As loss of body weight and death of mice after influenza A virus infection. In B6 mice, infection with a high titer (105 pfu/head i.n.) of PR/8 virus dramatically decreased the body weight of mice at 2,5DPI (Fig. 2A, closed triangle) and all mice 
were dead at 8 DPI (Fig. 2B, closed triangle). On the contrary, in the mice infected with a low titer of the virus (102 pfu/ head, i.n.), reduction of body weight was slightly observed at 5,6 DPI, and all these mice survived until 19 DPI (Fig. 2A and B, open square). By AKT inhibitor 2 supplier plaque assay, at 1DPI, the virus titer in the lungs of mice infected with a high titer was shown to be significantly higher, but was lower compared to that with a low titer of the virusafter 2DPI (Fig. 2C). As shown in a previous report [6], these findings suggested that the initial infected but not propagated virus titer in the lungs of mice correlate with the severity of symptoms or mortality of mice after influenza A virus infection. To clarify the correlation of the function of Fas or FasL gene with the severity of illness in this model, their MedChemExpress BTZ043 expression in the lungs of these mice were assessed by quantitative real time PCR (QPCR) methods using specific primer sets for these genes. In a high virus titer infection (lethal condition, 105 pfu/head i.n.), a very high expression of FasL gene was observed at 2DPI and this expression level was sustained until the mice died (Fig. 3A). Compared with FasL gene, expression level of Fas gene was slightly increased during the infection (Fig. 3B). In a low virus titer infection (non-lethal condition, 102 pfu/head i.n.), induction of FasL gene expression was observed after 4DPI (Fig. 3C) and Fas gene expression was not changed (Fig. 3D). It has been demonstrated that the induction level of FasL gene expression is correlated with body weight loss in both lethal and 
non-lethal conditions (compared with Fig. 3A versus 3E, and Fig. 3C versus 3F). These findings suggested that the gene expression level of FasLImportance of Type I IFN and FasL in InfluenzaFigure 3. Induction of FasL gene in the lungs of mice infected with the PR/8 virus. B6 mice were infected with the PR/8 virus at the indicated virus titer. These mice were sacrificed at the indicated day, and mRNA expression of FasL (A and C) or Fas (B and D) in the lungs of these mice were assessed as described in Materials and Methods. The mean value of GAPDH was used for the internal control. Changes in body weight of mice infected with a lethal (E) or a non-lethal (F) virus titer were shown as percentage of the reduction compared with the original body weight (N = 3/each group). doi:10.1371/journal.pone.0055321.gbut not Fas is important to determine the severity of illness in mice infected with PR/8 virus.Type-I Interferon Signal is Essential for the Induction of FasL Protein Expression in the Lungs of MiceRegarding the mechanism for regulating FasL protein induction by virus infection, there are two possibilities. One is that a virus component, such as viral RNA or protein should directly activate an intracellular signaling, which induces FasL expression. The other is that some cytokines including type-I interferon (IFN), which is produced by virus infected cells, should induce FasL expression. To clarify these possibilities, we assessed the effect of shut down on a type-I interferon (IFN) signal on FasL expression induced with the viral infection. Control B6 mice or B6-IFNR-KO were infected with a lethal virus titer of the PR/8 virus (10.As loss of body weight and death of mice after influenza A virus infection. In B6 mice, infection with a high titer (105 pfu/head i.n.) of PR/8 virus dramatically decreased the body weight of mice at 2,5DPI (Fig. 2A, closed triangle) and all mice were dead at 8 DPI (Fig. 2B, closed triangle). On the contrary, in the mice infected with a low titer of the virus (102 pfu/ head, i.n.), reduction of body weight was slightly observed at 5,6 DPI, and all these mice survived until 19 DPI (Fig. 2A and B, open square). By plaque assay, at 1DPI, the virus titer in the lungs of mice infected with a high titer was shown to be significantly higher, but was lower compared to that with a low titer of the virusafter 2DPI (Fig. 2C). As shown in a previous report [6], these findings suggested that the initial infected but not propagated virus titer in the lungs of mice correlate with the severity of symptoms or mortality of mice after influenza A virus infection. To clarify the correlation of the function of Fas or FasL gene with the severity of illness in this model, their expression in the lungs of these mice were assessed by quantitative real time PCR (QPCR) methods using specific primer sets for these genes. In a high virus titer infection (lethal condition, 105 pfu/head i.n.), a very high expression of FasL gene was observed at 2DPI and this expression level was sustained until the mice died (Fig. 3A). Compared with FasL gene, expression level of Fas gene was slightly increased during the infection (Fig. 3B). In a low virus titer infection (non-lethal condition, 102 pfu/head i.n.), induction of FasL gene expression was observed after 4DPI (Fig. 3C) and Fas gene expression was not changed (Fig. 3D). It has been demonstrated that the induction level of FasL gene expression is correlated with body weight loss in both lethal and non-lethal conditions (compared with Fig. 3A versus 3E, and Fig. 3C versus 3F). These findings suggested that the gene expression level of FasLImportance of Type I IFN and FasL in InfluenzaFigure 3. Induction of FasL gene in the lungs of mice infected with the PR/8 virus. B6 mice were infected with the PR/8 virus at the indicated virus titer. These mice were sacrificed at the indicated day, and mRNA expression of FasL (A and C) or Fas (B and D) in the lungs of these mice were assessed as described in Materials and Methods. The mean value of GAPDH was used for the internal control. Changes in body weight of mice infected with a lethal (E) or a non-lethal (F) virus titer were shown as percentage of the reduction compared with the original body weight (N = 3/each group). doi:10.1371/journal.pone.0055321.gbut not Fas is important to determine the severity of illness in mice infected with PR/8 virus.Type-I Interferon Signal is Essential for the Induction of FasL Protein Expression in the Lungs of MiceRegarding the mechanism for regulating FasL protein induction by virus infection, there are two possibilities. One is that a virus component, such as viral RNA or protein should directly activate an intracellular signaling, which induces FasL expression. The other is that some cytokines including type-I interferon (IFN), which is produced by virus infected cells, should induce FasL expression. To clarify these possibilities, we assessed the effect of shut down on a type-I interferon (IFN) signal on FasL expression induced with the viral infection. Control B6 mice or B6-IFNR-KO were infected with a lethal virus titer of the PR/8 virus (10.
