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

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Reagents and Culture Medium
  • Animals
  • Bacteria
  • Preparation of Cell Components
  • Isolation of Peritoneal Macrophages and Culture
  • Microscopic Analysis of Phagocytosis
  • Flow Cytometric Analysis of Phagocytosis
  • N-Acetylmuramidase Treatment
  • ELISA for Cytokines
• Results
  • IL-12–Inducing Ability of Lactobacillus Type Strains and LcS
  • Importance of Bacterial Phagocytosis and TLR Signaling in IL-12 Induction
  • Phagocytosis and Lysis of Lactobacilli by Macrophages
  • Sensitivity of Lactobacilli to In Vitro N-Acetylmuramidase Treatment
  • Potent IL-12–Inducing Ability Is Conserved in Strains of the L. casei Group and in L. fermentum
  • Loss of IL-12–Inducing Ability in LcS After Lysis of the Cell Wall
  • IL-12–Inducing Ability of Cell Wall Components of LcS
• Discussion
• Conclusions
• Acknowledgements
• Supplementary data
• References
• Copyright

Abstract 

Some strains of lactobacilli can stimulate macrophages and dendritic cells to secrete IL-12, which plays a key role in activating innate immunity. We examined the IL-12–inducing ability of 47 Lactobacillus strains belonging to 10 species in mouse peritoneal macrophages, and characterized the properties important for the induction of IL-12. Although considerable differences in IL-12–inducing ability were observed among the strains tested, almost all strains belonging to the Lactobacillus casei group (L. casei, Lactobacillus rhamnosus, and Lactobacillus zeae) or to Lactobacillus fermentum induced high levels of IL-12. Phagocytosis of lactobacilli was necessary for IL-12 induction, and the strains with strong IL-12 induction were relatively resistant to lysis in the macrophages. The sensitivity of Lactobacillus strains to in vitro treatment with M-1 enzyme, a member of the N-acetylmuramidases, was negatively correlated with IL-12–inducing ability. Using a probiotic strain, L. casei strain Shirota (LcS), we showed that the cell wall of LcS could be digested by long-term treatment with a high dose of M-1 enzyme and that the IL-12–inducing ability was diminished according to the duration of the enzyme treatment. The soluble polysaccharide–peptidoglycan complex released from the cell wall of LcS did not induce IL-12, whereas the insoluble intact cell wall of LcS induced IL-12. These results suggest that the intact cell wall structure of lactobacilli is an important element in the ability to induce IL-12 and that Lactobacillus strains having a rigid cell wall resistant to intracellular digestion effectively stimulate macrophages to induce IL-12.

Key words: interleukin-12, lactobacilli, phagocytosis, lysis

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Introduction 

Lactobacilli are one of the major constituents of normal human indigenous flora and are believed to play important roles in the development and maintenance of both the mucosal and systemic immune system of the host (Björkstén, 1999). These gram-positive lactic acid bacteria have long been used in the preparation of fermented foods such as yogurt, cheese, and fermented vegetables and meats. Recently, much attention has been paid to the beneficial functions of lactobacilli in addition to their importance in the preparation process of fermented foods. Lactobacilli and bifidobacteria are now the bacteria most often used as probiotics, which are defined as live microbial food ingredients that are beneficial to human health (Salminen et al., 1998). Among several health-promoting effects of probiotic lactobacilli, immunoregulatory functions have been studied with great interest (Dugas et al., 1999;Reid et al., 2003). Some specific strains of probiotic lactobacilli were recently shown to exhibit antitumor and anti-infectious activity through activation of innate immunity, such as augmentation of the functions of macrophages and NK cells (Kato, 2000;Perdigón et al., 2001). Some probiotics have antiallergic activity, which may be explained by regulation of the T helper (Th)1–Th2 balance (Shida et al., 2002;Kalliomäki and Isolauri, 2004).

Interleukin-12 is well known to play a critical role both in inducing a Th1-dominant immune response and in enhancing cellular immunity (Trinchieri, 1994). Many researchers have discussed the importance of IL-12 induced by lactobacilli in relation to their immunoregulatory functions. We have clearly documented that Lactobacillus casei strain Shirota (LcS) can promote the development of Th1 cells from naïve CD4-positive T cells through secretion of IL-12 by macrophages (Shida et al., 1998). Some studies have shown that neutralization of IL-12 with monoclonal antibodies can at least partially reduce the immunoregulatory activity of lactobacilli (Yasutake et al., 1999;Shida et al., 2002). Therefore, IL-12 is considered to play key roles in the functions of probiotics, especially in enhancing the Th1 response and cellular immunity. Accordingly, the ability of a strain to induce IL-12 could be a critical index of immunostimulatory activity.

Although studies comparing the IL-12–inducing ability of Lactobacillus strains have shown that some specific strains belonging to the species of L. casei–Lactobacillus paracasei and Lactobacillus rhamnosus are potent IL-12 inducers (Miettinen et al., 1998;Hessle et al., 1999;Christensen et al., 2002), it has not been clear why these specific strains can strongly induce IL-12. Little information has been available on the active components or structures in lactobacilli or on the properties important for IL-12 induction. Active components have previously been revealed from other bacteria that induce IL-12 secretion. Lipopolysaccharide (Salkowski et al., 1997) from gram-negative bacteria, lipoarabinomannan (Yoshida and Koide, 1997) from mycobacteria, lipoteichoic acid (Cleveland et al., 1996) from gram-positive bacteria, and bacterial lipopeptide (Thoma-Uszynski et al., 2000) and unmethylated CpG DNA (Halpern et al., 1996) have been reported to induce IL-12. These bacterial components are recognized by corresponding Toll-like receptors (TLR), followed by cytokine secretion (Takeda and Akira, 2005). On the other hand, researchers have often observed that the ability of these soluble bacterial components to induce IL-12 is not particularly strong compared with preparations of intact bacterial cells (Cleveland et al., 1996;Huang et al., 1999; Hessle et al., 2000). Underhill et al. (1999) showed that phagocytosis of bacteria elicits recruitment of TLR to phagosomes to recognize bacterial components effectively, and that bacterial phagocytosis and subsequent recognition of TLR cooperate to induce proinflammatory cytokines. This may be why soluble bacterial components show weaker induction of IL-12 than do intact bacterial cell preparations. Thus, further studies investigating the active components or structures of bacteria responsible for IL-12 induction should focus on the interaction between whole bacterial cells and phagocytic cells, in addition to that between bacterial cell components and their receptors on phagocytic cells, considering the importance of bacterial phagocytosis.

In this study, we examined the phagocytosis and lysis of lactobacilli by macrophages and evaluated properties related to the IL-12–inducing ability of lactobacilli. The results obtained indicate the importance of a rigid cell wall structure resistant to intracellular digestion in the effective induction of IL-12.

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Materials and Methods 

Reagents and Culture Medium 

Lysozyme from egg white, trypsin, and cytochalasin D were purchased from Sigma (St. Louis, MO). The M-1 enzyme (N-acetylmuramidase SG) from Streptomyces globisporus was obtained from Seikagaku Corp. (Tokyo, Japan), Benzon nuclease was from Merck (Darmstadt, Germany); and DNase, RNase, and pronase were from Roche Diagnostics (Indianapolis, IN). Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma), supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, and 100μg/mL of streptomycin, was used to culture macrophages.

Animals 

Female BALB/c mice and male C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Male MyD88-, TLR2-, and TLR4-deficient mice with a C57BL/6 genetic background were obtained from Oriental Bioservice (Kyoto, Japan). Animals were used at 8 to 11 wk of age. Experiments were performed in accordance with the guidelines for the care and use of laboratory animals of the Yakult Central Institute (Tokyo, Japan).

Bacteria 

The Lactobacillus strains used in this study are summarized in Table 1. A probiotic strain, LcS, was originally isolated from healthy human intestine at the Yakult Central Institute, based on acid and bile tolerance and survival in the gastrointestinal passage. The other bacteria were initially obtained from the American Type Culture Collection (ATCC, Rockville, MD), the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM, Braunschweig, Germany), the Japan Collection of Microorganisms (JCM, Wako, Japan), the National Collection of Dairy Organisms (NCDO, Reading, UK), the National Collection of Food Bacteria (NCFB, Reading, UK), the National Collection of Industrial, Food and Marine Bacteria (NCIMB, Aberdeen, UK), the National Institute for Research in Dairying (NIRD, Reading, UK), and the Nodai Research Institute Culture Collection (NRIC, Tokyo, Japan). These bacteria were renumbered as YIT (Yakult Institute, Tokyo) strains at the Yakult Central Institute. Preparation of heat-killed bacteria has been described elsewhere (Shida et al., 1998). In brief, the bacteria were cultured at 37°C for 20h in lactobacilli–MRS broth (Difco, Detroit, MI), washed with sterile distilled water, heated at 100°C for 30min, and then lyophilized.

Table 1. Lactobacillus strains used in this study

Fluorescein isothiocyanate (FITC)-labeled lactobacilli were prepared with FITC isomer-I (Dojindo Lab., Kumamoto, Japan). Heat-killed lactobacilli were suspended at a concentration of 5mg/mL in 50mM carbonate buffer (pH 9.6), reacted with FITC isomer-I (4.5μg/mL) at 37°C for 60min, and then washed with sterile PBS.

Preparation of Cell Components 

Bacterial cell components were prepared from heat-killed LcS. Isolation of the protoplast and the polysaccharide–peptidoglycan complex (PS-PG) was described previously (Nagaoka et al., 1990;Matsuguchi et al., 2003). In brief, heat-killed LcS was exhaustively digested with M-1 enzyme for 16h. After centrifugation, the precipitate was washed with distilled water and then collected as the protoplast. The supernatant was treated with DNase, RNase, and trypsin. The digest was dialyzed against distilled water, lyophilized, and used as PS-PG. Intact cell wall (ICW) was prepared as described elsewhere (Matsuguchi et al., 2003). Briefly, heat-killed cells were suspended in 0.3% SDS solution and boiled for 15min. After centrifugation, the precipitate was washed with acetone. The cells were treated with pronase and delipidated by successive refluxing with methanol, methanol–chloroform–water (1:1:1), and methanol–chloroform (1:1). The delipidated materials were treated with Benzon nuclease and pronase. The insoluble material was washed with distilled water, lyophilized, and then used as ICW. To release the polysaccharide moiety from the linkage region of ICW, the ICW was treated with 47% hydrogen fluoride at 4°C for 20h (Mort and Lamport, 1977). Hydrogen fluoride-treated ICW (HF-ICW) was washed with deionized water and lyophilized.

Isolation of Peritoneal Macrophages and Culture 

Peritoneal macrophages were isolated from BALB/c mice, unless indicated otherwise. Mice were injected intraperitoneally with 2mL of 4% thioglycollate. After 4 d, peritoneal exudate cells were isolated as macrophages by washing the peritoneal cavity with Hanks’ balanced salt solution containing 10mM HEPES. Peritoneal macrophages (2×10⁵ cells) were cultured with heat-killed bacteria (0.1 to 100μg/mL) in 200μL of RPMI 1640 medium in a 96-well culture plate. Supernatants were collected at 24h for determination of IL-12p70 and IL-10. To examine the importance of bacterial phagocytosis, cytochalasin D (5μg/mL) was added 30min before stimulation with bacteria (10μg/mL; Shibata, 1995).

Microscopic Analysis of Phagocytosis 

Peritoneal macrophages (2×10⁵ cells) were plated on round 12-mm collagen type I-coated cover glasses (Asahi Techno Glass, Tokyo, Japan) in a 24-well culture plate in 2mL of RPMI 1640 medium. After preincubation for 16h, bacteria were added at 10μg/mL. The macrophages were harvested after 4 and 24h cultures, washed with PBS, fixed with methanol for 10min, and then stained with Giemsa. The phagocytosis of bacteria by macrophages was observed by light microscopy.

Flow Cytometric Analysis of Phagocytosis 

Peritoneal macrophages (1×10⁶ cells) were cultured with FITC-labeled lactobacilli (10μg/mL) in 2mL of RPMI 1640 medium in a 24-well culture plate for 2, 4, 8, and 24h. The macrophages were dislodged by treatment with 10mM EDTA–PBS for 10min, washed with 3mM EDTA–PBS, and then suspended in 1.5mM EDTA–1.25% formalin–PBS. Analysis was performed on an EPICS Altra flow cytometer with Expo32 software (Beckman Coulter, Miami, FL).

N-Acetylmuramidase Treatment 

To evaluate the sensitivity of bacteria to N-acetylmuramidase, heat-killed bacteria suspended at 2mg/ mL in 50mM Trismaleate buffer (pH 7.0) containing 4mM MgCl₂ were treated with lysozyme (50μg/mL) for 120min or with M-1 enzyme (10μg/mL) for 10min. After denaturing the enzymes by heat treatment (100°C, 5min), 10% SDS solution was added to the reaction mixtures at a final concentration of 2%, and the mixture was stirred vigorously to dissolve protoplasts resulting from digestion of the cell wall. Optical density at 600nm (OD600) was measured. Sensitivity of bacteria to the enzymes was calculated using the following formula:

To prepare LcS with cell wall structures at levels of various degradation, heat-killed LcS was treated with 50μg/mL of M-1 enzyme for 10, 30, 60, or 120min. Enzyme-treated LcS was washed 3 times with sterile PBS before addition to the cell cultures. The extent of lysis of the cell wall was evaluated on the basis of the reduction of OD600 after SDS treatment, as described above. Cell wall degradation was also confirmed by microscopic analysis after Giemsa staining, which showed that the color of cells had changed from blue to red.

ELISA for Cytokines 

Determination of IL-12p70 and IL-10 levels in culture supernatants was performed by sandwich ELISA. Rat antimouse IL-12 (clone 9A5) and IL-10 (clone JES5-SXC1) monoclonal antibodies were used as the capture antibodies, and biotinylated rat antimouse IL-12 (clone C17.8) and IL-10 (clone JES5-2A5) monoclonal antibodies, respectively, were used as the detection antibodies. These antibodies and recombinant mouse IL-12 and IL-10 were purchased from BD Pharmingen (San Diego, CA).

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Results 

IL-12–Inducing Ability of Lactobacillus Type Strains and LcS 

Mouse peritoneal macrophages were cultured with 10 type strains of common Lactobacillus species or a probiotic strain, LcS, for 24h, and the concentrations of IL-12 in the supernatants were measured. Considerable differences in IL-12–inducing ability were observed among the strains (Figure 1). Lactobacillus casei strain Shirota and the type strains of L. casei, L. rhamnosus, Lactobacillus zeae, and Lactobacillus fermentum induced high levels of IL-12, whereas Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, and Lactobacillus plantarum induced very low levels of IL-12.

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• Figure 1. 

  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.

We also tested IL-10–inducing ability. Lactobacillus delbrueckii ssp. bulgaricus and L. plantarum, in addition to L. rhamnosus, L. zeae, and L. fermentum, were able to induce high levels of IL-10. Interestingly, the optimal bacterial doses that induced IL-12 and IL-10 were different in most Lactobacillus strains (10μg/mL for IL-12; 100μg/mL for IL-10).

Importance of Bacterial Phagocytosis and TLR Signaling in IL-12 Induction 

The phagocytosis of bacteria and the recognition of bacterial components by TLR are important in the effective induction of proinflammatory cytokines. We therefore examined whether the step of phagocytosing lacto-bacilli is critical in inducing IL-12 by using cytochalasin D, an inhibitor of actin polymerization. The addition of cytochalasin D to macrophage cultures markedly diminished the IL-12–inducing ability of lactobacilli (Table 2), indicating that the phagocytosing step is important for IL-12 induction by lactobacilli.

Table 2. Effect of cytochalasin D on IL-12 induction by lactobacilli¹

Lactobacillus	IL-12, ng/mL
	Control	With cytochalasin D
LcS	79.3±5.7	1.8±0.1
L. casei	128.7±13.1	1.3±0.2
L. rhamnosus	75.7±8.9	1.3±0.4
L. zeae	112.4±10.2	1.2±0.3
L. fermentum	52.9±5.6	1.8±0.2

Macrophages prepared from mice deficient in MyD88, an adapter molecule for TLR signaling, could not secrete IL-12 in response to LcS stimulation (Figure 2A). The lack of IL-12 induction was also observed when MyD88-deficient macrophages were cultured with the type strains of L. casei, L. rhamnosus, L. zeae, and L. fermentum (data not shown). These data suggest the essential role of TLR signaling in the induction of IL-12. In contrast, macrophages deficient in either TLR2 or TLR4, as well as wild-type macrophages, secreted IL-12 in response to LcS stimulation (Figure 2B).

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• Figure 2. 

  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.

Phagocytosis and Lysis of Lactobacilli by Macrophages 

Peritoneal macrophages were cultured with Lactobacillus strains for 4 and 24h and then stained with Giemsa. The phagocytosis and lysis of lactobacilli by macrophages were observed by light microscopy. The pictures in Figure 3 indicate that the phagocytosis and lysis of lactobacilli could be divided into 3 patterns: group R (containing LcS and the type strains of L. casei, L. rhamnosus, L. zeae, and L. fermentum), in which 24h was required for the phagocytosis of large numbers of bacteria and in which phagocytosed bacteria maintained their morphology throughout this period; group S (containing L. acidophilus, L. delbrueckii ssp. bulgaricus, L. helveticus, and L. plantarum), in which less than 4h was required for the phagocytosis of numerous bacteria, which were rapidly lysed in macrophages; and group I (containing L. gasseri and L. johnsonii), in which 24h was required for the phagocytosis of many bacteria and in which phagocytosed bacteria were lysed. These results strongly suggest that phagocytosis of lactobacilli and their subsequent digestion are closely related to IL-12 induction.

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• Figure 3. 

  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.

Next, peritoneal macrophages were cultured with FITC-labeled L. casei (belonging to group R), L. johnsonii (belonging to group I), or L. plantarum (belonging to group S) for 2, 4, 8, and 24h, and phagocytosis was analyzed by flow cytometry. The quantitative analysis indicated that 22% of macrophages phagocytosed L. casei at 2h of incubation, and the ratio of cells phagocytosing L. casei gradually increased and reached 66% at 24h (Figure 4). In the case of L. johnsonii, the ratio of macrophages phagocytosing bacteria was 39% at 2h and gradually increased until 8h to 62%. At 24h of incubation, although most macrophages were considered to have phagocytosed bacteria, the intensity of fluorescence for those cells slightly decreased, probably because of decomposition of the FITC molecules. In L. plantarum, the ratio of macrophages phagocytosing bacteria had already reached 62% at 2h, and the intensity of fluorescence decreased slightly after 8h. The results obtained by flow cytometric analysis corresponded with those from the microscopic analysis shown in Figure 3.

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• Figure 4. 

  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 of Lactobacilli to In Vitro N-Acetylmuramidase Treatment 

N-Acetylmuramidases play a critical role in digesting bacteria in phagolysosomes. To evaluate the lysis of lactobacilli quantitatively, we investigated the sensitivity of Lactobacillus strains to in vitro N-acetylmuramidase (lysozyme and M-1 enzyme) treatment. As shown in Figure 5, LcS and the type strains of L. casei, L. rhamnosus, L. zeae, L. fermentum, and L. gasseri showed strong resistance to treatment with both lysozyme and M-1 enzyme under the reaction conditions examined (lysozyme treatment: 50μg/mL for 120min; M-1 enzyme treatment: 10μg/mL for 10min). In contrast, L. acidophilus, L. delbrueckii ssp. bulgaricus, L. helveticus, and L. plantarum were sensitive to the enzymes. Lactobacillus johnsonii was sensitive to M-1 enzyme but resistant to lysozyme.

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• Figure 5. 

  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.

Potent IL-12–Inducing Ability Is Conserved in Strains of the L. casei Group and in L. fermentum 

The IL-12–inducing ability of 36 additional reference strains was then investigated. As shown in Figure 6, 8 out of 9 strains belonging to L. casei, L. rhamnosus, or L. zeae induced high levels of IL-12, and all strains of these species were relatively resistant to M-1 enzyme treatment. Lactobacillus casei, L. rhamnosus, and L. zeae are closely related species in taxonomy and belong to the L. casei group. An examination of the results for LcS and the type and reference strains of the L. casei group species (Figures 1 and 6) revealed that 12 out of 13 strains belonging to this group were potent IL-12 inducers, suggesting that potent IL-12–inducing ability is conserved in strains of the L. casei group. In addition to the strains of the L. casei group, the 3 reference strains, as well as the type strain, of L. fermentum induced high levels of IL-12. All strains belonging to the other 6 species induced low levels of IL-12, as did the corresponding type strains. Most strains of these 6 species were sensitive to M-1 enzyme treatment.

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• Figure 6. 

  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).

The sensitivity of these Lactobacillus strains to M-1 enzyme and the ability of the strains to induce IL-12 are plotted in Figure 7. A statistically significant negative correlation (r=−0.747, P<0.001) was observed by Pearson's correlation coefficient test between sensitivity to M-1 enzyme and IL-12–inducing ability. These findings suggest that the resistance of lactobacilli to intracellular digestion is a very important factor in determining the ability to induce IL-12, although other unknown factors may also play a role.

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• Figure 7. 

  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.

Loss of IL-12–Inducing Ability in LcS After Lysis of the Cell Wall 

Using LcS, we examined the involvement of cell wall structure in the induction of IL-12. Although LcS was resistant to treatment with lysozyme or a low dose of M-1 enzyme (Figure 5), previous studies showed that extended treatment with a high dose of M-1 enzyme could digest the cell wall of LcS, resulting in the release of soluble PS-PG (Nagaoka et al., 1990). Thus, we treated LcS with 50μg/mL of M-1 enzyme for 10 to 120min, and the degree of digestion was evaluated both in terms of the decrease in OD600 and the reactivity to Giemsa staining. Optical density at 600nm decreased according to the duration of treatment with a high dose of M-1 enzyme and resulted in 80% loss after 120min of treatment (Figure 8A), and the reactivity to Giemsa stain was also reduced accordingly (Figure 8B). The pictures show the generation of protoplasts after lysis of the cell wall by M-1 enzyme. The IL-12–inducing ability of digested LcS was also examined. The IL-12–inducing ability of LcS diminished according to the duration of treatment with the enzyme (Figure 8C), indicating that the cell wall is essential for induction of IL-12.

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• Figure 8. 

  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.

IL-12–Inducing Ability of Cell Wall Components of LcS 

To identify the cell wall components responsible for IL-12 induction, PS-PG and an insoluble cell wall prepa ration having an intact structure (ICW) were also prepared from LcS, and their ability was tested. As shown in Figure 9A, ICW, as well as whole LcS, could induce IL-12 effectively, but soluble PS-PG could not. Elimination of the polysaccharide moiety from ICW by hydrogen fluoride treatment resulted in the loss of IL-12–inducing ability. These results indicate the importance of an intact cell wall structure for LcS to induce IL-12.

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• Figure 9. 

  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.

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Discussion 

Using mouse peritoneal macrophage cultures, 47 Lactobacillus strains belonging to 10 species were tested for the ability to induce IL-12. There were considerable differences in IL-12–inducing ability among the strains tested, and strains belonging to a single species exhibited similar IL-12–inducing abilities. Almost all strains belonging to the L. casei group could potently induce IL-12, and these results were consistent with previous observations showing that some specific strains belonging to the species of L. casei–L. paracasei and L. rhamnosus were strong IL-12 inducers (Miettinen et al., 1998;Hessle et al., 1999;Christensen et al., 2002). These findings suggest that potent IL-12–inducing ability may be conserved in certain species, namely, the L. casei group, to slightly varying degrees among strains belonging to this group.

To characterize the properties important for the induction of IL-12, we examined the phagocytosis and lysis of lactobacilli by macrophages. The pattern of phagocytosis and lysis of lactobacilli could be divided into 3 groups: group R, in which bacteria were gradually phagocytosed and were not easily lysed; group S, in which bacteria were rapidly phagocytosed and lysed; and group I, in which bacteria were gradually phagocytosed and were easily lysed. Potent IL-12–inducing strains, such as LcS and the type strains of L. casei, L. rhamnosus, L. zeae, and L. fermentum, belonged to group R. A negative correlation was revealed between sensitivity of lactobacilli to in vitro digestion with M-1 enzyme and IL-12–inducing ability. In addition, the IL-12–inducing ability of LcS was diminished by digestion of its cell wall. Taken together, these results demonstrate that phagocytosis of lactobacilli and their subsequent digestion are closely related to IL-12 induction, suggesting that the resistance of the cell wall to intra-cellular digestion is one of the critical factors in determining the ability of lactobacilli to induce IL-12.

Some strains were exceptions to the rule that Lactobacillus strains resistant to in vitro treatment with M-1 enzyme have strong IL-12–inducing ability. Lactobacillus casei YIT 0210, L. gasseri YIT 0075, L. johnsonii YIT 0284, L. acidophilus YIT 0200, and L. plantarum IT 0220 were relatively resistant to M-1 enzyme treatment but did not induce large amounts of IL-12 (Figure 6). The following explanation is possible. These strains might have digested in the macrophages, although they were resistant to in vitro M-1 enzyme treatment, as shown in the type strain of L. gasseri (Figures 3, 5). In contrast, L. fermentum YIT 0129 was relatively sensitive to M-1 enzyme treatment but could induce IL-12 effectively (Figure 6). Such an unusual strain may have cell components that can induce IL-12 production even after cell wall digestion within macrophages, or that might show different digestivity in macrophages from that toward M-1 enzyme treatment. Despite these exceptional cases, analyzing the sensitivity of Lactobacillus strains to M-1 enzyme treatment can be considered to provide valuable information on their IL-12–inducing ability.

Our findings can be considered to indicate that the host should activate the innate immune system to clear particular bacteria in the event of difficulty in digesting these bacteria within macrophages. That intracellular pathogenic bacteria such as Listeria monocytogenes induce IL-12 and activate the host immune system is well known (Trinchieri, 1994). These intracellular pathogens can escape from digestion, survive, and multiply in macrophages. On the other hand, lactic acid bacteria, including lactobacilli, are considered safe. Asahara et al. (2003) reported that live LcS, which differs from L. monocytogenes, showed a rapid decrease in viability after phagocytosis by macrophages, probably because of the activity of bactericidal nitric oxide. In addition, we confirmed that the LcS phagocytosed by macrophages was ultimately digested (data not shown). Considering these findings together with the long history of safe use of lactobacilli, it can be said that specific strains of lactobacilli are safe inducers of IL-12.

It is possible that the ability of bacteria to induce IL-12 is due to the skeletal structure of the cell wall. The Lactobacillus cell wall skeleton has peptidoglycan as a major component, polysaccharide covalently bound to peptidoglycan, and lipoteichoic acid extending from the cell membrane (Figure 9B). The present study showed that the intact cell wall structure of LcS was important in inducing IL-12, and that soluble PS-PG released from the LcS cell wall by M-1 enzyme treatment could not induce IL-12. Yasutake et al. (1984) showed that the antitumor activity of LcS was reduced by digestion of the cell wall through treatment with M-1 enzyme. Sato et al. (1988) reported that a cell wall preparation from LcS or another strain of L. casei activated host resistance against listeria, but neither the polysaccharide fraction nor a polysaccharide-depleted peptidoglycan fraction prepared from the cell wall of these strains could effectively enhance anti-infectious activity. These observations are consistent with our results showing that an intact LcS cell wall structure is necessary to induce IL-12. Although the results presented by Sato et al. and by our group indicate the importance of the polysaccharide moiety in the cell wall, the role of the polysaccharide moiety in inducing IL-12 is still unclear. Regarding this issue, we would like to propose 2 possible explanations. One possibility is that the polysaccharide moiety is directly recognized by a corresponding receptor mediating a cytokine response. In this case, large numbers of polysaccharides may need to be present on a certain insoluble matrix such as the peptidoglycan polymer for effective induction of IL-12 (Dixon et al., 2001), because soluble PS-PG could not induce IL-12. Alternatively, the polysaccharide moiety may act indirectly on IL-12 induction by protecting the peptidoglycan structure from enzyme digestion. Zhang et al. (2001) proposed that the polysaccharide moiety of the bacterial cell wall may protect peptidoglycan from enzyme degradation, leading to inflammatory reactions. We also ascertained that polysaccharide-depleted ICW (HF-ICW) could be easily digested by treatment with N-acetylmuramidase (data not shown).

Toll-like receptors are considered key players in the induction of cytokines by microbes and microbial components, and TLR2 and TLR4 are likely to be involved in recognizing the bacterial cell wall components (Takeda and Akira, 2005). The experiments using macrophages deficient in TLR2, TLR4, or MyD88 indicated that MyD88-dependent signaling was indispensable for IL-12 production induced by LcS, but neither TLR2 or TLR4 deficiency reduced IL-12 production. Both these TLR convey signals through MyD88 to produce cytokines, although TLR4 has another signaling pathway independent of MyD88. In recognizing LcS whole cells, stimulatory signals via 2 or more TLR recognizing cell components such as peptidoglycan and cell wall polysaccharides may lead to IL-12 secretion. Accordingly, because of redundancy of the signal via TLR2 and TLR4, our data do not exclude the possibility that TLR2 and TLR4 play a role in recognizing cell wall components of LcS. Alternatively, receptors other than TLR2 and TLR4 may be involved in inducing IL-12 in a MyD88-dependent manner. On the other hand, most studies examining the ligands and functions of TLR have been performed using various soluble cell components purified from bacterial cells, and the research on cell components responsible for IL-12 induction has been rather unsuccessful. Although some soluble bacterial components did induce IL-12, their ability to induce IL-12 was weaker than that of the intact bacterial cells (Cleveland et al., 1996;Huang et al., 1999;Hessle et al., 2000). Dixon et al. (2001) showed that lipopolysaccharide as a part of intact Neisseria meningitides, but not soluble lipopolysaccharide, can induce IL-12 effectively. Underhill et al. (1999) found that phagocytosis of bacteria by macrophages elicits recruitment of TLR2 to phagosomes to recognize bacterial components effectively. Furthermore, Uronen-Hansson et al. (2004) showed that effective induction of IL-12 required TLR2 and TLR4 within dendritic cells to recognize N. meningitides cells after phagocytosis. Our results also indicate that the insoluble ICW of LcS, but not the solubilized cell wall, could induce IL-12 and that the phagocytosing step is indispensable for IL-12 induction. Taken together, these findings could explain why soluble bacterial components show weaker induction of IL-12 than do intact bacterial cells.

Finally, this study demonstrates that particular strains of lactobacilli with resistance to digestion in macrophages can be potent inducers of IL-12. The findings not only provide valuable information on the bacterial characteristics necessary for effective induction of IL-12, but also propose a novel strategy for selecting new probiotic strains with the ability to augment innate immunity. Examining the sensitivity of Lactobacillus strains to N-acetylmuramidase can be useful for primary screening of IL-12–inducing strains. In fact, the well-known probiotic strain LcS is a potent IL-12 inducer with a rigid cell wall that is resistant to treatment with the enzyme. Interestingly, LcS was originally selected based on tolerance to gastric juices, bile acid, and intestinal digestive enzymes rather than on its immunostimulatory activity. The ability to survive gastrointestinal passage is considered to be a special feature of probiotic strains and may result from the rigid structure of the cell wall. If the probiotic feature of survival in the gut correlates with resistance to digestion in macrophages, it may be no wonder that some probiotic Lactobacillus strains are potent immunostimulators.

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Conclusions 

The intact cell wall structure is an important element in the ability of lactobacilli to induce IL-12, and the resistance of the cell wall to intracellular digestion is one of the critical factors in determining the IL-12–inducing ability of lactobacilli. These findings not only provide important information on the bacterial characteristics necessary for effective induction of IL-12, but also present a novel strategy for selecting new immunostimulatory probiotic strains.

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Acknowledgements 

We are grateful to Teruo Yokokura and Koji Nomoto for critical reading of the manuscript, and to Ikuo Kato and Shinichiro Shimada for valuable discussions. We also gratefully acknowledge the staff of the animal facility of the Yakult Central Institute for their expertise in breeding mice.

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Supplementary data 

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References 

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