Effect of intranasal administration of Lactobacillus casei Shirota on influenza virus infection of upper respiratory tract in mice

Abstract

In mice administered Lactobacillus casei strain Shirota (LcS) intranasally, potent induction of interleukin 12, gamma interferon, and tumor necrosis factor alpha, which play a very important role in excluding influenza virus (IFV), was evident in mediastinal lymph node cells. In this model of upper respiratory IFV infection, the titers of virus in the nasal wash of mice inoculated with 200 microg of LcS for three consecutive days (LcS 200 group) before infection were significantly (P < 0.01) lower than those of mice not inoculated with LcS (control group) (10(0.9 +/- 0.6) versus 10(2.1 +/- 1.0)). The IFV titer was decreased to about 1/10 of the control level. Using this infection model with modifications, we investigated whether the survival rate of mice was increased by intranasal administration of LcS. The survival rate of the mice in the LcS 200 group was significantly (P < 0.05) greater than that of the mice in the control group (69% versus 15%). It seems that the decrease in the titer of virus in the upper respiratory tract to 1/10 of the control level was important in preventing death. These findings suggest that intranasal administration of LcS enhances cellular immunity in the respiratory tract and protects against influenza virus infection.

Figures

FIG. 1

Effect of intranasal administration of LcS to mice on production of IL-12, IFN-γ, TNF-α and IL-4 by MLN cells. MLN cells from mice administered LcS 0 (control) (□), LcS 20 (), or LcS 200 (■) intranasally were cultured in the absence (−) or presence (+) of ConA. Supernatants were collected 72 h after the initiation of culture, and the concentrations of various cytokines were determined. Each bar represents the mean ± SD of triplicate samples. Double asterisk, statistically significant difference from the control by Dunnett's test at P < 0.01.

FIG. 2

Effect of intranasal administration of LcS to mice on production of IL-12, IFN-γ, TNF-α, and IL-4 by MLN cells stimulated with purified PR8. MLN cells from mice administered LcS 0 (control) (□) or LcS 200 (■) intranasally were cultured in the absence (0 μg/ml) or presence of purified PR8 (0.2 to ∼20 μg of protein/ml). Collection of supernatants and determination of various cytokines were performed in the same manner as described in the legend to Fig. 1. Each bar represents the mean ± SD of triplicate samples. Single, double, and triple asterisk, statistically significant difference from the control by Student's t test at P < 0.05, P < 0.01, and P < 0.001, respectively.

FIG. 3

Effect of intranasal administration of LcS on the titer of virus in nasal washes. Mice were administered 20 μl of LcS solution at the concentration of 0 (control) (□), 1 (LcS 20) (▴), or 10 (LcS 200) (●) mg/ml intranasally for three consecutive days. One day thereafter, the mice were intranasally infected with PR8, and 3 days later the titers of virus in the nasal washes from the infected mice were measured. Each bar represents the mean of values for 14 mice. Double asterisk, statistically significant difference from values for the control by Dunnett's test at P < 0.01.

FIG. 4

Survival of mice infected with PR8 in the upper respiratory tract. Mice were inoculated with 2 μl of fluid containing 103.5 EID50 of PR8, and PBS was administered intranasally at 3 days postinfection (n = 8) (▵). Infected mice not administered PBS intranasally served as controls (n = 6) (■). After nasal inoculation with PR8, the mice were observed for 14 days to assess the survival rates. Double asterisk, statistically significant difference from the control by Fisher's exact test at P < 0.01.

FIG. 5

Protection against mortality due to IFV infection in mice administered LcS intranasally. Mice administered LcS (●) (n = 13) intranasally and mice not administered LcS (▵) (n = 13) were inoculated with 2 μl of fluid containing 103.5 EID50 of PR8, and PBS was administered intranasally at 3 days postinfection. Assessment of the survival was performed in the same manner as described in the legend to Fig. 4. Asterisk, statistically significant difference from the control by Fisher's exact test at P < 0.05.