Background Response to the bacterial lipopolysaccharide (LPS), a potent secondary danger signal for NLRP3 activation, varies after exposure to different uric acid (UA) concentrations . Although monosodium urate (MSU) crystals are also recognised as a danger signal by the innate immune system through the NLRP3 inflammasome-interleukin (IL)-1b pathway , whether UA is also able to enhance MSU driven soluble inflammation is an attractive possibility that has not been tested so far.
Objectives To assess whether cytokine response to monosodium urate (MSU) crystals is conditioned by the UA level in an in vitro model, and to replicate this phenomenon in gouty patients.
Methods A) In vitro: Primary human neutrophils were cultured with increasing concentrations of UA (1, 2, 4, 6, 7, and 8mg/dL). Cells were stimulated with MSU crystals at a constant dose (0.4ug/mL). The production of IL-2, IL-4, IL-6, IL-10, tumour necrosis factor-alpha (TNFa), interferon-gamma (IFNg), and IL-17 was measured by duplicate. B) For the in vivo study, consecutive patients with gout seen in our clinics and under urate-lowering therapy (ULT) were screened. Those whose ULT dose adjustment was considered by the clinician after long-term treatment were selected. Blood tests were taken at two different time-points: time 1, during the standard ULT; and time 2, after ULT dose adjustment (discontinuation or dose reduction). Serum levels of UA, IL-1b, TNFa, and IL-6 were measured at both time-points. Wilcoxon signed-rank test was used to compare matched samples, and Spearman's rho to assess correlation between serum UA and cytokines levels.
Results A) In vitro: At increasing UA concentrations, TNFa, IL-6, IFNg, and IL-2 levels showed a progressively rising after MSU crystal stimulation, while IL-4, IL-10, and IL-17 levels did not modify [Figure, up]. Interestingly, highest cytokine levels were achieved with UA at 6mg/dL, and further increases in UA were not followed by further parallel cytokine levels increase. For the B) in vivo study, 14 gouty patients were enrolled, median (p25-75) aged 64.5 (60.0, 70.3) years, 92.9% males. Median disease duration was 10.0 (5.8, 17.8) years, being 21.4% tophaceous. Blood tests showed a significant increase in serum UA after ULT dose modification/discontinuation (p=0.001), TNFa (p=0.004), and IL-6 (p=0.03), and a non-significant trend for IL-1b (p=0.055) [Figure, down]. TNFa levels strongly correlated with SUA levels (r=0.77, p=0.001), while the correlation of the other cytokines with SUA was found to be moderate (r=0.45, p=0.13 for IL-6; r=0.31, p=0.30 for IL-1b).
Conclusions Both in vitro and in vivo studies confirm that UA is able to enhance MSU-induced cytokine inflammatory response in a concentration-dependent manner.
Acknowledgements This study was funded by Menarini.
Disclosure of Interest None declared