Abstract Body: Introduction: Macrophage (Mφ) CD36 is the oxidized LDL (oxLDL) receptor critical for foam cell formation. CD36 has a single extracellular domain with six conserved cysteines, classically viewed to form inert disulfide bonds. Emerging evidence indicates that extracellular disulfides can act as redox-sensitive switches, undergoing cleavage or modifications in response to environmental cues. Itaconate (ITA), a metabolite produced by mitochondrial IRG1 enzyme in activated Mφs, modulates immunity through alkylation of cytosolic proteins and can be exported, suggesting potential paracrine signaling functions.
Hypothesis: We hypothesized that Mφ CD36 harbors free extracellular cysteine thiols that function as environmental sensors. ITA alkylation of these thiols acts as a paracrine “metabolite checkpoint," regulating CD36-oxLDL interactions.
Methods: Peritoneal Mφs were isolated. Extracellular free cysteine residues on CD36 were detected using 3-(N-maleimidopropionyl)-biocytin labeling. ITA alkylation (itaconated cysteine) of CD36 in murine Mφs and human aorta was analyzed by immunoblots. Apoe null mice underwent lethal irradiation and were reconstituted with bone marrow (BMs) from Apoe null or Apoe/Cd36 double null donors, then fed a high-fat diet (HFD). The plaque burdens in the aortic arch and sinus were quantified.
Results: CD36 displayed accessible free cysteine residues on the surface of peritoneal Mφs in vivo (n = 4). In the 10-week HFD-induced atherosclerosis model (Apoe null), Mφs exhibited reduced surface CD36 free cysteine and elevated IRG1 protein expression compared to those from chow diet-fed mice (n = 3-6). Immunoprecipitation of CD36 followed by immunoblotting with the itaconated cysteine antibody (CPTC-2MeSC-2) revealed that CD36 is itaconated in both mouse Mφs and human aorta (n = 3). Treatment with ITA derivative 4-OI reduced plaque formation by 87.8% in the aortic arch and 39.2% in the aortic sinus of Apoe null BMs-transplanted HFD mice. No plaque reduction occurred in Apoe/Cd36 double null BMs-transplanted mice (n=5-8).
Conclusions: We identify extracellular cysteines of CD36 as covalent targets of Mφ-derived itaconate, linking mitochondrial metabolism to the regulation of lipid uptake and foam cell formation. This mechanism defines a new paradigm for immune regulation and inspires novel therapeutic strategies to suppress atherosclerosis.