Abstract
BACKGROUND: Interleukin-6 (IL-6) is a pleiotropic cytokine, with specific effects depending on the immune microenvironment. Extensive research has confirmed the pathological roles of the IL-6/JAK2/STAT1/3 signaling pathway in inflammation, autoimmunity, and cancer, as well as its involvement in the pathogenesis of various rheumatic diseases. However, the role and impact of IL-6 as an upstream regulator of the JAK2-STAT1/3 pathway in gout have seldom been reported. This study explores the influence and role of upstream IL-6 in regulating the JAK2-STAT1/3 signaling pathway on gout inflammation, offering new insights for targeted therapeutic interventions and drug development in gout management. METHODS AND RESULTS: Clinical data and peripheral blood specimens were collected from gout patients and healthy individuals. In vitro and in vivo models of acute gout inflammation were established by stimulating PBMCs, THP-1 cells, and mice with MSU crystals. IL-6 expression was manipulated using IL-6 agonists and IL-6 knockout (KO) mouse technology to investigate the role and impact of the IL-6-mediated JAK2-STAT1/3 signaling pathway in gout models. RT-qPCR, WB, and ELISA were utilized to assess gene and protein expression levels. Paw swelling in mice was measured using a caliper gauge, while HE and IHC staining were conducted to evaluate the inflammatory status of mouse paw pad synovial tissues and detect the positive expression of relevant proteins. Serum IL-6 protein expression levels were significantly elevated in patients with gouty arthritis (GA) compared to healthy individuals, with multifactor logistic regression revealing an odds ratio (OR) of 2.175 for IL-6. In GA patients, mRNA expression of IL-6, JAK2, STAT1/3, and IL-1β was notably lower in the gout group compared to the healthy control (HC) group. Moreover, IL-6, JAK2, STAT1/3, p-JAK2, p-STAT1/3, and IL-1β proteins were markedly higher in the acute gout (AG) group compared to the intercritical gout (IG) and HC groups. Within the IG group, IL-6, JAK2, STAT3, and IL-1β proteins were significantly elevated compared to the HC group, whereas STAT1, p-JAK2, and p-STAT1/3 proteins were significantly lower. The expression of IL-6 protein and JAK2 mRNA showed positive correlations with certain inflammatory markers. In the 2h human blood in vitro gout inflammation model, expressions of IL-1β, IL-6, JAK2 mRNA, and IL-1β, IL-6, JAK2, STAT1/3, p-JAK2, p-STAT1/3 proteins were significantly higher compared to both the blank control and PBS-negative control groups. In the acute gout THP-1 cell model, The 6-hour model group showed significantly higher levels of IL-1β, IL-6, JAK2, STAT1/3 mRNA, and corresponding proteins, including their phosphorylated forms, compared to the blank control group. Additionally, treatment with an IL-6 agonist further increased these expression levels compared to the untreated model group. In the acute gout mouse model, IL-6 KO mice exhibited significantly reduced footpad swelling and swelling index compared to wild-type (WT) mice. HE staining revealed decreased inflammatory cell infiltration in IL-6 KO mice. Furthermore, Compared to 12-hour gout model WT mice, IL-1β, IL-6, JAK2, STAT1/3 mRNA, protein expression, and phosphorylated protein levels were notably decreased in IL-6 KO mice. IHC staining showed reduced positive expression of p-JAK2 and p-STAT1/3 in IL-6 KO mice. At the 24-hour mark, IL-6 mRNA and protein expression levels did not differ significantly between IL-6 KO and WT mice; however, IL-1β mRNA and protein expression, as well as JAK2 and STAT3 mRNA expression, were reduced in IL-6 KO mice, while STAT1 mRNA expression remained similar. CONCLUSION: IL-6 emerges as a potential risk factor for acute gout attacks, with its involvement in the JAK2-STAT1/3 signaling pathway contributing to the inflammation and pathogenesis process of acute gout through positive feedback mechanisms.