Abstract Body: Sustained weight loss decreases cardiovascular disease (CVD) risk. In mice, short-term caloric restriction (stCR) -induced weight loss also causes accumulation of a phagocytic macrophage (MΦ) in atherosclerotic plaques and adipose tissue, marked by high expression of the Fc γ receptor FCGR4 (murine orthologue of human FCGR3A, also known as CD16A). We have previously shown that MΦ FCGR4 plays a functional role in efferocytosis and reduces necrotic core size during stCR-induced atherosclerosis resolution. Here we investigate the regulation of Fcgr4 expression in MΦ.
scRNAseq data from human adipose tissue Fcgr3A⁺ MΦ as well as stCR murine plaque and adipose tissue Fcgr4⁺ MΦ identified transcription factor TCF7L2, downstream of β -catenin dependent canonical Wnt signaling, as a potential regulator of Fcgr4 expression. We confirmed the TCF7L2-dependent expression of Fcgr4 and other efferocytosis-associated genes in the Fcgr4⁺ MΦ cluster, such as Mertk and Wdfy3, by treating bone marrow (BM) cells with a control or the canonical Wnt pathway activator CHIR99021, which increased both Fcgr4 mRNA (0.095 ±0.05 vs 0.484 ±0.28, p<0.001), and FCGR4 protein (1.57% ±0.88 vs. 13.93% ±8.61, p<0.0001, n=6-15). Notably, Fcgr4 and Mertk mRNA and FCGR4 protein levels were significantly reduced when the BM cells were additionally treated with Macrocycle 13, a direct inhibitor of the interaction between β -catenin and TCF7L2 (Fcgr4 mRNA 0.53 ±0.09 vs. 0.33 ±0.09 p=0.01, Mertk mRNA 0.244 ±0.10 vs. 0.175 ±0.11, p=0.006, FCGR4 protein 1701.4 ±560.93 vs. 578.4 ±230.60, p=0.006, n=4-5). ChIP studies are in progress to validate a direct interaction of TCF7L2 with the Fcgr4 promoter region.
Inducing a Fcgr4⁺ MΦ phenotype through activation of TCF7L2 in vitro also significantly increased their efferocytotic activity compared to vehicle control treated cells (6.32 ±2.56 vs. 10.27 ±3.78, p=0.01, n=8). Furthermore, we demonstrate that Fcgr4⁺ MΦ are of circulating monocytic origin, rather than resident MΦ-derived.
In summary, TCF7L2, in concert with β-catenin, forms a key transcriptional module required for induction of FCGR4/FCGR3A in MΦs and for the enhanced efferocytotic function that accompanies stCR-driven atherosclerosis resolution.