Staphylococci planktonic and biofilm environments differentially affect osteoclast formation.

INTRODUCTION: The pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction. OBJECTIVE AND METHOD: Investigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM). RESULTS: Priming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM. CONCLUSION: Our data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

Bacterial and Metabolic Factors of Staphylococcal Planktonic and Biofilm Environments Differentially Regulate Macrophage Immune Activation.

Biofilm formation is a leading cause for chronic implant-related bone infections as biofilms shield bacteria against the immune system and antibiotics. Additionally, biofilms generate a metabolic microenvironment that shifts the immune response towards tolerance. Here, we compared the impact of the metabolite profile of bacterial environments on macrophage immune activation using Staphylococcus aureus (SA) and epidermidis (SE) conditioned media (CM) of planktonic and biofilm cultures. The biofilm environment had reduced glucose and increased lactate concentrations. Moreover, the expression of typical immune activation markers on macrophages was reduced in the biofilm environment compared to the respective planktonic CM. However, all CM caused a predominantly pro-inflammatory macrophage cytokine response with a comparable induction of Tnfa expression. In biofilm CM, this was accompanied by higher levels of anti-inflammatory Il10. Planktonic CM, on the other hand, induced an IRF7 mediated Ifnb gene expression which was absent in the biofilm environments. For SA but not for SE planktonic CM, this was accompanied by IRF3 activation. Stimulation of macrophages with TLR-2/-9 ligands under varying metabolic conditions revealed that, like in the biofilm setting, low glucose concentration reduced the Tnfa to Il10 mRNA ratio. However, the addition of extracellular L-lactate but not D-lactate increased the Tnfa to Il10 mRNA ratio upon TLR-2/-9 stimulation. In summary, our data indicate that the mechanisms behind the activation of macrophages differ between planktonic and biofilm environments. These differences are independent of the metabolite profiles, suggesting that the production of different bacterial factors is ultimately more important than the concentrations of glucose and lactate in the environment.