Background/Purpose: Systemic juvenile idiopathic arthritis (SJIA) is a chronic childhood arthropathy with features of autoinflammation. New-onset SJIA is associated with expansion and activation of neutrophils with a sepsis-like phenotype, but neutrophil phenotypes in longstanding and clinically inactive disease (CID) is unknown. The objective of this study was to examine activated neutrophil subsets, S100 alarmin release, and gene expression signatures in children with a spectrum of SJIA disease activity.

Methods: This protocol was approved by the Institutional Review Board, and informed consent was obtained from all patients and/or their legal guardians. Highly-purified neutrophils were isolated using a two-step procedure of density-gradient centrifugation followed by magnetic-bead based negative selection using the MACSexpress kit. Neutrophils were then stained for imaging flow cytometry, or RNA isolated was using MagMax Total RNA Isolation Kit. Alternatively, neutrophils were cultured for 4hr with or without PMA to quantify S100 protein release. Gene expression analysis was performed with the Ampliseq Transcriptome kit using the Ion Torrent platform.

Results: Patients with SJIA and active systemic features demonstrated a higher number of the immunosuppressive subset CD16⁺CD62L^lo neutrophils compared to controls. This neutrophil subset was not seen in patients with inactive disease or those with active arthritis only without systemic features. Using imaging flow cytometry, CD16⁺CD62L^lo neutrophils from patients with active SJIA had mildly increased nuclear hypersegmentation compared to CD16⁺CD62L⁺ neutrophils. Serum levels of S100A8/A9 and S100A12 were strongly correlated with peripheral blood neutrophil count (R=0.53 and R=0.64, respectively; p<0.001). Neutrophils from active SJIA patients cultured in vitro did not show enhanced resting S100 protein release compared to inactive disease or control neutrophils. However, regardless of disease activity, neutrophils from SJIA patients did show enhanced S100A8/A9 release upon PMA stimulation compared to control neutrophils (P<0.05). Whole transcriptome analysis of highly purified neutrophils from children with active SJIA identified 214 differentially expressed genes compared to control neutrophils (fold change >2.0, p<0.05). The most significantly upregulated gene pathway was Immune System Process (adjusted p=3.4×10^-16) including AIM2, IL18RAP, NLRC4, TLR2, TLR5, and TNFAIP3. Interestingly, this gene set showed intermediate levels of expression in neutrophils from patients with long-standing inactive disease but persistent serum IL-18 elevation. Indeed, all patient samples regardless of disease activity demonstrated elevated inflammatory gene expression, including inflammasome components and S100A8.

Conclusion: We identify features of neutrophil activation in SJIA, including a proinflammatory gene expression signature in patients with longstanding CID but elevated serum IL-18, reflecting persistent innate immune activation. Taken together, these studies expand understanding of neutrophil function in chronic autoinflammatory disorders such as SJIA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophils-from-children-with-systemic-jia-exhibit-persistent-proinflammatory-activation-despite-long-standing-clinically-inactive-disease/