gms | German Medical Science

Objective: At the onset of an infection, human cells are confronted with bacterial bodies and secreted bacterial products. These diffusible products are first detected by host sensors like Toll-like receptors (TLR) or tumor necrosis factor receptors (TNFR) that induce expression of chemokines like CXCL8. Chemokines are important mediators of the immune reaction in the innate immune system response. The main objective of this study was to characterize the response of glioblastoma cells (GBM) to products secreted by S. aureus wild type and a mutant unable to produce toxins s during infection.

Methods: To prepare Staphylococcus aureus culture supernatants (SaS) DMEM was seeded with 0.5ml of the bacterial overnight culture and cultivated again for 8 h. Protein concentration of the SaS was determined after acetone precipitation. 10 µg/ml SaS in stimulation medium (DMEM/5%FBS) were added to GBM cells and incubated for 96 h to determine cell viability. For analysis of cytokine expression, mRNA was extracted at different time points of incubation. The mRNA expression of the pro-inflammatory chemokines IL-1ß, IL-6, and CXCL8 was determined by real time PCR.

Results: The viability of three different GBM cell cultures was reduced by 30% after 96 h of incubation with SaS from the strongly hemolytic S. aureus strain 6850. No reduction in viability was visible after cultivation with SaS from the mutant S. aureus strain LS1 Δagr/ΔsarA which is unable to produce toxins. SaS from wild type and mutant strain induced an increased expression of IL-1ß and CXCL8 within the first two hours of incubation, which decreased over time. Expression of IL-6 was not affected.

Conclusion: Secreted products from hemolytic S. aureus strains are mainly proteases and toxins like alpha-hemolysin, PSMs, etc. These toxins induce the observed cell death. However, these toxins are not responsible of cytokines induction. Our results with wild type and mutant strains suggest that other components like lipoteichoic acid (LTA) and protein A released by the bacteria are the real inducers for cytokines. Protein A and LTA activate NF-κB and AP-1 pathways via TLR2 and TNFR1 to induce cytokines. These results indicate that products secreted by S. aureus induce necrotic cell death and chemokine expression in GBM cells that potentially result in an activation of the innate immune system.