[Single-cell transcriptomic analysis of the impact of circadian rhythm disruption on immune function in zebrafish]

The impact of circadian rhythm disruption on immune system function has been widely studied; however, the precise mechanisms by which it modulates immune system structure and function at the single-cell level remain poorly understood. This study was conducted from April to November 2024 in the Department of Pediatrics, the Third Xiangya Hospital, Central South University. In this study, a zebrafish model of circadian rhythm disruption was established by exposing larvae to constant light (24 h LL) for three days, while control groups were maintained under a standard 14-hour light/10-hour dark (14∶10 h LD) cycle. Single-cell RNA sequencing was performed to comprehensively profile the immune cells from both groups. Immune cell subpopulations, their proportional shifts, and developmental trajectories were characterized through UMAP-based clustering, marker gene annotation, and Monocle pseudotime trajectory analysis. A total of seven immune-related cell subtypes were identified, including hematopoietic progenitors, T cell progenitor-like cells, activated T cell-like cells, macrophages, neutrophils, NK cells, and migratory/repair-associated cells. Notably, the proportion of activated T cells was increased in the circadian rhythm-disrupted group (18.11%) compared with the control group (7.61%), accompanied by enhanced expression of immune activation markers such as cd40lg, il2rb,tnfrsf9b (log₂FC=7.49, 5.58, 3.76, adjusted P<0.01). Pseudotime analysis revealed bifurcated differentiation paths along myeloid and lymphoid lineages. Although the core developmental trajectories remained intact under circadian rhythm disturbance, alterations were observed in the distribution and maturation pace of terminal lymphoid cells. Overall, the findings demonstrate that circadian rhythm disruption induces structural and functional remodeling of the zebrafish immune system, characterized by enhanced activation of T and NK cells and a shift in immune response status. These results provide single-cell-level insights into the immunological mechanisms underlying sleep-related disorders.