Induction of Interleukin-12 by Lactobacillus Strains Having a Rigid Cell Wall Resistant to Intracellular Digestion

Induction of IL-12 and IL-10 by Lactobacillus type strains and L. casei strain Shirota (LcS). Peritoneal macrophages were cultured with 10 type strains of Lactobacillus species or LcS (0.1 to 100μg/mL) for 24h, and the levels of IL-12 (top panel) and IL-10 (bottom panel) in culture supernatants were determined by ELISA. Data are means±SD of triplicate cultures.

Importance of Toll-like receptor (TLR) signaling in IL-12 induction by L. casei strain Shirota (LcS). (A) Macrophages from MyD88-deficient (MyD88-KO) or wild-type (WT) C57BL/6 mice were cultured with LcS (1μg/mL, open bars; 10μg/mL, gray bars) for 24h. (B) Macrophages from TLR2-deficient (TLR2-KO), TLR4-deficient (TLR4-KO), or WT C57BL/6 mice were cultured with LcS for 24h. The levels of IL-12 in culture supernatants were determined by ELISA. Data are means±SD of triplicate cultures.

Microscopic analysis of phagocytosis and lysis of lactobacilli by macrophages. Peritoneal macrophages on collagen type I-coated cover glasses were cultured with 10 type strains of Lactobacillus species or L. casei strain Shirota (LcS; 10μg/mL) for 4 and 24h and stained with Giemsa. Phagocytosis and lysis of bacteria by macrophages were observed by light microscopy.

Flow cytometric analysis of phagocytosis of lactobacilli. Peritoneal macrophages were cultured with fluorescein isothiocyanate-labeled Lactobacillus type strains (10μg/mL) for 2, 4, 8, and 24h and then analyzed by flow cytometry. The percentage of fluorescence positive cells is shown.

Sensitivity to N-acetylmuramidase treatment of lactobacilli. Ten type strains of Lactobacillus species or L. casei strain Shirota (LcS) were treated with lysozyme (50μg/mL, open bars) for 120min or M-1 enzyme (10μg/mL, gray bars) for 10min. The sensitivity of bacteria to the enzymes was calculated as described in the Materials and Methods section. Data are means±SD of 3 independent experiments.

Interleukin-12–inducing ability and sensitivity to M-1 enzyme in reference strains of lactobacilli. (Top panel) Peritoneal macrophages were cultured with 36 Lactobacillus reference strains (10μg/mL) for 24h, and the levels of IL-12 in culture supernatants were determined by ELISA. Data are means±SD of triplicate cultures. (Bottom panel) Lactobacillus strains were treated with M-1 enzyme (10μg/mL) for 10min. The sensitivity of bacteria to M-1 enzyme was calculated as described in the Materials and Methods section. Data are means of 2 independent experiments. YIT=Yakult Institute, Tokyo (Tokyo, Japan).

Negative correlation between the sensitivity of lactobacilli to in vitro N-acetylmuramidase treatment and IL-12–inducing ability. The sensitivity of 36 Lactobacillus reference strains to M-1 enzyme and the IL-12–inducing ability (the IL-12 level induced by 10μg/mL bacteria in Figure 6) of these strains are plotted. The logarithmic curve and r value are shown.

Importance of the cell wall in IL-12 induction by L. casei strain Shirota (LcS). Lactobacillus casei strain Shirota was treated with M-1 enzyme (10μg/mL, open circles; 50μg/mL, closed circle) or lysozyme (50μg/mL, open triangles) for 10, 30, 60, and 120min. (A) The extent of lysis of the cell wall was evaluated with a reduction of optical density at 600nm (OD600). (B) Lactobacillus casei strain Shirota treated with 50μg/mL of M-1 enzyme was stained with Giemsa. (C) Peritoneal macrophages were cultured with LcS (10μg/mL) treated with 50μg/mL of M-1 enzyme for 24h, and the levels of IL-12 in culture supernatants were determined by ELISA. Data are means±SD of triplicate cultures.

Effect of cell wall components prepared from L. casei strain Shirota (LcS) on IL-12 production. (A) Peritoneal macrophages were cultured with or without (Med) 10μg/mL of LcS, polysaccharide–peptidoglycan complex (PS-PG), protoplast (PP), intact cell wall (ICW), or hydrogen fluoride-treated ICW (HF-ICW) for 24h, and the levels of IL-12 in culture supernatants were determined by ELISA. Data are means±SD of triplicate cultures. (B) Diagrams of the structure of cell wall components are shown.