Epigenetic coding of long-term heat acclimation on DNA through cytosine hydroxy-methylation vs. methylation in mouse blood mononuclear cells

Our purpose was to identify epigenetic biomarkers of long-term heat acclimation in an accessible blood phenotype that could be used to identify acclimatization state. We heat-acclimated (ACC) groups of adult male and female diversity outbred (DO) mice (n = 8-10/group) by continuous incubation at 34°C for 30 days. Results were compared to sham control mice (CNTRL) exposed to 28°C (i.e. within mouse thermoneutral zone). After 48 h of recovery at 28°C, the mice were sacrificed and whole blood samples taken for isolation of peripheral blood mononuclear cells (PBMCs). DNA was isolated and analyzed for global cytosine methylation (5mC) and hydroxy-methylation (5HmC) genomic content using ELISA (Epigentek, MethylFlashTM). RESULTS: Total 5mC content was significantly higher (by ~2 fold) in CNTRL females compared to CNTRL males (P< 0.0001) and lower in acclimatized ACC vs. CNTRL females (P< 0.05). Total 5HmC content was also higher (~2-3 fold) in CNTRL females compared to CNTRL males (P=0.006), and male ACC mice had higher 5HmC content compared to CNTRL (~ 3 fold; P=0.0006). The ratio of 5HmC/5mC content was ~ 1.5 fold higher in ACC mice of both sexes compared to their respective CNTRLs (P< 0.0001 for each sex). SUMMARY and CONCLUSIONS. Both methylation and hydroxy-methylation appear to be important in epigenetic programming responses to long-term heat acclimatization with the most consistent signals across both sexes being the level of 5HmC/5mC. Since 5hmC is an oxidation product of 5mC via ten-eleven translocation (TET) methylcytosine dioxygenase enzymes, the increased ratio of 5HmC/5mC suggests that sustained heat exposure initiates broad 5mC to 5HmC conversion on multiple gene loci. These likely contribute to gene regulation of immune cells in the acclimatized state. Since these signals remain following days of thermoneutral recovery and blood leukocyte turnover is rapid, it is likely that this conversion occurs in hematopoietic cells that are known to harbor epigenetic memories of environmental stress. This initial analysis provides a roadmap for future studies, suggesting that 5mC-5HmC conversion at a nucleotide level may provide a robust pathway of future discovery in epigenetic environmental adaptation. Funded by the Department of Defense US Army Medical Research and Development Command, BA220200 HT94252410275 (TLC). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.