Characterization of Alive and Impaired NET-Releasing Neutrophils in A Model of the Blood-Cerebrospinal Fluid Barrier after Streptococcus suis Infection

Objectives: Streptococcus suis (S. suis) is a worldwide occurring pathogen in pigs and humans that can cross the blood-cerebrospinal fluid barrier (BCSFB) to cause meningitis, while host neutrophils counter infection through mechanisms including the release of neutrophil extracellular traps (NETs). NET-formation involves the release of nuclear DNA with antimicrobial components, which can bind and kill bacteria. We aimed to characterize the host-pathogen interaction between S. suis and neutrophils within the CSF compartment, focusing on NET-formation. Methods: A 3D cell culture model of the porcine BCSFB was used by cultivating a porcine choroid plexus epithelial cell line (PCP-R) on filter inserts. Following S. suis infection, porcine neutrophils were added to the basolateral side. The study specifically focused on the quantification of alive and impaired NET-releasing neutrophils. Transmigrated neutrophils in the CSF compartment were stained for NETs and with Live-or-Dye NucFix™ and analyzed by confocal microscopy to quantify NET release and cell viability. Results: In uninfected samples, most transmigrated neutrophils remained NET-negative (96.9% SD ± 1.9%). In contrast, 46.9% (SD ± 12.9%) of neutrophils infiltrating the S. suis-infected CSF compartment exhibited NET formation. Only a minor fraction of NET-releasing cells remained alive (9.8% SD ± 8.7%), whereas the majority became impaired (Live-or-Dye positive). This process correlated with increased S. suis colony-forming units in the CSF compartment. However, Live-or-Dye positive neutrophils retained the ability to produce reactive oxygen species (ROS) in response to S. suis, although ROS production decreased after 4 h. Conclusion: Using a novel staining approach to distinguish NET formation and cell viability, we demonstrated that S. suis infection predominantly induces NET release in the CSF compartment in an in vitro model of the BCSFB after four hours. The majority of these NET-releasing neutrophils are impaired. This likely leads to a loss of the defense mechanisms of the neutrophils.