Background/Purpose: Macrophage activation syndrome (MAS) is a life-threatening complication of rheumatic diseases including systemic juvenile idiopathic arthritis (SJIA). SJIA shows prominent neutrophil activation with expansion of proinflammatory neutrophils in the active phase and persistent activation even in clinically inactive disease, but how neutrophils contribute to the pathogenesis of SJIA and MAS remains unclear. Repeated toll like receptor (TLR)-9 induced MAS mouse model is the most characterized model of MAS; however, the dynamics of neutrophils in this system are largely unexplored. The objective of this study is to define neutrophil activation in the TLR9-induced mouse model in both acute MAS and MAS resolution.

Methods: Wild type C57BL/6 mice were injected with CpG intraperitoneally five times in 10 days and sacrificed one day after last injection (acute MAS) or after 21 days (resolution). Neutrophils were identified as Ly6G+CD11b+, and maturity was assessed using CD101 and CXCR2. For gene expression analysis, neutrophils from peripheral blood were isolated via magnetic negative selection. Total RNA extracted from neutrophils were processed using the Ampliseq Transcriptome system and Ion Torrent S5 next-generation sequencing system. Sequence data was analyzed and visualized using AltAnalyze.

Results: During acute MAS, pancytopenia, splenomegaly and elevated plasma IL-18 were observed, which largely normalized at resolution. Neutrophil counts were significantly decreased during acute MAS and still low at resolution. Immature neutrophils markedly increased during acute MAS only.

We performed gene expression profiling on purified neutrophils during acute MAS and resolution. Compared to control neutrophils, we found that during acute MAS, 167 genes were upregulated and 196 genes were down regulated, while in resolution phase 3 genes were upregulated and 412 genes were down regulated (rawp < 0.05, fold change >2.0). The most enriched upregulated gene ontology (GO) pathways at MAS included Defense response (Z score=14.83, adjp=2.18×10^-18) and Cellular Response to IFNγ(Z score=14.28, adjp=1.75×10^-6), including CXCL9 and CXCL10, while there were no significantly enriched downregulated pathways.

During MAS resolution, there were no significantly upregulated GO pathways, while the most enriched downregulated GO included Protein binding (Z score=8.75, adjp=2.27×10^-13) and Immune System Process (Z score=7.26, adjp=8.39×10^-7), including C1QB, CXCL2,IRF4 and TLR9. When we compared upregulated genes during experimental MAS to our previous study on human neutrophils from patients with active SJIA, which showed numerous upregulated inflammasome related genes, we found very little overlap.

Conclusion: TLR9-induced experimental MAS induced overall neutropenia but increased immature neutrophil production and IFNγ-driven neutrophil transcriptional activation, while resolution shows persistent neutropenia and transcriptional profiles of downregulated chemokine production. Together, this model demonstrates long-standing alternations in the neutrophil compartment that are markedly different than seen in children with SJIA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dynamics-of-neutrophil-activation-in-repeated-tlr-9-induced-mouse-model-of-macrophage-activation-syndrome/