Abstract
Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation and immune dysregulation, including expansion of pro-inflammatory monocytes and effector T cells, alongside reduced regulatory T cells (Tregs). Bacillus subtilis natto, a spore-forming probiotic, has shown anti-atherosclerotic effects, though its systemic immunomodulatory mechanisms remain unclear. In this study, we employed an AAV-mPCSK9-induced murine atherosclerotic model to investigate the effects of daily B. subtilis natto NTU-18 administration over 16 weeks. High-dimensional flow cytometry using two 13-marker panels enabled longitudinal profiling of 18 peripheral immune cell subsets across lymphoid and myeloid compartments. While no significant changes in serum cholesterol and mild decrease of body weight were observed, B. subtilis natto NTU-18-treated mice presented a significant reduction in aortic lesion area compared to PBS-treated controls. Immune profiling revealed a transient expansion of peripheral myeloid cells and CD44⁺ trained CD8⁺ T cells, followed by increased frequencies of naïve CD8⁺ T cells and reduced central/effector memory subsets at longer time point treatment. In the CD4⁺ T cell compartment, a transient increase in trained cells was accompanied by a sustained enrichment of CD25⁺CD4⁺ Tregs throughout the daily B. subtilis natto NTU-18 treatment. In contrast, no significant differences were observed in Ly6C⁻ or Ly6C⁺ monocytes, neutrophils, or eosinophils. These findings suggest that B. subtilis natto NTU-18 attenuates atherosclerosis progression not through lipid lowering or broad myeloid modulation, but via targeted reprogramming of peripheral T cell responses. This work provides mechanistic insight into the immunotherapeutic potential of B. subtilis natto NTU-18 in atherosclerosis prevention and treatment. KEY POINTS: • B. subtilis natto NTU-18 significantly reduces aortic plaque burden in atherosclerotic mice without affecting serum cholesterol levels. • B. subtilis natto NTU-18 induces transient immune remodeling, marked by early expansion of trained CD8⁺ and CD4⁺ T cells, followed by increased naïve and regulatory T cells. • The atheroprotective effect is primarily mediated through adaptive immunity as myeloid subsets remain unchanged throughout B. subtilis natto NTU-18 treatment.