Tert-Butylhydroquinone (TBHQ) Suppresses LPS- and Poly (I:C)-Induced RAW 264.7 Macrophage Activation Through Reduced NF-κB/Type 1 Interferon and Enhanced Antioxidant-Related Pathways

The global demand for processed foods has increased reliance on synthetic phenolic antioxidants (SPAs), including tert-butylhydroquinone (TBHQ), a widely used additive to prevent lipid oxidation and extend shelf life. TBHQ is considered safe at present regulated levels; however, studies suggest potential adverse effects, including oxidative stress, genotoxicity, and impacts on immune function, raising concerns about human health and ecological risks. Herein, we investigated the immunomodulatory effects of TBHQ on RAW 264.7 murine macrophages pre-exposed to 0.1, 1, and 5 µM TBHQ and then stimulated with lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly I:C, PIC) to model bacterial and viral immune challenges. We then used functional assays and transcriptomic profiling to assess inflammatory responses and oxidative stress signaling. TBHQ reduced nitric oxide production and IL-10 secretion at the highest non-cytotoxic dose, and enhanced phagocytosis and IL-6 secretion at the lowest concentrations. Overall, transcriptomics revealed significant downregulation of proinflammatory pathways and induction of glutathione and xenobiotic metabolism. Pre-treatment with TBHQ increased gene transcript counts of key metabolic genes/transporters such as <i>Cbr3</i>, <i>Adh7</i>, <i>Gstp1/3</i>, <i>Gsta3</i>, <i>Hmox1</i> and <i>Gclm</i>. Following treatment with LPS or PIC several genes for classical proinflammatory chemokines and cytokines such as <i>Cxcl2</i>, <i>Ccl2</i>, <i>Ccl12</i>, <i>Acod1</i>, <i>Ptgs2</i>, <i>Nos2</i>, and <i>Il6</i> were downregulated. Genes involved in NF-κB signaling, such as <i>Nfkbia</i>, <i>Nfkb1</i>, and <i>Ikbke</i> were also downregulated. Our study suggests that the induction of Nrf2-related antioxidant pathways by TBHQ is the main driver for reduced inflammatory signaling in macrophages.