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

TL;DR

This study shows that inhibiting IRAK4 reduces inflammation in immune cells and liver cells, offering a potential new treatment for gout.

Contribution

The study demonstrates IRAK4 inhibition's anti-inflammatory effects in macrophages and HepG2 cells under MSU and xanthine stimulation.

Findings

IRAK4 inhibition reduced pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in macrophages.

IRAK4 inhibition promoted an anti-inflammatory M2-like macrophage phenotype and reduced NF-κB and MAPK activation.

In HepG2 cells, IRAK4 inhibition attenuated xanthine-induced xanthine dehydrogenase and cytokine expression.

Abstract

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.

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.

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.

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.

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