Laura M. Merlo Pich (1), Viola Klück (1), Liesbeth van Emst(1), Ruiqi Liu(1), Martin Jaeger (1), Tania Crisan (2), Mihai G. Netea (1,3), Yang Li (1,4), Leo A.B. Joosten (1,2)
Affiliation(s):
1. Department of Internal Medicine, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
2. Department of Medical Genetics, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
3. Department for Immunology and Metabolism, Life and Medical Sciences Institute (LIMES), University of Bonn, 53115 Bonn, Germany
4. Department of Computational Biology of Individualised Medicine, Centre for Individualised Infection Medicine (CiiM), a Joint Venture Between the Hannover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
Background: Gout is one of the most common forms of inflammatory arthritis worldwide. Among the risk factors for gout, chronic hyperuricemia is the most relevant one. Nonetheless, a minority of people experiencing hyperuricemia will develop MSU crystals in the joints and symptomatic gout flares. This suggests that other factors may induce a predisposition to gout, including variations in the immune responses and their inflammatory pathways. These variations are to date not completely understood. Therefore, the aim of this study is to assess the genetic variations in human primary myeloid cells highlighted by a change in inflammatory cytokine production induced by acute stimulation with MSU crystals, and further explore the role of RUNX1 in these cells.
Methods: PBMCs obtained from two cohorts of healthy volunteers were stimulated for 24h with palmitic acid (C16.0) and monosodium urate (MSU) crystals. The obtained supernatants were used to measure cytokine production with ELISA assays (R&D). The same volunteers were also genotyped using the Illumina Human OmniExpress Exome-8 v1.0 SNP chip. A meta-analysis of genome wide cQTL mapping in each cohort was performed. Next, we associated all annotated RUNX1 SNPs with cQTL data form the largest cohort. For in vitro validation, RUNX1 expression in PBMCs and U937 cells was assessed by qPCR and, to assess functional consequences, both a RUNX1 knock down cell line was generated and a RUNX1 inhibitor was tested on healthy donor PBMC.
Results. rs4817731, 500kb upstream of RUNX1, was identified as the top SNP associated with IL-6 production after C16.0 and MSU crystal stimulation (meta p-value p=1,37e-6). 618 other SNPs annotated to RUNX1 also influenced either IL-1β or IL-6 release after stimulation (p<0.05). Transcription of RUNX1 is upregulated in human PBMCs by stimulation with TLR ligand and/or MSU crystals or recombinant human IL-1β. RUNX1 knockdown in a monocytic cell line resulted in a reduction of cytokine production after stimulation with MSU and toll-like receptor agonists.
Discussion. cQTL analysis revealed that RUNX1 could play a relevant role in MSU crystal-induced cytokine production and potentially in gout. This was supported by our in vitro experiments in which RUNX1 acts as a positive feedback loop for IL-1β. Further studies are warranted to assess the relevance of RUNX1 in gout in vivo and the prevalence among gout patients of the identified variation.