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. 2004 Jul;11(4):675–679. doi: 10.1128/CDLI.11.4.675-679.2004

Reduction of Influenza Virus Titer and Protection against Influenza Virus Infection in Infant Mice Fed Lactobacillus casei Shirota

Hisako Yasui 1,*, Junko Kiyoshima 1, Tetsuji Hori 1
PMCID: PMC440622  PMID: 15242940

Abstract

We investigated whether oral administration of Lactobacillus casei strain Shirota to neonatal and infant mice ameliorates influenza virus (IFV) infection in the upper respiratory tract and protects against influenza infection. In a model of upper respiratory IFV infection, the titer of virus in the nasal washings of infant mice administered L. casei Shirota (L. casei Shirota group) was significantly (P < 0.05) lower than that in infant mice administered saline (control group) (102.48 ± 100.31 and 102.78 ± 100.4, respectively). Further, the survival rate of the L. casei Shirota group was significantly (P < 0.05) higher than that of the control group (14.3 versus 40.0%). One day after infection, pulmonary NK cell activity and interleukin-12 production by mediastinal lymph node cells of mice in the L. casei Shirota group were significantly greater than those of mice in the control group. These findings suggest that oral administration of L. casei Shirota activates the immature immune system of neonatal and infant mice and protects against IFV infection. Therefore, oral administration of L. casei Shirota may accelerate the innate immune response of the respiratory tract and protect against various respiratory infections in neonates, infants, and children, a high risk group for viral and bacterial infections.

Influenza is an acute viral respiratory infection that results in high morbidity and significant mortality (3, 10). In particular, influenza infection sometimes causes pneumonia in older adults and encephalitis or encephalopathy in infants and children (9). It is important to reduce excess influenza deaths in older adults, infants, and children. It has been reported that, since the immune response, particularly cellular immunity, declines in older adults and the immune system in infants and children is immature, these individuals have increased susceptibility to influenza virus (IFV) infection.

Lactic acid bacteria and their products have beneficial health effects on host homeostasis, including activation of the immune system (7, 8). Lactobacillus casei strain Shirota, a member of the lactic acid bacteria, was originally isolated from the human intestine and has been used commercially for a long time to produce fermented milk. Various aspects of the effects of L. casei Shirota have been studied intensively. L. casei Shirota exhibits marked antitumor activity against transplantable tumors and 3-methylcholanthrene-induced tumors (17, 23) and anti-infectious activity against various pathogens such as Listeria monocytogenes and herpes simplex virus (20, 28). In a human study, it was demonstrated that oral administration of L. casei Shirota to surgical patients suppressed the recurrence of superficial bladder cancer in a double-blind clinical trial (2). Further, we have previously reported that oral administration of L. casei Shirota to aged mice activates their weakened cellular immune system and reduces the nasal IFV titer in a model of upper respiratory IFV infection.

The purpose of the present study was to investigate whether oral administration of L. casei Shirota to neonatal and infant mice, whose immune system is immature, activates the immune system of the respiratory tract and whether it ameliorates IFV infection in the upper respiratory tract and protects against IFV infection. Special attention was focused on the possibility of inhibiting IFV infection in infants and children through oral administration of L. casei Shirota.

MATERIALS AND METHODS

Mice.

Pregnant BALB/c mice were obtained from CLEA Japan (Tokyo, Japan), and their 2-day-old (male and female) and 2-, 3-, 5-, 7-, and 13-week-old (female) offspring were used for the experiments.

L. casei Shirota.

L. casei Shirota was originally isolated from human feces at Yakult Central Institute for Microbiological Research (Tokyo, Japan). L. casei Shirota cells were cultured for 24 h at 37°C in MRS broth (DIFCO Laboratories, Detroit, Mich.), collected by centrifugation, washed several times with sterile saline, and used for oral administration to neonatal and infant mice.

Virus.

Influenza A/PR/8/34 (PR8, H1N1) virus was grown in the allantoic sacs of 11-day-old chicken embryos for 2 days at 34°C by a previously reported method (30). The allantoic fluid was removed and stored at −80°C. The titer of virus in allantoic fluid was expressed as the 50% egg-infecting dose (EID50) (29). Serial 10-fold dilutions of allantoic fluid were injected into five embryonated eggs, and the presence of virus in the allantoic fluid of each egg was determined on the basis of hemagglutinating capacity 2 days after injection. The titer of virus of allantoic fluid was 109.2 EID50/ml.

Virus infection.

Lower respiratory infection was induced by dropping 10 μl of fluid containing 103.5 EID50 of PR8 into each nostril (20 μl per mouse), and upper respiratory infection was induced by dropping 1 μl of fluid containing 103.5 EID50 of PR8 into each nostril (2 μl per mouse) after the mice were anesthetized by intraperitoneal injection of sodium amobarbital (0.25 μg per mouse) (26).

Population of DX5+ cells and NK activity in pulmonary cells.

Three- and 13-week-old mice and L. casei Shirota- or saline-administered 3-week-old mice were anesthetized with diethyl ether and killed by exsanguination. The lungs were removed, minced finely, and incubated for 90 min with 150 U of collagenase (Yakult Honsha Co., Tokyo, Japan) in 15 ml of medium. To dissociate tissue into single cells, collagenase-treated lungs were gently tapped in a plastic dish. After removal of debris, erythrocytes were depleted by hypotonic lysis. The cells were washed with RPMI 1640 medium (Sigma) supplemented with 100 U of penicillin/ml and 100 μg of streptomycin/ml and then resuspended in medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (14).

These cells were stained with biotin-labeled anti-mouse pan-NK cell antibody (DX5; PharMingen, San Diego, Calif.) for 30 min at 4°C and then reacted with phycoerythrin-labeled streptavidin (PharMingen). The rate of DX5+ cells was measured with a flow cytometer (Epics Altra; Beckman Coulter).

Appropriate numbers of cells from the lungs were added to 2 × 104 51Cr-labeled YAC-1 cells in 96-half-area-well microtiter plates (Corning, Corning, N.Y.) in a total volume of 0.1 ml of medium containing 10% FCS. The plates were gently centrifuged for 5 min at 50 × g and then incubated for 4 h at 37°C in 5% CO2. After incubation, the plates were centrifuged for 5 min at 50 × g, and a 50-μl sample was removed from each well for gamma scintillation counting (14). The percentage of specific release of 51Cr was calculated with the following formula (cpm, counts per minute):

graphic file with name M1.gif

Protocol.

Neonatal and infant mice were given live L. casei Shirota cells (≒108.6 CFU/mouse) or saline via a stomach tube 5 times/week for about 3 weeks (total, 17 times), from 2 to 24 days old, before induction of upper respiratory infection. The recovery of live L. casei Shirota was 107.8 CFU/g of feces. One day after infection, the NK activity of the lung and interleukin-12 (IL-12) production by the mediastinal lymph nodes (MLN) in infant mice were measured. Further, 3 days after induction of upper respiratory infection, the viral titer of nasal washings was measured. At the same time, 20 μg of phosphate-buffered saline (PBS) was administered intranasally to disseminate the increased virus from the nasal cavity to the lower respiratory tract and the accumulated symptom rate and survival rate of the mice were determined (Fig. 1) (13). The symptoms monitored were erect hair and crouch.

FIG. 1.

FIG. 1.

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Protocol for administering L. casei Shirota (LcS) to neonatal and infant mice.

Viral titer of nasal washings.

Mice were anesthetized with diethyl ether and killed by exsanguination. The head of the mouse was removed, and the lower jaw was cut off. A syringe needle was inserted into the posterior opening of the nasopharynx, and a total of 1 ml of PBS containing 0.1% bovine serum albumin was injected. This procedure was repeated three times to collect the outflow as nasal washings. The nasal washings were centrifuged to remove cellular debris and used for the determination of the viral titer (27). The viral titer of each specimen, expressed as the EID50, was calculated from the lowest dilutions for eggs with virus. The viral titer in each experimental group was presented as the mean ± standard deviation of the viral titer of each washing specimen from all mice in each group.

IL-12 production of MLN cells.

Mice were anesthetized with diethyl ether and killed by exsanguination. MLN were aseptically removed and isolated as a single cell suspension. After depletion of erythrocytes, the single cell suspension (2.5 × 105) was cultured in the absence or presence of concanavalin A (ConA; Sigma) at 2 μg/ml in 0.1 ml of RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U of penicillin/ml, and 100 μg of streptomycin/ml in a 96-half-area-well culture plate (Corning). Supernatants were collected on day 3 for IL-12 determination and stored at −80°C for further analysis. Determination of IL-12 was performed by sandwich enzyme-linked immunosorbent assay. Rat anti-mouse IL-12 monoclonal antibody (C15.6; PharMingen) was used as the capture antibody, and biotinylated rat anti-mouse IL-12 monoclonal antibody (clone C17.8; PharMingen) was used as the detection antibody (13). Standard recombinant mouse IL-12 was purchased from PharMingen.

Statistical analyses.

Comparisons of NK cell activity, DX5+ cell rate, IL-12 production, and viral titer were performed by means of Student's t test. The differences in survival rate and accumulated symptom rate were examined by means of the Cox-Mantel test. Probability values of less than 5% were considered significant.

RESULTS

Age dependence of influenza infection in BALB/c mice after nasal inoculation with IFV.

The 2-, 3-, 5-, 7-, and 13-week-old mice were inoculated nasally with a single IFV dose of 103.5 EID50/20 μl, and the survival rate was monitored every day after inoculation. As shown in Fig. 2, all 2- and 3-week-old mice died (survival rate, 0%), the survival rate of 5-week-old mice was 40%, and that of 7- and 13-week-old mice was 70%. Therefore, infant mice were more susceptible than adult mice to IFV infection.

FIG. 2.

FIG. 2.

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Age-related survival rate of BALB/c mice after inoculation of lower respiratory tract with IFV (PR8). Mouse age groups (n = 5): open circle, 2 weeks old; filled square, 3 weeks old; dotted circle, 5 weeks old; filled circle, 7 weeks old; open square, 13 weeks old.

Changes in pulmonary NK cells with age.

In the next study, we observed differences in the population of DX5+ cells and NK activity in pulmonary cells between 3- and 13-week-old mice. The population of DX5+ cells and the NK activity of pulmonary cells in 3-week-old mice were significantly lower than those in 13-week-old mice (Fig. 3). These findings suggested that the innate immune system, specifically NK activity, of infant mice was immature compared to that of adult mice.

FIG. 3.

FIG. 3.

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Changes in pulmonary NK cells with age. Pulmonary cells were prepared from 3-week-old mice (n = 3) (black bar and filled circles) and 13-week-old mice (n = 3) (white bar and open circles). (A) The cells were stained with biotin-labeled anti-mouse pan-NK cell antibody, and the proportion of DX5+ cells was measured with a flow cytometer. (B) NK activity in these cells was examined by using a short-term 51Cr-release assay. Each bar and circle represent the mean ± standard deviation for the results from 3 mice. The double and single asterisks represent statistically significant differences from the control value by Student's t test at P values of <0.01 and <0.05, respectively. E:T ratio, effector-to-target cell ratio; wks, weeks.

Effect of oral administration of L. casei Shirota on viral titer in nasal washings of mice inoculated nasally with IFV.

We investigated the effect of oral administration of L. casei Shirota on the viral titers of infant mice inoculated with IFV. As shown in Fig. 4, the viral titers in the control and L. casei Shirota groups were 102.78 ± 100.40 and 102.48 ± 100.31, respectively. The viral titer in the L. casei Shirota group was significantly (P < 0.05) lower than that in the control group.

FIG. 4.

FIG. 4.

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Effect of oral administration of L. casei Shirota (LcS) on viral titer in nasal washings. Neonatal mice were administered L. casei Shirota (○) or saline (•) orally for 3 weeks. Then these mice were intranasally infected with PR8, and 3 days later, the viral titer in nasal washings from the infected mice was measured. Each bar represents the mean value for the results from 18 or 16 mice, respectively. The asterisk represents a statistically significant difference from the control value by Student's t test at a P value of <0.05.

Effect of oral administration of L. casei Shirota on accumulated symptom rate and survival rate of mice inoculated nasally with IFV.

We investigated whether oral administration of L. casei Shirota for 3 weeks in neonatal and infant mice affects the accumulated symptom rate and survival rate of mice infected nasally with IFV, by using a method to disseminate the virus from the nasal cavity to the lower respiratory tract. The accumulated symptom rate and survival rate in the L. casei Shirota group were significantly lower (73.3 versus 92.9%; P < 0.05) and higher (40.0 versus 14.3%; P < 0.05) than those in the control group, respectively (Fig. 5).

FIG. 5.

FIG. 5.

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Protection against morbidity and mortality due to IFV infection in mice administered L. casei Shirota orally. Mice administered L. casei Shirota (○) (n = 15) or saline (•) (n = 14) orally were inoculated with 2 μl of fluid containing 103.5 EID50 of PR8, and then PBS was administered intranasally at 3 days postinfection. These mice were observed for 17 days to assess the accumulated symptom rate and survival rate. The asterisk represents a statistically significant difference from the control by the Cox-Mantel test at a P value of <0.05.

Effect of oral administration of L. casei Shirota on pulmonary NK cell activity.

We investigated the difference in pulmonary NK cell activity 1 day after IFV infection between the control and L. casei Shirota groups. NK cell activity in the control and L. casei Shirota groups at an effector-to-target cell ratio of 6.25:1 was 100.42 ± 100.02 and 101.02 ± 100.44, respectively, with a significant difference between the groups (Fig. 6).

FIG. 6.

FIG. 6.

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Effect of oral administration of L. casei Shirota (LcS) on pulmonary NK cell activity. One day after viral inoculation, pulmonary cells from mice administered L. casei Shirota (○) (n = 6) or saline (•) (n = 5) were prepared and their NK activity was determined by using a short-term 51Cr-release assay. The effector-to-target cell ratio was 6.25:1. The asterisk represents a statistically significant difference from the control (Cont) value by Student's t test at a P value of <0.05.

Effect of oral administration of L. casei Shirota on production of IL-12 by MLN cells.

We measured IL-12 production by MLN in the control and L. casei Shirota groups 1 day after infection with IFV cultured in the absence or presence of ConA. In spite of the addition of ConA, the mean IL-12 concentration in the L. casei Shirota group was significantly higher than that in the control group (Fig. 7).

FIG. 7.

FIG. 7.

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Effect of oral administration of L. casei Shirota on production of IL-12 by MLN. One day after viral inoculation, MLN cells from mice administered L. casei Shirota (white bars) (n = 6) or saline (black bars) (n = 5) were prepared and cultured in the absence (−) or presence (+) of ConA. The collection of supernatants and determination of IL-12 were performed. The triple and single asterisks represent statistically significant differences from the control by Student's t test at P values of <0.01 and <0.05, respectively.

DISCUSSION

As the immune system in infancy and childhood is immature and immune function low, infants and children belong to one of the high risk groups for respiratory infection, as they do for diarrheal disease (9). We found that all 2- and 3-week-old mice, some 5-week-old mice, and a few 7- and 13-week-old mice died after lower respiratory infection with IFV. Comparing innate immunity, specifically the NK property, of 3- and 13-week-old mice, the number of NK cells and NK activity of pulmonary cells in 3-week-old mice were significantly lower than those in 13-week-old mice. Therefore, it was shown that mouse pups are a suitable animal model of IFV infection in the neonatal and childhood periods.

It has been found that L. casei Shirota administered orally augmented NK cell activity of splenocytes and pulmonary cells in aged mice (14, 15). Takagi et al. (24) also reported augmentation of NK cell activity in splenocytes by oral administration of L. casei Shirota in tumor-bearing mice. In a human study, Nagao et al. (19) reported that oral administration of L. casei Shirota enhanced NK cell activity of peripheral blood mononuclear cells in individuals with low levels of NK. These results suggest that L. casei Shirota normalizes declining cellular immunity in mice and humans. In this study, we demonstrated that L. casei Shirota administered orally augmented the activity of NK cells in the lungs and enhanced innate immunity in the respiratory tract.

It has been suggested that both humoral and cellular immunity are important for protection against IFV infection (4, 6, 30). We have been studying two strains of lactic acid bacteria which modulate different immune systems, each in its own way (31). One strain is Bifidobacterium breve YIT 4064, which enhances adaptive humoral immunity. It was previously reported that oral administration of this strain augmented production of antigen-specific immunoglobulin G in serum and protected against IFV infection in mice (30). The other strain is L. casei Shirota, which enhances innate cellular immunity. It was previously reported that intranasal administration of this strain enhanced cellular immunity in the respiratory tract and protected against IFV infection in mice (13), and oral administration of L. casei Shirota activated not only the systemic immune system but also the local immune system and ameliorated IFV infection in aged mice (14).

Influenza is a respiratory tract disease, and it is important to enhance local cellular immunity in the respiratory tract for protection against this infection (22). In this study, we also found that L. casei Shirota administered orally enhanced IL-12 production of MLN and NK activity in the lung. It has been reported that MLN cells in the respiratory tract play an important role in preventing IFV infection (1, 11). Cytokines are important in the development of various immune cells. IL-12 potently stimulates cytotoxic T cells and NK cells and enhances the production of Th1 cytokines and the proliferation of Th1 cells. Shida et al. reported that L. casei Shirota induces IL-12 production in splenocytes cultured in vitro (21a). IL-12 is secreted by B cells, dendritic cells, and macrophages. Kato et al. reported that L. casei Shirota activates X-ray-irradiated splenocytes, probably macrophages, and that these cells secreted IL-12 (16).

In this study, we observed a significant decrease in the titer of IFV in the respiratory tract of mice administered L. casei Shirota. Furthermore, we showed that the survival rate of mice administered L. casei Shirota was significantly higher than that of mice administered saline, with a murine model of IFV infection in which virus moves from the upper respiratory tract to the lower respiratory tract (13). It is assumed that the possible mechanism of protection against IFV infection by oral administration of L. casei Shirota is activation of the innate immune response by IL-12, particularly NK cells, in the respiratory tract. Further, it was reported that IFV infection depressed IL-2 production (5) and significantly reduced NK cell activity (21a). Although the mechanism by which IFV induces immunosuppression is not completely understood, there is evidence that induction of an immunosuppressed state plays a role in the pathogenesis of the disease (18). It can be speculated that oral administration of L. casei Shirota inhibited the decrease of cellular immunity after IFV infection. Regarding this point, further study is needed.

The mechanism inhibiting the decrease of pulmonary cellular immunity after IFV infection upon oral administration of L. casei Shirota is not clear, but there has been a study to determine the route by which L. casei Shirota is recognized by host immune systems (25). L. casei Shirota administered orally was taken up by M cells in Peyer's patches and was hardly taken up by epithelial cells on the lamina propria of the intestine. Therefore, it was presumed that L. casei Shirota was taken up by M cells in Peyer's patches and stimulated macrophages, T cells, NK cells, and other cells, and these cells migrated to mesenteric lymph nodes and then migrated via the thoracic duct and bloodstream to the spleen, lungs, and MLN.

Recently, it has been reported that one of the lactobacilli, Lactobacillus GG, reduced respiratory infections and their severity in children (12), but the mechanism is not clear. It is expected that L. casei Shirota activates innate immune responses and protects against respiratory infection in children.

Acknowledgments

We thank S. Tamura and T. Kurata (National Institute of Health, Tokyo, Japan) for help with the study.

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