Downregulation of type I interferon signalling pathway by urate in primary human PBMCs

Abstract

Type I interferons (IFN1s) mediate innate responses to microbial stimuli and regulate interleukin (IL)-1 and IL-1 receptor antagonist (Ra) production in human cells. This study explores interferon-stimulated gene (ISG) alterations in the transcriptome of patients with gout and stimulated human primary cells in vitro in relation to serum urate concentrations. Peripheral blood mononuclear cells (PBMCs) and monocytes of patients with gout were primed in vitro with soluble urate, followed by lipopolysaccharide (LPS) stimulation. Separately, PBMCs were stimulated with various toll-like receptor (TLR) ligands. RNA sequencing and IL-1Ra cytokine measurement were performed. STAT1 phosphorylation was assessed in urate-treated monocytes. Cytokine responses to IFN-β were evaluated in PBMCs cultured with or without urate and restimulated with LPS and monosodium urate (MSU) crystals. Transcriptomics revealed suppressed IFN-related signalling pathways in urate-exposed PBMCs or monocytes which was supported by diminishment of phosphorylated STAT1. The stimulation of PBMCs with IFN-β did not modify the urate-induced inflammation. Interestingly, in vivo, serum urate concentrations were inversely correlated to in vitro ISG expression upon stimulations with TLR ligands. These findings support a deficient IFN1 signalling in the presence of elevated serum urate concentrations, which could translate to increased susceptibility to infections.

Keywords: IL‐1; gout; hyperuricemia; soluble urate; type I interferons.

FIGURE 1

RNA‐Seq reveals downregulation of type I interferon signalling pathways in human PBMCs upon urate priming. Schematic representation of in vitro experiments used for transcriptomic analysis (n = 52) (a); Pathway enrichment analysis of down‐regulated genes at padj <0.05 and log2FC < −1 using Gene Ontology—Biological processes (top 20 terms) (b). Enrichment map of GO Biological Processes terms of down‐regulated genes at padj <0.05 and log2FC < −1 (c). Curve of Gene Set Enrichment Analysis (GSEA) enrichment scores for type I interferon signalling pathways (padj = 242022E − 05; NES = −2128) (d). Heatmap of differentially expressed genes (down‐regulated) that overlap the GO BP terms, response to type I interferon, cellular response to type I interferon and type I interferon signalling pathway (variance stabilized normalized counts corrected for batch effect); The relative value for each gene is shown by the colour intensity of the Z score, with red indicating up‐regulated and blue indicating downregulated genes (e). Network Visualizations with ChEA3 of transcription factors (TFs) according to the similarity in their co‐expression patterns using differentially expressed gene set of urate 50 mg/dL + LPS 10 ng/mL at padj <0.05; logFC < −1 (f).

FIGURE 2

RNA‐Seq confirms downregulation of type I interferon in primary human monocytes. Schematic representation of in vitro experiments used for transcriptomic analysis (n = 4) (a); Pathway analysis of down‐regulated differentially‐expressed genes at padj <0.05 and log2FC < −1 for GO Biological processes (top 20 terms) (b). Heatmap of Interferon Stimulated Genes treated with urate 50 mg/dL and RMPI medium for control. The relative value for each gene is shown by the colour intensity of the Z score, with red indicating up‐regulated and blue indicating downregulated genes (c). Network visualizations with ChEA3 of transcription factors according to the similarity in their co‐expression patterns using differentially expressed gene set of urate 50 mg/dL at padj <0.05; logFC < −1 (d).

FIGURE 3

Phosphorylated STAT1 is decreased upon urate treatment in monocytes. Schematic representation of in vitro experiments used for flow cytometry experiment; PBMCs primed in vitro with urate 50 mg/dL for 24 h and restimulated with LPS 10 ng/mL + MSU crystals 300 μg/mL for 4 h (a). Phosphorylated STAT1 in gated HLA‐DR+ monocytes (b) and classical monocytes (c) (n = 5); Wilcoxon test; Data presented as mean with SEM and representative of two independent experiments.

FIGURE 4

Cytokine production given by IFN‐β stimulation in the presence of urate. Schematic representation of in vitro experiments used for cytokine measurements (n = 9) (a). Coincubation of IFN‐β (100 IU/mL) with urate (50 mg/dL) followed by restimulation with LPS (10 ng/mL) + MSU (300 μg/mL) in PBMCs of healthy volunteers (b). IL‐1Ra, IL‐1β and IL‐6 cytokine production in supernatants from at least three independent experiments; Wilcoxon test; Data presented as median with 95% CI.

FIGURE 5

Serum urate levels in patients are inversely correlated with gene expression of ISGs. Schematic representation of in vitro experiments used for transcriptomic analysis (a). ComplexHeatmap for Spearman rho and p‐values of serum urate levels in correlation with gene expression (variance stabilized normalized counts) of 24 h treated PBMCs with Poly: IC (n = 47), CpG (n = 47) and heat‐killed Candida albicans (n = 46) (b). Scatter plots of genes that inversely correlate with serum urate concentrations in 24 h stimulation of PBMCs with C. albicans (r = −0.3755, p = 0.0093) and Poly: IC (r = −0.3715, p = 0.0101)—gene expression on the y‐axis as variance stabilized normalized counts and on the x‐axis serum urate levels in mg/dL along with the fitted linear regression line in blue (c). Correlation of serum urate levels with IL‐1Ra cytokine production in 24 h treated PBMCs with PBMCs Poly: IC, CpG and heat‐killed C. albicans (d).