2,4-Dichlorophenoxyacetic acid potentiates LPS-induced inflammatory responses via NF-κB p65 and ERK1/2 signaling in RAW 264.7 macrophages.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a widely used herbicide, yet its immunomodulatory properties remain insufficiently characterized, raising concerns about potential adverse outcomes. This study examined whether 2,4-D, at environmentally and biologically relevant concentrations, influences inflammatory responses in RAW 264.7 macrophages under basal and lipopolysaccharide (LPS)-stimulated conditions. Under basal conditions, 2,4-D had no effect on inflammatory mediator production or activation of nuclear factor-kappa B (NF-κB) p65, extracellular signal-regulated kinase (ERK1/2), or p38 signaling pathways. However, 2,4-D markedly potentiated LPS-induced responses, increasing pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β), COX-2 expression, nitric oxide production, and CD80 surface expression without cytotoxicity. Consistently, 2,4-D enhanced LPS-induced activation of NF-κB p65, ERK1/2, and p38 pathways. Comparative Toxicogenomics Database analysis revealed 167 overlapping genes associated with both 2,4-D and LPS exposure. Integrative pathway, PPI, and Metascape analyses identified RELA (NF-κB p65) and MAPK1 (ERK2) as key mediators. Pharmacological inhibition confirmed that IL-6 and COX-2 responses depended mainly on NF-κB p65 and ERK1/2, whereas CD80 up-regulation was predominantly NF-κB p65 dependent. Collectively, these findings provide new mechanistic insight into the amplification of macrophage inflammatory responses by 2,4-D under immune-challenged conditions via NF-κB p65 and ERK1/2 signaling pathways.