Abstract Body: Despite decades of progress into antibacterial approaches and new insights into its pathophysiology, sepsis remains fatal for nearly 1 in 3 individuals. Of those surviving, nearly half do not return to the same quality of life prior to their infection. Methicillin-resistant Staphylococcus aureus (MRSA) sepsis is particularly nefarious, with the kidney frequently affected leading to acute injury or chronic complications.

Neutrophil extracellular traps (NETs) in sepsis have been described to promote immunothrombosis, a protective mechanism by which leukocytes eject their DNA in an attempt to contain infection in the circulation. This process also drives pathology. Using a mouse model of MRSA bacteremia induced by intravenous injection of live S. aureus, we have demonstrated that prior neutrophilia and NET-priming through repeated granulocyte colony-stimulating factor (G-CSF) administration protected mice from clinical sepsis. This was due to peptidylarginine deiminase 4 (PAD4)-mediated NET release, as seen in mice lacking neutrophil PAD4 (MRP8Cre+PAD4fl/fl) vs their littermate controls (PAD4fl/fl).

We performed deep immunophenotyping using spectral flow cytometry during sepsis progression and found that MRSA induced immune cell repertoire changes in the spleen, including evidence of extramedullary hematopoiesis, maturation of B cells to plasma cells, and differentiation of T cells toward TCRγΔ and Th17 cells. There was no impact on macrophage polarization upon MRSA infection, despite an increase in CD11b+ cells in the spleen and kidney of which neutrophils comprised the majority in the kidney. NETs had surprisingly little impact on the immune cell repertoire other than increased plasma cells, with no evidence of T cell activation effect. G-CSF administration prevented MRSA spread within kidney as seen by lack of abscess formation compared to mice that received vehicle injections and had neutrophilia only induced by the infection itself. G-CSF-induced neutrophils reached S. aureus and also accelerated CCR2+ monocyte recruitment to the kidney, and promoted exacerbated NET-rich immunothrombosis in the liver. Without neutrophil PAD4 and NET release, neutrophils had limited capacity to prevent dissemination of bacteria throughout the kidney.

Whether higher neutrophil numbers or their increased NETting capacity is the protective factor in this model remains to be determined, and may inform future clinical applications targeting neutrophils to protect the kidney.