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 TNFβ 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

Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients

Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient's study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections

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

COVID-19; Monocytes; Single-cell transcriptomicsCOVID-19; Monocitos; Transcriptómica unicelularCOVID-19; Monòcits; Transcriptòmica unicel·lularBackground 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.This study has been funded by R+D+i project of the Spanish Ministry of Science and Innovation (grant number PID2020-117212RB-I00/ MICIN/AEI/10.13039/501100011033). We also thank the Chan Zuckerberg Initiative (grant 2020–216799) and Wellcome Sanger core funding (WT206194). This publication has also been supported by the Unstoppable campaign of the Josep Carreras Leukaemia Foundation. Additional funding comes from Project PI18/00346 (Instituto de Salud Carlos III and co-funded by European Union (ERDF/ESF ). A.B. received additional support from a Gates Cambridge Scholarship

Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease

© The Author(s) 2021.Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.We thank CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (grant numbers SAF2014-55942-R and SAF2017-88086-R), co-funded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe, and a Senior Research Award from the Multiple Myeloma Research Foundation (MMRF). C.O.-d.-S. was funded by the Spanish Ministry of Science, Innovation and Universities, under grant RTI2018-094494-B-C22 (MCIU/AEI/FEDER, UE). M.G. received financial support from the Spanish FIS-ISCIII (PI15/02156 and PI19/01384) and FEDER. A.G.G is funded by a postdoctoral contract of the Asociación Española Contra el Cáncer (AECC). F.P. was funded by grants from Instituto de Salud Carlos III (ISCIII), PI17/00701 and PI19/01352, TRASCAN (EPICA and Immunocell), Fundació La Marató de TV3, the Accelerator award CRUK/AIRC/AECC joint funder-partnership, CIBERONC (CB16/12/00489) and co-financed with FEDER funds and Fundación Ramón Areces (PREMAMM)

Targeting aberrant DNA methylation in mesenchymal stromal cells as a treatment for myeloma bone disease

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD. Mesenchymal stromal cells (MSCs) have been shown to support multiple myeloma (MM) development. Here, MSCs isolated from the bone marrow of MM patients are shown to have altered DNA methylation patterns and a methyltransferase inhibitor reverts MM-associated bone loss and reduces tumour burden in MM murine models

COVID-19 progression and convalescence in common variable immunodeficiency patients shows incomplete adaptive responses and persistent inflammasome activation

Patients with common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, are characterized by hypogammaglobulinemia, poorly protective vaccine titers and increased susceptibility to infections. New pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), might constitute a particular threat to these immunocompromised patients since many of them experience a slower recovery and do not achieve full response to SARS-CoV-2 vaccines. To define the molecular basis of the altered immune responses caused by SARS-CoV-2 infection in CVID patients, we generated longitudinal single-cell datasets of peripheral blood immune cells along viral infection and recovery. We sampled the same individuals before, during and after SARS-CoV-2 infection to model their specific immune response dynamics while removing donor variability. We observed that COVID-19 CVID patients show defective canonical NF-κB pathway activation and dysregulated expression of BCR-related genes in naïve B cells, as well as enhanced cytotoxic activity but incomplete cytokine response in NK and T cells. Moreover, monocytes from COVID-19 CVID patients show persistent activation of several inflammasome-related genes, including the pyrin and NLRC4 inflammasomes. Our results shed light on the molecular basis of the prolonged clinical manifestations observed in these immunodeficient patients upon SARS-CoV-2 infection, which might illuminate the development of tailored treatments for COVID-19 CVID patients.We thank the CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This publication is part of the Human Cell Atlas: www.humancellatlas.org/publications. This study was funded by ”la Caixa” Foundation under the grant agreement LCF/PR/HR22/52420002, Spanish Ministry of Science and Innovation (grant number PID2020-117212RB-I00/AEI/10.13038/501100011033) (E.B.), by the Wellcome Trust Grant 206194 and 108413/A/15/D (R.V.-T.), Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program) (E.B.), and the Chan Zuckerberg Initiative (grant 2020-216799) (R.V.-T. and E.B.). This publication has also been supported by the Unstoppable campaign of the Josep Carreras Leukaemia Foundation. We are indebted to the donors for participating in this research.N

Epigenetics of Dendritic Cells in Tumor Immunology

Dendritic cells (DCs) are professional antigen-presenting cells with the distinctive property of inducing the priming and differentiation of naïve CD4+ and CD8+ T cells into helper and cytotoxic effector T cells to develop efficient tumor-immune responses. DCs display pathogenic and tumorigenic antigens on their surface through major histocompatibility complexes to directly influence the differentiation of T cells. Cells in the tumor microenvironment (TME), including cancer cells and other immune-infiltrated cells, can lead DCs to acquire an immune-tolerogenic phenotype that facilitates tumor progression. Epigenetic alterations contribute to cancer development, not only by directly affecting cancer cells, but also by their fundamental role in the differentiation of DCs that acquire a tolerogenic phenotype that, in turn, suppresses T cell-mediated responses. In this review, we focus on the epigenetic regulation of DCs that have infiltrated the TME and discuss how knowledge of the epigenetic control of DCs can be used to improve DC-based vaccines for cancer immunotherapy

Epigenetics of Dendritic Cells in Tumor Immunology

Dendritic cells (DCs) are professional antigen-presenting cells with the distinctive property of inducing the priming and differentiation of naïve CD4+ and CD8+ T cells into helper and cytotoxic effector T cells to develop efficient tumor-immune responses. DCs display pathogenic and tumorigenic antigens on their surface through major histocompatibility complexes to directly influence the differentiation of T cells. Cells in the tumor microenvironment (TME), including cancer cells and other immune-infiltrated cells, can lead DCs to acquire an immune-tolerogenic phenotype that facilitates tumor progression. Epigenetic alterations contribute to cancer development, not only by directly affecting cancer cells, but also by their fundamental role in the differentiation of DCs that acquire a tolerogenic phenotype that, in turn, suppresses T cell-mediated responses. In this review, we focus on the epigenetic regulation of DCs that have infiltrated the TME and discuss how knowledge of the epigenetic control of DCs can be used to improve DC-based vaccines for cancer immunotherap

Direct Quantitative Immunochemical Analysis of Autoinducer Peptide IV for Diagnosing and Stratifying Staphylococcus aureus Infections

An immunochemical strategy to detect and quantify AIP-IV, the quorum sensing (QS) signaling molecule produced by Staphylococcus aureus agr type IV, is reported here for the first time. Theoretical calculations and molecular modeling studies have assisted on the design and synthesis of a suitable peptide hapten (AIPIVS), allowing to obtain high avidity and specific antibodies toward this peptide despite its low molecular weight. The ELISA developed achieves an IC50 value of 2.80 ± 0.17 and an LOD of 0.19 ± 0.06 nM in complex media such as 1/2 Tryptic Soy Broth. Recognition of other S. aureus AIPs (I-III) is negligible (cross-reactivity below 0.001%), regardless of the structural similarities. A pilot study with a set of clinical isolates from patients with airways infection or colonization demonstrates the potential of this ELISA to perform biomedical investigations related to the role of QS in pathogenesis and the association between dysfunctional agr or the agr type with unfavorable clinical outcomes. The AIP-IV levels could be quantified in the low nanomolar range in less than 1 h after inoculating agr IV-genotyped isolates in the culture broth, while those genotyped as I-III did not show any immunoreactivity after a 48 h growth, pointing to the possibility to use this technology for phenotyping S. aureus. The research strategy here reported can be extended to the rest of the AIP types of S. aureus, allowing the development of powerful multiplexed chips or point-of-care (PoC) diagnostic devices to unequivocally identify its presence and its agr type on samples from infected patients.This work has been funded (SAF2015-67476-R, RTI2018-096278-B-C21, both to M.-P. Marco) by MICIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, and to C. Prat through the Instituto de Salud Carlos III (PI17/01139). M.-P. Marco has also received funding from the Fundació Marató de TV3 (TV32018-201825-30-31). Enrique-J. Montagut wishes to thank the FPI fellowship (BES-2016-076496) from the MICIN/AEI/10.13039/501100011033 and ESF investing in your future. We also thank the denomination of consolidated group from the Generalitat de Catalunya: 2017 SGR 1441 (M.-P. Marco) and 2017 SGR 1439 (M. Royo). CIBER Actions, CB06/01/0036 (M.-P. Marco), CB06/06/0031 (C. Prats) and CB06/01/0074 (M. Royo) have been financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund (ERDF). The U2 (Custom Antibody Service (CAbS) and the U3 (Synthesis of Peptide Unit) of the ICTS “NANBIOSIS” at the IQAC-CSIC, are acknowledged for the assistance and support related respectively to the development of the immunoreagents and the synthesis of the different peptide haptens used in this work.Peer reviewe

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

We are very grateful to Dr. José Luis Sardina for useful feedback. We thank CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (MICINN ; grant number PID2020-117212RB-I00/AEI/10.13038/501100011033). E.M.-C. is funded with RESTORE project (EU H2020 Research and Innovation Programme, number 779316) and Spanish projects PI17/01521 and PI20/01313, integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirección General de Evaluación and FEDER. O.M.-P. holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acción Estratégica en Salud 2013-2016 ISCIII, co-financed by Fondo Social Europeo. F.F. holds a PhD fellowship from the INsTRuCT Consortium, which receives. Innovative Training Network subsidy from the EU H2020 program.We are very grateful to Dr. Jos? Luis Sardina for useful feedback. We thank CERCA Programme/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. E.B. was funded by the Spanish Ministry of Science and Innovation (MICINN; grant number PID2020-117212RB-I00/AEI/10.13038/501100011033). E.M.-C. is funded with RESTORE project (EU H2020 Research and Innovation Programme, number 779316) and Spanish projects PI17/01521 and PI20/01313, integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirecci?n General de Evaluaci?n and FEDER. O.M.-P. holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acci?n Estrat?gica en Salud 2013?2016 ISCIII, co-financed by Fondo Social Europeo. F.F. holds a PhD fellowship from the INsTRuCT Consortium, which receives. Innovative Training Network subsidy from the EU H2020 program. F.C.-M. and E.B. conceived and designed the study; F.C.-M. A.G.F.-B. G.G.-T. O.M.-P. L.C. L.B. F.F. and T.L. performed the differentiation, chromatin immunoprecipitation, co-immunoprecipitation experiments, and immunological assays; F.C.-M. performed the bioinformatic analyses; F.C.-M. A.G.F.-B. G.G.-T. E.M.-C. and E.B. analyzed results; J.R.-U. and E.B. supervised the study; F.C.-M. T.L. and E.B. wrote the manuscript; all authors participated in discussions and interpreting the results. The authors declare no competing interests.UDTRIASThe 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