Publication: Efferocytosis of oxidized low-density lipoprotein generated apoptotic bodies by primary m1 and m2 macrophages: a comparison between non-obstructive and obstructive coronary artery disease patients
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Date
2024-12
Authors
Idrus, Fatin Najiah Mohd
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Abstract
Efferocytosis; an immunological process of macrophage clearing apoptotic bodies, is invincible in the human physiology. Nonetheless, unresolved apoptotic bodies are abundant in atherosclerotic plaque, putting it risky of instability and rupture. Dysregulated efferocytosis in coronary artery disease (CAD) may be linked to macrophage phenotypes, stages of apoptosis, or inflammatory cytokines. This study measured the efferocytosis rate of M1 and M2 macrophages from non-obstructive and obstructive CAD patients towards early and late apoptotic bodies. Importantly, the implication of the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α) and anti-inflammatory cytokine, tumor growth factor-beta (TGF-β) in dysregulating efferocytosis was investigated. 27 non-obstructive and 29 obstructive CAD patients were recruited, and 30 mL of peripheral venous blood was collected to isolate monocytes. M1 and M2 macrophages were polarized from the monocytes using respective stimulants (M1: 20 ng/µL GM-CSF, 20 ng/µL IFN-γ, and 10 ng/µL LPS; M2: 10 ng/µL M-CSF, 20 ng/µL IL-4, and 20 ng/µL IL-13). Early and late apoptotic bodies were generated by incubating macrophages with 100 µg/mL oxidized low-density lipoprotein for 72 hours and 168 hours, respectively. M1 and M2 macrophages were subsequently transfected with TNF-α siRNA and TGF-β siRNA to investigate the implication of cytokine level towards efferocytosis. The mRNA expression of the efferocytosis receptor, MER proto-oncogene, tyrosine kinase (MERTK), and its potential protease, ADAM metallopeptidase domain 17 (ADAM17) was measured using quantitative real-time polymerase chain reaction. Independent t-test/Mann Whitney or ANOVA/Kruskal Wallis test was performed to determine the significant difference between two or more than two parameters accordingly. As expected from previous research, the expression of MERTK in M2 was higher compared to M1 macrophages (11.88 ± 4.86 vs. 5.89 ± 1.69, p=0.002). Adding to the depth of knowledge, this study for the first time showed that efferocytosis of early apoptotic bodies was most efficiently performed by M2 macrophages from non-obstructive CAD (11.92% ± 5.54%, p=0.049), while late apoptotic bodies were most efficiently resolved by M2 macrophages from obstructive CAD (6.70% ± 3.68%, p=0.036). Interestingly, this study revealed that upon the silencing of TNF-α, efferocytosis significantly improved in the respective group of M2 macrophages (M2 non-obstructive-early apoptotic bodies: 46.78% ± 0.76%, p<0.001; M2 obstructive-late apoptotic bodies: 29.24% ± 2.46%, p<0.001). This study suggests that macrophage origin and phenotype, stages of apoptotic bodies, and inflammatory cytokines contribute to efferocytosis dysregulation, revealing potential therapeutic targets for CAD patients.