Distinct Neutrophil Populations in the Spleen During PICS

While mortality after acute sepsis has decreased, the long-term recovery for survivors is still poor, particularly those developing persistent inflammation, immunosuppression, and catabolism syndrome (PICS). While previously thought that activated neutrophils responding to the acute phase of sepsis migrate to the spleen to undergo cell death and contribute to immunosuppression, our data show a significant accumulation of distinct, yet functional, neutrophil populations in the spleen in a murine model of PICS. The exact role and function of neutrophils in this response is still unclear. The objective of our study was to better define the immune function of splenic neutrophils to determine if this could give insight into the pathogenesis of PICS. Using a murine model of cecal ligation and puncture (CLP), which demonstrates all characteristics of PICS by 8 days, spleens were harvested, and neutrophils were identified by Ly6G and CD11b expression via flow cytometry. Nearly all splenic neutrophils expressed CD54, but there were distinct CD54^hi and CD54^lo cells, with the majority being CD54^lo cells during PICS. The CD54^hi population showed traditional, proinflammatory properties, but a relatively decreased chemotactic response, while CD54^lo cells had significantly higher chemotaxis, yet significantly decreased proinflammatory functions. Using 5-ethynyl-2'-deoxyuridine (EdU) incorporation, we found that the CD54^hi population on day 2 after CLP may be participating in emergency myelopoiesis. However, the vast majority of the CD54^lo population were paused in the G₁ phase at this time point and not proliferating. By day 8 after CLP, most of the CD54^hi cells in the spleen were no longer proliferating, while the CD54^lo cells were, indicating that CD54^lo dominate in extramedullary myelopoiesis at later time points. Almost none of the neutrophils produced arginase or inducible nitric oxide synthase (iNOS), indicating that these are not suppressor cells. Overall, our data demonstrate that neutrophil accumulation in the spleen during PICS is related to extramedullary myelopoiesis, leading to the production of immature neutrophils. While not suppressor cells, the majority have greater chemotactic function but less inflammatory responsiveness, which may contribute to the immunosuppression seen in PICS. Attention to these distinct neutrophil populations after septic or other systemic inflammatory responses is therefore critical to understanding the mechanisms of PICS.