Unraveling the immunological roles of murine serum amyloid A3 in aortic immune cell subsets during atherosclerosis progression

Atherosclerosis is a growing concern in developed nations, necessitating the identification of therapeutic targets for advancing personalized medicine. Serum amyloid A3 (Saa3) has been linked to accelerated plaque progression by affecting cholesterol metabolism and modulation of inflammation. We hypothesize that knocking out Saa3 (Saa3-/-) could mitigate plaque development by regulating aortic immune cell subsetscompositions during atherosclerosis progression. Using a murine model, we induced atherosclerosis via a gain-of-function mutant PCSK9-encoding adeno-associated viral vector (AAVmPCSK9) in wild-type (WT) and Saa3-/- mice. Single-cell RNA sequencing revealed that Saa3-/- mice developed smaller plaques than WT mice, with and single-cell RNA sequencing revealed significant differences in aortic immune cell populations, particularly among aortic macrophages. Saa3-/-Trem2hi macrophages, characterized by high Gpnmb, Lpl, and Spp1 expressions, predominated over the typical resident foamy macrophages in WT mice.Saa3-/- compared to WT mice. SAA3 regulates cholesterol metabolism and inflammatory responses in foamy macrophages. Notably, aortic immune cells in atherosclerotic Saa3-/- mice also showed enhanced intercellular immune communication and increased signaling interactions, suggesting a shift towards an anti-inflammatory and tissue-repairing phenotype. Our study highlights the potential of Saa3 as a therapeutic target forkey modulator of aortic immune cells that impact atherosclerosis progression. 12-week-old Wild-type C57BL/6J mice wild-type (WT) and Saa3-/- littermates used for induction of murine atherosclerosis by injecting intraperitoneally once with AAVmPCSK9 (ssAAV8/hAAT-mPCSK9D377Y, AAV core, Institute of Biomedical Sciences, Academia Sinica, using a plasmid acquired from Addgene (Plasmid #58376) and kindly provided by Dr. Mi-Hua Tao) at 5×1011viral particles/mouse and fed on Western diet (Research Diets, Cat# D12079Bi) for 18 weeks. Single-cell RNA sequencing was then performed on the sorted aortic immune cells from atherosclerotic WT and Saa3-/- mice. Bone marrow cells isolated from wild-type C57BL/6J mice were differentiated into bone marrow-derived macrophages (BMDMs) by culturing in RPMI 1640 with 10% heat-inactivated FBS, 1% P/S/A, and 20 ng/mL M-CSF for seven days. On day 7, adherent macrophages were detached and seeded into two groups: one left untreated (Control) and one treated with 100 ng/mL SAA3 for 24 hours. Single-cell RNA sequencing (scRNAseq) was then performed on both untreated and SAA3-treated BMDMs.