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.

Enhanced Responsive Formation of Extracellular Traps in Macrophages Previously Exposed to Porphyromonas gingivalis.

Tolerance is defined to be a hyporesponsive state following repeated stimulations with bacteria or their virulence factors and has potential impacts on the development of periodontitis. Recently, macrophages have been reported to release chromatin and antimicrobial peptides to form extracellular traps upon bacterial or chemical stimulations. Thus, we explored the roles and mechanisms of tolerance induced by Porphyromonas gingivalis (P. gingivalis) in macrophage extracellular traps (METs). Tolerance in peritoneal macrophages from mice was triggered by repeated P. gingivalis stimulation. METs were observed using fluorescence microscopy, and the levels of extracellular DNA were determined by microplate reader assays. The expression of p-RAF, p-MEK, and p-ERK was examined by Western blot, and reactive oxygen species (ROS) production was explored using flow cytometry. Moreover, the levels of intracellular Ca2+ were also determined by confocal microscopy to identify the possible mechanisms related to the changes in METs in P. gingivalis-pretreated macrophages. Repeated P. gingivalis stimulation contributed to the formation of METs and increased levels of extracellular DNA (p < 0.05). ROS generation and RAF/MEK/ERK phosphorylation were decreased in P. gingivalis-pretreated macrophages compared with non-pretreated cells (p < 0.05), which was inconsistent with the changes in METs. However, in P. gingivalis-pretreated macrophages, the levels of intracellular Ca2+ were significantly increased compared with the single stimulation group. Additionally, inhibition of intracellular Ca2+ resulted in a decrease in the levels of extracellular DNA in P. gingivalis-pretreated cells (p < 0.05). Taken together, P. gingivalis-pretreated macrophages released more METs, possibly related to the increased levels of intracellular Ca2+.