Evaluation of mRNA expression and DNA methylation status of immune checkpoints in the peripheral blood of COVID-19 patients

BACKGROUND: COVID-19 immunopathology involves dysregulated immune responses, partly mediated by inhibitory immune checkpoints (ICs). Epigenetic mechanisms, particularly DNA methylation by DNA methyltransferases (DNMTs), regulate gene expression. However, the interplay between IC transcription, promoter methylation, and DNMT expression across disease severities remains unclear. RESEARCH DESIGN AND METHODS: This cross-sectional study enrolled 208 COVID-19 patients (mild to critical) and 93 healthy controls. mRNA expression of PDCD-1, HAVCR-2, LAG-3, CTLA-4, DNMT-1, DNMT-3a, and DNMT-3b was measured by RT qPCR after covariate adjustment. Promoter methylation of the four IC genes was assessed by MS-HRM in 90 patients and 24 controls. RESULTS: COVID-19 patients showed significantly lower mRNA levels of PDCD-1, LAG-3, and CTLA-4 versus controls, with further reductions in hospitalized, ICU-admitted, and non-survivor subgroups. DNMT-1, DNMT-3a, and DNMT-3b transcripts were also markedly decreased. No significant methylation changes were observed for any of the four IC genes after FDR correction (all q >0.05). CONCLUSIONS: Reduced DNMT transcripts suggest possible global hypomethylation, but no significant promoter methylation changes were detected for the IC genes examined. Whole-blood analysis and lack of protein/functional validation limit interpretation. This exploratory study provides a foundation for future cell-specific and prospective investigations into epigenetic-transcriptional crosstalk in COVID-19.