PMC

(a, b) The expression of cytokines/chemokines in mouse lungs is shown separately for the experiments described in . Expression was visualized by using a heatmap and by using hierarchal clustering with the UPGMA method in TIBCO Silver Spotfire ver. 3.2. Expression of the representative cytokines in distinct clusters is shown: (c) IL-10, (d) GM-CSF, (e) IL-5, and (f) G-CSF. All values were normalized to the mean value of the saline-treated, PBS-inoculated mice at 14 days post-b240 administration (−7 days post-infection). The values are means ± SD (n = 3). Asterisk: P value < 0.05, significant difference compared with the control group (two-way ANOVA).

Mice were administered heat-killed b240 at a dose of 10 mg/mouse daily for 21 days prior to infection and for 14 days after infection. Mice in the control group received saline. They were then intranasally infected with 0.3 or 10 MLD₅₀ of mouse-adapted CA04 virus on day 21 post-b240 administration. Mortality and morbidity were monitored daily for 14 days post-infection. Percent survival (a) and body weight (c) are shown for each group of ten mice infected with 0.3 MLD₅₀ of virus. Percent survival (b) and body weight (d) are shown for b240- or saline-treated groups of 24 or 25 mice infected with 10 MLD₅₀ of virus, respectively. The body weight values are means ± SD for the mice that were alive at each time point. Asterisk: P value < 0.05, significant difference compared to the virus-infected group not treated with b240 (log-rank test).

We conducted three types of experiments to determine the prophylactic effects of b240 administration on pathogenesis and host responses in mice. In all experiments, mice were orally mock-administered with saline or administered with heat-killed Lactobacillus pentosus b240 at a dose of 10 mg/mouse daily for 21 days prior to infection and for 14 days after infection or mock infection. (a) To define the prophylactic effect of b240 administration on mouse survival, mice were infected with 0.3 or 10 MLD₅₀ of mouse-adapted CA04 virus on day 21 post-b240 administration and mortality and morbidity were observed for 14 days post-infection. (b) To define the effects of b240 administration on virus replication, histopathology, cytokine expression, and gene expression, mice were infected with 10 MLD₅₀ of CA04 virus on day 21 post-b240 administration and euthanized on days 1, 3, and 6 post-infection. Their lungs were subjected to virus titration, histopathological examination, cytokine measurement, and microarray analysis. (c) To investigate the immune responses induced by b240 administration in the lungs of mice, mice were mock-infected with PBS on day 21 post-b240 administration and euthanized on days −7, 0, 1, 3, and 6 post-infection (14, 21, 22, 24, and 27 post-b240 administration). Their lungs were subjected to cytokine measurement and microarray analysis.

Animal experiments were conducted as described in . Genes that were differentially regulated following b240 administration were identified by comparing the gene expression in the lungs of mice treated with b240 and those given saline. (a) Twenty-nine genes were selected by using two-way ANOVA (treatment effect, P < 0.05) and by filtering the genes whose expression changed by at least 1.5-fold relative to the level in the saline-treated group at at least one time point. Gene expression was visualized by means of a heatmap and by using hierarchal clustering with the UPGMA method in TIBCO Silver Spotfire ver. 3.2. The heatmap was generated by using the mean expression values for the b240-treated animals, which were normalized to the values of the time-matched, saline-treated controls in the PBS-inoculated group (n = 3). (b) Validation of microarray findings by using qRT-PCR. The expression of the following eight genes was measured by using qRT-PCR: Acot1, Acot2, Acot5, Cyr61, Egr1, Fos, Rsad2, and Stfa1. The gene expression levels of three biological replicates from three animals per group are represented as mean values. The microarray data presented are those from .