Background and Objectives<br />Periodontitis is an inflammatory disease of tooth supporting tissues, which is initiated by pathogenic oral biofilms. Alteration of biofilm content from symbiotic to pathogenic is currently considered as a major etiological agent of periodontitis and leads to inappropriate and destructive immune response. The components of biofilms are recognized by specific receptors of innate immune system, particularly by toll-like receptors (TLRs). TLRs are a family of proteins, which recognize specific conserved parts of bacteria and viruses. Activation of all TLRs excepting TLR3 leads to inflammatory response through MyD88-dependent mechanisms, whereas TLR3 works exclusively through MyD88-independent pathways. TLR4 is the only member of TLRs family, which acts through both MyD88 dependent and MyD88-independent pathways. <br />Since pathogenic biofilms contain numerous bacterial species and viruses, it can activate different TLRs simultaneously. However, the response of host cells to simultaneous stimulation of different TLRs is investigated rather poorly. Therefore in the present study we investigated the response of human periodontal ligament cells (hPDLCs) to simultaneous stimulation with TLR2 agonist Pam3CSK4, which acts exclusively through MyD88-dependent pathway, and TLR3 agonist Poly-I:C, which acts exclusively through MyD88-independent pathway. Simultaneous effect of these two substances was compared with that of TLR4 agonists E. coli LPS, which is known to activate both signaling pathways.<br />Material and methods<br />Human periodontal ligament cells were isolated from five different donors. Cells were stimulated with TLR2 agonist Pam3CSK4 (0.01, 1 g/ml), TLR3 agonist Poly-I:C (0.1, 1 g/ml) and their different combinations for 24 h. The resulting production of pro-inflammatory mediators interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP1) was measured on gene and protein levels by qPCR and ELISA, respectively. The response to simultaneous stimulation with TLR2 and TLR3 agonists was compared to that of E. coli LPS (1 g/ml).<br />Results<br />Both TLR2 and TLR3 agonists induce production of pro-inflammatory mediators by hPDLCs. The response of hPDLCs to simultaneous stimulation with TLR2 and TLR3 agonists was generally significantly higher compared to stimulation with single stimuli. In addition, the response of hPDLCs to simultaneous stimulation with TLR2 and TLR3 agonists was significantly higher than that to TLR4 agonist E. coli LPS.<br />Conclusion<br />Our data show that response of hPDLCs to simultaneous stimulation with TLR2 and TLR3 agonists is an addition of their responses to single stimulation and results in extremely high production of pro-inflammatory mediators. The response of hPDLCs to E. coli LPS was substantially lower than that to combination of TLR2 and TLR3 agonists, which suggest that the response to TLR4 might activate some additional pathways resulting in dampening of inflammatory response.