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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.
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.
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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.