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Updated: Jun 25, 2025
Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
[Prdx1 regulates macrophage polarization by maintaining mitochondrial homeostasis]
Xiang Zhang1, Ziyue Zhang1, Yiming Qi1
1Medical College, Yan'an University, Yan'an 716000, Shaanxi, China.
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.
Insights
Peroxiredoxin-1 (Prdx1) knockout impairs macrophage mitochondrial function, promoting M1 polarization and inhibiting M2 polarization by affecting oxidative phosphorylation.
Area of Science:
- Immunology
- Cell Biology
- Mitochondrial Biology
Background:
- Macrophage polarization is crucial for immune responses.
- Peroxiredoxin-1 (Prdx1) is an antioxidant enzyme with potential roles in cellular processes.
- Understanding Prdx1's role in macrophage polarization is essential for immune modulation.
Purpose of the Study:
- To investigate the role of Prdx1 in M1 and M2 macrophage polarization.
- To elucidate the impact of Prdx1 knockout on macrophage mitochondrial function and metabolism.
- To determine the mechanisms by which Prdx1 influences macrophage polarization.
Main Methods:
- RAW264.7 cells, including Prdx1 knockout variants, were polarized into M1 and M2 macrophages using LPS+IFNγ or IL-4.
- Flow cytometry and ELISA were used to assess M1/M2 markers and cytokines.
- Mitochondrial function was evaluated by measuring oxygen consumption rate (OCR), extracellular acidification rate (ECAR), mitochondrial membrane potential, and ATP production.
- Oxidative stress markers and mitochondrial protein expression were analyzed.
Main Results:
- Prdx1 knockout led to increased reactive oxygen species (ROS) and oxidative damage (8-OHDG).
- Mitochondrial dysfunction was evident, with decreased ATP production, membrane potential, and mitochondrial DNA copy number.
- Prdx1 knockout inhibited M2 polarization (reduced arginase-1 activity) and promoted M1 polarization (increased iNOS activity).
- Downregulation of TIM23 and HSP60 was observed in Prdx1 knockout cells.
Conclusions:
- Prdx1 knockout impairs mitochondrial function in macrophages.
- This impairment leads to altered macrophage polarization, favoring M1 over M2 phenotypes.
- Prdx1 plays a critical role in maintaining macrophage mitochondrial homeostasis and regulating polarization.
