The role of staphylococcal Panton-Valentine leukocidin during lung inflammation invivo

Abstract number: O291

Zivkovic A., Sharif O., Stich K., Biaggio M., Doninger B., Mesteri I., Knapp S.

Objective: Panton-Valentine Leukocidin (PVL) positive Staphylococcus aureus is an emerging pathogen associated with highly lethal necrotizing pneumonia. PVL is a bi-component b-barrel pore-forming toxin that has been shown to cause cell death in neutrophils. However, the precise mechanisms leading to necrotizing pneumonia are not fully understood and the role of PVL herein is controversial. We therefore aimed to investigate the mechanism by which PVL might contribute to the development of severe lung inflammation.

Methods: Recombinant PVL was generated and its cytotoxic properties were verified by patch clamp and FACS. The inflammatory potential of PVL was tested on alveolar macrophages using mciroarray profiling, PCR and ELISA. Signaling pathways were investiagted using western blots and electromobility shift assays. The in vivo role of PVl was then studied in a murine pneumonitis model, in which lung tissue and lavage fluid was assessed for cell-influx and cytokine/chemokine release.

Results: PVL rapidly induces pore formation and death of neutrophils. In contrast, alveolar macrophages were found less sensitive to the toxic effects and only succumbed after prolonged incubation. Microarray assays performed 1 h after addition of PVL to alveolar macrophages revealed the selective induction of 29 genes upon stimulation. Bioinformatic analysis disclosed the upregulation of NFkB target genes. These data were verified by PCR and on protein levels and suggest that PVL selectively induces NFkB associated inflammation in vitro. Inhibitor studies and gene reporter assays further confirmed this finding. Furthermore, PVL-induced lung inflammation in mice confirmed the inflammatory potential of PVL, as illustrated by a rapid influx of neutrophils and enhanced levels of pro-inflammatory cytokines and chemokines within the pulmonary compartment.

Conclusions: Our data demonstrate that PVL, beside its pore-forming properties on neutrophils, is able to strongly induce inflammation via NFkB activation in alveolar macrophages, leading to pneumonitis in vivo.