Endotoxin tolerance induced by Porphyromonas gingivalis lipopolysaccharide alters macrophage polarization.

Endotoxin tolerance refers to a state refractory to subsequent lipopolysaccharide (LPS) stimulations following a primary LPS exposure. To study the relationship between endotoxin tolerance and macrophage polarization, endotoxin tolerance was induced by 1 µg/mL LPS from the periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs). Repeated P. gingivalis LPS challenges increased the quantities of CD206+ PMs, while the number of CD86+CD206+ PMs was reduced compared with the non-tolerant group (p < 0.05). However, there were no changes in BMDMs (p > 0.05). Down regulations of TNF-α, IL-12, nitric oxide and MMP-2 production, and upregulated IL-10, MMP-9 levels and arginase-1 activities occurred in tolerant PMs and BMDMs (p < 0.05). P. gingivalis LPS-tolerant PMs and BMDMs also enhanced scrape-wound healing abilities of 15p-1 cells (p < 0.05). Expressions of phospho-signal transducer and activator of transcription 6 (p-STAT6) and protein tyrosine phosphatase 1B (PTP1B) were increased, while p-MEK1/2 levels were downregulated in tolerant PMs and BMDMs (p < 0.05). IL-10 production in tolerant Stat6 knockdown RAW264.7 cells was lower than tolerant control cells (p < 0.05). P. gingivalis LPS-tolerant macrophages represented an intermediate state between M1/M2 polarization, which functioned as M2-like cells, and led to limited inflammatory responses and enhanced wound healing activities. The PTP1B-MEK1/2-STAT6 signaling pathway might be involved in the polarization of tolerant macrophages.

Effects of Endotoxin Tolerance Induced by Porphyromonas gingivalis Lipopolysaccharide on Inflammatory Responses in Neutrophils.

Periodontitis is a dental plaque-induced chronic inflammatory disease. Long-term exposure of the host to periodontal pathogens leads to a hyporesponsive state to the following stimulations, which is described as endotoxin tolerance. Neutrophils are the most abundant innate immune cells in the body. To clarify the roles of endotoxin tolerance in periodontitis, inflammatory responses in Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)-tolerized neutrophils were explored in this study. Here, apoptosis and respiratory burst in neutrophils upon single or repeated P. gingivalis LPS stimulations were explored by flow cytometry. Cytokine production (TNF-α, IL-8, and IL-10) in tolerized neutrophils or neutrophils co-cultured with peripheral blood mononuclear cells was determined by ELISA. Phagocytosis of P. gingivalis by tolerized neutrophils was also assayed by flow cytometry. In addition, quality and quantitation of neutrophil extracellular trap (NET) formation were detected using immunofluorescence microscope and microplate reader, respectively. The protein expressions of extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) were examined to identify possible mechanisms for the abovementioned changes. Tolerance induced by P. gingivalis LPS significantly suppressed apoptosis, reactive oxygen species (ROS) generation, and phagocytosis in neutrophils (p < 0.05). In both neutrophils alone and co-culture system, repeated P. gingivalis LPS stimulations significantly decreased TNF-α production, but increased IL-10 secretion (p < 0.05). Moreover, in tolerized neutrophils, NET formations were strengthened and there were more released extracellular DNA (p < 0.05). In P. gingivalis LPS-tolerized neutrophils, phosphorylation of ERK1/2 was suppressed compared with that in non-tolerized cells. Taken together, immune responses in neutrophils were reprogrammed by P. gingivalis LPS-induced tolerance, which might be related with the development of inflammation in periodontal tissues. Moreover, ERK1/2 might play important roles in endotoxin tolerance triggered by P. gingivalis LPS.

Tolerance induced by Porphyromonas gingivalis may occur independently of TLR2 and TLR4.

OBJECTIVE: 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. Porphyromonas gingivalis (P. gingivalis) 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 P. gingivalis. METHODS: THP-1-derived macrophages were pretreated with 1x108 colony-forming units/ml P. gingivalis ATCC 33277 or 21 clinical isolates from moderate to severe chronic periodontitis patients (24 h), washed (2 h) and treated with P. gingivalis ATCC 33277 or the same clinical isolates again (24 h). Levels of pro-inflammatory cytokines TNF-α and IL-1ß 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. RESULTS: After repeated challenge with P. gingivalis ATCC 33277 or clinical isolates, production of TNF-α and IL-1ß 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 P. gingivalis ATCC 33277 or clinical isolate-tolerized cells (p>0.05). In addition, there was interstrain variability in the ability to induce IL-1ß and IL-10 production after repeated P. gingivalis stimulation. However, no significant changes in TLR2 or TLR4 were detected in macrophages that were repeatedly treated with P. gingivalis ATCC 33277 or clinical isolates compared with those stimulated with P. gingivalis only once (p>0.05). CONCLUSIONS: Repeated P. gingivalis stimulation triggered tolerance, which might contribute to limiting periodontal inflammation. However, tolerance induced by P. gingivalis might develop independently of TLR2 and TLR4 and be related to molecules in signaling pathways downstream of TLR2 and TLR4.