Functional Characteristics of Distinct Neutrophil Subsetsand Their Potential for Granulocyte Transfusion

This study aimed to compare the functional characteristics and ex vivo survival of distinct neutrophil subsets, and to evaluate the effects of an optimized culture system on neutrophil viability, functional maintenance, and the efficacy of granulocyte transfusion. The study employed flow cytometric sorting to isolate CXCR2⁺and CXCR2⁻ neutrophils derived from mouse bone marrow. Single-cell transcriptomic analysis was performedto compare functional features and cell death-related pathways between the two subsets. Phagocytosis assays andROS (reactive oxygen species) measurements were used to assess effector functions. An MSC-conditioned medium plus G-CSF culture system (MSC+G) was further optimized, and its effects on neutrophil survival, apoptoticstatus, functional maintenance, and post-treatment survival after 24 h of culture were evaluated. In addition, usinga cyclophosphamide-induced neutropenic mouse model, both total and subset-defined neutrophils were examinedfor in vivo recruitment in an LPS-induced pulmonary inflammation model and for antibacterial protection in an E. coli (Escherichia coli)-induced pneumonia model. Single-cell transcriptomic analysis showed that, compared withCXCR2⁻ neutrophils, CXCR2⁺ neutrophils were significantly enriched for chemotaxis, migration, and phagocytosis-related pathways, while pyroptosis-, inflammasome-, and ferroptosis-associated pathways were also markedlyactivated (P<0.001). In vitro functional assays demonstrated that CXCR2⁺ neutrophils exhibited significantly higher phagocytic positivity, phagocytic index, and ROS levels than CXCR2− neutrophils. Under conventional cultureconditions, the survival rates of CXCR2⁺ neutrophils on day 1, 2, and 3 were 27.4%, 12.3%, and 4.3%, respectively,which were significantly lower than those in the optimized MSC+2G group (i.e., a culture system combining thesupernatant from 3-day-cultured mesenchymal stem cells with G-CSF and GM-CSF), whose corresponding survival rates were 84.7%, 81.8%, and 77.5% (P<0.05). The MSC+2G system continuously preserved the phagocyticactivity and ROS-generating capacity of CXCR2+ neutrophils, with effects comparable to those of the CLON-Gculture system. After 24 h of MSC+2G treatment, CXCR2+ neutrophils maintained survival and functional statusclose to those of freshly isolated cells even after transfer to conventional culture conditions. In vivo, total neutrophils cultured in MSC+2G for 24 h retained substantial recruitment capacity and antibacterial activity, with nosignificant differences from fresh neutrophils or CLON-G-treated cells. Further subset analysis showed that, in theLPS-induced pulmonary inflammation model, the absolute number of MSC+2G-cultured CXCR2+ neutrophils recruited to the lungs reached 3.2×10⁴, significantly higher than that of CXCR2⁻ neutrophils (5×10³, P<0.000 1). In the E. coli-induced neutropenic pneumonia model, MSC+2G-cultured CXCR2⁺ neutrophils significantly increasedthe survival rate of recipient mice, whereas no significant improvement in recipient mouse survival was observedin the CXCR2⁻ group. The study concludes that CXCR2⁺ neutrophils possess stronger immune effector functionscompared to the CXCR2⁻ neutrophils. The optimized MSC+2G culture system significantly prolongs their ex vivosurvival while preserving both in vitro and in vivo immune functions, with an overall performance approaching thatof the reported benchmark culture system, CLON-G. The combination of CXCR2+ neutrophils and the MSC+2Gculture system provides experimental and theoretical support for short-term neutrophil preservation and effectivegranulocyte transfusion, with potential relevance for the management of neutropenia and severe infection.

CSTR: 32200.14.cjcb.2026.06.0002