Immunostimulating properties of intragastrically administered Acetobacter-derived soluble branched (1,4)-beta-D-glucans decrease murine susceptibility to Listeria monocytogenes

Abstract

We previously found that AC-1, an extracellular polysaccharide, produced by Acetobacter xylinum and composed of (1,4)-beta-D-glucan with branches of glucosyl residues, showed a strong activity to induce production of interleukin-12 (IL-12) p40 and tumor necrosis factor alpha by macrophages in vitro via Toll-like receptor 4 (TLR-4) signaling. In the present study, we examined the effect of oral administration of AC-1 on protective immunity against Listeria monocytogenes. Mice were given AC-1 or phosphate-buffered saline (PBS) intragastrically 2 days before, on the day of, and 2 days after an intraperitoneal inoculation of L. monocytogenes. The survival rate of AC-1-treated mice was significantly improved and bacterial growth in AC-1-treated mice was severely retarded compared to those of PBS-treated mice after infection with L. monocytogenes. IL-12 p40 levels in serum and magnitudes of CD4+ Th1 and CD8+ Tc1 responses against Listeria antigen were significantly higher in AC-1-treated mice than in PBS-treated mice. The effect of AC-1 on antilisterial activity was diminished in C3H/HeJ mice carrying mutated TLR-4. Thus, AC-1, a potent IL-12 inducer through TLR-4, enhanced protective immunity against L. monocytogenes via augmentation of Th1 responses. These results suggest that infectious processes driven by intracellular microorganisms could be prevented to develop by the (1,4)-beta-D-glucan.

FIG. 1.

Effect of oral administration of AC-1 on survival rates after i.p. inoculation with a lethal dose of L. monocytogenes. C57BL/6 mice were inoculated i.p. with 10⁶ CFU L. monocytogenes in 0.2 ml of PBS on day 0. Mice were challenged i.g. with a suspension of 200 μl of AC-1 (10 mg/ml) or PBS 2 days before, on the day of, and 2 days after inoculation with L. monocytogenes. *, Significantly different from the value for PBS-treated mice (P < 0.01 by the generalized Wilcoxon's test).

FIG. 2.

Effect of oral administration of AC-1 on bacterial growth in the spleen and liver after L. monocytogenes infection. C57BL/6 mice were challenged i.p. with 0.2 ml of a single cell suspension containing a sublethal dose of L. monocytogenes, and bacterial growth was measured in the spleen and liver on day 6 after infection. The data were obtained from three separate experiments and are expressed as means + the standard deviation (SD) for five mice at each point. Statistically significant differences between AC-1-treated and PBS-treated mice are indicated (**, P < 0.01).

FIG. 3.

IL-12 production in the sera of AC-1-treated mice after Listeria infection. Sera were collected from AC-1-treated and PBS-treated mice at 24, 48, and 72 h after Listeria infection, and IL-12 protein levels were determined by ELISA. The data were obtained from three independent experiments and are expressed as the means of triplicate determinations ± the SD (**, P < 0.01).

FIG. 4.

Generation of Listeria antigen-specific T cells in AC-1-treated mice. Nylon wool-enriched T cells from AC-1-treated mice or PBS-treated mice on day 6 after Listeria infection were cultured without any stimulation or with HKL and immobilized CD3 MAb in the presence of APC for 2 days. Cytokine activity in the culture supernatants was determined by ELISA. The data are representative of three independent experiments with pooled cells from three AC-1-treated or PBS-treated mice and are shown as the means of triplicate determinations + the SD. Statistically significant differences between AC-1-treated mice and PBS-treated mice are shown (*, P < 0.05). ND, not detectable.

FIG. 5.

Intracellular expression of IFN-γ by CD4⁺ T cells from AC-1-treated mice infected with L. monocytogenes. Splenocytes or PEC were pooled from three mice of each group on day 6 after infection and cultured with or without HKL in the presence of brefeldin A for 4 h at 37°C. After culture, the cells were surface stained with anti-CD4 and anti-CD44 MAb. Intracellular cytokine-producing cells were examined by using a flow cytometer and analyzed by gating on CD4⁺ T cells. The data are representative of two independent experiments with pooled cells from three mice and are shown as typical two-color profiles.

FIG. 6.

Generation of OVA_257-264-specific CD8⁺ T cells in AC-1-treated mice infected with rLM-OVA. (A) AC-1-treated or PBS-treated mice were inoculated i.p. with 5 × 10⁵ CFU rLM-OVA. On day 6 after infection, spleen cells or PEC from these mice were stained with OVA_257-264 K^b tetramer, anti-CD44 MAb and anti-CD8 MAb. The results of flow cytometry are presented as typical profiles after an analysis gate had been set on CD8⁺ cells. (B) Spleen cells or PEC from AC-1-treated or PBS-treated mice were prepared on day 6 after rLM-OVA infection and cultured with or without OVA_257-264 peptide in the presence of brefeldin A for 4 h at 37°C. Intracellular cytokine-producing cells were examined by using a flow cytometer and analyzed by gating on CD8⁺ T cells.

FIG. 7.

Effect of oral administration of AC-1 on bacterial growth in the spleen and liver from C3H/HeJ mice after L. monocytogenes infection. C3H/HeN and C3H/HeJ mice were challenged i.p. with a sublethal dose of L. monocytogenes (2 × 10⁵ CFU), and bacterial growth was measured in the spleens and livers on day 6 after infection. The data were obtained from three separate experiments and are expressed as means + the SD for five mice at each point. Statistically significant differences between AC-1-treated and PBS-treated mice are shown (*, P < 0.05; **, P < 0.01).