Down’s syndrome-associated arthritis (DA) is an aggressive form of arthritis and the most common childhood rheumatic disease. Based on previous findings, DA is clinically distinct from juvenile idiopathic arthritis. Our aims involve elucidating the mechanisms DA uses to drive their hostile invasive and migratory properties.
Aim
Down’s syndrome-associated arthritis (DA) is a more common and clinically distinct disease compared to juvenile idiopathic arthritis (JIA). This study aims to identify the underlying mechanisms involved in driving synovial fibroblasts (FLS) activation and destructive capacity.
Materials and methods
Primary DA FLS were isolated from synovial biopsies from children with DA. To examine the potential role of cytokine synergy, DA FLS were cultured with IL-17a (20ng/ml and 50ng/ml), IFN-g (10ng/ml and 50ng/ml) and GM-CSF (20ng/ml and 100ng/ml) in the presence or absence of TNF-a (0.1ng/ml). Culture supernatants were harvested and IL-6, IL-8, MCP-1 and RANTES quantified by ELISAs. Leukocyte adhesion was assessed by leukocyte-DA FLS adhesion assays. Flow cytometric analysis was used to examine DA FLS adhesion molecules (VCAM-1, ICAM-1), chemokine receptors (CCR3, CXCR4) and IFN-g receptor expression. The two major energy pathways glycolysis (ECAR) and oxidative phosphorylation (OCR) were quantified by the Seahorse XFe96 Analyser following the same stimulations as before.
Results
TNF-a, IL-17a and IFN-g induced IL-6, IL-8, RANTES and MCP-1 with no effect observed for GM-CSF. TNF-a IL-17a, IFN-g and GM-CSF increased leukocyte adhesion to DA FLS. TNF-a and IFN-g upregulated cell-surface expression of ICAM-1, VCAM-1, CXCR3 and CXCR4 with no effect observed for IL-17a and GM-CSF. IFN-g potentiated the effects of TNF-a on IL-6 and MCP-1 while decreasing IL-8. This synergy was also demonstrated for ICAM-1, VCAM-1, CXCR3, CXCR4 expression. No synergistic relationships were observed for either IL-17a and GM-CSF. Finally, IFN-g downregulated IFN-g receptor expression with no effect observed for TNF-a, IL-17a and GM-CSF. Furthermore, synergy between TNF-a and IL-17a deters DA FLS from OCR and switches to ECAR instead.
Discussion
DA is a more common and aggressive form of inflammatory arthritis compared to JIA. DA FLS function is regulated by differential cytokine stimulation, with TNF-g and IFN-g demonstrating potent synergistic induction of adhesion, inflammatory and chemokine receptor expression, suggesting complex cytokine signalling pathway mediate these effects.

Down’s syndrome-associated arthritis (DA) is an aggressive form of arthritis and the most common childhood rheumatic disease. Based on previous findings, DA is clinically distinct from juvenile idiopathic arthritis. Our aims involve elucidating the mechanisms DA uses to drive their hostile invasive and migratory properties.
**Aim**
Down’s syndrome-associated arthritis (DA) is a more common and clinically distinct disease compared to juvenile idiopathic arthritis (JIA). This study aims to identify the underlying mechanisms involved in driving synovial fibroblasts (FLS) activation and destructive capacity.
**Materials and methods**
Primary DA FLS were isolated from synovial biopsies from children with DA. To examine the potential role of cytokine synergy, DA FLS were cultured with IL-17a (20ng/ml and 50ng/ml), IFN-g (10ng/ml and 50ng/ml) and GM-CSF (20ng/ml and 100ng/ml) in the presence or absence of TNF-a (0.1ng/ml). Culture supernatants were harvested and IL-6, IL-8, MCP-1 and RANTES quantified by ELISAs. Leukocyte adhesion was assessed by leukocyte-DA FLS adhesion assays. Flow cytometric analysis was used to examine DA FLS adhesion molecules (VCAM-1, ICAM-1), chemokine receptors (CCR3, CXCR4) and IFN-g receptor expression. The two major energy pathways glycolysis (ECAR) and oxidative phosphorylation (OCR) were quantified by the Seahorse XFe96 Analyser following the same stimulations as before.
**Results**
TNF-a, IL-17a and IFN-g induced IL-6, IL-8, RANTES and MCP-1 with no effect observed for GM-CSF. TNF-a IL-17a, IFN-g and GM-CSF increased leukocyte adhesion to DA FLS. TNF-a and IFN-g upregulated cell-surface expression of ICAM-1, VCAM-1, CXCR3 and CXCR4 with no effect observed for IL-17a and GM-CSF. IFN-g potentiated the effects of TNF-a on IL-6 and MCP-1 while decreasing IL-8. This synergy was also demonstrated for ICAM-1, VCAM-1, CXCR3, CXCR4 expression. No synergistic relationships were observed for either IL-17a and GM-CSF. Finally, IFN-g downregulated IFN-g receptor expression with no effect observed for TNF-a, IL-17a and GM-CSF. Furthermore, synergy between TNF-a and IL-17a deters DA FLS from OCR and switches to ECAR instead.
**Discussion**
DA is a more common and aggressive form of inflammatory arthritis compared to JIA. DA FLS function is regulated by differential cytokine stimulation, with TNF-g and IFN-g demonstrating potent synergistic induction of adhesion, inflammatory and chemokine receptor expression, suggesting complex cytokine signalling pathway mediate these effects.

Cytokine synergy and glycolysis used to promote aggressive phenotype in fibroblasts of children with Down's syndrome-associated arthritis

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