Disease activity drives divergent epigenetic and transcriptomic reprogramming of monocyte subpopulations in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is characterised by systemic inflammation involving various immune cell types. Monocytes, pivotal in promoting and regulating inflammation in SLE, differentiate from classic monocytes into intermediate and non-classic monocytes, assuming diverse roles and changing their proportions in inflammation. In this study, we investigated the epigenetic and transcriptomic profiles of these and novel monocyte subsets in SLE in relation to activity and progression. METHODS: We obtained the DNA methylomes and transcriptomes of classic, intermediate, non-classic monocytes in patients with SLE (at first and follow-up visits) and healthy donors. We integrated these data with single-cell transcriptomics of SLE and healthy donors and interrogated their relationships with activity and progression. RESULTS: In addition to shared DNA methylation and transcriptomic alterations associated with a strong interferon signature, we identified monocyte subset-specific alterations, especially in DNA methylation, which reflect an impact of SLE on monocyte differentiation. SLE classic monocytes exhibited a proinflammatory profile and were primed for macrophage differentiation. SLE non-classic monocytes displayed a T cell differentiation-related phenotype, with Th17-regulating features. Changes in monocyte proportions, DNA methylation and expression occurred in relation to disease activity and involved the STAT pathway. Integration of bulk with single-cell RNA sequencing datasets revealed disease activity-dependent expansion of SLE-specific monocyte subsets, further supported the interferon signature for classic monocytes, and associated intermediate and non-classic populations with exacerbated complement activation. CONCLUSIONS: Disease activity in SLE drives a subversion of the epigenome and transcriptome programme in monocyte differentiation, impacting the function of different subsets and allowing to generate predictive methods for activity and progression.

Interactive DNA Methylation Array Analysis with ShinyÉPICo.

Arrays provide a cost-effective platform for the analysis of human DNA methylation. ShinyÉPICo is an interactive, web-based, and graphical tool that allows the user to analyze Illumina DNA methylation arrays (450 k and EPIC), from the user's own computer or from a server. This tool covers the analysis entirely, from the raw data input to the final list of differentially methylated positions or regions. Here, we describe the steps of the analysis, the different parameters available, and useful information to understand and select the best options in each step.

Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines.

BACKGROUND: COVID-19 manifests with a wide spectrum of clinical phenotypes, ranging from asymptomatic and mild to severe and critical. Severe and critical COVID-19 patients are characterized by marked changes in the myeloid compartment, especially monocytes. However, little is known about the epigenetic alterations that occur in these cells during hyperinflammatory responses in severe COVID-19 patients. METHODS: In this study, we obtained the DNA methylome and transcriptome of peripheral blood monocytes from severe COVID-19 patients. DNA samples extracted from CD14 + CD15- monocytes of 48 severe COVID-19 patients and 11 healthy controls were hybridized on MethylationEPIC BeadChip arrays. In parallel, single-cell transcriptomics of 10 severe COVID-19 patients were generated. CellPhoneDB was used to infer changes in the crosstalk between monocytes and other immune cell types. RESULTS: We observed DNA methylation changes in CpG sites associated with interferon-related genes and genes associated with antigen presentation, concordant with gene expression changes. These changes significantly overlapped with those occurring in bacterial sepsis, although specific DNA methylation alterations in genes specific to viral infection were also identified. We also found these alterations to comprise some of the DNA methylation changes occurring during myeloid differentiation and under the influence of inflammatory cytokines. A progression of DNA methylation alterations in relation to the Sequential Organ Failure Assessment (SOFA) score was found to be related to interferon-related genes and T-helper 1 cell cytokine production. CellPhoneDB analysis of the single-cell transcriptomes of other immune cell types suggested the existence of altered crosstalk between monocytes and other cell types like NK cells and regulatory T cells. CONCLUSION: Our findings show the occurrence of an epigenetic and transcriptional reprogramming of peripheral blood monocytes, which could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these patients.

Vitamin C enhances NF-κB-driven epigenomic reprogramming and boosts the immunogenic properties of dendritic cells.

Dendritic cells (DCs), the most potent antigen-presenting cells, are necessary for effective activation of naïve T cells. DCs' immunological properties are modulated in response to various stimuli. Active DNA demethylation is crucial for DC differentiation and function. Vitamin C, a known cofactor of ten-eleven translocation (TET) enzymes, drives active demethylation. Vitamin C has recently emerged as a promising adjuvant for several types of cancer; however, its effects on human immune cells are poorly understood. In this study, we investigate the epigenomic and transcriptomic reprogramming orchestrated by vitamin C in monocyte-derived DC differentiation and maturation. Vitamin C triggers extensive demethylation at NF-κB/p65 binding sites, together with concordant upregulation of antigen-presentation and immune response-related genes during DC maturation. p65 interacts with TET2 and mediates the aforementioned vitamin C-mediated changes, as demonstrated by pharmacological inhibition. Moreover, vitamin C increases TNFß production in DCs through NF-κB, in concordance with the upregulation of its coding gene and the demethylation of adjacent CpGs. Finally, vitamin C enhances DC's ability to stimulate the proliferation of autologous antigen-specific T cells. We propose that vitamin C could potentially improve monocyte-derived DC-based cell therapies.

Vitamin D receptor, STAT3, and TET2 cooperate to establish tolerogenesis.

The active form of vitamin D, 1,25-dihydroxyvitamin D3, induces a stable tolerogenic phenotype in dendritic cells (DCs). This process involves the vitamin D receptor (VDR), which translocates to the nucleus, binds its cognate genomic sites, and promotes epigenetic and transcriptional remodeling. In this study, we report the occurrence of vitamin D-specific DNA demethylation and transcriptional activation at VDR binding sites associated with the acquisition of tolerogenesis in vitro. Differentiation to tolerogenic DCs associates with activation of the IL-6-JAK-STAT3 pathway. We show that JAK2-mediated STAT3 phosphorylation is specific to vitamin D stimulation. VDR and the phosphorylated form of STAT3 interact with each other to form a complex with methylcytosine dioxygenase TET2. Most importantly, pharmacological inhibition of JAK2 reverts vitamin D-induced tolerogenic properties of DCs. This interplay among VDR, STAT3, and TET2 opens up possibilities for modulating DC immunogenic properties in clinics.

Coordinated glucocorticoid receptor and MAFB action induces tolerogenesis and epigenome remodeling in dendritic cells.

Glucocorticoids (GCs) exert potent anti-inflammatory effects in immune cells through the glucocorticoid receptor (GR). Dendritic cells (DCs), central actors for coordinating immune responses, acquire tolerogenic properties in response to GCs. Tolerogenic DCs (tolDCs) have emerged as a potential treatment for various inflammatory diseases. To date, the underlying cell type-specific regulatory mechanisms orchestrating GC-mediated acquisition of immunosuppressive properties remain poorly understood. In this study, we investigated the transcriptomic and epigenomic remodeling associated with differentiation to DCs in the presence of GCs. Our analysis demonstrates a major role of MAFB in this process, in synergy with GR. GR and MAFB both interact with methylcytosine dioxygenase TET2 and bind to genomic loci that undergo specific demethylation in tolDCs. We also show that the role of MAFB is more extensive, binding to thousands of genomic loci in tolDCs. Finally, MAFB knockdown erases the tolerogenic properties of tolDCs and reverts the specific DNA demethylation and gene upregulation. The preeminent role of MAFB is also demonstrated in vivo for myeloid cells from synovium in rheumatoid arthritis following GC treatment. Our results imply that, once directly activated by GR, MAFB plays a critical role in orchestrating the epigenomic and transcriptomic remodeling that define the tolerogenic phenotype.

JAK2-STAT Epigenetically Regulates Tolerized Genes in Monocytes in the First Encounter With Gram-Negative Bacterial Endotoxins in Sepsis.

Microbial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.

shinyÉPICo: a graphical pipeline to analyze Illumina DNA methylation arrays.

SUMMARY: Illumina DNA methylation bead arrays provide a cost-effective platform for the simultaneous analysis of a high number of human samples. However, the analysis can be time-demanding and requires some computational expertise. shinyÉPICo is an interactive, web-based, and graphical tool that allows the user to analyze Illumina DNA methylation arrays (450k and EPIC), from the user's own computer or from a server. The tool covers the entire analysis, from the raw data to the final list of differentially methylated positions and differentially methylated regions between sample groups. It allows the user to test several normalization methods, linear model parameters, including covariates, and differentially methylated CpGs filters, in a quick and easy manner, with interactive graphics helping to select the options in each step. shinyÉPICo represents a comprehensive tool for standardizing and accelerating DNA methylation analysis, as well as optimizing computational resources in laboratories studying DNA methylation. AVAILABILITY AND IMPLEMENTATION: shinyÉPICo is freely available as an R package at the Bioconductor project (http://bioconductor.org/packages/shinyepico/) and GitHub (https://github.com/omorante/shinyepico) under an AGPL3 license.

Tolerogenic Dendritic Cells in Autoimmunity and Inflammatory Diseases.

Dendritic cells (DCs), the most efficient antigen-presenting cells, are necessary for the effective activation of naïve T cells. DCs can also acquire tolerogenic functions in vivo and in vitro in response to various stimuli, including interleukin (IL)-10, transforming growth factor (TGF)-ß, vitamin D3, corticosteroids, and rapamycin. In this review, we provide a wide perspective on the regulatory mechanisms, including crosstalk with other cell types, downstream signaling pathways, transcription factors, and epigenetics, underlying the acquisition of tolerogenesis by DCs, with a special focus on human studies. Finally, we present clinical assays targeting, or based on, tolerogenic DCs in inflammatory diseases. Our discussion provides a useful resource for better understanding the biology of tolerogenic DCs and their manipulation to improve the immunological fitness of patients with certain inflammatory conditions.

Understanding the Relevance of DNA Methylation Changes in Immune Differentiation and Disease.

Immune cells are one of the most complex and diverse systems in the human organism. Such diversity implies an intricate network of different cell types and interactions that are dependently interconnected. The processes by which different cell types differentiate from progenitors, mature, and finally exert their function requires an orchestrated succession of molecular processes that determine cell phenotype and function. The acquisition of these phenotypes is highly dependent on the establishment of unique epigenetic profiles that confer identity and function on the various types of effector cells. These epigenetic mechanisms integrate microenvironmental cues into the genome to establish specific transcriptional programs. Epigenetic modifications bridge environment and genome regulation and play a role in human diseases by their ability to modulate physiological programs through external stimuli. DNA methylation is one of the most ubiquitous, stable, and widely studied epigenetic modifications. Recent technological advances have facilitated the generation of a vast amount of genome-wide DNA methylation data, providing profound insights into the roles of DNA methylation in health and disease. This review considers the relevance of DNA methylation to immune system cellular development and function, as well as the participation of DNA methylation defects in immune-mediated pathologies, illustrated by selected paradigmatic diseases.

SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages.

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.

Inflammatory cytokines shape a changing DNA methylome in monocytes mirroring disease activity in rheumatoid arthritis.

OBJECTIVE: 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.