V. Klück 1, L. Mies 1, R. Bakker 2, T. O. Crișan 3, L. Joosten 1,3
1. Department of Internal Medicine, Radboudumc, Nijmegen, The Netherlands 2. Departement of Rheumatology, Radboudumc, Nijmegen, The Netherlands 3. Department of Medical Genetics, Luliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca, Romania.
Introduction: Hyperuricemia, elevated serum urate levels, is the main risk factor for gout, but is also associated with higher incidence of comorbidities such as cardiovascular disease, type 2 diabetes, metabolic syndrome and chronic kidney disease . Crisan et al. showed that urate leads to increased production of interleukin (IL)-1β, a pro-inflammatory cytokine, and downregulation of IL-1 receptor antagonist (IL-1Ra), the natural inhibitor of IL-1, in human monocytes . This imbalance between IL-1β and IL-1Ra is mediated by epigenetic reprogramming of innate immune cells . RNA sequencing in urate-treated monocytes demonstrated that the TGF-β signalling pathway was differentially expressed . The objective of this study is to further explore the role of TGF-β in urate induced priming of human monocytes.
Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers, adhered to a flat bottom plate, and treated for 24h with a dosing range of urate after which mRNA was isolated. For validation experiments, PBMCs from 9 gout patients and 7 healthy controls were isolated and adhered to a flat bottom plate for 4h after which cells were stored for RNA isolation. qPCR primers designed for TGF-β, TGF-β receptor I and II, MMP9, SMAD7 and ITGAV were used to assess expression levels of the TGF-β pathway in these adherent monocytes. For priming experiments, adherent monocytes were primed for 24h with urate and/or recombinant TGF-β1 (R&D systems) with or without a TGF-β receptor II antibody (R&D systems), cells were washed and restimulated with LPS for 24h. Cytokine levels in supernatant were determined by ELISA for IL-1β, IL-6 and IL-1Ra.
Findings: mRNA expression of TGF-β and its downstream targets were upregulated in urate treated monocytes and in gout patients compared to healthy controls. Moreover, urate levels significantly correlated to TGF-β in individuals with gout. Both urate and TGF-β priming increased the release of IL-1β and IL-6 after LPS stimulation in human monocytes. We did not observe a synergistic effect between the two and therefore hypothesized that urate induced inflammation is mediated via TGF-β. Blocking the TGF-β receptor II partly reversed the urate induced phenotype: lowered IL-1β and IL-6 production and restored levels of IL-1Ra. Further validation experiments are ongoing.
Significance of the project: This study contributes to the understanding of the pathways involved in urate induced inflammatory status and might in the future provide a mechanistic explanation for the occurrence of some comorbidities in patients with gout. Additionally, as TGF-β is a major player in the pathogenesis of systemic sclerosis, this study might give a rationale for treatment of hyperuricemia in this population.
References: 1. Bardin, T. and P. Richette, Impact of comorbidities on gout and hyperuricaemia: an update on prevalence and treatment options. BMC Med, 2017. 15(1): p. 123. 2. Crisan, T.O., et al., Soluble uric acid primes TLR-induced proinflammatory cytokine production by human primary cells via inhibition of IL-1Ra. Ann Rheum Dis, 2016. 75(4): p. 755-62. 3. Crisan, T.O., et al., Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway. Proc Natl Acad Sci U S A, 2017. 114(21): p. 5485-5490.