Splenic extramedullary granulopoiesis and tissue microenvironments shape functionally distinct neutrophil subsets in autoimmunity

Neutrophils are increasingly recognized as key regulators of autoimmune responses and exhibit substantial functional diversity. However, the understanding of neutrophil heterogeneity in autoimmune diseases remains limited. Here, using experimental autoimmune encephalomyelitis (EAE), we identify three morphologically and transcriptionally distinct neutrophil subsets with tissue-specific distributions. Trajectory analysis reveals a stepwise maturation from proliferative precursors (N0) to immature (N1) and mature neutrophils (N2), with Ki67⁺, Acvrl1⁺, and CD300ld⁺ identified and validated as subset markers, respectively. Notably, pro-inflammatory CD300ld⁻ immature neutrophils preferentially infiltrate the central nervous system, whereas immunosuppressive CD300ld ⁺ mature neutrophils expand in the lung during EAE. Mechanistically, splenic extramedullary granulopoiesis contributes to the expansion of the neutrophil pool, while disease-associated and tissue-specific microenvironments reshape their functional states. In addition, we identify previously uncharacterized subclusters within mature neutrophils, suggesting that autoimmune disease may generate distinct developmental lineages and expand the established architecture of the neutrophil compartment. Importantly, analogous neutrophil subsets are identified in mouse models of rheumatoid arthritis and systemic lupus erythematosus, with immature neutrophil infiltration into target tissues, while neutrophil subset reprogramming is also observed in patients with multiple sclerosis. Collectively, these findings show that splenic extramedullary granulopoiesis drives neutrophil heterogeneity and, together with tissue microenvironments, shapes their functional plasticity in autoimmunity, providing a framework for targeting defined subsets for immunomodulation.