Effects of Co-exposure to AhR Ligands and Lipopolysaccharides on RAW 264.7 Macrophage Polarization

Document Type : Original Article

Authors

1 Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

2 Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Abstract

Background: Co-exposure to an aryl hydrocarbon receptor (AhR) agonist, such as benzo[a]pyrene (BaP) and lipopolysaccharides (LPS), commonly occurs through environmental pollution or smoking. The AhR plays a crucial role in macrophage polarization, influencing immune responses within the tumor microenvironment (TME). This study aimed to investigate how co-exposure to AhR ligands and LPS affects the balance between pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. 
Materials and Methods: RAW264.7 macrophages were treated with LPS (100 nM), BaP (2.5 µM), and CH-223191 (AhR antagonist, 1 µM), both individually and in combination. The polarization of RAW264.7 macrophages was assessed using flow cytometry and real-time polymerase chain reaction (PCR), focusing on the analysis of surface markers CD80 and CD206, as well as the gene expression levels of iNOS, Arg1, and CD206. Enzyme-linked immunosorbent assay (ELISA) was utilized to measure IL-10 and TNF-α secretion.
Results: Combining LPS with BaP or CH-223191 altered macrophage surface marker expression, increasing a population positive for both M1 and M2 markers. LPS significantly upregulated iNOS expression, whereas co-treatment with BaP or CH-223191 attenuated this effect. Co-exposure to BaP and LPS reduced Arg1 expression relative to LPS alone, while co-treatment with CH-223191 and LPS increased CD206 expression. LPS treatment alone, or in combination with BaP or CH-223191, increased TNF-α levels. IL-10 secretion increased only in the CH-223191+LPS group. 
Conclusion: These findings suggest that co-exposure to BaP and LPS promotes a complex macrophage activation state characterized by strong pro-inflammatory cytokine secretion but altered marker expression, whereas CH-223191 modulates this response differently.

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