*Purpose: Neutrophils are a key cellular component of innate immunity and play an essential role in the defense against microbial infection. Emerging evidence suggests that emergency granulopoiesis could occur in the spleen during systemic inflammation. However, the mechanisms underlying splenic granulopoiesis and the plasticity of spleen-produced neutrophils are unknown. In this study, we aimed to determine the process of splenic granulopoiesis and the function of spleen-produced neutrophils using spleen transplantation models and intravital microscopy.
*Methods: Sepsis was induced in ubiquitin-GFP mice or neutrophil reporter mice (MRP8^Crex Rosa26^mTmG) in B6 background via the intraperitoneal injection of 200 uL cecal slurry. After 3 days, the spleen from septic mice or non-septic controls were transplanted into syngeneic B6 WT recipients. The native spleen was removed. Flow cytometry was performed to phenotype the circulating neutrophils released from the spleen at post-operative day (POD) 7 to POD 60. Intravital imaging was used to visualize the process of splenic granulopoiesis. RNAseq was used to analyze gene profiles of sorted neutrophils (GFP⁺vs GFP^-) in mice receiving septic spleens.
*Results: In mice receiving non-septic control spleens, less than 1% of blood neutrophils were GFP⁺ at POD 7-14, indicating that at steady-state conditions, the spleen has minimal granulopoiesis capacity. However, in mice receiving septic spleens, 10-30% of blood neutrophils were GFP⁺ at POD 7-14 and this population was maintained around 10% even at POD 60, suggesting that the strong granulopoiesis capacity in the septic spleen is long-lasting. Moreover, intravital imaging showed the number of neutrophils migrating into blood vessels was significantly higher in the septic spleen than the control. Phenotypically, compared to other blood-circulating neutrophils, septic spleen-released neutrophils had an increased expression of CXCR4, MHC Class I, and decreased expression of CD11b. Functional experiments suggested that they had increased ROS production and decreased phagocytosis capacity. RNAseq data revealed that the pathways related to neutrophil degranulation and activation were downregulated, and the pathways of positive regulation of macrophage chemokines and migration were upregulated.
*Conclusions: These data indicate that the septic spleen has long-lasting granulopoiesis capacity and releases neutrophils with distinct functional and transcriptomic signatures into the blood.

Longhui Qiu

University of California San Francisco

Appearances