Background/Purpose:

Gout, the leading cause of inflammatory arthritis, is becoming increasingly prevalent worldwide, affecting both developed and developing countries. This condition results from the deposition of uric acid crystals in joints and bursal tissues, particularly in individuals with hyperuricemia, causing intense inflammation and severe pain. In the joints, macrophages engulf MSU crystals, activating TLR2/4, which subsequently triggers the NLRP3 inflammasome and NF-κB activation. IRAK4, a crucial kinase in the TLR/IL-1R signaling pathway, interacts with Myd88 to form the myddosome, initiating the production of proinflammatory cytokines via NF-κB activation. This leads to IL-1β secretion and neutrophil influx, resulting in inflammation and joint damage. In this study, we aimed to elucidate the anti-inflammatory mechanism of the IRAK4 inhibitor (PF06650833) by examining MSU-induced pro-inflammatory mediators in human PBMCs.

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 was extracted for qPCR to quantify levels of IL-1β, IL-18, TNF-α, IL-6, IL-8, and TGFβ. 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.

Results: Pretreatment with IRAK4 inhibitor (IRAK4i) reduced MSU-induced inflammation in PBMC by 60-70%, as evidenced by decreased levels of IL-1β, IL-18, TNF-α, IL-6, and IL-8, and a 300% increase in TGFβ (p< 0.01; n=4). These findings were also reflected at the mRNA level. Additionally, IRAK4i suppressed MSU-induced phagocytosis, potentially preventing macrophages from engulfing MSU crystals, and activating TLRs, thus reducing inflammation. Immunoblot and in-cell western assays in PBMC showed inhibition of IRAK4 phosphorylation. Furthermore, MSU stimulation led to phosphorylation of P38, JNK, and ERK which was significantly diminished or completely inhibited by IRAK4i, effectively dampening downstream proinflammatory signaling pathways.

Conclusion: Our study demonstrated that IRAK4 inhibitor (IRAK4i) modulates inflammation induced by monosodium urate (MSU) in peripheral blood mononuclear cells (PBMC). Additionally, we observed effective inhibition of phagocytosis and confirmed the signaling mechanisms potentially disrupted by IRAK4i in MSU-induced pathways in PBMC. Therefore, IRAK4i appears to be a promising therapeutic strategy for gout patients by counteracting MSU and TLR-mediated inflammation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeting-irak4-in-monosodium-urate-crystals-induced-inflammation/