P024 LPS-P. gingivalis stimulate interleukin-6, −8 and CCL-20 release, and NF-kB translocation in human periodontal ligament mesenchymal stem cells

Abstract

Introduction Periodontal diseases are clinically characterized by inflammation of the periodontal connective tissue that ultimately induces the destruction of periodontal tissue and the loss of alveolar bone. In chronic periodontitis, the anaerobic gram-negative bacterium Porphyromonas gingivalis (P. gingivalis) is implicated. The pathogenicity of P. gingivalis is exerted by a wide variety of factors, among which the lipopolysaccharides (LPSs). In this study, the expression of TLR-4, the cell growth, the cytokines release, and the nuclear factor-KB (NF-kB) transcription factor expression in response to LPS-P.Gingivalis (LPS-G) were examined in Human Periodontal Ligament Mesenchymal Stem Cells (PDL-MSCs). In this study, to understand the role and the response to LPS-G we examined cell growth, the IL-6, IL-8 and CCL-20 release and the NF-kB signaling pathway in periodontal stem cells. Methods PDL-MSCs were derived from the periodontal ligament of 10 young donors. After in vitro isolation, PDL-MSCs were incubated in the absence (controls) or presence of 5 μg/mL of LPS from P. gingivalis and after 12, 24, 48, and 7 h of incubation, their morphological features were analysed by light and confocal microscopy. PDL-MSCs were characterized by flow cytometry using against TRL-4 PE-conjugated monoclonal antibody.To evaluate the cell growth an MTT assay was performed. The release of IL-6, IL-8, and CCL-20 was analysed by ELISA test. Differences in the cell growth and in the interleukin secretion were analysed for statistical significance with two-way anova tests and the Holm-Sidak method for multiple comparisons. Results In basal conditions, human PDL-MSCs express high levels of TLR-4. In inflammatory conditions mimicked by LPS-Gchallenge, the MTT assay carried out at different treatment times evidenced the decrease of the cell growth. Moreover, the recognition of P. gingivalis components by TLR-4 culminated with the activation of secretion of inflammatory mediators such as: IL-6, IL-8 and CCL-20, and with the up-regulation of NF-kB, which was translocated into the nucleus. Our data intended to specify that TLR-4 expressed by PDL-MSCs is functional and plays a key role in inflammation. Conclusion The data obtained evidenced that the periodontal stem cells express a functional TLR-4 receptor, and the binding of LPS receptor activates the NF-kB signaling pathways. In fact, an increased level of expression and a nuclear translocation of the transcription factor is evident in treated cells. Moreover, a substantial increase of pro-inflammation cytokines IL-6 and IL-8 and CCL-20 chemokine production is visible after 24 h of stimulation with 5μg/ml LPS.In synthesis, our cells model seems to be a valuable tool for the evaluation of the response of stem cells to LPS-G, compounds that almost certainly are responsible for influencing the periodontal diseases. The distinct ability of the periodontal ligament cells to secrete IL-6, IL-8 and CCL-20 emphasizes that these cells may contribute to the release of cytokines in LPS-challenged periodontium and offer the opportunity to better comprehend the degenerative processes in periodontal diseases.