B from B6 or B6-IFNR-KO mice lethally infected, respectively. Upper panel shows results by the assay using anti-FasL specific Ab and lower shows that by the assay using anti-Fas specific Ab. doi:10.1371/journal.pone.0055321.ghistogram). In non-infected B6-IFNR-KO mice, it was observed that the pattern of Fas protein expression in the indicated cell types was similar to that in non-infected B6 mice (Fig. 4 lower panel, dark green color compared with orange color histogram). It should be noted that the expression levels of Fas protein on the cells in lethally infected conditions were slightly decreased by the specific targeting of IFNR1 gene expression (Fig. 4 lower panel, black color compared with light green color histogram), but remained at a similar level in non-infected B6 control or B6-IFNR-KO mice (Fig. 4 lower panel, black color compared with dark green or orange color histogram). Therefore, these observations have prompted the suggestion that type-I IFN signal specifically associates with the change of Fas expression Title Loaded From File induced by the viral ?infection but not in the naive condition. All these findings indicated that a type-I interferon signal is essential for FasL protein expression to be induced by viral infection on surface of cells in the lungs of mice.Difference in Time-course Kinetics of Type-I Interferon Amount in Bronchoalveolar Lavage Fluid in Lethally and Non-lethally Infected MiceIn the above study, it was shown that FasL mRNA expression in the lung of lethally infected mice was detected at earlier than in non-lethally infected mice (Fig. 3A and C). To clarify the detail of the differences in non-lethal or lethal infected conditions, the time dependent kinetics of production of type-I interferon in the lungs of mice infected non-lethally and lethally were evaluated. The amounts of type-I interferon in the broncho alveolar lavage uid (BALF) in the lungs of these mice were assessed. Murine IFN-b specific ELISA showed that production of IFN-b protein in the BALF of mice infected with a lethal titer of the PR/8 virus was induced at 3DPI and this production level was slightly decreased at 5DPI (Fig. 5). In the case of non-lethal infection, IFN-b production was not detected in the BALF at 3DPI, but was slightly detected at 5DPI (Fig. 5). These findings indicate that the time dependent kinetics of IFNb production is different between the lethal and non-lethal infections of the virus in the lungs of mice.Importance of Type I IFN and FasL in InfluenzaFigure 5. Production of IFN- ?in the lungs of mice infected lethally or non-lethally with the PR/8 virus. B6 mice were intranasally infected with 105 (closed triangle) or 102 (open square) pfu/head of the PR/8 virus. At 0, 3 or 5 DPI, the BALF of these mice were isolated. The amount of IFN- ?or total protein contained in these samples was assessed by mouse IFN- ?specific ELISA or BCA protein 24786787 assay, respectively. The amounts of IFN- ?were normalized by that of the total protein in each Title Loaded From File sample. “N.D.” means not detected. doi:10.1371/journal.pone.0055321.gDiscussionIn this study, we proposed that type-I IFN production highly induces the expression of FasL on several cells in the lung which leads to the reduction of the survival rate after a lethal infection of PR/8 virus. Previously, it was reported that intranasal administration of anti-Fas specific agonistic antibody induces acute lung inflammation [7,8]. We also found that functional mutation of the FasL gene protects mice from a let.B from B6 or B6-IFNR-KO mice lethally infected, respectively. Upper panel shows results by the assay using anti-FasL specific Ab and lower shows that by the assay using anti-Fas specific Ab. doi:10.1371/journal.pone.0055321.ghistogram). In non-infected B6-IFNR-KO mice, it was observed that the pattern of Fas protein expression in the indicated cell types was similar to that in non-infected B6 mice (Fig. 4 lower panel, dark green color compared with orange color histogram). It should be noted that the expression levels of Fas protein on the cells in lethally infected conditions were slightly decreased by the specific targeting of IFNR1 gene expression (Fig. 4 lower panel, black color compared with light green color histogram), but remained at a similar level in non-infected B6 control or B6-IFNR-KO mice (Fig. 4 lower panel, black color compared with dark green or orange color histogram). Therefore, these observations have prompted the suggestion that type-I IFN signal specifically associates with the change of Fas expression induced by the viral ?infection but not in the naive condition. All these findings indicated that a type-I interferon signal is essential for FasL protein expression to be induced by viral infection on surface of cells in the lungs of mice.Difference in Time-course Kinetics of Type-I Interferon Amount in Bronchoalveolar Lavage Fluid in Lethally and Non-lethally Infected MiceIn the above study, it was shown that FasL mRNA expression in the lung of lethally infected mice was detected at earlier than in non-lethally infected mice (Fig. 3A and C). To clarify the detail of the differences in non-lethal or lethal infected conditions, the time dependent kinetics of production of type-I interferon in the lungs of mice infected non-lethally and lethally were evaluated. The amounts of type-I interferon in the broncho alveolar lavage uid (BALF) in the lungs of these mice were assessed. Murine IFN-b specific ELISA showed that production of IFN-b protein in the BALF of mice infected with a lethal titer of the PR/8 virus was induced at 3DPI and this production level was slightly decreased at 5DPI (Fig. 5). In the case of non-lethal infection, IFN-b production was not detected in the BALF at 3DPI, but was slightly detected at 5DPI (Fig. 5). These findings indicate that the time dependent kinetics of IFNb production is different between the lethal and non-lethal infections of the virus in the lungs of mice.Importance of Type I IFN and FasL in InfluenzaFigure 5. Production of IFN- ?in the lungs of mice infected lethally or non-lethally with the PR/8 virus. B6 mice were intranasally infected with 105 (closed triangle) or 102 (open square) pfu/head of the PR/8 virus. At 0, 3 or 5 DPI, the BALF of these mice were isolated. The amount of IFN- ?or total protein contained in these samples was assessed by mouse IFN- ?specific ELISA or BCA protein 24786787 assay, respectively. The amounts of IFN- ?were normalized by that of the total protein in each sample. “N.D.” means not detected. doi:10.1371/journal.pone.0055321.gDiscussionIn this study, we proposed that type-I IFN production highly induces the expression of FasL on several cells in the lung which leads to the reduction of the survival rate after a lethal infection of PR/8 virus. Previously, it was reported that intranasal administration of anti-Fas specific agonistic antibody induces acute lung inflammation [7,8]. We also found that functional mutation of the FasL gene protects mice from a let.