Valentin Nica, Georgiana Cabău, Orsolya Gaal, Medeea Badii, Andreea-Manuela Mirea, Ioana Hotea, Hint Consortium, Cristina Pamfil, Simona Rednic, Radu A. Popp, Tania O. Crișan, Leo A.B. Joosten
Affiliation(s):
Department of Medical Genetics, UMF “Iuliu Hatieganu”, Cluj-Napoca, Romania
Introduction: Gout is an autoinflammatory disease characterized by the deposition of monosodium urate (MSU) crystals in joints, preceded by hyperuricemia, an asymptomatic condition associated with increased risk for multiple comorbidities. In the present study we perform large-scale transcriptomic analysis to identify mechanistic pathways related to inflammation in gout and hyperuricemia.
Methodology: Bulk transcriptomic analysis was performed in multiple study designs : (1) 219 samples of freshly isolated PBMCs (115 normouricemia, 24 hyperuricemia, 80 gout); (2) 70 isolated CD14+ Monocytes (35 controls and 35 gout); (3) 10 before-after pairs of samples (PBMCs and monocytes) isolated from patients that underwent urate lowering therapy and had a drop in urate level within 2 months;(4) in vitro priming of PBMCs with solubilized urate (50 mg/dL, 12,5 mg/dL or control medium), for 24 hours, followed by washing and further stimulation with LPS for 24 hours (n=52). RNA sequencing was performed using the DNBseq platform. After filtering, the reads were mapped to unigene datasets with Bowtie2 and the counts were calculated with RSEM. The DEseq2 package was used for normalization and identification of differentially expressed genes (DEGs). Pathway enrichment analysis was performed with GSEA and transcription factor enrichment with Rcistarget.
Findings: PBMCs of subjects with hyperuricemia show an upregulation of - pathways involved in inflammation, such as production of IL-1 IL-4 IL-6 and IL-10, lysosomal activity and reactive oxygen species production. These findings remain consistent when a linear model tracking the serum UA level is used. Several pathways related to atherosclerosis and cancer in response to urate increase were identified. The comparison between PBMCs from gout versus hyperuricemia revealed more discrete changes - 13 upregulated and 19 downregulated genes (log2foldChange > 1 or < -1 and padj < 0.05). The main pathway that was identified is the upregulation of lysine 27 trimethylation in histone 3 (H3K27me3). Only minor differences were observed when comparing CD14+ monocytes in gout versus controls. Pathway analysis identified downregulation of oxidative phosphorylation and complement pathways. In vitro urate priming experiments revealed 45 upregulated and 89 downregulated genes. We observe a stronger activation of pro-inflammatory pathways through the NF-kB, REL, FOS transcription factors, increased FGF signaling and IL-6, IL-19, IL-24 production, the downregulation of the Interferon and Complement pathways. Multiple metabolic pathways were also found to be upregulated.
Conclusions: Based on the experimental urate priming data, we confirm previously known transcriptomic reprogramming upon urate stimulation in a dose-dependent manner and in a larger group of samples. This was corroborated by upregulation of inflammatory pathways observed in unstimulated freshly isolated cells of patients with gout and in individuals with elevated urate levels. The identified genes and pathways are relevant for the validation of mechanisms that could lead to a urate-induced inflammatory signature or to provide molecular links for urate-associated comorbidities.