Background/Purpose: Although metabolic reprograming and its regulation in macrophages after TLR activation is well established, little is known about the regulation and the role of metabolic changes after monosodium urate (MSU) crystal stimulation. Here, we evaluated the transcriptional and epigenetic changes, and its regulation, in macrophages after MSU stimulation.

Methods: RNA-Seq and ATAC-Seq of bone marrow derived macrophages (BMDM) or human monocyte-derived macrophages (mDM) after MSU (0.25 mg/ml) or LPS (100ng/ml) activation. ChIP-Seq for H3K27ac and H3K4me3 was also performed in BMDM. NMR was conducted on sera from patients with acute gout flare and in BMDM after MSU activation. Cytokine production was assessed by ELISA. Experiments with and without pretreatment of SP600125 (5uM), a JNK inhibitor, were also performed. For in vivo experiments, murine air pouch and intraarticular MSU injection models of acute gouty inflammation were performed to assess effects of inhibiting JNK (using SP600125, 15mg/kg) and glycolysis inhibition (using BAY-876, a GLUT1 inhibitor, 7.5mg/kg). Infiltrating leukocytes and cytokines from the lavage were quantified.

Results: RNA-seq results highlighted divergence of BMDM or mDM treated with MSU from those treated with LPS (Figure 1). Genes that were up-regulated only in MSU belonged to inflammatory -but not interferon activation- and metabolic pathways, including glycolysis. NMR analysis showed low levels of glucose in sera from patients with acute attack, and an increase of glycolytic products in supernatants of BMDM after MSU activation. GSEA showed that whereas up-regulated genes in LPS with increased H3K27ac or H3K4me3 signal belong to inflammatory pathways, up-regulated genes in MSU with increased H3K27ac or H3K4me3 signal belong to inflammation and metabolic pathways, including metabolism of carbohydrates and transport of small molecules (Figure 2). Motif analysis showed that regions of H3K27ac or H3K4me3 up-regulated signal with up-regulated gene transcript in MSU are majorly enriched in AP-1 motifs (Figure 3). Treatment of BMDM with JNK inhibitor ameliorated the up-regulation of p-JUN^Ser63 and reduced the of 88.8% of genes up-regulated by MSU, including inflammatory cytokines (Ccl2, Ccl4, Tnf and Cxcl1), metabolic genes (Slc2a1, Eno2, Pfkfb3) and AP-1 members (Jun, Junb, Fosl1). This reduction in gene expression was accompanied by reduction on H3K27ac and H3K4me3 signal over regions of open chromatin. Finally, SP600125 significantly lowered numbers of infiltrating leukocytes in the air pouch gout flare model (1.34±0.45×10⁶ vs 0.49±0.48×10⁶, p< 0.01, in the MSU group after PBS or JNK inhibitor respectively) and amounts of CXCL1 and IL-1b. BAY-876 markedly reduced leukocyte infiltration after knee intraarticular injection of MSU (inflammation score: 1.5±0.9 vs 0.8±0.7, p< 0.03, in the MSU group after PBS or BAY-876 respectively).

Conclusion: These results indicated that up-regulation of JUN and p-JUN^Ser63 via JNK is necessary for up-regulation of the transcriptional program and epigenetic changes induced by MSU in macrophages. Monocyte and macrophage JNK activation are novel targets for blunting metabolic-inflammatory response to MSU crystals in gout.

Figure 1: RNA sequencing (RNA-Seq) of bone marrow derived macrophages (BMDM) or human monocyte-derived macrophages (mDM) treated with MSU (0.25 mg/ml) or LPS (100ng/ml) for 2 and 5h. Principal component (PC) and of RNA-seq results highlighted the divergence of BMDM or mDM treated with MSU from those treated with LPS or untreated

Figure 2: Chromatin immunoprecipitation (ChIP-Seq) for H3K27ac and H3K4me3 was performed to examine alteration in the activity of regions of open chromatin. Up-regulated genes in MSU with increased H3K27ac or H3K4me3 signal belong to inflammation and metabolic pathways, including metabolism of carbohydrates and transport of small molecules.

Figure 3: Motif analysis showed that regions of H3K27ac or H3K4me3 up-regulated signal with up-regulated gene transcript in MSU are majorly enriched in AP_1 motifs

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/urate-crystals-regulate-a-distinct-jnk-dependent-metabolic-and-inflammatory-response/