M. Badii 1, T. O. Crișan 1, V. Klück 2, G. Cabău 1, B. Novakovic 3, H. Stunnenberg 3, M. G. Netea 2, R. A. Popp 1, L. A. B. Joosten 1,2 and The Hint Consortium
1. Department of Medical Genetics, Iuliu Hatieganu University of Medicine And Pharmacy, Cluj-Napoca, Romania 2. Department of Internal Medicine And Radboud Institute for Molecular Life Sciences (Rimls), Radboud, University Medical Center, Nijmegen, The Netherlands 3. Department of Molecular Biology And Radboud Institute for Molecular Life Sciences (Rimls), Radboud University Medical Center, Nijmegen, The Netherlands
Introduction: The induction of type 1 interferons (IFN) and interferon-stimulated genes (ISGs) is essential for the host immune response to viral stimuli and other PRR ligands, being an important component of innate immune responses and generating the transition to adaptive immunity. Interestingly, type I IFNs dampen IL-1 mediated inflammation as IFN-β was shown to inhibit IL-1β production in vitro and it is known that type I IFN therapy has been effective in the treatment of a number of autoinflammatory and autoimmune diseases. A previous report showed transcriptional upregulation of type I IFN pathway genes following urate lowering by rasburicase in whole blood of healthy individuals challenged with uric acid infusion. Moreover, in vitro monocyte priming with soluble urate results in high IL-1β and IL-6, but low IL-1Ra. In this study we assessed whether urate treatment inhibits the type 1 IFN signaling pathway in mononuclear cells.
Material and methods: Primary monocytes were treated for 20h in vitro with high concentrations of urate solubilized in RPMI or control, followed by stimulation with LPS for another 4h. RNA sequencing was performed in monocytes at 20h and 24h. STAT1 and STAT3 phosphorylation was assessed by flow cytometry in PBMCs and monocytes treated with urate 10 or 50 mg/dL. Cytokine response to Poly I:C 50 μg/mL (type I IFN inducer) was assessed by ELISA in the supernatants of PBMCs cultured for 24 h in the presence or absence of soluble urate (50, 10, 5 mg/dL ) and restimulated 24h with LPS 10ng/mL and LPS(10ng/mL)+MSU(300µg/mL). The cytokine response was further validated using IFN-β (100 IU/mL) to directly induce type 1 IFN pathway. Steady-state levels of IFN-β mRNA have been measured in normouricemic and hyperuricemic patients.
Results: Differentially expressed genes were interrogated using gene enrichment analysis according to GO Biological process and the type 1 IFN signaling pathway was the most significant GO term associated with down-regulated genes. Motif enrichment analysis revealed down-regulation of binding sites for IFN-regulatory factor 1 and 2 and IFN sensitive regulatory element. Uric acid 10 mg/dL or 50 mg/dL resulted in lower levels of phosphorylated STAT1 or STAT3. qPCR data indicates a lower steady-state level of IFN-β mRNA in the hyperuricemic patients compared to controls. The stimulation of cells with Poly I:C in presence of urate resulted in lower IL-6 cytokine production compared to Poly:IC alone and this was observed to be dose dependent. The proinflammatory effects of urate pre-treatment of cells were not modified by type 1 IFN inducers Poly I:C or IFN-β.
Conclusions: Pathway analysis of differentially expressed genes and transcription factor motif enrichment in uric acid treated monocytes showed downregulation of type 1 IFN signaling pathway. This was confirmed by inhibition of Poly I:C induced cytokine production, diminished STAT1 and STAT3 phosphorylation in presence of uric acid in PBMCs, and lower steady state level of IFN-β mRNA in hyperuricemic versus normouricemic people. However, inducing type 1 IFN with either Poly I:C or IFN-β did not reverse the proinflammatory effect of urate, suggesting that most likely type 1 IFN signaling is not a mechanisms for urate priming, but another potentially detrimental consequence of urate pre-exposure. This could be a potential new mechanism linking soluble urate to deficient type I IFN signaling or poor responses to IFN immunotherapy.