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Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT.

Kawamoto D, Ando-Suguimoto ES, Bueno-Silva B, DiRienzo JM, Mayer MP - Front Cell Infect Microbiol (2016)

Bottom Line: This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant.After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells.Therefore, the CDT may impair host defense mechanisms in periodontitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo São Paulo, Brazil.

ABSTRACT
The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP(+) cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP(+) cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis.

No MeSH data available.


Related in: MedlinePlus

Effect of AaCDT on the number of TRAP+ multinucleated cells. In (A) a representative photomicrograph illustrating TRAP stained BMMC cells after 6 days of incubation with 50 μg/ml RANKL, and different concentrations of AaCDT. Image magnification is 40x using Olympus BX60 microscope and NIS-Elements F capture system (Nikon, Center Valley, PA, USA). Cells added with osteoprotegerin (OPG; 100 ng/ml) were used as negative controls or RANKL (100 ng/ml) used as positive control. In (B) without, in (C) with the addition of 50 ng/ml of RANKL the average of TRAP+ cells per well. Controls (without AaCDT): (0) negative control; (OPG) cells added with OPG 100 ng/ml; (100 ng/ml RANKL) cells added with optimal RANKL concentration for osteoclastogenesis. Statistically significant difference when compared with negative control (ANOVA-Tukey), *p < 0.05, **p < 0.01 and ***p < 0.001.
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Figure 2: Effect of AaCDT on the number of TRAP+ multinucleated cells. In (A) a representative photomicrograph illustrating TRAP stained BMMC cells after 6 days of incubation with 50 μg/ml RANKL, and different concentrations of AaCDT. Image magnification is 40x using Olympus BX60 microscope and NIS-Elements F capture system (Nikon, Center Valley, PA, USA). Cells added with osteoprotegerin (OPG; 100 ng/ml) were used as negative controls or RANKL (100 ng/ml) used as positive control. In (B) without, in (C) with the addition of 50 ng/ml of RANKL the average of TRAP+ cells per well. Controls (without AaCDT): (0) negative control; (OPG) cells added with OPG 100 ng/ml; (100 ng/ml RANKL) cells added with optimal RANKL concentration for osteoclastogenesis. Statistically significant difference when compared with negative control (ANOVA-Tukey), *p < 0.05, **p < 0.01 and ***p < 0.001.

Mentions: The addition of 12.5 and 25.0 μg/ml AaCDT led to an increase in the number TRAP+ cells harboring one or two nuclei in cultures lacking RANKL (Figure 2B), and in presence of RANKL the AaCDT. The exposure to increasing concentrations of AaCDT resulted in increased numbers of TRAP+ cells containing 3–4 nuclei, in a dose dependent way (Figure 2C), although these differences were not statistically significant. TRAP+ cells containing ≥3 nuclei as shown in Figure 2A, indicated that pre-osteoclasts differentiated to osteoclasts. The exposure to AaCDT in the presence of RANKL resulted also in increased numbers of TRAP+ cells containing ≥5 nuclei (not significant).


Alteration of Homeostasis in Pre-osteoclasts Induced by Aggregatibacter actinomycetemcomitans CDT.

Kawamoto D, Ando-Suguimoto ES, Bueno-Silva B, DiRienzo JM, Mayer MP - Front Cell Infect Microbiol (2016)

Effect of AaCDT on the number of TRAP+ multinucleated cells. In (A) a representative photomicrograph illustrating TRAP stained BMMC cells after 6 days of incubation with 50 μg/ml RANKL, and different concentrations of AaCDT. Image magnification is 40x using Olympus BX60 microscope and NIS-Elements F capture system (Nikon, Center Valley, PA, USA). Cells added with osteoprotegerin (OPG; 100 ng/ml) were used as negative controls or RANKL (100 ng/ml) used as positive control. In (B) without, in (C) with the addition of 50 ng/ml of RANKL the average of TRAP+ cells per well. Controls (without AaCDT): (0) negative control; (OPG) cells added with OPG 100 ng/ml; (100 ng/ml RANKL) cells added with optimal RANKL concentration for osteoclastogenesis. Statistically significant difference when compared with negative control (ANOVA-Tukey), *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4815040&req=5

Figure 2: Effect of AaCDT on the number of TRAP+ multinucleated cells. In (A) a representative photomicrograph illustrating TRAP stained BMMC cells after 6 days of incubation with 50 μg/ml RANKL, and different concentrations of AaCDT. Image magnification is 40x using Olympus BX60 microscope and NIS-Elements F capture system (Nikon, Center Valley, PA, USA). Cells added with osteoprotegerin (OPG; 100 ng/ml) were used as negative controls or RANKL (100 ng/ml) used as positive control. In (B) without, in (C) with the addition of 50 ng/ml of RANKL the average of TRAP+ cells per well. Controls (without AaCDT): (0) negative control; (OPG) cells added with OPG 100 ng/ml; (100 ng/ml RANKL) cells added with optimal RANKL concentration for osteoclastogenesis. Statistically significant difference when compared with negative control (ANOVA-Tukey), *p < 0.05, **p < 0.01 and ***p < 0.001.
Mentions: The addition of 12.5 and 25.0 μg/ml AaCDT led to an increase in the number TRAP+ cells harboring one or two nuclei in cultures lacking RANKL (Figure 2B), and in presence of RANKL the AaCDT. The exposure to increasing concentrations of AaCDT resulted in increased numbers of TRAP+ cells containing 3–4 nuclei, in a dose dependent way (Figure 2C), although these differences were not statistically significant. TRAP+ cells containing ≥3 nuclei as shown in Figure 2A, indicated that pre-osteoclasts differentiated to osteoclasts. The exposure to AaCDT in the presence of RANKL resulted also in increased numbers of TRAP+ cells containing ≥5 nuclei (not significant).

Bottom Line: This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant.After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells.Therefore, the CDT may impair host defense mechanisms in periodontitis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo São Paulo, Brazil.

ABSTRACT
The dysbiotic microbiota associated with aggressive periodontitis includes Aggregatibacter actinomycetemcomitans, the only oral species known to produce a cytolethal distending toxin (AaCDT). Give that CDT alters the cytokine profile in monocytic cells, we aimed to test the hypothesis that CDT plays a role in bone homeostasis by affecting the differentiation of precursor cells into osteoclasts. Recombinant AaCDT was added to murine bone marrow monocytes (BMMC) in the presence or absence of RANKL and the cell viability and cytokine profile of osteoclast precursor cells were determined. Multinucleated TRAP(+) cell numbers, and relative transcription of genes related to osteoclastogenesis were also evaluated. The addition of AaCDT did not lead to loss in cell viability but promoted an increase in the average number of TRAP(+) cells with 1-2 nuclei in the absence or presence of RANKL (Tukey, p < 0.05). This increase was also observed for TRAP(+) cells with ≥3nuclei, although this difference was not significant. Levels of TGF-β, TNF-α, and IL-6, in the supernatant fraction of cells, were higher when in AaCDT exposed cells, whereas levels of IL-1β and IL-10 were lower than controls under the same conditions. After interaction with AaCDT, transcription of the rank (encoding the receptor RANK), nfatc1 (transcription factor), and ctpK (encoding cathepsin K) genes was downregulated in pre-osteoclastic cells. The data indicated that despite the presence of RANKL and M-CSF, AaCDT may inhibit osteoclast differentiation by altering cytokine profiles and repressing transcription of genes involved in osteoclastogenesis. Therefore, the CDT may impair host defense mechanisms in periodontitis.

No MeSH data available.


Related in: MedlinePlus