AB0930 URATE INDUCED PRIMING OF HUMAN MONOCYTES IS MEDIATED VIA THE TGF-Β PATHWAY

Background: 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[1]. 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[2]. This imbalance between IL-1β and IL-1Ra is mediated by epigenetic reprogramming of innate immune cells[2]. RNA sequencing in urate-treated monocytes demonstrated that the TGF-β signalling pathway was differentially expressed[3]. Objectives: 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 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. Results: 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. Conclusion: Urate induced priming of human monocytes is at least partly mediated via the TGF-β pathway. 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. Disclosure of Interests: Viola Klück: None declared, Linda Mies: None declared, René Bakker: None declared, Tania Crisan: None declared, Leo Joosten Consultant of: SAB member of Olatec Therapeutics LLC