Molecular interactions between circadian gene TIMELESS and BCG immunization

Tuberculosis (TB) remains a major global health issue, causing 1.3 million deaths each year. Although BCG vaccination coverage is high, its effectiveness in preventing adult TB is limited, especially with the rise of drug-resistant strains. Circadian rhythms, regulated by clock proteins such as TIMELESS, influence immune responses and other biological functions. This study explores TIMELESS's role in shaping immune responses to BCG stimulation and considers whether aligning vaccination with circadian cycles might improve efficacy HEK293T cells were genetically edited using CRISPR/Cas9 to knock out the TIMELESS gene. These cells were then stimulated with BCG, and gene expression was assessed using the RT² Profiler™ PCR Array (Human Innate & Adaptive Immune Responses). Additionally, in silico tools were used to analyze TIMELESS's evolutionary relationships, structural features, and miRNA interactions.BCG stimulation led to a 90% increase in immune gene expression. Most immune genes were downregulated in TIMELESS knockout cells without BCG, except CD8A, which increased 1.97-fold. When knockout cells were stimulated with BCG, CD40, CD40LG, and SLC11A1 increased expression, indicating enhanced T-cell and macrophage activation. These findings suggest that TIMELESS is crucial for proper immune function during BCG stimulation. Its absence alters key immune pathways, particularly CD40-CD40LG and SLC11A1-related responses. Understanding TIMELESS's role may support the development of circadian-based vaccine strategies to improve TB control and vaccine effectiveness.