Synergistic effects triggered by simultaneous Toll-like receptor-2 and -3 activation in human periodontal ligament stem cells

Abstract

Background: Although periodontitis is associated with disruption of the host-microbial homeostasis, viruses are currently discussed to influence disease progression. Viral pathogens are recognized by Toll-like receptor (TLR)-3, which engages a different signaling pathway than other TLRs. This study aimed to investigate the effect of TLR-3 agonist polyinosinic:polycytidylic acid (Poly I:C) on the expression of inflammatory markers and bone metabolism proteins by human periodontal ligament stem cells (hPDLSCs) compared with TLR-2 agonist Pam3CSK4, which mimics the effect of bacterial lipoproteins. To assess potential combined effects of bacterial and viral infections, hPDLSCs response to simultaneous TLR-2 and TLR-3 activation was investigated. Methods: HPDLSCs were stimulated with Poly I:C (0.0001-1 µg/mL), Pam3CSK4 (1 µg/mL), and their combinations for 24 hours. Gene expression and protein levels of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, and osteoprotegerin (OPG) were measured with qPCR and ELISA. Results: Production of IL-6, IL-8, MCP-1, and OPG was significantly increased by Poly I:C or Pam3CSK4 to a similar extent. The levels of all inflammatory mediators induced by simultaneous stimulation with Poly I:C and Pam3CSK4 were significantly higher compared with single stimuli as well as to their summed response. Gene expression and protein levels of OPG were enhanced by Poly I:C, but by lesser extent than by Pam3CSK4. OPG levels upon simultaneous stimulation with Pam3CSK4 and Poly I:C were significantly lower compared with Pam3CSK4 stimulation alone. Conclusions: Simultaneous TLR-2 and TLR-3 activation synergistically triggers IL-6, IL-8, and MCP-1 production, which was not observed for OPG. These findings suggest that TLR-3 activation by viral infections might promote periodontitis progression.