Fermentation Metabolites from Lactobacillus gasseri and Propionibacterium freudenreichii Exert Bacteriocidal Effects in Mice

Abstract

Lactobacillus gasseri OLL 2716 promotes the elimination of Helicobacter pylori and is utilized in yogurts that are specifically labeled as health foods. On the other hand, milk whey fermented by Propionibacterium freudenreichii ET-3, which increases the numbers of Bifidobacterium, is effective for intestinal disorders. We previously demonstrated that oral administration of L. gasseri and P. freudenreichii fermentation metabolites (LP-FM) improved calf intestinal microflora and reduced the incidence of diarrhea. However, the detailed immunological mechanisms responsible for these effects remain to be fully understood. In this study, we investigated whether LP-FM stimulates the innate immune response and promotes the elimination of Listeria monocytogenes in mice. The C57BL/6 female mice that were treated with LP-FM or L. gasseri fermentation metabolites alone for 4 weeks had more peripheral white blood cells than the untreated control mice. In particular, LP-FM-treated mice had higher CD4- and CD8-positive T-cell counts. The levels of reactive oxygen and nitrogen species produced by peritoneal macrophages were also higher in LP-FM-treated mice. Furthermore, LP-FM-treated mice that were infected with L. monocytogenes exhibited significant enhancement of the elimination of Listeria from the spleen and the liver in comparison with untreated control mice infected with Listeria. The activation of innate immunity by LP-FM was increased by the combination of fermentation metabolites from P. freudenreichii. These results suggest that LP-FM, which contains metabolites from L. gasseri and P. freudenreichii, stimulates the function of the innate immune system, thereby significantly promoting the elimination of L. monocytogenes in mice.

Introduction

F ermented products from beneficial bacteria modulate a variety of immune functions such as an increased resistance against infectious diseases and the attenuation of allergic responses.^1,2 Certain strains of lactic acid bacteria, which are effective probiotics, reduce the incidence of diarrhea,³ prevent infection by pathogenic Escherichia coli (O157),⁴ stimulate the production of specific immunoglobulin A,^4
–6 and activate macrophages.^7

–10

Probiotics, including live bacterial cells, can improve the intestinal microflora and modulate immune functions in beneficial ways.^1,11 Probiotics have also been shown to function as antimicrobial effectors,¹² and oral administration of certain lactic acid bacteria can prevent pathogenic infection by microbes such as Klebsiella pneumoniae, Listeria monocytogenes,¹³ E. coli,¹⁴ and Salmonella enteritidis ¹⁵ through the regulation of inflammatory cytokines. Although the immunomodulatory effects of fermented products have been reported elsewhere,^16

–19 the mechanisms by which cell-free products, i.e., “fermentation metabolites,” stimulate the immune system remains poorly understood.

Lactobacillus gasseri OLL 2716 promotes the elimination of Helicobacter pylori in humans,²⁰ exerts a protective effect against the generation of lesions in a rat gastric ulcer model,²¹ and is utilized in yogurts that are specifically labeled as health foods.²² L. gasseri also has strong immunomodulatory effects via the activation of Toll-like receptor-9 and nuclear factor κB.²³ We previously demonstrated that fermentation metabolites containing nonviable L. gasseri bacteria increased the number of Lactobacillus in the calf intestine while reducing the incidence of diarrhea.^24,25

Propionibacterium freudenreichii ET-3 (milk whey ferment by P. freudenreichii ET-3), which contains bifidogenic growth factor,^26,27 increased the numbers of Bifidobacterium ²⁸ and decreased the numbers of Clostridium in the intestinal microflora of calves.²⁹ Bifidogenic growth factor could potentially be applied for the treatment of ulcerative colitis^26,30 by not only altering the intestinal microflora, but also by modifying the expression of cytokines related to inflammation.³¹

Taken together, these data suggest that these fermentation metabolites act as immune modulators. To further explore this possibility, we fed these fermentation metabolites to mice and examined their immune function and the elimination of Listeria from an infected mouse model. Listeria is an intracellular and Gram-positive bacterium. It is a food-borne pathogen that is ingested via dairy and meat products.³² Listeria has been widely used in experimental studies on infectious immunity in mice. Innate immunity plays an important role in the clearance of Listeria from a host, especially the activation of macrophages.³³ Therefore, we used a Listeria-infected mouse model to evaluate the efficacy of immune stimulation by L. gasseri fermentation metabolites alone (L-FM) or in the presence of P. freudenreichii fermentation metabolites (LP-FM).

Materials and Methods

Animals

Female C57BL/6 mice (5–6 weeks old) were purchased from CLEA Japan Inc. (Tokyo, Japan) and were housed under pathogen-free conditions. In total, 60 mice were divided into two groups: one was for immunological monitoring, and the other was for an infection study. All experiments were carried out according to the Animal Control Guidelines of Rakuno Gakuen University (Hokkaido, Japan).

Preparation of fermentation metabolites

Culture supernatants from L. gasseri OLL 2716 and P. freudenreichii ET-3 were obtained from Meiji Dairies Corp. (Tokyo). They were powdered using dextrin for the preparation of fermentation metabolites; therefore, we used the same dose of dextrin as a control reagent. In total, 30 mice were divided into three groups. The first group of mice was orally administered water containing 5% fermentation metabolite from L. gasseri (L-FM), the second group was administered water containing 5% fermentation metabolites from L. gasseri and 0.2% fermentation metabolites from P. freudenreichii (LP-FM), and the last group was administered water containing 5% dextrin as a control. Each group was given its assigned water solution and a commercial pellet feed ad libitum for 4 weeks, and then we monitored immunological properties.

Analysis of intestinal microflora

The populations of intestinal microflora were monitored according to standard methods.^34,35 In brief, freshly collected individual fecal samples were immediately diluted 10-fold with fecal dilution buffer (33 mM KH₂PO₄, 42 mM Na₂HPO₄, 3 mM l-cysteine hydrochloride anhydrous, 0.05% Tween 80, and 0.1% Bacto™ agar [BD Bioscience, Franklin Lakes, NJ, USA]). Tenfold serial dilutions were prepared, and 50-μL aliquots were spread onto selective agar plates: LBS agar (BD Biosciences) was used for Lactobacillus, BL agar (Nissui, Tokyo) was used for Bifidobacterium, and modified CW agar (Nissui) was used for the detection of Clostridium. LBS, BL, and CW agar plates were incubated anaerobically at 37°C for 48 hours in an anaerobic cultivation jar (AnaeroPack™ rectangular jar, Mitsubishi Gas Chemical Co., Inc., Tokyo). After incubation, colonies were enumerated, and the numbers of colony-forming units (CFU)/mg of feces were determined for the previously mentioned bacteria.

Infection studies

L. monocytogenes was aerobically cultured on PALCAM plates (Merck KGaA, Darmstadt, Germany), a selective media for Listeria, and the bacteria were suspended in phosphate-buffered saline (PBS) at 1.0 × 10⁵ CFU/mL. After 4 weeks of administration of the fermentation metabolites, all groups of mice were subjected to infection with Listeria by intraperitoneal injection (1.0 × 10⁴ CFU per head). To evaluate the bacteriocidal effect in fermentaion metabolites-administered mice, the bacterial count in each mouse was measured 72 hours postinfection. The livers and spleens of the mice were weighed and homogenized in ice-cold PBS. Serial dilutions of homogenates were prepared, and 100 μL of each dilution was plated onto a PALCAM plate and aerobically incubated at 37°C. After a 24-hour incubation, colony numbers were counted, and the number of CFU of Listeria/g in each organ was determined.

Analysis of lymphocyte subsets

To elucidate the effect of fermentation metabolites administration on lymphocyte subsets in mice, CD4 and CD8 populations were analyzed using flow cytometry. A total of 1.0 × 10⁶ lymphocytes isolated from mouse blood and splenocytes were incubated with fluorescein isothiocyanate-conjugated rat anti-mouse CD4 (Beckman Coulter, Fullerton, CA, USA) and phycoerythrin-conjugated rat anti-mouse CD8 (Beckman Coulter) antibodies for 30 minutes at room temperature. After incubation, the cells were washed twice with PBS and fixed in 500 μL of 0.5% formalin in PBS. Flow cytometry was performed on an Epics XL™ flow cytometer (Beckman Coulter), and analysis was conducted using Expo 32 software (Beckman Coulter).

Cytokine gene expression in spleens

To examine the expression of cytokine genes, we used splenocytes isolated from each group of mice. The isolated cells were dissolved in 350 μL of RLT buffer (Qiagen, Hilden, Germany), and total RNA was extracted using an RNeasy^® Mini kit (Qiagen). First-strand cDNA was synthesized from 1 μg of total RNA using an oligo(dT) primer and a Transcriptor First Strand cDNA synthesis kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. Then 1/20th the amount of the synthesized first-strand cDNA was used for polymerase chain reaction (PCR) amplification. Cytokine expression was evaluated using real-time PCR (LightCycler^® 2.0, Roche) using a QuantiTect primer assay kit (Qiagen) for interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and glyceraldehyde 3-phosphate dehydrogenase. These kits include specific primer pairs designed and bioinformatically validated specifically for real-time reverse transcription-PCR. The standard cycling program used for real-time PCR was as follows: denaturation for 15 minutes at 94°C, then 45 cycles of denaturation for 10 seconds at 94°C, annealing for 10 seconds at 60°C, and extension for 10 seconds at 72°C. The expression of each cytokine was normalized according to the expression of the glyceraldehyde 3-phosphate dehydrogenase gene.

Preparation of macrophages

Peritoneal macrophages were collected from each group of mice by washing out the peritoneal cavity with 5 mL of PBS. Macrophages were centrifuged and washed twice with PBS and then cultured or used for the following experiments.

Chemiluminescence analysis of reactive oxygen species production

Peritoneal macrophages and splenocytes from each group of mice were subjected to chemiluminescence analysis as a means of monitoring their production of reactive oxygen species (ROS). Freshly prepared peritoneal macrophages (5.0 × 10⁵ cells) and splenocytes (1.0 × 10⁶ cells) in 100 μL of RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) were first incubated with 400 μL of Hanks buffer (Nissui) with luminol (1.0 × 10⁻⁴ M; Sigma-Aldrich) for 5 minutes and then with fresh plasma and zymosan (925.9 μg/mL) (Sigma-Aldrich) at 37°C in a Luminescencer PSN luminometer (ATTO, Tokyo). The incubation time was 20 minutes for macrophages and 30 minutes for splenocytes. The production of ROS in each cell type was measured in terms of the chemiluminescence counts accumulated during the entire incubation period for that cell type, and the units were designated as relative light units (RLUs).

Nitric oxide production

Peritoneal macrophages isolated from each group of mice were cultured in 24-well plates with 1.0 × 10⁵ cells per well in RPMI-1640 medium supplemented with 10% fetal calf serum at 37°C in an atmosphere of 5% CO₂. After a 3-hour incubation, conditioned media from the macrophage cultures were collected. Nitric oxide (NO) levels in these media were assessed via the measurement of nitrate accumulation using a Griess reagent system (Promega, Madison, WI, USA).³⁶ In brief, 50 μL of conditioned medium was transferred into a 96-well plate, and 50 μL of sulfanilamide solution was added. After a 10-minute incubation, 50 μL of N-(1-naphthylethyl)enediamine dihydrochloride solution was added. The optical density of the samples was measured at 550 nm, and the NO concentrations were determined using a nitrate reference curve from the serial dilution of a positive standard nitrate solution (sodium nitrate).

Statistical analysis

The data were analyzed using analysis of variance-least significant difference, and the results were expressed as mean ± SE values. P values < .05 were considered statistically significant.

Results

Animal conditions and intestinal microflora

There was no difference in the caloric intake or health status among the groups of mice during the administration period. The average body weight of L-FM-treated mice was 20.0 ± 0.2 g, and that of LP-FM-treated mice was 20.5 ± 0.4 g after oral administration; that of control mice was 19.9 ± 0.3 g (data not shown). Fecal bacterial counts were as follows: for Lactobacillus, 9.36 ± 0.30 log CFU/g in L-FM-treated mice, 9.20 ± 0.24 log CFU/g in LP-FM-treated mice, and 9.69 ± 0.37 log CFU/g in control mice; for Bifidobacterium, 4.41 ± 0.25 log CFU/g in L-FM-treated mice, 5.17 ± 0.23 log CFU/g in LP-FM-treated mice, and 4.96 ± 0.17 log CFU/g in control mice; and for Clostridium, 3.48 ± 0.04 log CFU/g in L-FM-treated mice, 3.09 ± 0.10 log CFU/g in LP-FM-treated mice, and 3.81 ± 0.50 log CFU/g in control mice (data not shown). There were no significant differences in fecal microflora between the groups.

Protective effect of the fermentation metabolites against Listeria in mice

Mice were intraperitoneally infected with L. monocytogenes (1.0 × 10⁴ CFU per head), and bacterial loads in the spleen and the liver were measured 72 hours postinfection. Serial dilutions of organ homogenates were plated onto a PALCAM plate. After a 24-hour incubation, colony numbers were counted, and the log CFU of Listeria in each organ was determined. In spleens, the bacterial loads were 4.7 ± 0.2 log CFU per organ in L-FM-treated mice, 3.7 ± 0.5 log CFU per organ in LP-FM-treated mice, and 5.1 ± 0.1 log CFU per organ in control mice. Listeria was significantly eliminated from the LP-FM-treated mice (P < .05, Fig. 1). The bacterial loads in the liver were also decreased by treatment with fermentation metabolites, and it was lower in the LP-FM-treated group (3.2 ± 0.3 log CFU per organ) than in the L-FM-treated group (3.4 ± 0.3 log CFU per organ) or control group (3.8 ± 0.2 log CFU per organ, Fig. 1).

FIG. 1.

Elimination of L. monocytogenes from mice treated with L. gasseri fermentation metabolites alone (L-FM) or in the presence of P. freudenreichii fermentation metabolites (LP-FM). The mice were infected intraperitoneally with 1.0 × 10⁴ L. monocytogenes after a 4-week administration of fermentation metabolites. The bacterial loads in the spleen and liver were evaluated at 3 days postinfection. Data are mean ± SE values for 10 mice per group in repeated experiments. Significantly different from the control group: *P < .05. CFU, colony-forming units.

Population of immune cells in peripheral blood and spleens of mice

To examine the effect of treatment with fermentation metabolites on the immune system, peripheral white blood cells (WBCs) were counted. After a 4-week treatment, L-FM- and LP-FM-treated mice had significantly more peripheral WBCs (2.0 × 10³/μL and 2.3 × 10³/μL, respectively) than control mice (0.7 × 10³/μL) (Fig. 2a). The ratio of blood neutrophils to lymphocytes was also higher in L-FM- and LP-FM-treated mice (0.37 ± 0.06 and 0.29 ± 0.02, respectively) than in control mice (0.21 ± 0.01) (Fig. 2b).

FIG. 2.

Oral administration of fermentation metabolites (L-FM and LP-FM) changes the population of leukocytes in mice. After a 4-week administration of fermentation metabolites, (a) white blood cell (WBC) counts, (b) neutrophil/lymphocyte (N/L) ratio, and (c) CD4- and CD8-positive T-cells in the blood from the mice were examined. Data are mean ± SE values for 10 mice per group in repeated experiments. Significantly different from the control group: *P < .05, **P < .01.

The lymphocyte subpopulations of CD4- and CD8-positive T cells in the peripheral blood in both fermentation metabolites-treated groups were significantly higher than in the control group. The numbers of CD4- and CD8-positive T cells in the L-FM-treated mice were 214 ± 51 cells/μL and 221 ± 54 cells/μL, those in the LP-FM-treated mice were 232 ± 43 cells/μL and 273 ± 54 cells/μL, and those in the control mice were 77 ± 16 cells/μL and 71.5 ± 14 cells/μL, respectively (Fig. 2c).

The ratio of CD4/CD8-positive cells in the blood was 0.97 ± 0.02 and 0.86 ± 0.03 in L-FM-treated and LP-FM-treated mice, respectively, while that of control mice was 1.10 ± 0.08. This ratio was significantly lower in LP-FM-treated mice than in control mice (P < .05). Similar results were observed for the ratio of CD4/CD8-positive cells in the spleens of these mice (data not shown).

Cytokine gene expression in spleen

The gene expression levels of IFN-γ and TNF-α were analyzed in splenocytes from each group of mice. The gene expression levels of both cytokines were detectable using real-time PCR from all samples. Relative to their expression levels in control mice (= 1.0), the level of IFN-γ was 2.7 and 2.0 in L-FM- and LP-FM-treated mice, respectively (Fig. 3a). The level of TNF-α was 1.0 and 2.6 in L-FM- and LP-FM-treated mice, respectively (Fig. 3b). These expression levels were significantly higher in mice treated with fermentation metabolites than in control mice (P < .05). Although the cytokine levels in plasma were determined by Bio-Plex suspension array system (Bio-Rad, Hercules, CA, USA), there was no significant difference between the groups (data not shown).

FIG. 3.

Cytokine gene expression in mice orally administered fermentation metabolites (L-FM and LP-FM). After a 4-week administration of fermentation metabolites, gene expression levels of (a) interferon (IFN)-γ and (b) tumor necrosis factor (TNF)-α in splenocytes were evaluated by using real-time polymerase chain reaction. The expression levels are shown as relative values when the data from the control mice were assigned a value of 1. Data are mean ± SE values for 10 mice per group in repeated experiments. Significantly different from the control group: *P < .05, **P < .01.

Production of ROS and NO in peritoneal macrophages and splenocytes from mice

Peritoneal macrophages from LP-FM-treated mice produced 200.7 ± 90.8 × 10³ RLUs of accumulated ROS in 30 minutes, whereas those from control mice produced 105.8 ± 50.8 × 10³ RLUs. A similar tendency was observed in splenocytes: those from LP-FM-treated mice produced 52.4 ± 5.4 × 10³ RLUs of accumulated ROS in 20 minutes, whereas those from control mice produced 30.4 ± 12.6 × 10³ RLUs.

Peritoneal macrophages from L-FM-treated mice produced NO at 12.7 ± 0.8 μM/10⁴ cells, and those from LP-FM-treated mice produced NO at 16.2 ± 1.1 μM/10⁴ cells, whereas those from control mice produced 8.5 ± 3.0 μM/10⁴ cells. NO production was significantly higher in LP-FM-treated mice than in control mice (P < .05) (Fig. 4).

FIG. 4.

Oral administration of fermentation metabolites (L-FM and LP-FM) increases nitric oxide (NO) production in macrophages. Peritoneal macrophages were isolated after a 4-week administration of fermentation metabolites. The macrophages were examined for NO production by using a Griess reagent system. Data are mean ± SE values for five mice per group. Significantly different from the control group: *P < .05.

Discussion

In this study, we administered two types of cell-free fermentation metabolites to mice in order to examine their effect on immunological responses. Both of the fermentation metabolites increased the number of immune cells and stimulated their function. The level of up-regulation of these immunological functions was different according to the type of fermentation metabolite: LP-FM, the combined fermentation metabolites from L. gasseri and P. freudenreichii, had a stronger effect than L-FM alone. Therefore, it was considered that P. freudenreichii might significantly accelerate the clearance of Listeria.

The oral administration of whey products fermented with live lactic acid bacteria cells increases the number of circulating leukocytes, especially the number of neutrophils, in mice.³⁷ In the present study, we observed the same phenomenon as the number of WBCs in peripheral blood (Fig. 2) and the ratio of neutrophils/leukocytes increased in LP-FM-treated mice. LP-FM administration also increased the numbers of CD4- and CD8-positive cells (Fig. 2). The mechanism underlying the modulation of increasing neutrophils and lymphocyte subpopulations is obscure, but these results suggest that cell-free fermentation metabolites can modulate lymphocyte populations. Thus, the administration of these fermentation metabolites could enhance immunological responses by modulating the numbers of neutrophils and the functions of lymphocytes in peripheral blood.

Many viable lactic acid bacteria have bacteriocidal effects, e.g., the oral administration of Lactobacillus casei has an effect on the elimination of E. coli,^4,14 and Lactobacillus acidophilus has an effect on the elimination of Candida albicans.³⁸ In this study, we also evaluated the bacteriocidal effects associated with the oral administration of fermentation metabolites in mice infected with L. monocytogenes. At 72 hours postinfection, bacterial loads in the spleens and livers from LP-FM-treated mice were markedly lower than those from L-FM-treated mice and control mice (Fig. 1). These results imply that the growth of Listeria might be considerably inhibited in LP-FM-treated mice.

Listeria is an intracellular Gram-positive, food-borne pathogen that is ingested via dairy and meat products,³⁹ and listeriosis is one of the most critical zoonotic diseases in the field of food hygiene. Macrophages play a key role in eliminating intracellular bacteria, such as Listeria, as part of the innate immune response.^32,33 Clearance of and resistance to Listeria require immunological responses from host cells, specifically the secretion of T-helper 1-type cytokines (e.g., IFN-γ) followed by the activation of macrophages that subsequently produce TNF-α and ROS; therefore, the responses generated by host cells enhance the organism's ability to eliminate Listeria.^33,40
–42 Ishida-Fujii et al.¹⁴ reported that the oral administration of L. casei activated macrophages, resulting in an increased survival rate against pathogenic E. coli.

In order to evaluate the response of activated macrophages against microorganisms and the promotion of a bacteriocidal effect, we examined the production of ROS and NO in the mice treated with fermentation metabolites. In peritoneal macrophages, NO production was significantly higher in LP-FM-treated mice (Fig. 4). Similarly, ROS production was higher in LP-FM-treated mice than in control mice. Increases in the production of ROS and NO in macrophages enhance their bacteriocidal ability by promoting the fusion of phagocytes and lysosomes. Therefore, our findings indicate that the administration of LP-FM to mice increased the activation of macrophages, improving their listeriocidal ability.

Cell walls and cytoplasmic extracts from L. acidophilus, Lactobacillus bulgaricus, L. casei, L. gasseri, Lactobacillus helveticus, and Lactobacillus reuteri stimulate cloned macrophages to produce significant amounts of NO.⁸ Therefore, the levels of NO production in macrophages may vary in response to different Lactobacillus species.⁶ NO production by peritoneal macrophages was significantly higher in LP-FM-treated mice. The differences in NO production between the L-FM- and LP-FM-treated mice suggested that the components of P. freudenreichii could affect the degree of NO production, in the presence or absence of L. gasseri.

Discussions related to the immunological effects of dietary supplements are often focused on the expression of cytokines. For example, the expression level of IFN-γ in the spleens of mice increases when the mice are treated with Lactobacillus rhamnosus or L. acidophilus.⁴³ In the present study, likewise, both of the fermentation metabolites (L-FM and LP-FM)-treated mice exhibited increased gene expression of IFN-γ in their spleens, while the expression of TNF-α was markedly higher in LP-FM-treated mice (Fig. 3). Leenen et al. ⁴⁴ showed that IFN-γ and TNF-α were necessary to induce the activation of macrophages to kill Listeria in vitro. Taken together, these results suggest that both of the fermentation metabolites induce the gene expression of IFN-γ following the activation of macrophages. Moreover, LP-FM might induce the strong activation of macrophages that results in the enhanced elimination of Listeria in mice.

In this study, we demonstrated that the administration of two types of fermentation metabolites, in the absence of viable cells, activates immune functions in mice as evidenced by the increase in number of peripheral WBCs, IFN-γ gene expression, and ROS and NO production. The fermentation metabolites containing P. freudenreichii synergistically enhanced the listeriocidal effect in mice. However, it remains to be determined whether P. freudenreichii alone contributes to the elimination of Listeria from infected mice.

Although the fermentation metabolites did not significantly improve the intestinal microflora in mice, these fermentation metabolites play roles in the activation of immune cells without a probiotic pathway. Our research provides another application of fermentation metabolites in health care.

Footnotes

Acknowledgments

We thank Dr. Yutaka Tamura (Rakuno Gakuen University) for providing L. monocytogenes (from the human isolate L.m. #685). This study was partially supported by a grant-in-aid to the High Technological Research Center (Rakuno Gakuen University) and by a grant-in-aid for scientific research (project number S0891002) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Author Disclosure Statement

Y.K-M., T.O., and K.S. are employees of Meiji Feed Co. Ltd. Y.K., M.S., and K.H. declare no competing financial interests.

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