Endotoxin tolerance induction in human periodontal ligament fibroblasts stimulated with different bacterial lipopolysaccharides

Objective: Human periodontal ligament fibroblasts (hPDLFs) may play an important role in immune responses in the periodontal microenvironment by secreting proinflammatory cytokines. The present study aimed to explore the reaction of hPDLFs to a secondary lipopolysaccharide (LPS) challenge by examining cytokines and innate sensors. Methods: Primary cultures of hPDLFs were obtained and identified by immunochemistry. Cells were treated with a secondary LPS challenge followed by a primary LPS or no LPS as a control. The levels of cytokines were assayed using cytometric bead array (CBA) kits, and the protein levels of Toll-like receptors (TLRs) were determined using flow cytometry. The messenger RNA (mRNA) levels of TLRs were determined using real-time polymerase chain reaction (PCR). Results: We show that the initial LPS exposure significantly induced hPDLFs to produce cytokines interleuldn-8 (IL-8) and IL-6, whereas the secondary LPS challenge dramatically diminished the levels of cytolcines IL-8 and IL-6. The mRNA and protein levels of TLRs were increased by the initial LPS stimulus but decreased by the secondary LPS challenge. Conclusion: An LPS stimulus induces immune responses in hPDLFs, whereas an LPS challenge exerts endotoxin tolerance by downregulating proinflammatory cytokines and TLR mRNA and protein expression. This process may confer hPDLFs with their essential functions for maintaining oral mucosal immunity homeostasis. (C) 2014 Elsevier Ltd. All rights reserved.