Maria Muntiu (1), Andreea Manuela Mirea (1), Medeea Badii (1,2), Orsolya Gaal (1,2), Georgiana Cabău (1), Valentin Nica (1), Cristina Pamfil (3), Simona Rednic (3), Tania O. Crişan (1,2), Leo A.B. Joosten (1,2)
Affiliation(s):
1. Department of Medical Genetics, UMF “Iuliu Hatieganu”, Cluj-Napoca, Romania
2. Department of Internal Medicine, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands
3. Department of Rheumatology, UMF “Iuliu Hatieganu”, Cluj-Napoca, Romania
Introduction: Gout is a highly prevalent disease, caused by the deposition of monosodium urate (MSU) crystals in articular structures and leading to intense joint inflammation. IL-1β is a key mediator of inflammation in gout. A single-nucleotide polymorphism (rs16944) located 511 base pairs upstream of the first exon of the IL1B gene has been associated with altered expression of IL-1β and anti-inflammatory cytokine IL37 through a long noncoding RNA (AMANZI)- mediated regulatory mechanism. The aim of this study was to investigate whether rs16944 genotype is associated with cytokine production, IL1B mRNA transcription and frequency of gout flares.
Materials and Methods: We analyzed data from a previously characterized cohort of subjects included in the HINT project (Cluj-Napoca, Romania), that included gout patients, subjects with asymptomatic hyperuricemia and healthy controls. Genomic DNA was isolated from whole blood and genotyping was conducted using Infinium™ Global Screening Array-24 v3.0 BeadChip. Cytokine production was evaluated in vitro using freshly isolated PBMCs in multiple experiment designs: 24h direct stimulation with gout-relevant stimuli (e.g. palmitate, MSU crystals), TLR ligands or microbial stimuli (n=320). 2) 24h uric acid (UA) priming followed by 24 h LPS stimulation (n=304). 3) 7-day stimulation with TLR ligands or microbial stimuli (n=236). ELISA for IL-1β, IL-1Ra, IL-6, TNF, IL-17, IFNγ was performed on cell culture supernatants. Expression of IL1B mRNA in unstimulated PBMCs and monocytes, as well as in LPS-stimulated PBMCs following UA priming was evaluated (n=47). A retrospective analysis of the frequency of flares in gout patients was conducted, with subjects (n=243) stratified in three groups: 1 flare/ year (or fewer), 2-6 flares/year and 7 or more flares/year.
Results: After direct, short-term stimulation, we observed a change in IL-1β and IL-6 production, with levels tending to follow the pattern of elevated proinflammatory cytokines in G allele carriers though statistical significance was only reached in few comparisons. In contrast, IL-1Ra levels exhibited an inverse trend, which was consistent throughout all tested stimuli. IL-1β levels measured after UA priming and LPS stimulation also followed this pattern, as did IFNγ levels measured after 7-day stimulation with C. albicans (p<0.05) and heat killed S.aureus. Base level expression of IL1B mRNA in monocytes and PBMCs was not correlated with genotype, nor was IL1B expression in PBMCs after UA priming and LPS stimulation. No correlation between rs16944 genotype and frequency of gout flares was found.
Conclusion: This study showed a tendency towards higher proinflammatory and lower anti-inflammatory cytokine production in subjects with rs169644 G allele carriers highlighting the potential relevance of this SNP in the context of gout and hyperuricemia associated inflammation.