Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleosidebased DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G 0 to G 1 phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-␣ and interferon-␥. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versushost disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease. (Blood.
During vitellogenesis, one of the most tightly regulated processes in oviparous reproduction, vitellogenins are incorporated into the oocyte through vitellogenin receptor (VgR)‐mediated endocytosis. In this paper, we report the cloning of the VgR cDNA from Blattella germanica, as well as the first functional analysis of VgR following an RNA interference (RNAi) approach. We characterized the VgR, VgR mRNA and protein expression patterns in pre‐adult and adult stages of this cockroach, as well as VgR immunolocalization in ovarioles, belonging to the panoistic type. We then specifically disrupted VgR gene function using RNAi techniques. Knockdown of VgR expression led to a phenotype characterized by low yolk content in the ovary and high vitellogenin concentration in the haemolymph. This phenotype is equivalent to that of the yolkless mutant of Drosophila melanogaster, which have the yl (VgR) gene disrupted. The results additionally open the perspective that development genes can be functionally analyzed via systemic RNAi in this basal species.
Common variable immunodeficiency (CVID), the most frequent primary immunodeficiency characterized by loss of B-cell function, depends partly on genetic defects, and epigenetic changes are thought to contribute to its aetiology. Here we perform a high-throughput DNA methylation analysis of this disorder using a pair of CVID-discordant MZ twins and show predominant gain of DNA methylation in CVID B cells with respect to those from the healthy sibling in critical B lymphocyte genes, such as PIK3CD, BCL2L1, RPS6KB2, TCF3 and KCNN4. Individual analysis confirms hypermethylation of these genes. Analysis in naive, unswitched and switched memory B cells in a CVID patient cohort shows impaired ability to demethylate and upregulate these genes in transitioning from naive to memory cells in CVID. Our results not only indicate a role for epigenetic alterations in CVID but also identify relevant DNA methylation changes in B cells that could explain the clinical manifestations of CVID individuals.
BackgroundSepsis, 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.MethodsBead 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.ResultsHere, 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.ConclusionWe 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.
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