Gametes are highly specialized cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mice, germ cells are first specified in the developing embryo ...around embryonic day (E) 6.25 as primordial germ cells (PGCs). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming around E10.5-E11.5, including genome-wide loss of 5-methylcytosine. The underlying molecular mechanisms of this process have remained unclear, leading to our inability to recapitulate this step of germline development in vitro. Here we show, using an integrative approach, that this complex reprogramming process involves coordinated interplay among promoter sequence characteristics, DNA (de)methylation, the polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of TET1 to enable the activation of a critical set of germline reprogramming-responsive genes involved in gamete generation and meiosis. Our results also reveal an unexpected role for TET1 in maintaining but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will help to guide attempts to recapitulate complete gametogenesis in vitro.
Methylation at the 5 position of cytosine in DNA (5meC) is a key epigenetic mark in eukaryotes. Once introduced, 5meC can be maintained through DNA replication by the activity of 'maintenance' DNA ...methyltransferases (DNMTs). Despite their ancient origin, DNA methylation pathways differ widely across animals, such that 5meC is either confined to transcribed genes or lost altogether in several lineages. We used comparative epigenomics to investigate the evolution of DNA methylation. Although the model nematode Caenorhabditis elegans lacks DNA methylation, more basal nematodes retain cytosine DNA methylation, which is targeted to repeat loci. We found that DNA methylation coevolved with the DNA alkylation repair enzyme ALKB2 across eukaryotes. In addition, we found that DNMTs introduced the toxic lesion 3-methylcytosine into DNA both in vitro and in vivo. Alkylation damage is therefore intrinsically associated with DNMT activity, and this may promote the loss of DNA methylation in many species.
Almost 65 years have passed since the discovery of 5 methylcytosine (5mC). DNA methylation has since been shown to be involved in gene expression, genomic imprinting, X chromosome inactivation, the ...repression of retrotransposons and cell differentiation. In the context of mammalian embryonic development, DNA methylation undergoes genome-wide reprogramming in primordial germ cells (PGCs). Despite a huge research effort, the mechanism underlying DNA demethylation in PGCs is unclear. Limited progress is largely due to technical challenges. Many widely used analytical techniques require large amounts of biological material and are therefore inappropriate for studies using small numbers of cells isolated from developing embryos. Our understanding of the mechanisms behind the developmental reprogramming processes may therefore be greatly enhanced by the use of complementary model systems and alternative technologies. The experiments described herein focus on the development and optimisation of a number of in vitro and in vivo systems to explore the nature of epigenetic DNA modifications, including 5-methylcytosine, and the mechanisms that dictate dynamic changes in the epigenetic landscape. The development and validation of sensitive LC-MS methods facilitates the quantification of DNA modifications, potentially enables the discovery of novel modifications, and by combining LC-MS with isotope tracing, may fundamentally challenge our current understanding of the stable nature of DNA methylation. Ultimately, we envisage that these approaches will form a versatile and informative platform to study genome-wide epigenetic reprogramming processes in an in vivo context. In many respects, loss of 5-methylcytosine in migratory PGCs mirrors the loss of 5-methylcytosine in mESCs upon transition from serum-containing to 2i medium. Accordingly, I used this accessible system to probe the contribution of DNA methyltransferases, putative DNA demethylases, their cofactors and binding partners to the loss of 5-methylcytosine in nascent PGCs. I show that loss of Dnmt3a isoform 2 is necessary for 2i-induced DNA demethylation. Furthermore, this loss is dependent on RNA-induced silencing. More generally, miRNAs appear to be necessary for the epigenetic reprogramming of mESCs to the naive ground state. It is possible that the same miRNAs are involved in DNA demethylation in migratory PGCs. I also show that retinol has an unexpected effect on the rate and extent of DNA demethylation. How this finding translates in the context of primordial germ cell development is unknown. It does, however, highlight both a potential source of variation in the published data and the need to understand the interplay between cell metabolism and epigenetic reprogramming.
Critically ill patients with impaired neutrophil phagocytosis have significantly increased risk of nosocomial infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) improves ...phagocytosis by neutrophils ex vivo. This study tested the hypothesis that GM-CSF improves neutrophil phagocytosis in critically ill patients in whom phagocytosis is known to be impaired.
This was a multicentre, phase IIa randomised, placebo-controlled clinical trial. Using a personalised medicine approach, only critically ill patients with impaired neutrophil phagocytosis were included. Patients were randomised 1:1 to subcutaneous GM-CSF (3 μg/kg/day) or placebo, once daily for 4 days. The primary outcome measure was neutrophil phagocytosis 2 days after initiation of GM-CSF. Secondary outcomes included neutrophil phagocytosis over time, neutrophil functions other than phagocytosis, monocyte HLA-DR expression and safety.
Thirty-eight patients were recruited from five intensive care units (17 randomised to GM-CSF). Mean neutrophil phagocytosis at day 2 was 57.2% (SD 13.2%) in the GM-CSF group and 49.8% (13.4%) in the placebo group, p=0.73. The proportion of patients with neutrophil phagocytosis≥50% at day 2, and monocyte HLA-DR, appeared significantly higher in the GM-CSF group. Neutrophil functions other than phagocytosis did not appear significantly different between the groups. The most common adverse event associated with GM-CSF was fever.
GM-CSF did not improve mean neutrophil phagocytosis at day 2, but was safe and appeared to increase the proportion of patients with adequate phagocytosis. The study suggests proof of principle for a pharmacological effect on neutrophil function in a subset of critically ill patients.
Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired ...infection. Despite this, remarkably few neutrophil-specific treatments exist. Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β2 -agonists. Inhibitors and activators of cyclic AMP (cAMP)–dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. Results β2 -Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β2 -agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β2 -agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.
Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired ...infection. Despite this, remarkably few neutrophil-specific treatments exist. Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using beta 2-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. Results beta 2-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by beta 2-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by beta 2-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.
Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired ...infection. Despite this, remarkably few neutrophil-specific treatments exist. Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β2-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosisin vitro. Results β2-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β2-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β2-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.
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