Abstract Body: Background: Dysregulated lipid metabolism results in the formation of lipid-dense macrophages, termed foam cells. Foam cell formation and accumulation in the artery is a key process in atherosclerosis development. The scavenger receptor CD36 is the main responsible receptor in foam cell formation by mediating oxidized LDL (oxLDL) uptake. Itaconate, a byproduct of tricarboxylic acids cycle, possesses various functions such as anti-inflammation and anti-oxidants through protein alkylation, a posttranslational modification. Recently, it has been reported that the itaconate derivative, 4-octyl-itaconate (4-OI) alleviates atherosclerosis and reduces foam cell formation in vivo. However, the mechanism of how 4-OI reduces macrophage lipid accumulation is unknown.
Hypothesis: We hypothesize that 4-OI modifies CD36 and reduces foam cell formation by inhibiting CD36-mediated oxLDL uptake.
Methods: Mouse peritoneal macrophages or human monocyte-derived macrophages were treated with oxLDL and 4-OI. Foam cell formation was evaluated by Oil red O staining and cholesterol assay. Quantitative RT-PCR and Western blot were used to analyze CD36 expression. Surface CD36 expression and oxLDL binding and uptake assay were observed by flow cytometry. To detect modified CD36 protein, itaconate-alkyne (Italk) probe was applied to macrophages and followed by a pull-down assay and Western blot.
Results: After 4-OI 24h treatment, foam cell numbers and cholesterol content induced by oxLDL were reduced by half both in mouse peritoneal macrophages and human monocyte-derived macrophages (n=4). After 4-OI treatment with WT macrophages, oxLDL binding to the cell surface reduced by 25% and oxLDL uptake by 40%, despite no change in surface CD36 (n=6). However, 4-OI didn't change oxLDL binding and uptake in CD36 knockout cells. Finally, using Italk probe, we showed that 4-OI alkylated macrophage CD36 (n=3).
Conclusion: We identify a novel regulatory mechanism that 4-OI inhibits foam cell formation by alkylating CD36 protein and reducing oxLDL uptake in macrophages. These findings may have new therapeutic implications on atherosclerosis.