Single-cell RNA sequencing uncovers neutrophil clusters associated with autoimmune neuroinflammation

Abstract Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) characterized by multifocal inflammation and axonal degeneration, driven by innate and adaptive immune cells. The Janus Kinase (JAK)/Signal Transducers and Activators of Transcription (STAT)/Suppressors Of Cytokine Signaling (SOCS) pathway regulates immune cell activity, with SOCS proteins functioning as negative regulators. Using the Experimental Autoimmune Encephalomyelitis (EAE) model of MS, our prior work demonstrated that mice lacking Socs3 in myeloid cells (Socs3 ΔLysM) developed severe, brain-targeted EAE (btEAE), with increased cerebellar infiltration of activated neutrophils. To define neutrophil-specific roles, we generated mice with Socs3 deletion restricted to neutrophils (Socs3 ΔLy6G). Following MOG-induced EAE, these mice exhibited clinical features identical to Socs3 ΔLysM mice, including severe cerebellar demyelination, increased cerebellar infiltration of activated neutrophils and CD4+ T-cells, and clinical symptoms of both btEAE and classical EAE (cEAE), the latter involving the spinal cord (SC). Cerebellar neutrophils from Socs3 ΔLy6G mice exhibited a primed, inflammatory phenotype with elevated reactive oxygen species, neutrophil extracellular traps (NETs) and heightened production of pro-inflammatory cytokines/chemokines. Neutrophil depletion alleviated btEAE, confirming their pathogenic role. Single-cell RNA Sequencing (scRNA-Seq) of cerebellum (CB) and SC neutrophils revealed five clusters in naïve and EAE mice, with expansion of two clusters (Neu2 and Neu4) in Socs3 ΔLy6G mice with EAE. Neu2, Neu3 and Neu4 clusters showed high expression of Saa3, Il1b and Cxcl2, with Neu4 enriched in cytokine signaling pathways and inflammatory responses. Strikingly, Saa3 mRNA and protein expression were markedly increased in the CB and SC of Socs3 ΔLy6G mice with EAE compared to controls. Translationally, the human orthologue SAA1 was significantly elevated in plasma from MS patients relative to healthy controls. Collectively, these findings demonstrate that Socs3 deficiency unleashes pathogenic neutrophil activity in Socs3 ΔLy6G mice with EAE. They further demonstrate neutrophil heterogeneity within the inflamed CNS and define inflammatory transcriptional states, with Saa3/SAA1 as a potential biomarker and/or target in autoimmune neuroinflammation.