Widespread genomic de novo DNA methylation occurs following CD8+ T cell activation and proliferation.

This research investigates the intricate dynamics of DNA methylation in the hours following CD8+ T cell activation, during a critical yet understudied temporal window. DNA methylation is an epigenetic modification central to regulation of gene expression and directing immune responses. Our investigation spanned 96-h post-activation and unveils a nuanced tapestry of global and site-specific methylation changes. We identified 15,626 significant differentially methylated CpGs spread across the genome, with the most significant changes occurring within the genes ADAM10, ICA1, and LAPTM5. While many changes had modest effect sizes, approximately 120 CpGs exhibited a log2FC above 1.5, with cell activation and proliferation pathways the most affected. Relatively few of the differentially methylated CpGs occurred along adjacent gene regions. The exceptions were seven differentially methylated gene regions, with the Human T cell Receptor Alpha Joining Genes demonstrating consistent methylation change over a 3kb window. We also investigated whether an inflammatory environment could alter DNA methylation during activation, with proliferating cells exposed to the oxidant glycine chloramine. No substantial differential methylation was observed in this context. The temporal perspective of early activation adds depth to the evolving field of epigenetic immunology, offering insights with implications for therapeutic innovation and expanding our understanding of epigenetic modulation in immune function.

Site-specific decreases in DNA methylation in replicating cells following exposure to oxidative stress.

Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850 K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48 h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity.

Genome-wide impact of hydrogen peroxide on maintenance DNA methylation in replicating cells.

BACKGROUND: Environmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2. RESULTS: Sublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level. CONCLUSIONS: This research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression.

Regulation of the epigenetic landscape by immune cell oxidants.

Excessive production of microbicidal oxidants by neutrophils can damage host tissue. The short-term response of cells to oxidative stress is well understood, but the mechanisms behind long-term consequences require further clarification. Epigenetic pathways mediate cellular adaptation, and are therefore a potential target of oxidative stress. Indeed, there is evidence that many proteins and metabolites involved in epigenetic pathways are redox sensitive. In this review we provide an overview of the epigenetic landscape and discuss the potential for redox regulation. Using this information, we highlight specific examples where neutrophil oxidants react with epigenetic pathway components. We also use published data from redox proteomics to map out known intersections between oxidative stress and epigenetics that may signpost helpful directions for future investigation. Finally, we discuss the role neutrophils play in adaptive pathologies with a focus on tumour initiation and progression. We hope this information will stimulate further discourse on the emerging field of redox epigenomics.

Cutaneous squamous cell carcinomas with markers of increased metastatic risk are associated with elevated numbers of neutrophils and/or granulocytic myeloid derived suppressor cells.

BACKGROUND: A subset of presenting cutaneous squamous cell carcinomas (CSCC) is high risk with respect to their high rates of recurrence, metastasis and patient death. The identification of such high risk CSCC is problematic. Neutrophil and granulocytic myeloid derived suppressor cell (G-MDSC) numbers are elevated in a number of cancers, but their association with current markers of high risk tumors in the setting of CSCC is unknown. OBJECTIVES: To assess circulating and tumor-localised neutrophil and G-MDSC populations for associations with high-risk tumor characteristics and overall survival (OS) in CSCC patients. METHODS: A retrospective clinical audit was performed of patients who had hospital operations for primary CSCC and did not have other malignancies or HIV. Therapeutically immuno-suppressed individuals (TII, n=129) and non-TII (n=29) were analysed separately with respect to the presence of high-risk tumor features and OS. In addition, 47 patients with prospectively collected blood and primary CSCC tumor samples were analysed to determine frequencies of circulating G-MDSC and tumor localised CD66b+ and CD8+ leukocytes. RESULTS: In the clinical audit of non-TII, high circulating neutrophil counts were associated with tumor thickness≥5mm, Clark level V and high T-stage. Univariate analysis showed elevated neutrophil count was a significant marker of poor OS, whilst tumor thickness remained the only independent histological predictor of OS after adjusting for age and immuno-suppression. The prospective study demonstrated that tumors≥5mm thick had significantly increased total and peri-tumorally localised CD66b+ leukocytes (comprising neutrophils and/or G-MDSC) and that elevated circulating G-MDSC numbers were associated with high T-stage tumors. CONCLUSIONS: This study demonstrates that the presence of high risk CSCC is associated with increased numbers of both circulating and tumor resident populations of neutrophils and/or G-MDSC. These cell types therefore merit further investigation with respect to their functional and prognostic significance in CSCC.