Cytotoxicity of DATS
The cytotoxicity of DATS and AMT on A549 cells was examined using the MTT based cell viability assay. Results showed that 375 µM of DATS and 500 µM of AMT had little or no cytotoxicity to A549 cells (Figure 1). Therefore, 93.75 µM-375 µM of DATS, and 500 µM of AMT were used for subsequent experiments.
Antiviral activity of DATS
To characterize anti-H9N2 AIV activity of DATS, its effects on various stages of infection were examined. DATS was added to A549 cell cultures to a final concentration of 93.75, 187.5, or 375 µM before (pretreatment), or after (post-infection) H9N2 AIV infection. H9N2 AIV-infected A549 cells were treated with 500 µM AMT as positive anti-viral control in the same manner. As AMT was dissolved in 0.1% DMSO, cells in the untreated control groups were treated with 0.1% DMSO before or after infection to serve as negative controls.
In the pretreatment experiment, the viability of untreated H9N2 AIV infected- cells was 43%, and that of AMT-treated infected group was 61%. The viability of infected cells treated with 93.75, 187.5, or 375 µM of DATS was 47%, 57%, and 57%, respectively (Figure 2A), suggesting that pretreatment of A549 cells with DATS rendered them less susceptible to H9N2 AIV infection. In the post-infection experiment, the viability of untreated H9N2 AIV infected-cells was 53%, and that of AMT-treated infected cells was 82%. The viability of infected cells treated with 93.75, 187.5, or 375 µM of DATS was 68%, 73%, and 79%, respectively. These results suggested that DATS inhibited both infection and replication of H9N2 AIV. The effect of DATS was dose dependent, and the protection was more profound when DATS was added 1 hour post-infection.
The antiviral effect of DATS was also examined by the HA assay. As shown in Figure 2B, DATS exhibited remarkably inhibit the virus hemagglutination in pre- or post-treatment, but no dose dependent was observed in the assay. To further assess the effect of DATS on H9N2 AIV replication, the expression of the M gene of H9N2 AIV was examined by qRT-PCR. As seen in Figure 2C, M gene expression in AMT-treated infected cells was 0.71 and 0.54-fold of the level of untreated H9N2 AIV-infected cells in pretreatment and post-infection experiments, respectively. M gene expression in DATS pretreated A549 cells was about 0.6-fold (40% decrease) that of untreated H9N2-infected cells for all 3 DATS doses. The effect of DATS on H9N2 AIV replication was most profound in the post-infection experiment as M gene expression in A549 cells treated with 93.75, 187.5, or 375 µM of DATS after infection was 0.27, 0.18, and 0.16-fold that of untreated cells, indicating that DATS at 93.75, 187.5, and 375 µM inhibited H9N2 AIV replication by 73, 82, and 84% when it was added to the cells 1 hour after infection. Based on MTT and HA assay results, the protection was more effective when DATS was added 1-hour post-infection, so the 375µM of DATS post-infection were used in the following experiments.
DATS diminishes H9N2 AIV induced inflammation
To investigate the effect of DATS on H9N2 AIV-induced inflammation, the expression levels of inflammatory cytokines TNF-α and IL-6 in cells of post-infection were measured by qRT-PCR at two different time points.
In the experiment, A549 cells were infected with H9N2 virus for 1h, and then washed twice with PBS, followed by treatment with 375µM of DATS for 24 and 48h, respectively. For IL-6, H9N2 AIV infection increased its expression by 15.8-fold or 9.6-fold compared to uninfected control (set as 1.0) at the two indicated time points (Figure 3 A). Treatment of H9N2 infected cells with 500 µM AMT resulted in decreased expression (5.6-fold and 2.5-fold of control, respectively). Treatment of H9N2 infected cells with 375 µM of DATS reduced its expression to 9.0-fold and 2.3- fold of control, respectively. Treatment of H9N2 infected cells with 500 µMAMT also resulted in IL-6 decreased expression (5.6-fold and 2.5-fold, respectively of control). For TNF-α, H9N2 AIV infection increased its expression by 11.3-fold and 23.7-fold compared to uninfected control (set as 1.0), respectively (Figure 3B). Treatment of H9N2 infected cells with 500 µM AMT resulted in decreased its expression (2.9-fold and 2.3-fold of control, respectively). Treatment of H9N2 infected cells with DATS reduced its expression from 11.3-fold to 4.8-fold, 23.7-fold to 8.6-fold of control, respectively. These results showed that DATS could down-regulate proinflammatory response induced by H9N2 AIV infection.
DATS increases the antiviral immune response during H9N2 AIV infection
Type I IFN play an important role in the innate immune system, and IRF3 can regulate its production. In this study, the effects DATS treatment on the expression of RIG-I, IRF-3 and IFN-β in A549 cells were investigated by qRT-PCR. As shown in Fgure 3, H9N2 AIV infection increased RIG-1 expression 14.8-fold and 12.9-fold compared to uninfected control (set as 1.0) at two different time points. Treatment of A549 cells after H9N2 AIV infection with 500 µM AMT increased RIG-1 expression to 29.8 and 17.9-fold of control, respectively. Treatment of cells after infection with 375 µM of DATS increased RIG-1 expression to 26.9, and 15.3-fold of control, respectively. For IRF3, H9N2 AIV infection increased its expression 19.8-fold and 3.3-fold compared to uninfected control. Treatment of A549 cells after H9N2 AIV infection with 500 µM AMT increased its expression to 62.8 and 24.4-fold of control, respectively. Treatment of cells with 375 µM of DATS increased its expression to 51.8, and 34.2-fold of control, respectively. For IFN-β, H9N2 AIV infection increased its expression 6.1-fold and 4.7-foldd compared to uninfected control. Treatment of A549 cells after H9N2 AIV infection with 500 µM AMT increased its expression to 13.6 and 9.7-fold of control, respectively. Treatment of cells with DATS also increased its expression to 9.2, and 8.2-fold of control, respectively. These results suggested that DATS might exert its anti-H9N2 AIV by up-regulating the innate immune response.
DATS protects mice from H9N2 AIV infection
Based on results of in vitro experiments, we hypothesized that DATS can attenuate the symptoms of H9N2 AIV infection in mice. As reported previously, H9N2 AIV-infected mice exhibited marked inactivity, emaciation, ruffled fur, lack of appetite, labored breathing, respiratory distress, and decreased body weight (Gui et al, 2017). The decrease in body weight caused by H9N2 AIV infection was most profound 3 days after infection, from 100% to 87.2%. Treatment of infected mice with DATS (30 mg/kg) diminished such loss, from 100% to 91.8% on 3 days after infection. The body weight of DATS treated control group and uninfected control group was not significantly changed (Figure 4).
To investigate pulmonary edema caused by H9N2 virus infection, lung index (weight lung to body weight ratio) was determined at different time points during infection. As shown in Figure 5A, the lung index increased in the two infected groups on days 2-6 after H9N2 virus infection, but it was lower in the DATS treated group than in the infected group. At days 6, DATS effect was not significant. These results indicated that H9N2 infection caused edema, thus increasing the lung index, and DATS treatment could reduce the severity of edema. Similar patterns of DATS effect were observed at the titration of virus in lungs. A significant reduced lung titer on treatment with DATS on 2- and 4-days post infection (Figure 5B). Since influenza M protein can reflect the levels of virus replication, so we also checked the effect of DATS treatment on influenza M protein mRNA levels. The results showed that a significantly reduced the levels of M gene after treatment with DATS on 2- and 4-days post infection (Figure 5C). To further examine the lung lesion, HE staining of lung sections was carried out. As shown in figure 6, lungs of H9N2 AIV-infected mice showed collapse of alveolar spaces, infiltration of inflammatory cells, interstitial and alveolar edema, and hemorrhage. These pathological changes were relatively mild in DATS treatment. These results all suggested that DATS treatment might reduce the severity of edema and the virus titers in lungs.
The expression levels of lung inflammatory cytokines (IL-6 and TNF-α) and antiviral cytokines (RIG-1 and IFN- β) induce by H9N2 AIV infection were then determined. As shown in Figure 7, the effects of H9N2 AIV infection and DATS treatment on the expression of TNF-α and IL-6 expression were most profound at 6 days post infection. At that time, H9N2 AIV infection increased TNF-α expression by 6.5-fold, and DATS treatment reduced it to 3.9-fold of uninfected control. For IL-6, H9N2 AIV infection increased its expression by 20-fold, and DATS treatment reduced it to 18-fold of uninfected control. The effect on RIG-I expression was most profound at day 6 post-infection. At which, H9N2 AIV infection increased its expression 16.9-fold, and DATS treatment further increased it to 29-fold of uninfected control. The effect on IFN-β expression was most profound at day 2 post-infection. H9N2 AIV infection increased its expression 10-fold, and DATS treatment further increased it to 12-fold of uninfected control at that time. These results showed that H9N2 AIV infection caused a profound increase in the expression of inflammatory and antiviral cytokines. DATS treatment of infected mice reduced the expression of TNF-α and IL-6, but increased the expression of RIG-1 and IFN-β.