noted that, conversely, both CD163 and CD163L1 presented higher levels Funding This work was supported by the HELICAL Innovative Training Network, a European Commission funded project under the Horizon 2020 research and of methylation in active patients compared with patients in innovation programme under the Marie Skłodowska-­Curie grant agreement remission with treatment that correlated with a decreased gene number 813 545 and, by the Cooperative Research Thematic Network programme expression, thus supporting the anti-­inflammatory role of these (RD16/0012/0013). AM is recipient of a Miguel Servet fellowship (CP17/00008) molecules. Remarkably, CD163 has been found to be signifi- from ISCIII (Spanish Ministry of Economy, Industry and Competitiveness). LO-­F was supported by Juan de la Cierva Incorporación fellowship (IJC2019-­040746-­I) funded cantly increased in temporal artery biopsies from patients with by MCIN/AEI /10.13039/501100011033. GCA treated with GC.54 Competing interests None declared. It has been described that GC can act on naïve monocytes inducing monocytes with an anti-­ inflammatory profile that Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. may suppress T cell activation, present an increased phagocytic capacity and release anti-­ inflammatory mediators.55 Consis- Patient consent for publication Not applicable. tently, our results indicate that, in addition to its role promoting Ethics approval The study was approved by the ethical committees of all the expression of GR-­target genes involved in the suppression of institutions involved in this study. However, these institutions do not provide a reference number or ID for the ethics approval. Participants gave informed consent to inflammation, GC treatment may also promote the expansion Protected by copyright. participate in the study before taking part. of monocytes with an anti-­inflammatory phenotype in GCA. Provenance and peer review Not commissioned; externally peer reviewed. In addition, the large differences observed between patients in remission with and without GC treatment suggest that the alter- Data availability statement Data are available in a public, open access repository. DNA methylation and expression data for this publication have been ations derived from GC therapy could be reversed in the absence deposited in the NCBI Gene Expression Omnibus and are accessible through GEO of treatment. SuperSeries accession number GSE201754. The code used for the analyses is Besides contributing to the elucidation of the pathogenic available from https://github.com/lterroncamero/GCA_CD14_Analysis.git. mechanisms involved in GCA, our study has revealed the exis- Supplemental material This content has been supplied by the author(s). It tence of specific methylation and transcription profiles in active has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have and GC-­treated patients that could potentially improve the clin- been peer-­reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and ical care of this vasculitis. In this regard, evaluation of the molec- responsibility arising from any reliance placed on the content. Where the content ular pattern of GCA monocytes could be especially relevant for includes any translated material, BMJ does not warrant the accuracy and reliability early and differential diagnosis as well as for therapy moni- of the translations (including but not limited to local regulations, clinical guidelines, toring, thus avoiding a delay in treatment and the use of inef- terminology, drug names and drug dosages), and is not responsible for any error fective drugs. Moreover, we have provided a significant number and/or omissions arising from translation and adaptation or otherwise. of molecules that could be targeted in future functional studies Open access This is an open access article distributed in accordance with the and potentially used as biomarkers. For example, CD163, which Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any is upregulated after GC treatment in circulating monocytes and purpose, provided the original work is properly cited, a link to the licence is given, temporal artery biopsies, represents an interesting candidate to and indication of whether changes were made. See: https://creativecommons.org/​ assess the molecular response to this therapy. licenses/by/4.0/. In conclusion, we have performed an exhaustive analysis of ORCID iDs the methylome and transcriptome of one of the most relevant Elkyn Estupiñán-­Moreno http://orcid.org/0000-0002-5818-4389 cell types in GCA, exploring for the first time the contribution of Lourdes Ortiz-­Fernández http://orcid.org/0000-0002-0247-4280 epigenetic to the disease activity and molecular response to GC Maria Cinta Cid http://orcid.org/0000-0002-4730-0938 in a large cohort of patients. Nevertheless, despite our relevant Ana Márquez http://orcid.org/0000-0001-9913-7688 Esteban Ballestar http://orcid.org/0000-0002-1400-2440 findings, evaluation of methylation and transcription profiles in Javier Martín http://orcid.org/0000-0002-2202-0622 additional peripheral cell types and, specially, in temporal artery biopsies will be essential to obtain a clearer picture of the molec- REFERENCES ular network involved in this type of vasculitis. 1 Jennette JC, Falk RJ, Bacon PA, et al. 2012 revised international chapel Hill consensus conference Nomenclature of vasculitides. Arthritis & Rheumatism 2013;65:1–11. Author affiliations 2 Gonzalez-­Gay MA, Vazquez-­Rodriguez TR, Lopez-­Diaz MJ, et al. Epidemiology of giant 1 Institute of Parasitology and Biomedicine López-­Neyra (IPBLN), Spanish National cell arteritis and polymyalgia rheumatica. Arthritis Rheum 2009;61:1454–61. Research Council (CSIC), Granada, Spain 3 Koster MJ, Matteson EL, Warrington KJ. 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