Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

Abstract

Background: Staphylococcus aureus infection of normal osteoblasts induces expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Results: Normal osteoblasts were incubated in the presence of purified bacterial products over a range of concentrations. Results demonstrate that purified surface structures and a selected superantigen present in the extracellular environment are not capable of inducing TRAIL expression by osteoblasts. Osteoblasts were co-cultured with S. aureus at various multiplicities of infection utilizing cell culture chamber inserts. Results of those experiments suggest that direct contact between bacteria and osteoblasts is necessary for optimal TRAIL induction. Finally, S. aureus infection of osteoblasts in the presence of anti-TRAIL antibody demonstrates that TRAIL mediates caspase-8 activation and apoptosis of infected cells.

Conclusions: Collectively, these findings suggest a mechanism whereby S. aureus mediates bone destruction via induction of osteoblast apoptosis.

Figure 1

Western immunoblot analysis of TRAIL expression from normal human osteoblasts 24 h following incubation with LPS, peptidoglycan, teichoic acid (TA, 1 μg), staphylococcal enterotoxin A (SEA, 1 μg), or following infection with S. aureus at a MOI of 250:1 (S.a. 250). S. aureus infection was for 45 min, followed by washing and gentamicin treatment of osteoblasts for 24 h prior to protein isolation. Ten micrograms of total osteoblast protein were loaded for each lane.

Figure 2

Western immunoblot analysis of TRAIL expression from normal mouse osteoblasts co-cultured with S. aureus. Normal mouse osteoblasts were grown in the lower chamber of Transwell plates. S. aureus at a MOI of 25:1, 75:1, or 250:1 was added separately to Transwell^® cell culture chamber inserts. As a control, osteoblasts were directly infected with S. aureus at a MOI of 250:1. TRAIL expression was monitored at 45 minutes following the addition of bacteria. Twelve micrograms of total osteoblast protein was loaded for each lane.

Figure 3

FACS analysis of normal mouse osteoblasts infected with S. aureus at a MOI of 75:1 in the presence or absence of anti-human TRAIL antibody (1.0 or 2.0 μg/ml). Following a 45-minute incubation, osteoblasts were washed 3 times with HBSS and incubated in medium containing 25 μg of gentamicin per ml to kill extracellular bacteria. Cells were stained with annexin V and propidium iodide 24 hours post-infection and analyzed using FACS. Results are presented from one osteoblast culture, and are representative of results from three different osteoblast preparations.

Figure 4

Caspase-8 is activated in normal mouse osteoblasts infected with S. aureus at a MOI of 75:1, or following incubation with 10 μg/ml cycloheximide (CHX). Caspase-8 enzymatic activity was analyzed 45 min following infection. S. aureus-infected osteoblasts were incubated for 45 min in the presence of increasing concentrations of anti-human TRAIL antibody (0.5 μg/ml, 1.0 μg/ml, and 2.0 μg/ml). Values are presented as the mean +/- standard deviation from three independent osteoblast cultures. Data were analyzed utilizing the Student's t-test, *,# P < 0.05.