Killing of Treponema denticola by mouse peritoneal macrophages

Abstract

Treponema denticola has been identified as an important cause of periodontal disease and hypothesized to be involved in extra-oral infections. The objective of this study was to investigate the role of T. denticola cell length and motility during mouse peritoneal macrophages in vitro uptake. Macrophages, incubated under aerobic and anaerobic conditions, produced a similar amount of TNF-alpha when stimulated with Escherichia coli LPS. The uptake of FlgE- and CfpA-deficient mutants of T. denticola was significantly increased compared with the wild-type strain, due to cell size or lack of motility. Opsonization with specific antibodies considerably improved the treponemes' uptake. These results suggest that macrophages, in addition to other phagocytes, could play an important role in the control of T. denticola infection, and that the raising of specific antibodies could improve the efficacy of the immune response toward T. denticola, either at an oral site or during dissemination.

Figure 1.

Production of TNF-α by isolated murine peritoneal macrophages stimulated with 10 µg/mL of LPS, for up to 24 hrs, under anaerobic (●) and aerobic (▲) conditions of incubation. Production of TNF-α by isolated murine peritoneal macrophages in the absence of stimulus, under anaerobic (▼) and aerobic (♦) conditions of incubation. The data presented are from 3 independent experiments and are expressed as the means ± standard deviations (error bars) of 3 replicates.

Figure 2.

Percentages of motile T. denticola cells in the cell culture supernatants after macrophage challenge as observed by dark-field microscopy (400x). Bars indicate the percentages of motile treponemes under anaerobic conditions (dotted bars) and under aerobic conditions (white bars). The data presented are from 3 independent experiments and are expressed as the means ± standard deviations (error bars) for each condition of incubation.

Figure 3.

Macrophage uptake of T. denticola wild-type and mutant strains analyzed by immunofluorescence in both anaerobic (Part A) and aerobic conditions (Part B). Over-time uptake by isolated peritoneal murine macrophages of the wild-type cells (white bars), of the wild-type cells opsonized with anti-major-surface-protein (MSP) antibodies (bars with horizontal stripes), of the non-motile mutant cells (grey bars), and of the filamentous mutant cells (black bars). The results are expressed as a percentage of macrophages positive for antibodies against MSP. The differences observed between the number of positive macrophages challenged by the wild-type strain and the opsonized wild-type strain (p < 0.0001), or the mutant strains (p < 0.0001 for either strain), are statistically significant for both anaerobic (Part A) and aerobic conditions (Part B). The results are representative of 3 independent experiments, each done in triplicate, and are expressed as the means ± standard deviations (error bars) of 3 replicates.

Figure 4.

Macrophage uptake of T. denticola analyzed by real-time PCR. (A) Wild-type and mutant strains under anaerobic conditions. (B) Wild-type and mutant strains under aerobic conditions. The bars indicate the quantitative detection of DNA from the wild-type cells (white bars), the wild-type cells opsonized with anti-MSP antibodies (bars with horizontal stripes), the non-motile mutant cells (grey bars), and the filamentous mutant cells (black bars). The differences observed between the number of positive macrophages challenged by the wild-type strain and the opsonized wild-type strain (p < 0.05), or the mutant strains (p < 0.05 for either strain), are statistically significant in both anaerobic (A) and aerobic conditions (B). (C) Effects of different ratios of incubation between macrophages and the T. denticola wild-type strain under anaerobic conditions. (D) Effects of different ratios of incubation between T. denticola wild-type strain and macrophages under aerobic conditions. White bars indicate 10/1 ratio, grey bars, 50/1, and black bars, 100/1. The results are representative of 3 independent experiments, each done in triplicate, and are expressed as the means ± standard deviations (error bars) of 3 replicates.