Background: Still’s disease (SD) is an autoinflammatory syndrome characterized by profound innate immune dysregulation. The complement system, a key component of innate immunity, can drive inflammatory cascades through the classical, lectin or alternative pathway. Despite its central role in inflammation, its involvement in SD remains poorly defined.

Objectives: To determine whether the complement pathway is activated in SD.

Methods: Complement activation was assessed through transcriptomic, proteomic, and in vitro stimulation assays. RNA sequencing was performed on sorted CD14+ monocytes from healthy donors (HD, n=6), active non-systemic juvenile idiopathic arthritis (JIA, n=5), and SD patients across disease stages (onset n=13, remission n=11, macrophage activation syndrome (MAS) n = 2). In parallel, whole blood RNA from SD (active n=29, inactive n=26) and JIA patients (active n=639, inactive n=315) was analyzed by NanoString to quantify classical complement components C1QB and C1QC. Complement activation products (C1q, C3a, C5a, TCC) and inflammatory mediators (IL-18, CXCL9, CXCL10) were quantified using Luminex and ELISA. Functional classical complement activity was evaluated in sera of SD (active n=32, inactive n=66) and JIA patients (active n=12, inactive n=12). In vitro stimulation assays were conducted to identify stimuli inducing C1q expression in monocytes and to assess the effect of complement factors on CD8+ T cell activation.

Results: Gene set enrichment analysis of CD14 ⁺ monocytes from active SD patients compared with healthy donors revealed significant enrichment of the complement cascade, ranking among the top five upregulated pathways (hsa04160 Complement and coagulation cascades; NES = 2.3, padj < 0.01, 41 genes). More specifically, transcriptomic analyses demonstrated marked upregulation of classical complement components in active SD. Whole-blood NanoString analysis showed significantly higher C1q expression in active SD compared with inactive SD (median normalized counts: C1QB 30.4 vs 8.57, p <0.01; C1QC 15.1 vs 5.5, p <0.01) and active non-systemic JIA (C1QB 30.4 vs 10.6, p <0.01; C1QC 15.1 vs 6.03, p<0.01). Similarly, RNA-seq of CD14+ monocytes revealed increased C1q expression in active SD compared with inactive SD, active non-systemic JIA, and HDs. Notably, the highest C1q expression was observed in MAS patients (median C1QB normalized read counts: 463 [MAS; n=2] vs 202 [SD onset; n=13] vs 28 [SD remission; n=11]). At the protein level, active SD was characterized by significantly elevated C1q, C3a, C5a, and TCC, together with enhanced functional classical complement activity. Furthermore, whole-blood C1q gene expression positively correlated with markers of interferon signaling in active SD, including IL-18 (Spearman r = 0.50, p < 0.01), CXCL9 (r = 0.60, p < 0.01), and CXCL10 (r = 0.4, p = 0.0392). In vitro stimulation of healthy monocytes with IFN-γ robustly induced C1q expression, while exposure to C1q enhanced IFN-γ production by CD8 ⁺ T cells, suggesting a feed-forward inflammatory loop between C1q and type II IFN signaling.

Conclusions: Our findings indicate activation of the complement pathway in active SD, closely linked with type II IFN-signaling. Obtaining a better understanding of the role of the complement system, particularly the role of C1q-high monocytes, may open novel avenues for personalized therapeutic approaches in SD.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: Freya Huijsmans: None declared, Alejandra Bodelón de Frutos: None declared, Greta Rogani: None declared, Lyanne Siebers: None declared, Joost F. Swart: None declared, Susanne Benseler: None declared, Rae Yeung: None declared, Sebastian Vastert Consultant: Sobi and Novartis, Research Grant: Sobi, Jorg van Loosdregt: None declared