Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils

Abstract

Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells.

Fig 1. Effects of tolerized THP-1 cells on neutrophil migration.

Neutrophils were resuspended in the upper chamber of a transwell plate and supernatants from 1 μg/ml P.gingivalis LPS or 1 μg/ml E.coli LPS tolerized/non-tolerized THP-1 cells were added to the lower chamber. P.gingivalis LPS, E.coli LPS and IL-8 were served as controls. Following incubation for 90 min, neutrophils that migrated through transwell membrane were counted in 5 fields under a microscope (×400). Data are expressed as mean±SD (n = 5 per group). *p<0.05. One representative result of five independent experiments is shown in (1A).

Fig 2. Influences of tolerized THP-1 cells on ROS production in neutrophils.

Neutrophils were stimulated with supernatants from 1 μg/ml P.gingivalis LPS or 1 μg/ml E.coli LPS tolerized/non-tolerized THP-1 cells for 2 h. P.gingivalis LPS and E.coli LPS were served as controls. ROS production was measured by flow cytometry. Data are expressed as mean±SD (n = 5 per group). *p<0.05. One representative result of five independent experiments is shown in (2A).

Fig 3. Effects of tolerized THP-1 cells on the expressions of active Caspase 3 in neutrophils.

Neutrophils were challenged with supernatants from 1 μg/ml P.gingivalis LPS or 1 μg/ml E.coli LPS tolerized/non-tolerized THP-1 cells for 5 h. P.gingivalis LPS and E.coli LPS were served as controls. Levels of active Caspase 3 were measured by flow cytometry. Data are expressed as mean±SD (n = 5 per group). *p<0.05. One representative result of five independent experiments is shown in (3A).

Fig 4. Cytokine production in THP-1 cells stimulated with P.gingivalis LPS.

THP-1 cells were pretreated with medium or 1 μg/ml P.gingivalis LPS for 24 h, washed, and then incubated with medium or 1 μg/ml P.gingivalis LPS for another 24 h. Levels of IFN-γ (5A) and ENA-78 (5B) in the cultured supernatants were measured by ELISA. Data are expressed as mean±SD (n = 5 per group). *p<0.05.