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Paragraph: DNaseItreated RNA (0.2 g) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers AGG CAC CA-3  5 -CTC CTT AAT GTC ACG CAC GAT TTC-3  h IL-6  5 -CCT TCG GTC CAG TTG CCT TCT-3  5 -CCA GTG CCT CTT TGC TGC TTT C-3  h IFN were used: forward primer, 5 -CGC AGT ATT CAG AAG AAG CAAGAC-3 ; and reverse primer, 5 -TCC ATA AGG ATA GAC CAG CTA CCA-3 . The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95 ∘ C for 10 min, followed by 40 cycles of 95 ∘ C for 15 s, 56 ∘ C for 30 s, and 72 ∘ C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard. Relative qPCR was performed for other eight genes: hactin, h IL-6, h IFN-, and hTNF-for A549 cells; mactin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-, and mTNF-for Raw264.7 cells. The sequences of primers were shown in Table 1 . The reaction was run with 95 ∘ C for 10 min, followed by 40 cycles of denaturation at 95 ∘ C for 15 sec, annealing at 52 ∘ C for 30 s, and extension at 72 ∘ C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by 2 −ΔΔCT using -actin as an internal standard [20] . 2.8. ELISA. IL-6, TNF-, and IFN-levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer's protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10fold for ELISA. 2.10. Statistical Analysis. The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and < 0.05 was considered significant. Many botanical polysaccharides exhibit an immunomodulatory effect [11] . To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 g/mL) for 36 h. The mRNA levels of IL-6, TNF-, and IFN-were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-, and IFN-in a dosedependent manner both in A549 cells (Figures 1(a)-1(c) ) and Raw264.7 cells (Figures 1(d) -1(f)) compared with the PBS treatment group. To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2 (a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b) ). To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d) ). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model. The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-, and IFN-expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)-3(c) ). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-production in the EAP group was not significantly higher than that of the PBS group ( = 0.0599) (Figures 3(g)-3(i) ). These results suggest that EAP increases the IL-6, TNF-, and IFN-production. IL-6, TNF-, and IFN-expression at day 3 postinfection was determined by qPCR. In contrast, TNF-mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e) ), while IL-6 and IFN-expression were only slightly lower (not significant) (Figures 3(d) and 3(f) ). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice. Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d) ). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b) ). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice. Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-, IL-6, IL-8, and IL-12 [11] . This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a) ). In vitro assay showed similar trends. As shown in Figure 5 (b), Raw264.7 cells were treated with 200 g/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway. In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNFand IFN-) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized. The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22] . Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [12] [13] [14] [15] . The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-, and IFNand provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%). In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-, IL-6 and IFN-) were detected during H5N1 infection [23, 24] . This "cytokine storm" leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26] . However, it is unclear whether the lack of proinflammatory cytokines (such as TNFand IL-6) facilitates viral clearance. Interestingly, knockout 8

Evidence-Based Complementary and Alternative Medicine mice deficient in TNF-, TNF-receptor, IL-6, MIP-1 , and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28] . Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30] . These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-, and IFNare very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25] . This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30] . In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show). The antiinfluenza properties of IL-6, TNF-, and IFNhave been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31] . TNF-has been reported to exert a defensive effect against influenza infection in vitro [32] . IFN-treatment in the early stages of influenza infection improves the survival rate in mouse models [33] . In addition, high levels of IFN-secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12] . In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-, and IFNcompared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-mRNA levels were slightly lower, while TNF-production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important. The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11] . In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect. In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection

Question: What does this study demonstrate?
EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection