Background/Purpose: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (SJIA), characterized by activation and expansion of cytolytic lymphocytes and macrophages with hemophagocytic properties. Recent work has shown that emergence of MAS is associated with a surge in IFNg and IFN-induced chemokines; however, previous gene expression studies failed to demonstrate this IFN-induced signature in peripheral blood cells. However, these studies were limited by a failure to examine key myeloid effector cells, specifically activated macrophages which traffic to tissue including bone marrow during MAS.

Objective: Utilize single-cell RNA-sequencing to identify specific gene expression signatures of bone marrow macrophage populations in SJIA and MAS.

Methods: Macrophage single cell suspensions were obtained from unused portions of bone marrow aspirates using cell sorting for populations expressing the monocyte and macrophage surface markers CD14 and CD163 while excluding cells expression the granulocyte/monocyte marker CD15, prior to loading onto the Fluidigm C1 Single-Cell Auto Prep System. Extracted RNA was converted into cDNA and sequenced as a pooled library, and aligned to the human Ensembl transcriptome using Kallisto through AltAnalyze version 2.1.1.

Results: Three control samples yielded 180 single cells, and while there was substantial inter-individual variability, a core set of genes were identified that contributed to the heterogeneity of normal bone marrow macrophage population. Control macrophages formed at least three primary cellular clusters, which were distinguished based on expression of genes associated with proinflammatory receptors, GM-CSF signaling, and aurora B signaling. 61 single bone marrow macrophages were captured from a patient with newly diagnosed SJIA with active systemic features, arthritis, marked anemia, relative thrombocytopenia, but lacking other overt signs of MAS, but did have mild hemophagocytosis on diagnostic bone marrow aspiration. Expression profiles were broadly similar to control macrophages, and all macrophage clusters were represented. However, a distinct subpopulation of bone marrow macrophages from the SJIA patient was identified that exhibited markedly altered transcriptional profiles. Compared to other control and patient macrophages, this SJIA macrophage population showed alterations in gene pathways including cellular response to interferon gamma (p=1.35e-14), endocytic vesicle membranes (p=8.44E-14), phagosome (p=2.98e-9) and vesicle-mediated transport (p=1.05E-07). These cells showed a proinflammatory gene expression signature, including enrichment for genes regulated by NF-kB and STAT1.

Conclusion: We identify a distinct subpopulation of bone marrow macrophages in an SJIA patient with features associated with emergence of MAS, including interferon response, phagocytosis and vesicular transport. This validates a single-cell approach, and demonstrates the importance of studying these effector cells at the sites of inflammation, and suggests that tissue macrophages may be a key source of IFN-induced products during MAS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-rna-sequencing-of-bone-marrow-macrophages-identifies-a-distinct-subpopulation-in-systemic-jia-with-features-of-interferon-response-endocytic-vesicles-and-phagocytosis/