Extracellular Vesicles Suppress Basal and Lipopolysaccharide-Induced NFκB Activity in Human Periodontal Ligament Stem Cells

Periodontitis is an infectious disease characterized by chronic inflammation and progressive destruction of periodontal tissues. Chronic inflammatory environment may affect immunomodulatory function of periodontal ligament stem cells (PDLSCs) and promote shift toward proinflammatory phenotype contributing to propagation of periodontitis. Therefore, suppression of inflammatory response in PDLSCs represents a novel therapeutic approach. Extracellular vesicles (EVs) have been shown to display anti-inflammatory and immunosuppressive actions in different tissues and could represent a potent therapeutic tools against chronic inflammation during periodontitis. In the present study, we investigated the effects of EVs on the basal and lipopolysaccharide (LPS)-induced activity of NF kappa B signaling pathway in PDLSCs. We also examined the impact of EVs on the osteogenic differentiation and expression of osteogenesis-related genes. EVs were purified by differential ultracentrifugation from PDLSCs grown on gelatin-coated alginate microcarriers in a bioreactor. NF kappa B reporter assays demonstrated that EVs permanently suppressed basal and LPS-induced activity of NF kappa B in PDLSCs. Combined treatment with EVs and anti-TLR4 antibody (Ab) resulted in attenuation of the inhibitory effect on the NF kappa B activity, suggesting a possible interference through a competition for TLR4 signaling pathway. EVs also increased phosphorylation of Akt and its downstream target GSK3 beta (Ser 9) indicating that PI3K/Akt signaling pathway may act as suppressor of NF kappa B activity. LPS stimulated osteogenic mineralization of PDLSCs. Unexpectedly, anti-TLR4 blocking Ab per se significantly decreased osteogenic mineralization of PDLSCs. EVs did not affect osteogenic mineralization, but partially suppressed inhibitory effect of anti-TLR4 blocking Ab. Gene expression studies revealed significant effects of EVs on osteogenesis-related genes and possible interference with TLR4 signaling in PDLSCs. In conclusion, our study demonstrates that EVs suppress basal and LPS-induced activity of NF kappa B signaling pathway in PDLSCs and could potentially be used for targeting of chronic inflammation during periodontitis.