Single-cell transcriptomics reveal tissue-specific neutrophil heterogeneity and functional reprogramming in autoimmunity.

Neutrophils are increasingly recognized as key regulators of autoimmune responses; however, the mechanisms by which their heterogeneity is influenced across various tissues in autoimmunity remain insufficiently understood. Here, using experimental autoimmune encephalomyelitis (EAE), we show that splenic and bone marrow neutrophils acquire both CD4+ T cell suppressive and Th17-promoting capacities. In contrast, lung neutrophils exhibit predominantly CD4+ T cell suppressive activity. We further identify three distinct neutrophil subsets, comprising Ki67+ N0, Acvrl1+ N1, and CD300ld+ N2 neutrophils. Functionally, CD300ld- neutrophils are characterized by a pro-inflammatory phenotype, whereas CD300ld+ neutrophils demonstrate immunosuppressive activity, indicating tissue-specific properties of these cells. Mechanistically, EAE induces splenic extramedullary granulopoiesis, resulting in the generation of heterogeneous neutrophil subsets, with a notable enrichment of N2 neutrophils in the lung. Within this organ, a unique microenvironment further reprograms CD300ld+ neutrophils toward a more immunosuppressive state through pathways associated with lipid metabolism. Notably, analogous subsets are also observed in animal models of rheumatoid arthritis and systemic lupus erythematosus, as well as in patients with multiple sclerosis. Collectively, our findings illustrate that splenic extramedullary granulopoiesis creates a heterogeneous neutrophil landscape, which is further shaped by tissue-specific microenvironments, driving the functional plasticity of neutrophils in EAE. This framework defines distinct neutrophil subsets as potential targets for immunomodulatory intervention in autoimmune diseases.