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

• Previous stimuli human periodontal ligament fibroblasts (hPDLFs) with low levels of lipopolysaccharide (LPS) induce a state of tolerance.
• Ec LPS is better at initiating the responsiveness of hPDLFs than Pg LPS.
• Negative regulation of Toll-like receptors (TLRs) may be involved in mediating tolerance in hPDLFs.
• Very low doses of LPS pretreatment induce even a robust response to LPS challenge.

ObjectiveHuman 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.MethodsPrimary 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).ResultsWe show that the initial LPS exposure significantly induced hPDLFs to produce cytokines interleukin-8 (IL-8) and IL-6, whereas the secondary LPS challenge dramatically diminished the levels of cytokines 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.ConclusionAn 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.