Modulation of IRAK4 as a Therapeutic Strategy Against Monosodium Urate- and Xanthine-Induced Inflammation in Macrophages and HepG2 Cells

Background Interleukin-1 receptor-associated kinase 4 (IRAK4) is a pivotal mediator of toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling, critically involved in innate immune activation and pro-inflammatory cytokine production. Dysregulated IRAK4 activity contributes to chronic inflammation in both immune and non-immune cells. In this study, we evaluated the immunomodulatory potential of a selective IRAK4 inhibitor on monosodium urate (MSU) crystals-stimulated macrophages and xanthine-challenged HepG2 cells to assess its therapeutic potential. Methods Human PBMCs were pretreated with 1 µM IRAK4 inhibitor (IRAK4i) overnight, followed by stimulation with 100 µg/ml MSU for either 30 minutes or 24 hours. Conditioned medium was collected for ELISA and RNA for qPCR to quantify pro- and anti-inflammatory factors. Cell lysates were prepared to analyze various TLR/IL-1β signaling proteins, including phosphorylated IRAK4, P38, ERK, and JNK. Phagocytosis was assessed using a Vybrant™ phagocytosis assay kit in PBMCs. We also utilize HepG2 cells and pretreated with 1 µM IRAK4 inhibitor (IRAK4i) overnight, followed by stimulation with 2.5mM of xanthine for 24 hours to assess the expression of cytokine and xanthine oxidoreductase. Results Primary macrophages and HepG2 cells were treated with a potent IRAK4 inhibitor in the presence and absence of MSU or xanthine. In macrophages, IRAK4 inhibition significantly reduced the expression of TNF-α, IL-6, and IL-1β at both mRNA and protein levels, while promoting polarization toward an anti-inflammatory (M2-like) phenotype alongside reduced activation of NF-κB and MAPK pathways. In HepG2 cells, IRAK4 blockade attenuated xanthine-induced expression of xanthine dehydrogenase and inflammatory cytokines. Conclusion These findings demonstrate the dual anti-inflammatory effect of IRAK4 inhibition in both immune and hepatic cells and suggest a promising strategy to mitigate inflammation in gout.