Carnero-Montoro, E., Barturen, G., Povedano, E., Kerick, M., Martinez-Bueno, M.; PRECISESADS Clinical Consortium, Ballestar E, Martin, J., Teruel, M., Alarcón-Riquelme, M.E.
Epigenome-Wide Comparative Study Reveals Key Differences Between Mixed Connective Tissue Disease and Related Systemic Autoimmune DiseasesFrontiers in Immunology 10,1880 , .
Mixed Connective Tissue Disease (MCTD) is a rare complex systemic autoimmune disease (SAD) characterized by the presence of increased levels of anti-U1 ribonucleoprotein autoantibodies and signs and symptoms that resemble other SADs such as systemic sclerosis (SSc), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Due to its low prevalence, this disease has been very poorly studied at the molecular level. We performed for the first time an epigenome-wide association study interrogating DNA methylation data obtained with the Infinium MethylationEPIC array from whole blood samples in 31 patients diagnosed with MCTD and 255 healthy subjects. We observed a pervasive hypomethylation involving 170 genes enriched for immune-related function such as those involved in type I interferon signaling pathways or in negative regulation of viral genome replication. We mostly identified epigenetic signals at genes previously implicated in other SADs, for example MX1, PARP9, DDX60, or IFI44L, for which we also observed that MCTD patients exhibit higher DNA methylation variability compared with controls, suggesting that these sites might be involved in plastic immune responses that are relevant to the disease. Through methylation quantitative trait locus (meQTL) analysis we identified widespread local genetic effects influencing DNA methylation variability at MCTD-associated sites. Interestingly, for IRF7, IFI44 genes, and the HLA region we have evidence that they could be exerting a genetic risk on MCTD mediated through DNA methylation changes. Comparison of MCTD-associated epigenome with patients diagnosed with SLE, or Sjögren's Syndrome, reveals a common interferon-related epigenetic signature, however we find substantial epigenetic differences when compared with patients diagnosed with rheumatoid arthritis and systemic sclerosis. Furthermore, we show that MCTD-associated CpGs are potential epigenetic biomarkers with high diagnostic value. Our study serves to reveal new genes and pathways involved in MCTD, to illustrate the important role of epigenetic modifications in MCTD pathology, in mediating the interaction between different genetic and environmental MCTD risk factors, and as potential biomarkers of SADs.More information
Massoni-Badosa R, Iacono G, Moutinho C, Kulis M, Palau N, Marchese D, Rodríguez-Ubreva J, Ballestar E, Rodriguez-Esteban G, Marsal S, Aymerich M, Colomer D, Campo E, Julià A, Martín-Subero JI, Heyn H
Sampling time-dependent artifacts in single-cell genomics studies.Genome Biol. 11 May 2020, 21 (1) 112. Epub 11 May 2020
Robust protocols and automation now enable large-scale single-cell RNA and ATAC sequencing experiments and their application on biobank and clinical cohorts. However, technical biases introduced during sample acquisition can hinder solid, reproducible results, and a systematic benchmarking is required before entering large-scale data production. Here, we report the existence and extent of gene expression and chromatin accessibility artifacts introduced during sampling and identify experimental and computational solutions for their prevention.More information
Del Pino-Molina, L., Rodríguez-Ubreva, J., Torres Canizales, J., Coronel-Díaz, M., Kulis, M., Martín-Subero, J.I., van der Burg, M., Ballestar E, and López-Granados, E*
Impaired CpG Demethylation in Common Variable Immunodeficiency Associates with B Cell Phenotype and Proliferation RateFrontiers in Immunology 10, 878. , .
Common Variable Immunodeficiency (CVID) is characterized by impaired antibody production and poor terminal differentiation of the B cell compartment, yet its pathogenesis is still poorly understood. We first reported the occurrence of epigenetic alterations in CVID by high-throughput methylation analysis in CVID-discordant monozygotic twins. Data from a recent whole DNA methylome analysis throughout different stages of normal B cell differentiation allowed us to design a new experimental approach. We selected CpG sites for analysis based on two criteria: one, CpGs with potential association with the transcriptional status of relevant genes for B cell activation and differentiation; and two, CpGs that undergo significant demethylation from naïve to memory B cells in healthy individuals. DNA methylation was analyzed by bisulfite pyrosequencing of specific CpG sites in sorted naïve and memory B cell subsets from CVID patients and healthy donors. We observed impaired demethylation in two thirds of the selected CpGs in CVID memory B cells, in genes that govern B cell-specific processes or participate in B cell signaling. The degree of demethylation impairment associated with the extent of the memory B cell reduction. The impaired demethylation in such functionally relevant genes as AICDA in switched memory B cells correlated with a lower proliferative rate. Our new results reinforce the hypothesis of altered demethylation during B cell differentiation as a contributing pathogenic mechanism to the impairment of B cell function and maturation in CVID. In particular, deregulated epigenetic control of AICDA could play a role in the defective establishment of a post-germinal center B cell compartment in CVID.More information
Rodríguez-Ubreva, J., de la Calle-Fabregat, C., Li, T., Ciudad, L., Ballestar, M.L., Català-Moll, F., Morante-Palacios, O., Garcia-Gomez, A., Celis, R., Humby, F., Nerviani, A., Martin, J., Pitzalis, C., Cañete, J.D., and, Ballestar E
Inflammatory cytokines shape a changing DNA methylome in monocytes mirroring disease activity in rheumatoid arthritisAnnals of the Rheumatic Diseases 78,1505-1516 , .
bjective: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that mainly targets joints. Monocytes and macrophages are critical in RA pathogenesis and contribute to inflammatory lesions. These extremely plastic cells respond to extracellular signals which cause epigenomic changes that define their pathogenic phenotype. Here, we interrogated how DNA methylation alterations in RA monocytes are determined by extracellular signals.
Methods: High-throughput DNA methylation analyses of patients with RA and controls and in vitro cytokine stimulation were used to investigate the underlying mechanisms behind DNA methylation alterations in RA as well as their relationship with clinical parameters, including RA disease activity.
Results: The DNA methylomes of peripheral blood monocytes displayed significant changes and increased variability in patients with RA with respect to healthy controls. Changes in the monocyte methylome correlate with DAS28, in which high-activity patients are divergent from healthy controls in contrast to remission patients whose methylome is virtually identical to healthy controls. Indeed, the notion of a changing monocyte methylome is supported after comparing the profiles of same individuals at different stages of activity. We show how these changes are mediated by an increase in disease activity-associated cytokines, such as tumour necrosis factor alpha and interferons, as they recapitulate the DNA methylation changes observed in patients in vitro.
Conclusion: We demonstrate a direct link between RA disease activity and the monocyte methylome through the action of inflammation-associated cytokines. Finally, we have obtained a DNA methylation-based mathematical formula that predicts inflammation-mediated disease activity for RA and other chronic immune-mediated inflammatory diseases.
Keywords: DAS28; DNA methylation; TNFa; disease activity; rheumatoid arthritis.More information
Lorente-Sorolla, C., Garcia-Gomez, A., Català-Moll, F., Toledano, V., Ciudad, L., Avendaño-Ortiz, J., Maroun-Eid, C., Martín-Quirós, A. Martínez-Gallo, M., Ruiz-Sanmartín, A., García del Campo, A., Ferrer-Roca, R., Ruiz-Rodriguez, J.C. Álvarez-Errico, D., López-Collazo, E. and, Ballestar E
Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsisGenome Medicine 11, 66 , .
ackground: Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation.
Methods: Bead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming.
Results: Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance.
Conclusion: We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction.
Keywords: Cytokines; DNA methylation; Endotoxin tolerance; Monocytes; Sepsis.More information
Lu, Q., Wu, R., Zhao, M., Garcia-Gomez, A., and, Ballestar E
MicroRNAs as therapeutic targets in inflammatory diseaseTrends in Pharmacological Sciences 40, 853-865. , .
In the past decade, we have witnessed considerable developments in understanding the roles and functions of miRNAs. In parallel, the identification of alterations in miRNA expression in inflammatory disease indicates their potential as therapeutic targets. Pharmacological treatments targeting abnormally expressed miRNAs for inflammatory diseases are not yet in clinical practice; however, some small compounds and nucleic acids targeting miRNAs have shown promise in preclinical development. Here, we focus on recent advances in understanding miRNA deregulation in inflammatory diseases and provide an overview of the current development of miRNA-based therapeutics in these diseases with an emphasis on newly discovered miRNA therapeutic targets.More information
Li, T., Garcia-Gomez, A., Morante-Palacios, O., Ciudad, L., Özkaramehmet, S., Van Dijck, E., Rodríguez-Ubreva, J., Vaquero, A. and, Ballestar E
SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophageNucleic Acids Research 48, 665–681 , .
Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes.More information
Ballestar E, Sawalha, A.H. and Lu, Q
Clinical value of DNA methylation markers in autoimmune rheumatic diseases.Nature Reviews Rheumatology 16,514-524 , .
Methylation of cytosine residues in DNA, the best studied epigenetic modification, is associated with gene transcription and nuclear organization, and ultimately the function of a cell. DNA methylation can be influenced by various factors, including changes in neighbouring genomic sites such as those induced by transcription factor binding. The DNA methylation profiles in relevant cell types are altered in most human diseases compared with the healthy state. Given the physical stability of DNA and methylated DNA compared with other epigenetic modifications, DNA methylation is an ideal marker for clinical purposes. However, few DNA methylation-based markers have made it into clinical practice, with the notable exception of some markers used in the field of oncology. Autoimmune rheumatic diseases are genetically complex entities that can vary widely in terms of prognosis, subtypes, progression and treatment responses. Increasing reports showing strong links between DNA methylation profiles and different clinical outcomes and other clinical aspects in autoimmune rheumatic diseases reinforce the usefulness of DNA methylation profiles as novel clinical markers. In this Review, we provide an updated discussion on DNA methylation alterations in autoimmune rheumatic diseases and the advantages and disadvantages of using these markers in clinical practice.More information
Li, T., Ortiz, L., Andrés-León, E., Ciudad, L., Javierre, B.M., López-Isac, E., Guillén-Del-Castillo. A., Simeón-Aznar, C.P., Ballestar E, Martín, J.* / *co-corresponding/co-senior
Epigenomics and Transcriptomics of Systemic Sclerosis CD4+ T cells reveal Long Range Dysregulation of Key Inflammatory Pathways mediated by disease-associated Susceptibility LociGenome Medicine 12, 81 , .
Background: Systemic sclerosis (SSc) is a genetically complex autoimmune disease mediated by the interplay between genetic and epigenetic factors in a multitude of immune cells, with CD4+ T lymphocytes as one of the principle drivers of pathogenesis.
Methods: DNA samples exacted from CD4+ T cells of 48 SSc patients and 16 healthy controls were hybridized on MethylationEPIC BeadChip array. In parallel, gene expression was interrogated by hybridizing total RNA on Clariom™ S array. Downstream bioinformatics analyses were performed to identify correlating differentially methylated CpG positions (DMPs) and differentially expressed genes (DEGs), which were then confirmed utilizing previously published promoter capture Hi-C (PCHi-C) data.
Results: We identified 9112 and 3929 DMPs and DEGs, respectively. These DMPs and DEGs are enriched in functional categories related to inflammation and T cell biology. Furthermore, correlation analysis identified 17,500 possible DMP-DEG interaction pairs within a window of 5 Mb, and utilizing PCHi-C data, we observed that 212 CD4+ T cell-specific pairs of DMP-DEG also formed part of three-dimensional promoter-enhancer networks, potentially involving CTCF. Finally, combining PCHi-C data with SSc GWAS data, we identified four important SSc-associated susceptibility loci, TNIP1 (rs3792783), GSDMB (rs9303277), IL12RB1 (rs2305743), and CSK (rs1378942), that could potentially interact with DMP-DEG pairs cg17239269-ANXA6, cg19458020-CCR7, cg10808810-JUND, and cg11062629-ULK3, respectively.
Conclusion: Our study unveils a potential link between genetic, epigenetic, and transcriptional deregulation in CD4+ T cells of SSc patients, providing a novel integrated view of molecular components driving SSc pathogenesis.
Keywords: CTCF; DNA methylation; Epigenetics; Genetic susceptibility variants; Hi-C; Long-distance regulation; Systemic sclerosis.More information