Enhancement of natural killer cell activity by oral administration of a fermented soybean product in dogs

Abstract

Background: Probiotics are known for their ability to enhance cellular immunity, including the activation of macrophages, natural killer (NK) cells, and cytotoxic T lymphocytes. Natto is a Japanese traditional probiotic food made by fermenting soybean with bacteria Bacillus subtilis var. natto. Components of natto include spores of B. subtilis natto, poly-γ-glutamic acid, and levan, which have demonstrated their immunoadjuvant and anti-allergic effects through various in vitro and in vivo studies. However, it remains unclear whether oral administration of natto can modulate the immune activity in animals.

Aim: This study aimed to investigate the effects of oral administration of natto on the immune system of dogs.

Methods: Eight dogs were randomly divided into two groups: a natto-treated group and an untreated group. The dogs in the natto-treated group were fed with 10 g/head/day of a freeze-dried natto product in addition to a usual amount of regular dry food for 14 days, whereas the dogs in the untreated group were fed with the regular dry food alone. To determine cellular immune activity, the cell surface antigen analysis of peripheral blood lymphocytes and cytotoxicity analysis of peripheral blood mononuclear cells were carried out before and after the natto administration period. Additionally, a relative expression of inflammatory cytokines in peripheral blood monocytes after the introduction of antigen-stimulation was also examined.

Results: At the end of the administration period, a proportion of NK cells (CD^3- CD^5- CD^21- cells and CD³⁺ CD^5dim CD⁸⁺ cells) in peripheral blood lymphocytes were found to be significantly increased, and the cytotoxic effect of the peripheral blood mononuclear cells on canine tumor cells were greatly enhanced in the natto-treated group, but not in the untreated group. The expression of TNF-α in peripheral blood mononuclear cells following an antigen-stimulation was increased considerably in the dogs after administration of natto.

Conclusion: We conclude that oral administration of natto activated the cytotoxic activity of peripheral NK cells in dogs, and a daily intake of natto might be helpful in augmenting cellular immune activity.

Keywords: Bacillus subtilis natto; Cellular immune; Dog; Fermented soy bean; Natural killer cell.

Fig. 1.. Cell surface antigen analysis using flow cytometry. (A) Proportions of helper T cells (CD³⁺ CD⁴⁺ cells), cytotoxic T cells (CD³⁺ CD⁸⁺ cells), and NK cells (CD³⁻ CD⁵⁻ CD²¹⁻ cells and CD³⁺ CD^5dim CD⁸⁺ cells). (B) Representative plots of the detected NK cells. Before: before administration, after: after administration. Data represent mean ± SE. *p < 0.05 versus before administration.

Fig. 2.. Cytotoxicity against CTAC cells mediated by PBMC. The cytotoxicity of the PBMCs collected from the untreated group of dogs had no significant change between before and after administration. On the contrary, there was a significant increase in the PBMC cytotoxicity of the natto-treated group after administration when compared to that before administration at E:T ratios of 6:1, 3:1, and 1:1. Before: before administration, after: after administration. Data represent mean ± SE. *p < 0.05 versus before administration.

Fig. 3.. Expression levels of inflammatory cytokines in antigen-stimulated PBMCs. PBMCs were incubated in a culture medium containing 25 ng/ml of PMA and 1 μg/ml of ionomycin for 6 hours. Relative expression levels of the target genes were normalized against β-actin and calculated by the 2^-ΔΔCT method. Values were standardized to those of a dog in the untreated group. Before: before administration, after: after administration. Data represent mean ± SE. *p < 0.05 versus before administration.