Correspondence Yoshitaka Hirose, Food Science Research Center, House Wellness Foods Corporation, 3-20 Imoji, Itami 664-0011, Japan. Tel: +81 72 778 1127; fax: +81 72 778 0892; email: Hirose_Yoshitaka@house-wf.co.jp
Heat-killed cells of Lactobacillus plantarum L-137 are potent inducers of IL-12 in vitro as well as in vivo and have been shown to have antiallergic, antitumor, and antiviral effects through this induction, which leads to a Th1 type immune response. To determine why L-137 cells induce much greater IL-12 production than the type strain Lactobacillus plantarum JCM1149, we examined the differences in their CW components. The L-137 CW was found to have a higher alanine content and IL-12p40 induction was significantly greater in comparison with JCM1149 CW, whereas peptidoglycans isolated from both strains did not cause IL-12p40 induction. Because in purified CW preparations from gram-positive bacteria, the presence of LTA, the major proinflammatory structure on these bacteria, has been known to have high alanine content, we investigated the responsiveness of both strains to anti-LTA antibody by flow cytometry. L-137 cells reacted more with anti-LTA antibody than did JCM1149 cells. Furthermore, derivative strains of L-137, cured of a specific plasmid pLTK11 of the 15 endogenous plasmids in wild-type L-137, had poor responsiveness to anti-LTA antibody and showed lower IL-12p40 inducing activity than the wild-type L-137 with pLTK11. Our results suggest that LTA expression on the cell surface causes IL-12p40 induction, and that the above internal plasmid of L-137 influences LTA synthesis and expression on the cell surface.
The National Collection of Industrial and Marine Bacteria
Roswell Park Memorial Institute
sodium dodecyl sulfate
T helper 1
Toll-like receptor 2
wall teichoic acid(s)
LAB are the most common type of probiotic microbes. Reports of the beneficial roles of these bacteria in humans and animals have included effects on the immune system (1, 2). In this laboratory, HK-LP, a strain isolated from fermented food, has previously been shown to be a potent inducer of IL-12 in mice in vitro as well as in vivo (3). Administration of HK-LP suppressed not only IgE production against a natural antigen in a mouse model of food allergy, but also inhibited tumor growth in mice transplanted with syngeneic tumor cells (3, 4). Furthermore, it was demonstrated that, in healthy subjects, daily intake of HK-LP enhanced acquired immunity, especially enhancing Th 1-related immune functions, in addition to producing subsequent improvements in the health-related quality of life (5). These effects have been shown to be exerted through IL-12 induction, which leads to a Th1 type immune response (6). Thus, many researchers have focused on the importance of LAB-induced IL-12, and a great deal of evidence has indicated that LAB enhances the Th1 response through IL-12 induction. This ability of LAB to induce IL-12 induction is thus a key point to examine in studying LAB's strong immunoenhancing activity.
A number of studies comparing IL-12 induction ability among Lactobacillus species have shown that some strains of LAB, including L. plantarum, have strong IL-12 induction activity, but the reasons for their specific strong activities have not been clarified at the molecular or genetic level. On the other hand, the molecular mechanisms involved in the induction of cytokines by gram-positive bacteria, including LAB, have been partially characterized. The CW components in gram-positive bacteria, such as PGN, LTA, and unmethylated CpG DNA, have been shown to activate immune cells (7, 8).
TA, a component of the CW of gram-positive bacteria and accounting for >50% of CW dry weight, are known to stimulate the production of inflammatory mediators (9). L. plantarum contains two types of TA: LTA composed of polyglycerophosphate polymers anchored by a glycolipid to the membrane and highly substituted with D-alanyl esters (D-Ala:P ratio of 0.89:1) and, to a minor extent, with glucose (Glc:P ratio of 0.11:1) (10); and WTA consisting of polyribitolphosphates bound covalently to peptidoglycans via a linkage unit (11) and substituted with D-Ala and Glc residues in variable ratios among different Lactobacillus species (12). A number of studies have revealed that bacterial LTA are involved in the activation of innate immune functions (9, 13–18). For example, the extent of D-Ala substitution on LTA has been shown to be an important factor for cytokine induction through the synthesis of LTA substituted with L-Ala instead of D-Ala (15). Furthermore, the role of LTA D-alanylation in the anti-inflammatory properties of the probiotic strain L. plantarum NCIMB8826 has been evaluated (13). The immunomodulating capacity of a dltB mutant strain, L. plantarum NCIMB8826, which is deficient in LTA D-alanylation, was found to be significantly less than that of the parental strain, both in vivo and in vitro (13). These reports suggest that LTA composition is very important to the proinflammatory or anti-inflammatory properties of Lactobacillus cells.
The objective of this study was to investigate why strain L-137 is a more potent IL-12 inducer than the type strain L. plantarum JCM1149, paying particular attention to the phenotypic characteristics of their CW components, particularly LTA. The expression of LTA in both strains was elucidated by examining their reactivity to anti-LTA antibody as detected by flow cytometry. The results indicated that cell surface expression of LTA contributes to effective IL-12p40 induction, and that the plasmids of L-137 influence expression of LTA.
MATERIALS AND METHODS
Bacterial strains and growth media
L. plantarum L-137 was isolated from fermented food (19), and cured plasmid strains were derived from the wild-type strain L-137 by treatment with novobiocin (Sigma-Aldrich, St. Louis, MO, USA) as described by Ruiz-Barba et al. (20). L. plantarum JCM1149 were purchased from the JCM. HK LAB were prepared according to a method previously described (3). MRS medium (Difco, Detroit, MI, USA) was routinely used for cultivation of Lactobacillus strains at 30°C.
Specific pathogen-free female BALB/c mice were purchased from Charles River Japan (Hino, Japan). All experiments were performed with 6–12 wk old mice in accordance with the guidelines of the Animal Care and Use Committee of the House Wellness Foods Corporation.
Preparation of cell wall components
CW and PGN from Lactobacillus strains were prepared as described previously (21). In brief, 2 liters of LAB culture were centrifuged at 12 000 g at 4°C for 20 min, suspended in 160 ml of 0.1 M phosphate buffer (pH 7.2), and the cells disrupted on ice using an ultrasonic generator (Model US-300, Nihonseiki, Tokyo, Japan). Unbroken cells were removed by centrifugation at 5000 g at 4°C for 30 min, the supernatants subjected to high-speed centrifugation at 40 000 g at 4°C for 30 min, and the pellets resuspended in 30 ml of 4% SDS solution and boiled for 40 min. After cooling to room temperature, the solution was pelleted by centrifugation at 40 000 g at 25°C for 30 min and the pellets washed several times with sterilized water and lyophilized. PGN preparations were produced through suspension of CW preparations in 5% TCA, boiling for 20 min, cooling to room temperature, and washing with chloroform and several changes of sterilized water by centrifugation at 16 000 g at 25°C for 10 min to remove TCA. Finally, after successive washings with ethanol and ethyl ether, the pellets were lyophilized. Proteins, nucleic acids and endotoxins were not detected in either CW or PGN preparations.
Amino acid analysis
CW and PGN fractions were hydrolyzed in evacuated, sealed glass tubes using 6 N HCl at 100°C for 15 hr, and the amino acid composition analyzed through a o-phthalaldehyde fluorescence detection method using an amino acid analyzer (Hitachi 835, Hitachi, Tokyo, Japan).
Preparation of dendritic cell enriched fraction from spleen cells
An enriched fraction of splenic DC was isolated from mouse spleen cells as described previously (22). Briefly, spleen cell suspensions were prepared by mechanical dissociation, treated for 30 min with 400 U/ml collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA), and the resulting single-cell suspensions subjected to gradient centrifugation with Percoll (GE Healthcare, Uppsala, Sweden). Cells collected from the interface between the medium and 1.078 g/ml Percoll were then incubated on tissue culture plates for 90 min, non-adherent cells removed by extensive rinsing with medium, and adherent cells cultured overnight. Cells released during the second culture period were harvested and assessed as CD11c positive cells using FITC-conjugated hamster anti-mouse CD11c monoclonal antibody (clone HL3; BD Biosciences Pharmingen, San Diego, CA, USA) and flow cytometry. These cells were assessed at a purity of >70% splenic DC.
Cell culture and cytokine production
Spleen cells and splenic DC were suspended at 2.5 × 106/ml and 2.5 × 105/ml, respectively, in RPMI 1640 medium containing 10% FBS and cultured for 24 hr with or without one of several heat-killed bacteria. In some experiments, bacterial samples were preincubated with mouse anti-LTA monoclonal antibody (1:100, clone 55; HyCult Biotechnology, Uden, The Netherlands) at 37°C for 60 min prior to addition of spleen cells or splenic DC. At the end of the culture period, each supernatant was harvested to determine the cytokine production. IL-12p40 concentrations were determined by a sandwich ELISA, with rat anti-mouse IL-12 monoclonal antibody (clone 15.6; Biolegend, San Diego, CA, USA) used as the capture antibody and biotinylated goat anti-mouse IL-12 polyclonal antibody (R&D systems, Minneapolis, MN, USA) used as the detection antibody.
Western blot analysis
SDS-PAGE and Western blot analyses of cell-associated LTA were performed as described previously (23). Briefly, L. plantarum strains were cultured in MRS broth at 30°C for 18 hr, washed with 0.1 M PBS and the cells collected by centrifugation and suspended in 1 ml of 2% SDS-PAGE sample buffer, after normalization based on the optical density at 650 nm. Samples were heated at 80°C for 20 min, insoluble materials removed by centrifugation at 16 000 g for 20 min, and the supernatant samples subjected to 15% polyacrylamide gel electrophoresis by the method of Laemmli (24), followed by electrotransfer to PVDF membranes. LTA derived from Staphylococcus aureus (Sigma, St. Louis, MO, USA) was used as a positive control. Mouse anti-LTA primary antibody (clone 55) and horseradish peroxidase-linked goat anti-mouse IgG antibody (Anaspec, San Jose, CA, USA) were used at dilutions of 1:2500 and 1:5000, respectively. Immunoreactive LTA was detected with 1-Step TMB-Blotting (Pierce, Rockford, IL, USA), according to the manufacturer's instructions.
Analysis of surface LTA expression in L. plantarum by flow cytometer
HK LAB were suspended at a concentration of 5 μg/ml in PBS containing 2% FBS for 60min in the presence of 1:250 diluted rabbit anti-LTA antibody (clone 55), which can recognize L. plantarum LTA composed of polyglycerophosphate but does not recognize L. plantarum WTA composed of polyribitolphosphate. After washes with PBS containing 2% FBS, samples were probed with FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at dilutions of 1:250 at room temperature for 60 min, and analyzed using an EPICS XL ADC flow cytometer with EXPO32 software (Beckman Coulter, Miami, FL, USA).
Statistical differences of IL-12p40 production between L-137 and JCM1149 (Fig. 1 and Fig. 2) were analyzed by Student's unpaired t-test. Another statistical analysis (Fig. 4 and Fig. 5b) was performed with ANOVA, followed by Tukey-Kramer multiple comparisons test for comparison between groups using Statcel2 software (OMS Publishing, Tokorozawa, Japan). A probability of P < 0.05 was considered to be significant.
IL-12p40 induction by HK L. plantarum L-137 and JCM1149
IL-12p40 induction was examined in mouse spleen cells and splenic DC stimulated by HK L. plantarum L-137 (HK-LP) or type strain JCM1149 (HK-JCM1149) (Fig. 1). The HK-LP cells induced greater IL-12p40 production than the HK-JCM1149 cells. In addition, IFN-γ production in mouse spleen cells stimulated by HK-LP cells was greater than that induced by HK- JCM1149 cells (data not shown).
IL-12p40 induction by cell wall components
Many studies have shown that bacterial cell wall components induce inflammatory cytokines from mouse macrophages and bone marrow-derived DC (8, 25). The question of why L-137 is a more potent IL-12 inducer than JCM1149 was investigated by examining the IL-12p40-inducing activity of CW and PGN prepared from each strain in mouse spleen cells. IL-12p40 induction by L-137 CW was greater than by JCM1149 CW, whereas PGN from both strains did not induce IL-12p40 (Fig. 2). Amino acid composition analyses of each preparation confirmed their purity, the PGN amino acid composition being consistent with previous reports. Specifically, L. plantarum PGN consisted of Glu, Ala, and DAP at a molar ratio of about 1:1.5–2:1, respectively (26). However, the Ala content of L-137 CW was higher than in JCM1149 (Table 1), which suggests that the molecular nature of L-137 CW is different from that of JCM1149 CW.
Table 1. Amino acid composition of CW and PGN preparations from Lactobacillus strains
Molar ratio of amino acids
Analyses of LTA in L. plantarum using Western blot and flow cytometry
As the presence of LTA or WTA in purified CW preparations in gram-positive bacteria is known to have a high D-Ala/DAP ratio (27), it was considered that there might be phenotypic differences in LTA or WTA characteristics between the strains. This hypothesis was verified by investigating the reactivity of LTA from both strains to anti-LTA antibody using Western blot and flow cytometry. Cell-associated LTA extracted with SDS from both strains were detected in the molecular mass range of 20–45 kDa on SDS-PAGE gel (Fig. 3a). In comparison, HK-LP cells reacted more with anti-LTA antibody than did HK-JCM1149 (Fig. 3b), which suggests that LTA exists in both strains, but the chain lengths of polyglycerophosphate polymers or quantities of LTA are different between these strains.
Inhibitory effects of anti-LTA antibody on IL-12p40 induction by HK L. plantarum L-137
As L-137 cells showed higher reactivity to anti-LTA antibody, spleen cells stimulated by both strains with or without anti-LTA antibody were examined to investigate whether anti-LTA antibody affects IL-12 induction. The results showed that pretreatment with anti-LTA antibody partially inhibits IL-12p40 induction stimulated by HK-LP cells, but not that stimulated by HK-JCM1149 (Fig. 4).
Involvement of endogenous plasmid DNA in L-137 in responsiveness to anti-LTA antibody
Strain L-137 contains 15 endogenous plasmid DNA, and we previously obtained derivative strains which were cured of plasmids from the wild-type strain L-137 (28, 29). The possible involvement of internal plasmids in responsiveness to anti-LTA antibody was investigated by examination of the reactivity of L-137 derivatives to anti-LTA antibody as observed by flow cytometry. The cells of derivative strain L. plantarum H2–5, which contains 11 plasmids (pLTK1, pLTK2, pLTK3, pLTK4, pLTK6, pLTK8, pLTK9, pLTK10, pLTK11, pLTK13, and pLTK14), reacted more with anti-LTA antibody than did cells of L. plantarum NC14, whose plasmid pLTK11 has been deleted from derivative strain H2–5 (Fig. 5a). Another strain, L. plantarum NCL21-1, containing five plasmids (pLTK1, pLTK2, pLTK6, pLTK8, and pLTK11), also showed higher responsiveness to anti-LTA antibody than did L. plantarum NCL21-2, whose plasmid has been deleted from derivative strain NCL21-1 (Fig. 5a). These results suggest that the endogenous plasmid pLTK11 may be associated with cell surface expression of LTA, resulting in enhanced responsiveness to anti-LTA antibody.
Involvement of endogenous plasmid DNA in L-137 in IL-12p40 induction
As cell surface expression of LTA was considered a possible factor for high IL-12p40 inducing activity in L-137, IL-12p40 induction was assessed in spleen cells stimulated by derivative strains whose responsiveness to anti-LTA antibody were different. The cells of strain H2–5 and NCL21-1, which possess plasmid pLTK11 and show higher reactivity with anti-LTA antibody, showed significantly greater IL-12p40 induction than did those of NC14 and NCL21-2 (Fig. 5b) These findings suggest that IL-12p40 inducing activity of L-137 may be attributed to plasmid pLTK11, which may carrysome putative genes related to the synthesis of LTA.
In the present study, the importance of the cell surface expression of LTA was established. It was also shown that this expression, which leads to IL-12p40 induction in mouse spleen and splenic dendritic cells, may be attributable to putative LTA-synthesis related genes in an internal plasmid pLTK11 of L. plantarum L-137.
IL-12 is a heterodimeric cytokine composed of two subunits, p35 and p40. We have previously demonstrated that IL-12p40 induced by L. plantarum closely correlates with biological effects of IL-12, such as IFN-γ induction in vitro (3) as well as anti-tumor effect in vivo (4). When we examined the relationship between IL-12p40 production and IFN-γ production in all experiments as described in the results (Fig. 1, data not shown), we also found strong correlations between them. On the other hand, IL-12p40 subunit is shared by IL-23 which has its own unique light chain (p19). We measured IL-23 in some experiments, but could detect no production of IL-23 from mouse spleen cells stimulated by L. plantarum. Therefore the IL-12p40 detected in this study was considered to reflect bioactive IL-12.
HK cells of L-137 were initially shown to induce significantly greater IL-12p40 production than JCM1149 cells, a type strain L. plantarum, in mouse spleen cells (Fig. 1a). Strain L-137 was considered to act on dendritic cells because differences in IL-12p40-inducing activity was not observed between these two strains in mouse peritoneal macrophage and macrophage-derived cell lines (data not shown) but was observed in splenic DC (Fig. 1b).
Components of CW in gram-positive bacteria, such as PGN and LTA, or unmethylated CpG DNA have been shown to activate immune cells (7, 8). CW derived from L-137 was shown here to induce IL-12p40 production more strongly than JCM1149 CW, but PGN from both strains showed no IL-12p40 induction (Fig. 2). As the D-Ala/DAP ratios of L-137 CW are higher than those of JCM1149 (Table 1), it was considered that L-137 CW contains high proportions of LTA, which is known to induce production of inflammatory cytokines. These results correlate with previous observations that contamination of LTA in CW preparations from gram-positive bacteria contributes to the stimulation of TLR2, but highly purified PGN does not stimulate TLR2 (27).
LTA in LAB are cell membrane-bound polyglycerophosphate polymers anchored by a glycolipid and highly substituted with D-alanyl esters. A number of studies have revealed that bacterial LTA are involved in cytokine production, but the mechanisms of cytokine induction are not clear (30). Here, we confirmed that L-137 LTA is highly expressed on cell surfaces (Fig. 3b) and that the IL-12p40 inducing activity of L-137, but not of JCM1149, is partially neutralized by addition of anti-LTA antibody (Fig. 4). These results suggest that cell surface expression of LTA effectively induces IL-12p40 production. Several studies (16, 17, 31, 32), including an investigation employing chemically synthesized LTA analogs (15), have reported that LTA is a highly potent TLR2 ligand. In contrast, other researchers have proposed that the critical factor for gram-positive bacterial inflammation is not LTA, but lipoproteins which contaminate LTA preparations (33, 34). These conflicting observations could be explained by differences in the preparation procedures leading to differences in contaminating endotoxin and lipoprotein, structural damage to acyl residues, and loss of D-Ala (14, 33, 35, 36). In the present study, structural damage and contamination had little impact on the results because of the use of whole cells and an anti-LTA antibody which has been shown to specifically recognize LTA polyglycerophosphate residues (37).
The role of LTA D-alanylation in the anti-inflammatory properties of the probiotic strain L. plantarum NCIMB8826 has been described, including the observation that the composition of LTA in L. plantarum whole cells modulates proinflammatory or anti-inflammatory immune responses (13). As the D-Ala/DAP ratio of L-137 CW preparations is greater than that of JCM1149 CW preparations, D-Ala substitution of L-137 LTA may occur at greater frequencies, resulting in strong IL-12p40 inducing activities. On the other hand, many studies have indicated that the number and position of acyl chains of LTA is very important to the recognition of LTA through TLR2 (18). Previous reports have suggested that the existence of LTA D-Ala plays a role in environmental interactions, probably by modulating the net negative charge of the bacterial cell surface and, therefore, may be involved in the pathogenesis of this organism (38–40). Because microscopically enhanced adhesion of L-137 to macrophage/dendritic cells compared with JCM1149 was observed here (data not shown), the significantly greater expression of LTA and larger D-Ala/DAP ratio of L-137 LTA was implicated in these cells’ adhesion to immune cells. In the future, after purification of LTA from both strains, detailed descriptions of these structures need to be determined and explored in order to verify these hypotheses.
We next illustrated that the specific plasmid pLTK11, an L-137 internal plasmid, affects cell surface expression of LTA and induction activity of IL-12p40, as detected by Western blot and flow cytometry using derivative strains (Fig. 5). These results suggest that pLTK11 may contain genes encoding enzymes related to LTA production, such as synthesis (37), extension (37), D-alanylation (13, 41–43), or expression of polyglycerophosphate chains. Complete mapping of the L-137 genome and studies of the relevant DNA sequences are required in future studies of these genes.
In conclusion, our present study demonstrates that cell surface expression of LTA affects strong IL-12p40 induction in L. plantarum L-137, and that L-137 internal plasmids influence expression of LTA. These results suggest that expression of LTA on cell surfaces is a key factor in IL-12p40 induction by Lactobacillus strains, and that analyses of LTA components on cell surfaces are useful in screening Lactobacillus strains for potential IL-12 induction activity.
This research was supported in part by a grant program “Collaborative Development of Innovative Seeds” from the Japan Science and Technology Agency. The authors are grateful to Dr. Hiroshi Yamamoto, Osaka University, for critical comments on this work.