Single-Cell RNA Sequencing Uncovers Neutrophil Clusters Associated with Autoimmune Neuroinflammation

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) associated with multifocal inflammation and axonal degeneration, which is mediated by innate and adaptive immune cells. The Janus Kinase (JAK)/Signal Transducers and Activators of Transcription (STAT)/Suppressors of Cytokine Signaling (SOCS) pathway regulates the functionality of both innate and adaptive immune cells. We and others have documented dysregulation of the JAK/STAT/SOCS axis in T-cells and astrocytes in neuroinflammation, however, less is known about this axis in innate immune cells. Our previous studies using the Experimental Autoimmune Encephalomyelitis (EAE) model of MS demonstrated that mice lacking Socs3 in myeloid cells, both macrophages and neutrophils (Socs3?LysM), developed a severe, brain-targeted form of EAE (btEAE), with increased cerebellar infiltration of activated neutrophils. To examine the role of neutrophils specifically, we generated mice with Socs3 deletion exclusively in neutrophils (Socs3?Ly6G). EAE was induced in Socs3?Ly6G mice and Ly6G+/- control mice, and we observed that Socs3?Ly6G mice exhibit clinical signs of btEAE identical to Socs3?LysM mice, with more severe cerebellar demyelination and higher cerebellar infiltration of activated neutrophils and CD4+ T-cells compared to Ly6G+/- controls. The Socs3?Ly6G mice also exhibit clinical symptoms of classical EAE (cEAE), which is associated with spinal cord (SC) involvement. Cerebellar neutrophils from Socs3?Ly6G mice exhibited a primed phenotype with increased CD11b expression, reduced CD62L expression, increased reactive oxygen species, and formation of neutrophil extracellular traps (NETs). Depletion of neutrophils ameliorated btEAE clinical disease, demonstrating the pathogenic properties of these cells. Single-cell RNA Sequencing (scRNA-Seq) was performed to determine the transcriptional profiles of neutrophils from cerebellum (CB) and SC associated with EAE. Five neutrophil clusters were identified in naïve mice and those with EAE, with an expansion of two clusters (Neu2 and Neu4) in Socs3?Ly6G mice with EAE. Notably, high levels of Saa3, Il1b and Cxcl2 were detected in the Neu2, Neu3, Neu4 and Neu 5 clusters at the peak of EAE disease. Strikingly, Serum Amyloid A3 (Saa3) mRNA and protein expression was markedly enhanced in the CB and SC from Socs3?Ly6G EAE mice compared to Ly6G+/- mice. Human SAA1 is the homologue of mouse Saa3. SAA1 expression was also substantially elevated in plasma from MS patients compared to healthy controls. Collectively, our study demonstrates neutrophil heterogeneity in both the CB and SC of Socs3?Ly6G mice with EAE, transcriptional profiles associated with EAE disease, and potential mediators such as Saa3 that may serve as a therapeutic target to dampen autoimmune neuroinflammation. Overall design: We carried out single cell RNAseq on live immune cells (CD45+CD11b+) from the cerebellum (CB) and spinal cord (SC) of Socs3?Ly6G mice with EAE (n=3), SC of Ly6G+/- mice with EAE (n=3), combined SC of naive Ly6G+/- mice (n = 3), combinded SC (n = 4) and combined CB (n = 4) of naive Socs3?Ly6G mice.