Prdx1 regulates macrophage polarization by maintaining mitochondrial homeostasis

Abstract:

In order to investigate the role of Prdx1 in macrophage polarization, mouse leukemia cells of monocyte macrophage (RAW264.7) were treated with lipopolysaccharides (LPS)+ interferon gamma (IFNγ) or IL-4 to induce type 1 macrophage (M1) and type 1 macrophage (M2) macrophages, respectively. The Prdx1 gene knockout cells (Prdx1^-/-;) were used for the study. Flow cytometry was conducted to detect M1/M2 macrophage markers, and ELISA kits were used to measure M1/M2 cytokine levels. Inducible nitric-oxide synthase (iNOS) activity, arginase-1 (Arg-1) activity, and oxidative damage were also assessed. The Seahorse XFe24 Extracellular Flux Analyzer was employed to measure extracellular acidification rate and oxygen consumption rate. The mitochondrial membrane potential was analyzed using the mitochondrial membrane potential dye (JC-1) fluorescent probe, and mitochondrial superoxide was detected through fluorescence staining. Additionally, the impact of adding a mitochondrial reactive oxygen species (ROS) scavenger on RAW264.7 macrophage polarization was examined. The results demonstrated an increase in ROS, hydrogen peroxide, and 8-hydroxy-2 deoxyguanosine (8-OHDG). Cytotoxicity and mitochondrial toxic effects, including mitochondrial superoxide accumulation, decreased adenosine-triphosphate (ATP) production, reduced mitochondrial membrane potential, and decreased mitochondrial DNA copy number, were observed. Furthermore, down-regulation of translocase of inner mitochondrial membrane 23 (TIM23) mitochondrial protein and mitochondrial stress protein heat shock protein 60 (HSP60) was noted. The extra cellular acidification rate (ECAR) in M1 macrophage polarization in RAW264.7 cells was increased, while oxygen consumption rate (OCR) in M2 macrophages was reduced. These findings indicate that Prdx1 knockout in RAW264.7 cells can inhibit M2 macrophage polarization but promote M1 macrophage polarization by impairing mitochondrial function and reducing oxidative phosphorylation.