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Published on: July 14, 2016
Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway
Lee J Winchester1,1, Sudhakar Veeranki1,1, Srikanth Givvimani1,1
1Department of Physiology and Biophysics, School of Medicine, University of Louisville, Louisville, 500 South Preston Street, HSC Building A, KY 40202, USA.
Introduction:
Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases.
Methods:
murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry.
Results:
Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls.
Conclusion:
Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.
Insights
High homocysteine levels promote M1 macrophage differentiation, a key factor in inflammation. This process is partly driven by EMMPRIN, influencing macrophage behavior in disease.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Hyperhomocysteinemia (HHcy) is linked to inflammatory conditions and increased oxidative stress.
- Macrophage polarization into M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes is critical in disease pathogenesis.
- The specific impact of HHcy on macrophage differentiation remains unclear.
Purpose of the Study:
- To investigate the effect of HHcy on macrophage phenotype differentiation.
- To determine the role of extracellular matrix metalloproteinase inducer (EMMPRIN) in HHcy-induced macrophage changes.
Main Methods:
- Murine macrophages (J774A.1 and Raw 264.7) were treated with homocysteine (Hcy).
- Western blotting and immunocytochemistry were used to analyze protein expression.
- EMMPRIN function was blocked using an anti-EMMPRIN antibody.
Main Results:
- Hcy treatment increased M1 macrophage markers (CD40) and reactive oxygen species (ROS) production.
- Matrix metalloproteinase-9 (MMP-9) and EMMPRIN expression were elevated by Hcy.
- Blocking EMMPRIN significantly reduced CD40 expression, indicating its role in M1 differentiation.
Conclusions:
- HHcy promotes the differentiation of macrophages towards the M1 phenotype.
- EMMPRIN induction plays a partial role in mediating the effects of HHcy on macrophage polarization.
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