The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages

Macrophages play pivotal roles in the progression of atherosclerosis (AS) and their heterogeneous differentiation patterns have been studied extensively. The classical subtype of activated macrophage, M1, promotes the progression of AS. Conversely, the alternative subtype of activated macrophage, M2, is regarded as a repressor of AS. Homocysteine (Hcy) may influence macrophage subtype polarization both in vivo and in vitro. Homocysteinemia (HHcy) is an independent risk factor in coronary heart disease and the effect of Hcy on macrophage differentiation has not been studied until now.Different concentrations of Hcy in combination with a fixed concentration of lipopolysaccharide (LPS, 200 ng/mL) were used to treat RAW264.7 macrophages. Real-time PCR was used to detect and quantify RNA transcripts indicative of M1 and M2 differentiation. The efficacy and specificity for each chemical stimulant in inducing macrophage differentiation were also investigated. The M2 macrophages (anti-inflammatory subtype) induced using classical methods (IL-4, 10 ng/mL) were also treated with different concentrations of Hcy complemented with LPS. The synergistic effect of Hcy and LPS in the converting the M2 subtype to M1 was also studied.Macrophages can be induced to differentiate towards M1 by a combination of Hcy with LPS, with the strongest effect observed at an Hcy concentration of 50 μmol/L. After inducing macrophages to the M2 subtype using IL-4, treatment with both Hcy and LPS could elicit conversion from the M2 to M1 subtype.Combined treatment with Hcy and LPS can induce the polarization of cultured RAW264.7 macrophages into the pro-inflammatory subtype, as well as promote subtype conversion from anti-inflammatory to pro-inflammatory.