Brenda Kischkel (1), Twinu Wilson Chirayath (2), Tim L. Jansen (3), Mylène Zarka (2), Tania Criçan (4), Mihai G Netea(1), Pascal Richette (2), Leo A. B. Joosten(1,4) , Hang Korng Ea (2)
Affiliation(s):
1. Radboud University Medical Center, Department of Internal Medicine, 6500HB, Geert Grooteplein Zuid 10, Nijmegen, The Netherlands. Phone: +31-06-84452806, e-mail address: Brenda.Kischkel@radboudumc.nl. Secondary email: brendakischkel@gmail.com
2. Université de Paris, INSERM UMR 1132, Hôpital Lariboisière, AP-HP, Paris, France.
3. VieCuri Medical Centre, Department of Rheumatology – Complex Gout Expert Centre. Venlo, The Netherlands.
4 Iuliu Hatieganu University of Medicine and Pharmacy, Department of Medical Genetics, Cluj-Napoca, Romania
Background: Gout flares are characterized by severe attacks of pain, swelling, redness and tenderness that can last for 7 to 10 days, followed by an inter-critical period, which is an asymptomatic stage between flares. To prevent relapses of gout flares, urate lowering therapy (ULT) is used and can be linked to a treat-to-target (T2T) approach, in which the medication doses are adjusted until serum urate concentrations are below 0.36 mmol/L.
Objectives: In this study, we accessed the plasma proteomic profile of patients included in a prospective study (GOUTROS cohort) in order to characterize the systemic inflammatory profile of gout patients during flare (T1), inter-critical (T2) and T2T (T3) conditions.
Methods: Proximity extension assay technology (Olink) was used to measure 92 inflammation-related molecules in 71 enrolled patients. Our findings were validated in an independent cohort of patients, followed by in vitro and in vivo experiments to understand the involvement of a new biomarker in the context of gout flares.
Findings: We identified 19 proteins (26.8%) that were differentially expressed (FDR<0.05) between T1 (Flare) and T2 (inter-critical) and 13 proteins (18.3%) between T1 and T3 (T2T). Among them, IL-6, TNFSF14, CSF-1 and VEGFA were highly expressed during T1. TRANCE and Flt3L increased significantly during the T2 and T3, respectively, when compared with T1. Of interest, IL-6 expression was correlated to the presence of tophi, in which patients with high levels of IL-6 during the inter-critical period presented tophi (p=0.002, AUC 0.7). Next, we performed the same proteomic analysis on an independent cohort of patients to validate the expression of biomarkers observed during a gout flare. TNFSF14 and IL-6 were the most significantly expressed during flare in both cohorts. While the role of IL-6 in MSU crystal-induced inflammation is well known, the role of TNFSF14 has not been described before. Thus, we investigated its contribution in crystal-induced inflammation. To achieve this goal, we exposed human PBMCs to a TNFSF14 inhibitor, as well as the recombinant TNFSF14 protein followed by stimulation with MSU crystals and LPS. We observed that the production of proinflammatory cytokines, such as IL-6, TNF and IL-1β, significantly decreased in the presence of the inhibitor and increased in the presence of the recombinant protein. Of clinical importance, we were able to detect TNFSF14 being produced locally, in the synovial fluid of patients with gout and in air pouch membranes of mice injected with MSU crystals. Finally, using a cohort of 164 healthy individuals from the human functional genomics project (HFGP, 200FG) we demonstrated that PBMCs from individuals carrying variants of the TNFSF14 gene had the production of inflammatory cytokines affected in response to MSU crystals and LPS. Individuals carrying rs344559 exhibit decreased production of IL-1β, while individuals carrying rs2101765 showed increased production of IL-1β and IL-6.
Significance: This study helps to improve the understanding of how the human immune system reacts in different stages of gout, allowing the identification of TNFSF14 as a new biomarker associated with gout flares. Collectively, our results also suggest that TNFSF14 could modulate the inflammatory response during a gout flare and may be considered as a therapeutic target.