Rapamycin reduces atherosclerotic plaque inflammation in aged mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): 1. ERA-CVD. Beat ATHERO consortium. 2. Dutch Heart Foundation Aims Aging is the most dominant risk factor of atherosclerotic cardiovascular disease (CVD), the main underlying cause of death worldwide. Both aging and atherosclerosis are associated with changes in the composition and function of the immune system, such as an enlarged T cell effector memory pool and elevated pro-inflammatory cytokine secretion. Since the accumulation of these pro-inflammatory factors contribute to atherosclerosis progression, halting inflammation might be a promising strategy to combat CVD. Rapamycin is an immunosuppressant that was recently discovered to increase lifespan due to the inhibition of senescent cells and effector T cells. We therefore hypothesized that rapamycin treatment can attenuate pro-atherogenic immunity in aged mice. Methods To explore the effects of rapamycin treatment, we administrated 1 mg/kg rapamycin or control solvent to aged (22 months old) male atherosclerotic Ldlr-/- mice (n=16 per group) three times a week intraperitoneally. After eight weeks, mice were sacrificed and their organs were collected for flow cytometry, single-cell RNA sequencing and histological analysis. Results Using flow cytometry, we revealed that rapamycin treatment decreased total T cell numbers in the atherosclerotic aorta and spleen compared to control treatment and attributed to a 24% increase in the relative frequency of immunosuppressive regulatory T cells in the aorta (rapa: 70.6±3.0% vs. ctrl: 57.1±3.8%, p<0.05), indicating skewing towards anti-atherogenic immunity. Within the reduced effector T cell pool of rapamycin-treated mice, we observed increased percentages of IL-10 and IFN-γ-expressing CD4+ and CD8+ T cells. In addition, rapamycin diminished the percentage of pro-atherogenic age-associated B cells (rapa: 7.5±1.8% vs. ctrl: 9.2±1.5%, p<0.05) in the mediastinal lymph nodes, and the percentage of pro-atherogenic age-associated B cells in the mediastinal lymph nodes, and reduced the expression of the senescent markers p19 and BTG2 in the spleen. In line with attenuated inflammation we observed a 36% reduction in relative plaque macrophage content in rapamycin-treated mice (rapa: 14.0±3.6% vs. ctrl: 21.8±5.5%, p<0.05). Conclusions Collectively, our study demonstrates that rapamycin treatment in aged atherosclerotic mice promotes regulatory T cells and reduces systemic and plaque accumulation of T cells, ABCs and macrophages. This provides a new understanding of rapamycin in regulating age-associated atherogenic immunity, indicating rapamycin could be a potential therapeutic to halt CVD.