Tolerance induced by <i>Porphyromonas gingivalis</i> may occur independently of TLR2 and TLR4

<div><p>Objective</p><p>Periodontitis is a microbe-induced chronic inflammatory disease. Previous exposure of the host to bacteria or their virulence factors leads to refractory responses to further stimuli, which is called tolerance. <i>Porphyromonas gingivalis</i> (<i>P</i>. <i>gingivalis</i>) is one of the most important pathogenic microorganisms associated with periodontitis, and is a potent inducer of pro- and anti-inflammatory cytokines. The aim of this study was to explore the roles and possible mechanisms of tolerance induced by <i>P</i>. <i>gingivalis</i>.</p><p>Methods</p><p>THP-1-derived macrophages were pretreated with 1x10⁸ colony-forming units/ml <i>P</i>. <i>gingivalis</i> ATCC 33277 or 21 clinical isolates from moderate to severe chronic periodontitis patients (24 h), washed (2 h) and treated with <i>P</i>. <i>gingivalis</i> ATCC 33277 or the same clinical isolates again (24 h). Levels of pro-inflammatory cytokines TNF-α and IL-1β and anti-inflammatory cytokine IL-10 in supernatants were detected by ELISA. Moreover, to identify the possible mechanisms for the changes in cytokine secretion, Toll-like receptor 2 (TLR2) and TLR4 protein expressions were explored in these cells by flow cytometry.</p><p>Results</p><p>After repeated challenge with <i>P</i>. <i>gingivalis</i> ATCC 33277 or clinical isolates, production of TNF-α and IL-1β in macrophages was decreased significantly compared with that following a single stimulation (p<0.05), while only comparable levels of IL-10 were detected in <i>P</i>. <i>gingivalis</i> ATCC 33277 or clinical isolate-tolerized cells (p>0.05). In addition, there was interstrain variability in the ability to induce IL-1β and IL-10 production after repeated <i>P</i>. <i>gingivalis</i> stimulation. However, no significant changes in TLR2 or TLR4 were detected in macrophages that were repeatedly treated with <i>P</i>. <i>gingivalis</i> ATCC 33277 or clinical isolates compared with those stimulated with <i>P</i>. <i>gingivalis</i> only once (p>0.05).</p><p>Conclusions</p><p>Repeated <i>P</i>. <i>gingivalis</i> stimulation triggered tolerance, which might contribute to limiting periodontal inflammation. However, tolerance induced by <i>P</i>. <i>gingivalis</i> might develop independently of TLR2 and TLR4 and be related to molecules in signaling pathways downstream of TLR2 and TLR4.</p>