AUTHOR=Mayr Alexandra , Marciniak Jana , Eggers Benedikt , Blawat Kim , Wildenhof Jan , Bastos Craveiro Rogerio , Wolf Michael , Deschner James , Jäger Andreas , Beisel-Memmert Svenja 

TITLE=Autophagy Induces Expression of IL-6 in Human Periodontal Ligament Fibroblasts Under Mechanical Load and Overload and Effects Osteoclastogenesis in vitro

JOURNAL=Frontiers in Physiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2021.716441

DOI=10.3389/fphys.2021.716441

ISSN=1664-042X

ABSTRACT=<p><bold>Objective:</bold> Autophagy is an important cellular adaptation mechanism to mechanical stress. In animal experiments, inhibition of autophagy during orthodontic tooth movement triggered increased expression of inflammation-related genes and decreased bone density. The aim of this study was to investigate how autophagy affects cytokine levels of interleukin 6 (IL-6) in human periodontal ligament (hPDL) fibroblasts under mechanical pressure and the resulting influence on osteoblast communication.</p><p><bold>Methods:</bold> hPDL fibroblasts were subjected to physiologic mechanical load, constant overload, or rapamycin treatment for 16 to 24 h ± autophagy inhibitor 3-MA. Autophagosomes were quantified by flow cytometry. Gene expression of <italic>il-6</italic> as well as IL-6 levels in the supernatant were determined with rtPCR and ELISA. To investigate the influence of mechanically-induced autophagy on cell-cell communication, an osteoblast-culture was subjected to supernatant from stimulated hPDL fibroblasts ± soluble IL-6 receptor (sIL-6R). After 24 h, <italic>osteoprotegerin</italic> (<italic>opg</italic>) and <italic>receptor activator of nuclear factor</italic> κ<italic>B ligand</italic> (<italic>rankl</italic>) gene expressions were detected with rtPCR. Gene expression of <italic>a disintegrin and metalloproteinases</italic> (<italic>adam</italic>) <italic>10</italic> and <italic>17</italic> in stimulated hPDL fibroblasts was examined via rtPCR.</p><p><bold>Results:</bold> Autophagy was induced by biomechanical stress in hPDL fibroblasts in a dose-dependent manner. Mechanical load and overload increased IL-6 expression at gene and protein level. Autophagy inhibition further enhanced the effects of mechanical stimulation on IL-6 expression. Mechanical stimulation of hPDL fibroblasts downregulated <italic>adam10</italic> and <italic>adam17</italic> expressions. Inhibition of autophagy had stimulus-intensity depending effects: autophagy inhibition alone or additional application of physiological stress enhanced <italic>adam10</italic> and <italic>adam17</italic> expressions, whereas mechanical overload had adverse effects. Osteoblasts showed significantly reduced <italic>opg</italic> expression in the presence of supernatant derived of hPDL fibroblasts treated with autophagy inhibitor and sIL-6R.</p><p><bold>Conclusion:</bold> IL-6 levels were increased in response to pressure in hPDL fibroblasts, which was further enhanced by autophagy inhibition. This caused a decrease in <italic>opg</italic> expression in osteoblasts. This may serve as an explanatory model for accelerated tooth movement observed under autophagy inhibition, but may also represent a risk factor for uncontrolled bone loss.</p>