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.