Key Points

Tissue microenvironment influences microglial and leukocyte phagocytosis.

Phagocytic versus nonphagocytic cells exhibit transcriptional heterogeneity.

NOX2 prevents Staphylococcus aureus outgrowth during craniotomy infection.

Abstract

Craniotomies are performed to treat a variety of intracranial pathology. Surgical site infection remains a complication of craniotomy despite the use of prophylactic antibiotics and universal sterile precautions. Infections occur in 1–3% of procedures, with approximately half caused by Staphylococcus aureus that forms a biofilm on the bone flap and is recalcitrant to systemic antibiotic therapy. We used an S. aureus-dsRed construct to compare the phagocytic capacity of leukocytes and microglia in vitro and in vivo using a mouse model of craniotomy infection. In addition, single-cell RNA sequencing (scRNA-seq) was applied to determine whether a transcriptional signature could be identified for phagocytic versus nonphagocytic cells in vivo. S. aureus was phagocytosed to equivalent extents in microglia, macrophages, neutrophils, and granulocytic myeloid-derived suppressor cells in vitro; however, microglial uptake of S. aureus was limited in vivo, whereas the other leukocyte populations exhibited phagocytic activity. scRNA-seq comparing the transcriptional signatures of phagocytic (S. aureus-dsRed+) versus nonphagocytic (S. aureus-dsRed−) leukocytes identified classical pathways enriched in phagocytic cells (i.e., reactive oxygen species [ROS]/reactive nitrogen species, lysosome, iron uptake, and transport), whereas nonphagocytic populations had increased ribosomal, IFN, and hypoxia signatures. scRNA-seq also revealed a robust ROS profile, which led to the exploration of craniotomy infection in NADPH oxidase 2 knockout mice. S. aureus burden, leukocyte recruitment, and intracellular bacterial load were significantly increased in NADPH oxidase 2 KO compared with wild-type animals. Collectively, these results highlight the importance of ROS generation in phagocytes for S. aureus biofilm containment, but not clearance, during craniotomy infection.

Footnotes

This work was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke Grant R01 NS107369 (to T.K.). The University of Nebraska Medical Center (UNMC) DNA Sequencing Core receives partial support from the National Institute for General Medical Sciences (Institutional Development Award Program Networks of Biomedical Research Excellence, P20GM103427-14; and Centers of Biomedical Research Excellence, 1P30GM110768-01). Both the UNMC DNA Sequencing and Flow Cytometry Research Cores receive support from the Fred & Pamela Buffett Cancer Center Support Grant (P30CA036727).

J.M. and T.K. designed experiments; J.M., C.M.H., C.E.H., Z.V.R., and L.E.K. conducted experiments; J.M. wrote the manuscript; and all authors edited and approved the final manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article:

DEdifferential expressionG-MDSCgranulocytic myeloid-derived suppressor cellMdFImedian fluorescence intensityMOImultiplicity of infectionMono/Macmonocyte/macrophagemTORC1mammalian target of rapamycin complex 1NGFnerve growth factorNOX2NADPH oxidase 2O2−•superoxidePMNpolymorphonuclear neutrophilRNSreactive nitrogen speciesROSreactive oxygen speciesscRNA-seqsingle-cell RNA sequencingWTwild-typeReceived July 12, 2022.Accepted September 13, 2022.Copyright © 2022 by The American Association of Immunologists, Inc.