Open Access

ARTICLE

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

Laura Schaltz^1,2, Simon Lassnig^1,2, Karola Schlote^1,2, Christian Schwerk³, Horst Schroten³, Nicole de Buhr^1,2,*

1 Institute of Biochemistry, University of Veterinary Medicine Hannover, Hannover, Germany
2 Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany
3 Department of Pediatrics, Pediatric Infectious Diseases, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

* Corresponding Author: Nicole de Buhr. Email:

(This article belongs to the Special Issue: Neuroinflammation and Neuroprotection in CNS Diseases: From Mechanisms to Therapeutic Targets)

BIOCELL 2026, 50(8), 11 https://doi.org/10.32604/biocell.2026.079046

Received 13 January 2026; Accepted 20 April 2026; Issue published 27 July 2026

Abstract

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.

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