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Cranberry Proanthocyanidins Neutralize the Effects of Aggregatibacter actinomycetemcomitans Leukotoxin

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Cranberry Proanthocyanidins Neutralize the Effects of Aggregatibacter actinomycetemcomitans Leukotoxin

Amel Ben Lagha et al. Toxins (Basel).

Abstract

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that has been strongly associated with localized aggressive periodontitis. The capacity of A. actinomycetemcomitans to produce a leukotoxin (LtxA) that activates pyroptosis in macrophages and induces the release of endogenous danger signals is thought to play a key role in the disease process. The aim of the present study was to investigate the effects of cranberry proanthocyanidins (PACs) on gene expression and cytotoxic activities of LtxA. We showed that cranberry PACs dose-dependently attenuate the expression of genes making up the leukotoxin operon, including ltxB and ltxC, in the two strains of A. actinomycetemcomitans tested. Cranberry PACs (≥62.5 µg/mL) protected macrophages against the cytotoxic effect of purified LtxA. Moreover, cranberry PACs reduced caspase-1 activation in LtxA-treated macrophages and consequently decreased the release of both IL-1β and IL-18, which are known as damage-associated molecular patterns (DAMPs) and contribute to the progression of periodontitis by increasing cell migration and osteoclastogenesis. In addition, cranberry PACs reduced the expression of genes encoding the P2X7 receptor and NALP3 (NACHT, LRR and PYD domains-containing protein 3), which play key roles in pore formation and cell death. Lastly, cranberry PACs blocked the binding of LtxA to macrophages and consequently reduced the LtxA-mediated cytotoxicity. In summary, the present study showed that cranberry PACs reduced LtxA gene expression in A. actinomycetemcomitans and neutralized the cytolytic and pro-inflammatory responses of human macrophages treated with LtxA. Given these properties, cranberry PACs may represent promising molecules for prevention and treatment of the aggressive form of periodontitis caused by A. actinomycetemcomitans.

Keywords: Aggregatibacter actinomycetemcomitans; leukotoxin; macrophages; periodontitis; pyroptosis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of cranberry PACs on the expression of ltxB (A) and ltxC (B) mRNA in two strains of A. actinomycetemcomitans (Y4 and JP2). *, significant inhibition at p < 0.01.
Figure 2
Figure 2
Effects of the presence of cranberry proanthocyanidins (PACs) (62.5 µg/nL) in the culture medium of A. actinomycetemcomitans JP2 on the recovery of bioactive LtxA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (A) and cytotoxicity towards macrophages (B) of fractions obtained using the LtxA purification protocol. Note: *, significant inhibition at p < 0.01.
Figure 3
Figure 3
Effect of cranberry PACs on the real-time viability of macrophages exposed to purified LtxA (1 µg/mL). All values are significantly different from those of cells treated with LtxA (p < 0.01) (A). Immunofluorescence microscopy of macrophages treated (1 h) with purified LtxA in the presence or absence of cranberry PACs; the white bar corresponds to 300 µm (B).
Figure 4
Figure 4
Effect of cranberry PACs on LtxA-induced apoptotic death of macrophages. Cells were stained with annexin V/PI and were analyzed by flow cytometry. The percentage of each cell population is indicated.
Figure 5
Figure 5
Effect of cranberry PACs on LtxA-induced caspase-1 activation in macrophages. Active caspases were detected using the poly caspase reagent FAM-VAD-FMK FLICA after 60 min in the absence or presence of cranberry PACs.
Figure 6
Figure 6
Effect of cranberry PACs on intracellular and released caspase-1 (A), IL-1β (B), and IL-18 (C) from LtxA-treated macrophages. Note: Φ: significant increase in intracellular caspase-1 (p < 0.001) relative to control macrophages treated with LtxA alone. *: significant decrease in released caspase-1 (p < 0.001) relative to control macrophages treated with LtxA alone.
Figure 7
Figure 7
Effects of cranberry PACs on the LtxA-modulated CIAS (A) and P2X7 (B) expression in macrophages. Note: *, significant inhibition, p < 0.01.
Figure 8
Figure 8
Dose- and time-dependent effects of cranberry PACs on LtxA-induced increases in reactive oxygen species (ROS) (A) and superoxide (B) production by macrophages.
Figure 9
Figure 9
Effects of cranberry PACs on the binding of V-fluorescein isothiocyanate (FITC)–LtxA to macrophages. Cytotoxicity of FITC–LtxA against macrophages as assessed with an MTT test (A). Binding of FITC–LtxA to macrophages as determined by measuring the fluorescence using a Synergy 2 microplate reader (B) or a flow cytometer (C). The percentage of each cell population is indicated. Three independent assays were performed in triplicate.

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