Abstract Body (Do not enter title and authors here): Introduction: Clonal Hematopoiesis of Indeterminate Potential (CHIP) is associated with atherosclerotic cardiovascular disease (ASCVD) and is often caused by somatic mutations in the epigenetic modifier TET2. Reduced TET2 causes ASCVD through increased inflammatory signaling in tissue macrophages. Recently, macrophage lipid loading has been shown to potentiate inflammatory activation, particularly in the context of TET2 deficiency. Therefore, we sought to test the hypothesis that TET2 deficiency exerts a proinflammatory phenotype by directly increasing macrophage lipid uptake.

Methods: Using CRISPR/Cas9 genome editing, we modeled TET2-CHIP through monoallelic disruption of the TET2 open reading frame in THP-1 monocytes (TET2^+/-). We targeted the AAVS1 safe harbor locus to generate mock-edited control lines (AAVS1^+/-). After differentiation to macrophages, we assayed uptake of phrodo-labeled low density lipoprotein (phrodo-LDL) using live cell imaging and fluorescently conjugated oxidized LDL (DiI-oxLDL) using flow cytometry. RNA-sequencing libraries were constructed from TET2^+/- and AAVS1^+/- lines treated with or without oxLDL. Differentially expressed genes (DEGs) were identified with DESeq2. Pathway analysis was conducted using clusterProfiler. We used publicly available CHIP-seq data to assay TET2 binding at ENCODE candidate cis-regulatory elements (cCREs).

Results: We observed a two-fold increase of phrodo-LDL uptake in TET2^+/- cell lines compared to controls over a 24 hour period (p < 0.05). TET2^+/- lines treated with DiI-oxLDL exhibited significant increases in both mean fluorescence intensity (9774 vs. 7954, p < 0.001) and proportion of DiI⁺ foam cells (81.7% vs. 74.7%, p < 0.05). Compared to controls, TET2^+/- lines had four-fold increased expression of inflammatory cytokine CCL5 with and without oxLDL stimulation (p<0.001). The most significantly upregulated genes in TET2^+/- lines treated with oxLDL were VDLR (3.7-fold, p < 0.001) and LDLR (9-fold, p<0.001). Pathway analysis revealed significant enrichment of KEGG term ‘IL-17 signaling’ in ox-LDL treated TET2^+/- lines (p<0.001). CHIP-seq revealed TET2 occupancy at an LDLR cCRE.

Conclusion: We demonstrate that TET2 deficiency stimulates foam cell formation through increased LDL and oxLDL uptake, possibly due to aberrant activation of LDLR and VLDLR. These data suggest that therapeutic approaches targeting lipid metabolism may have an outsized benefit for patients with TET2 CHIP.