Interleukin-37 Ameliorates Influenza Pneumonia by Attenuating Macrophage Cytokine Production in a MAPK-Dependent Manner

Viral pneumonitis caused by influenza A (H1N1) virus leads to high levels of morbidity and mortality. Given the limited treatment options for severe influenza pneumonia, it is necessary to explore effective amelioration approaches. Interleukin-37 (IL-37) has been reported to inhibit excessive immune responses and protect against a variety of inflammatory diseases. In this study, by using BALB/c mice intranasally infected with A/California/07/2009 (H1N1), we found that IL-37 treatment increases the survival rate and body weight, and reduces the pulmonary index, impaired the lung injury and decreased production of pro-inflammatory cytokines in the BALF and lung tissue. Moreover, IL-37 administration enhanced not only the percentage of macrophages, but also the percentage of IL-18Rα+ macrophages, suggesting that enhancing the macrophages function may improve outcomes in a murine model of H1N1 infection. Indeed, macrophages depletion reduced the protective effect of IL-37 during H1N1 infection. Furthermore, IL-37 administration inhibited MAPK signaling in RAW264.7 cells infected with H1N1. This study demonstrates that IL-37 treatment can ameliorate influenza pneumonia by attenuating cytokine production, especially by macrophages. Thus, IL-37 might serve as a promising new target for the treatment of influenza A-induced pneumonia.


INTRODUCTION
In uenza H1N1 infection induced " u"-like illness or pneumonia depends on the infecting strain, host immune system, and environmental factors (Rice et al., 2012;Daoud et al., 2019). H1N1 pneumonia may progress rapidly, resulting in severe respiratory distress syndrome or refractory hypoxemia, is associated with a longer hospital stay with higher mortality compared to bacterial pneumonia (Perez-Padilla et al., 2009;Rello et al., 2009;Hermann et al., 2017).
Increasing evidence has demonstrated that it is an excessively activated immune response, not a direct viral infection that leads to the increasing in uenza pneumonia severity (Morita et al., 2013;Uematsu et al., 2015). Although various prevention and treatment methods have been used for viral diseases, the limited treatment options for severe in uenza pneumonia prioritize the need for the discovery of e ective therapies. October 2019 | Volume 10 | Article 2482 Interleukin-37 (IL-37), a novel member of the IL-1 family, inhibits systemic and local in ammation by reducing the levels of pro-in ammatory mediators (Nold et al., 2010;Boraschi et al., 2011;Al-Anazi et al., 2019). IL-37 binds to the IL-18Rα chain, and then recruits TIR-8/IL-1R8/SIGIRR to execute its anti-in ammatory e ects (Kumar et al., 2002;Boraschi and Tagliabue, 2013;Dinarello and Bu er, 2013;Lunding et al., 2015;Nold-Petry et al., 2015). IL-37 has been shown to increase the survival rate and body weight, and downregulated the production of IL-6 and IL-17A in a coxsackievirus B3-induced model of murine viral myocarditis . In addition, comparing with wild-type (WT) mice, a low dose of mouseadapted H1N1-induced morbidity and the decreases in body weight are signi cantly attenuated in IL-37tg mice, which express human IL-37 isoform b precursor transgene (Davis et al., 2017). Moreover, IL-37 signi cantly attenuates pulmonary eosinophilia, CCL11 production and airway hyper-reactivity in a murine asthma model (Lv et al., 2018). Increasing evidence suggests that IL-37 can inhibit excessive immune responses and protect against a variety of in ammatory diseases, autoimmune diseases, and tumors. However, little is known about the function of IL-37 in the in uenza-infected murine model, particularly the regulatory role in viral pneumonia induced by A/ California/07/2009 (H1N1) infection. us, in the present study, we focused on IL-37 treatment in H1N1-infected mice, to investigate the therapeutic e ect and the mechanisms by which IL-37 treatment ameliorates in uenza pneumonia.

Animals and Viruses
Speci c pathogen-free, 4-to 6-week-old female BALB/c mice were obtained from Vital River Laboratories (Beijing, China). e seasonal in uenza A virus strain A/California/07/2009 (H1N1) was provided by the Institute of Laboratory Animal Science, Peking Union Medical College, China. All experiments were performed in biosafety level 2 facilities in compliance with governmental and institutional guidelines. e experimental protocol was evaluated and approved by the Institute of Animal Use and Care Committee of the Institute of Laboratory Animal Science, Peking Union Medical College (BLL19004).

Therapeutic Treatments
Oseltamivir phosphate capsules were purchased from Roche Pharmaceutical Co., Ltd. (Shanghai, China). e pGEM-T-IL-37b plasmid was kindly supplied by Dr. RF. Wei., Institute of Laboratory Animal Science, Peking Union Medical College, China. Individual mice were anesthetized with tribromoethanol and inoculated intranasally with 50 μl (10 4.3 TCID 50 ) of allantoic uid containing in uenza A/California/07/2009 (H1N1) virus. Subsequently, the mice were chronically intragastrically administered oseltamivir phosphate (30 mg/kg) for 5 days, and the animals were inoculated with IL-37 (12.5 μg/kg) via intravenous or intranasal administration at three separate time points (2, 24, and 48 h post infection). Seven mice were selected randomly from each group for monitoring the disease signs, weight loss, and mortality daily up to 14 days post inoculation (d.p.i.). e remaining mice in each group were euthanized at 6 d.p.i. and blood samples, bronchoalveolar lavage uid (BALF), and lung tissues were collected for the assessment of lung histology, pro-in ammatory cytokines, and immune cell counts.

Preparation of Single Cell Suspensions From the Lung
Mice were anesthetized and the lung was ushed in situ with 20 ml of phosphate-bu ered saline (PBS) via cannulation of the heart to remove the intravascular blood pool. Minced lung tissues were incubated at 37°C for 1 h on a rocker with 200 μg/ ml collagenase D and 40 μg/ml DNase I (Roche Molecular Biochemicals) in 10 ml of DMEM supplemented with 10% FBS. Single cell suspensions from the digested lung were ltered through a 75-μm strainer and then collected through densitygradient centrifugation with lymphocyte separation solution.
e immune cells were washed twice with Hank's solution and suspended in Hank's solution.

Preparation of Lung Homogenate Supernatant
Lung homogenates were prepared by homogenizing perfused whole lung tissue using an electric homogenizer for 2 min 30 s in 1 ml of PBS. e homogenates were centrifuged at 3,000 rpm for 10 min at 4°C. e supernatant was collected and stored at −80°C.

Analysis of Bronchoalveolar Lavage Fluid
Bronchoalveolar lavage uid (BALF) was collected by washing the lungs of sacri ced mice twice with 1 ml PBS. e PBS was then recovered a er 1 min and centrifuged at 1,500 rpm for 10 min at 4°C. e supernatant was collected and stored at −80°C. Total cellular in ltration in the BALF was assessed using a hemocytometer; cytosine slides were xed and stained with Wright-Giemsa stain, and the composition was assessed in a blinded manner by counting 200 or more cells using a light microscope.

Clodronate Treatment
To deplete macrophages, ready-made clodronate liposomes and control liposomes (FormuMax; CA, USA) were intranasally administered to mice using the manufacturer's recommending dose 1 day before and 1 day a er A/California/07/2009 (H1N1) infection. Mice were monitored for signs of disease, weight loss, and mortality upto 14 d.p.i.

Hematoxylin and Eosin Staining
For each mouse, the whole right lung was xed in 10% formalin for 24 h and then embedded in para n for histological examination. e lung tissue sections (4 μm) were depara nized and hydrated using xylene and an alcohol gradient and then, stained with Hematoxylin and Eosin (H&E). e histopathology of the lung tissue was observed by light microscopy.

Quantitative Real-Time Polymerase Chain Reaction Analysis
Total RNA was isolated from individual samples using an RNeasy Mini kit, according to the manufacturer's instructions (Qiagen, Hilden, Germany). e RNA was reversely transcribed into cDNA using random primers and a SuperScript II reverse transcriptase reaction mixture (Invitrogen). e target gene mRNA transcripts were determined by RT-PCR using SYBR Green PCR Master Mix, speci c primers, and a 7500 PCR system (ABI, USA). Primer sets for individual genes are shown in Table 1.

Cells
Murine macrophage cell lines (RAW264.7) were maintained in Dulbecco's modi ed Eagle's medium (Gibco, Life Technologies, New York) supplemented with 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin and were incubated at 37°C with 5% CO 2 . RAW264.7 cells in six-well plates were infected with H1N1 at a multiplicity of infection (MOI) of 0.01 for 1 h absorption at 37°C. en, the cells were washed and cultured with 2 ml of serum-free DMEM containing TPCK-treated trypsin (0.5 mg/ml) antibiotics and 70 μM oseltamivir phosphate with or without IL-37b for 72 h. e cells were collected at 0, 12, and 24 h post infection to detect the expression of cytokines such as IL-6, MCP-1, TNF-α, IL-1β, and IL-1α. e protein levels of MAPKs and NLRP3 were detected by western blot assay at 0, 30, and 60 min a er infection.
Statistical Analysis e data are presented as the mean ± SEM. Analysis of variance (ANOVA) was used to analyze the di erences between three or more groups, and t tests were used to analyze the di erences between two groups. Statistical graphs were obtained using GraphPad Prism 5 so ware. Di erences were considered statistically signi cant at values of p < 0.05.

Interleukin-37 Treatment Reduces the Body Weight Recovery Time and Improves the Survival Rate in H1N1-Infected Mice
To explore the e cacy of the recombinant IL-37 protein, BALB/c mice challenged intranasally with 50 μl H1N1 were treated with oseltamivir phosphate for 5 days with/without recombinant IL-37 for 7 days at 2 h.p.i. As shown in Figure 1A, the body weights of mice treated for 7 days (oseltamivir+IL-37 7d) were not obviously di erent from those of mice in the oseltamivir phosphate group. Nevertheless, in this study, we extended the IL-37 administration time to 9 days (oseltamivir+IL-37 9d), and the death and body weight changes in the mice (n = 7) were monitored for 14 d.p.i. e results showed that the body weights of mice began to increase from 7 d.p.i., which was 2 days earlier than the body weights of mice in the oseltamivir phosphate group began to increase ( Figure 1A). A total of 71% of the mice from the oseltamivir+IL-37 9d group survived, whereas the survival rate in the oseltamivir phosphate group was 57% survival. All of the mice in the model group died (Figure 1B), suggesting that IL-37 treatment for 9 days advanced the onset of body weight recovery and improves the survival rate in H1N1-infected mice. combination with oseltamivir phosphate reduced the time to the onset of body weight recovery, and this reduction varied based on the di erent administration times. As shown in Figure 2A, the body weights of mice infected with H1N1 decreased and began to increase from the 7th day of IL-37 plus oseltamivir phosphate treatment at 2 h.p.i. (oseltamivir+IL-37 I.V 2 h), whereas the body weights of mice in the oseltamivir phosphate group began to increase on the 9th day; in the model group, the body weight continued to decrease until death. Moreover, animals that received PBS succumbed to infection 5-7 days a er viral challenge. When therapeutic treatment was started at 24 h.p.i, 57% of the infected mice survived, which was the same rate as the mice treated with oseltamivir phosphate only. e administration of IL-37 at 2 h.p.i. increased the mouse survival rate to 71%. However, in the group administered IL-37 at 48 h.p.i, the mortality rate increased (Figure 2B). Similar to the results shown in Figure 2, the survival rate of mice intranasally administered IL-37 at 2 h.p.i., was signi cantly enhanced compared to that of mice in the oseltamivir phosphate group (Figure 2D). However, the body weight in the intranasal IL-37 treatment group was not signi cantly increased compared with that in the oseltamivir phosphate group; however, the body weight decrease time was shortened and remained stable at 7-9 d.p.i (Figure 2C).
ese results demonstrate that intravenous IL-37 administration at 2 h.p.i signi cantly decreases the mortality of mice infected with H1N1 and shortens the recovery time of infected mice, so intravenous IL-37 administration at 2 h.p.i o ers enhanced protection against in uenza challenge in mice.

Interleukin-37 Treatment Reduces Lung Damage in Mice Infected With H1N1 Virus
In addition, to further validate the therapeutic e ect of IL-37, lung tissue was collected to monitor the pulmonary indexes and lung histology. As expected, the IL-37 combined with oseltamivir phosphate administration group had signi cantly lower pulmonary index values than the other groups ( Figure 3A). e BAL uid was gathered 6 days a er infection or IL-37 treatment.
e total cell number in the BALF was increased signi cantly a er infection ( Figure 3B). However, IL-37 treatment evidently diminished H1N1-induced neutrophilic and eosinophilic airway in ammation ( Figure 3C). Additionally, H&E staining of the lung tissue samples showed that the lungs in the model group exhibited many merged, in ated, or enlarged alveoli as well as an increase in the exudation of in ammatory proteins in the alveolar spaces at 6 d.p.i., which was largely decreased in the IL-37-treated group (Figure 3D). ese results further indicate that IL-37 could be a useful therapeutic agent in mice with H1N1 infection.

Interleukin-37 Inhibits the Production of In ammatory Cytokines in H1N1-Infected Mice
IL-37, as a potent inhibitor of innate immunity, can shi the cytokine equilibrium away from excessive in ammation (Teng et al., 2014). us, to more accurately assess the e cacy of IL-37 during H1N1 infection, the mRNA expression and the protein production levels of IL-6, TNF-α, MCP-1, IL-1α, IL-1β, MIP-1α, MIP-1β, IP-10, MIG, RANTES, IFN-γ and IL-10 were detected by RT-PCR and CBA in the lung tissue, BALF and serum samples on day 6 a er H1N1 infection. As expected, IL-37 treatment inhibited the increase in levels of MCP-1, IL-1β, MIP-1α, MIP-1β, MIG, IFN-γ and RANTES in the lungs of the model group ( Figure 4A). Paralleling the decreased production of cytokines, the upregulation of MCP-1, IL-1β, IL-6, IP-10, MIG, and RANTES mRNA expression was markedly reduced in the IL-37 treatment group ( Figure 4D). In addition, it is worth mentioning that the expression of MCP-1 and IL-1β, especially the level of MCP-1 in the oseltamivir group, was higher than that in the model group; however, oseltamivir plus IL-37 treatment corrected the increase in MCP-1 expression ( Figure 4D). Moreover, the mRNA expression of the anti-in ammatory cytokine IL-10 was downregulated in the IL-37-treated group ( Figure 4D); however, the production of IL-10 protein in the lungs was not signi cantly changed (data not shown). Furthermore, compared with that in the model and oseltamivir groups, the upregulated production of MCP-1, IL-6 and IFN-γ in the BALF ( Figure 4B) and serum ( Figure 4C) was markedly reduced in the IL-37 treatment group.
ese results indicate that IL-37 exerts a protective e ect by regulating the levels of in ammatory cytokines, particularly by regulating macrophage cytokine production.

Macrophage Percentages Are Increased in Interleukin-37 Treated Mice
To evaluate the roles of immune cells in the IL-37-mediated protection against in uenza A (H1N1) infection, the changes in the numbers of macrophages and T cells in the lungs of di erent groups at multiple time points were analyzed using ow cytometry. In fact, the percentages of CD4 + and CD8 + , IL-18Rα + CD4 + or CD8 + T cells in the IL-37 treatment group were not obviously di erent compared with those in the model group (Supplementary Figure S1). However, an increase in the percentage of macrophages, which were identi ed as CD45 + F4/80 + CD11b + cells, was observed in the lungs of mice in the IL-37 administration group, peaking at day 6 post infection ( Figure 5A). Paralleling the augmented macrophage percentage, the percentage of IL-18Rα + macrophages was markedly enhanced in the lungs of IL-37-treated mice ( Figure 5B).
ese results show that IL-37 administration impairs the decrease in the macrophage population in the lungs, indicating that macrophages may exert immunoprotective e ects in mice treated with IL-37 during H1N1 infection.

Depleting Macrophages Reduces the Protective Effect of Interleukin-37 During In uenza Virus Infection
As macrophages may exert immunoprotective e ects in H1N1infected mice treated with IL-37, we intranasally administered clodronate liposomes, which have been shown to deplete macrophages in the lungs (Tate et al., 2010;Wong and Smith, 2017), to mice during in uenza virus infection to test the dependency of the IL-37 treatment-induced reduction in mortality on macrophages. As expected, under the same conditions, the clodronate liposome administration group reached its lowest weight at 9 d.p.i. and the body weight change did not recover in the control liposome group (Figure 6A). Moreover, clodronate liposome treatment signi cantly increased the mortality rate ( Figure 6B). ese data further demonstrate that macrophages are critical for the protective e ect of IL-37 during in uenza virus infection.

Interleukin-37 Inhibits the Expression of In ammatory Cytokines in a MAPK-Dependent Manner in vitro
To clearly demonstrate the anti-in ammatory e cacy of IL-37, following H1N1 infection, murine macrophage RAW264.7 cells were treated with IL-37 for di erent times (12 and 24 h). e levels of in ammatory factors in the cells were determined by real-time PCR. As shown in Figure 7A, IL-6 mRNA expression at all time points was obviously downregulated in the IL-37 treatment groups compared with the oseltamivir group. Similar inhibitory e ects were also observed on TNF-α, IL-1β and MIP-1β expression ( Figure 7A).
To further explore the underlying mechanisms of IL-37, we examined the expression of PRR-related protein phosphorylation. For this purpose, western blot analyses were performed using the cell lysates of RAW264.7 cells treated with or without IL-37 to analyze the phosphorylation of MAPKs and GAPDH. In addition, NLRP3 protein production was detected. As shown in Figure 7B, compared with that in the infection and oseltamivir groups, the phosphorylation of ERK1/2 and p38 MAPK was signi cantly reduced in the IL-37 treated RAW264.7 cell group. Similarly, the ratio of NLRP3 in the IL-37-treated group was signi cantly decreased in vitro. ese results show that IL-37 treatment inhibits the production of macrophage in ammatory cytokines induced by H1N1 infection in a MAPK-dependent manner.

DISCUSSION
In uenza viruses cause seasonal epidemics and sporadic pandemics, and are a major burden on human health. e rapid development of viral pneumonitis induced by aggressive in ammation resulting in high morbidity and mortality, emphasizing the importance of exploring e ective approaches to ameliorate the viral pneumonia during H1N1 infection (Morita et al., 2013). IL-37 has been shown to block the deleterious e ects of pro-in ammatory stimuli or conditions in numerous models (Barbier et al., 2019;eoharides et al., 2019), it is essential for the inhibition of innate immunity and in ammation and plays a role in the inhibition of cytokine and chemokine production, and in ammatory cell in ltration ( eoharides et al., 2019). Increasing evidence suggests that the anti-in ammatory cytokine IL-37, improve functional outcomes in combination, including neuroprotection and reduced of lung infection burden (Zhang et al., 2019). In the present study, by using H1N1-infected BALB/c mice, we found that intravenous IL-37 treatment advanced the time to body weight recovery onset, improved the survival rate (Figures 1, 2), and ameliorated the increase in the exudation of in ammatory proteins in the alveoli (Figure 3). ese results demonstrate that IL-37 treatment can ameliorate viral pneumonia and a ord a better protection from A/California/07/2009 (H1N1) infection in the murine model. Furthermore, IL-37 treatment signi cantly reduced the production of the in ammatory cytokines and chemokines MCP-1, IL-1β, MIP-1, IFN-γ, MIG and RANTES, meanwhile the increased mRNA expression of MCP-1, IL-1β, IP-10, IL-10, MIG and RANTES in the lungs of the IL-37treatment group was obviously decreased compared with that in the oseltamivir group (Figure 4). Interestingly, most of the cytokines obviously decreased at both the transcriptional and translational levels were macrophage cytokines.
Indeed, IL-37 administration impaired the decrease in the percentage of macrophages in the lungs of H1N1-infected mice (Figure 5), and depleting macrophages reduced the protective e ect of IL-37 during in uenza virus infection (Figure 6). e anti-in ammatory endogenous ligand annexin A1 has been shown to attenuate pathology upon subsequent in uenza A virus infection, and reduction in lung damage severity is associated with an increase in the number of alveolar macrophages (AMs) in the murine model of in uenza A virus infection (Schloer et al., 2019). Numerous literatures have demonstrated that macrophage are critical for host defense in mice during in uenza viral infection (He et al., 2017;Wong and Smith, 2017). Our results are consistent with the results of these reports, showing that macrophages may exert immune protective e ects in H1N1-infected mice treated with IL-37.
IL-37 can strongly regulate macrophages to restrain the autoimmune response (Ye and Huang, 2015;Toulmin et al., 2017;Wang et al., 2018;Yang et al., 2019). It has been reported that IL-37 can promote macrophage polarization from the pro-in ammatory subtype (M1) to the anti-in ammatory subtype (M2) in atherosclerosis (McCurdy and Baumer, 2017). Moreover, IL-37 induces a phenotypic shi in THP1-derived macrophages toward a CD206 +high and CD86 +low macrophage subtype and enhanced the mRNA levels of IL-10, which are characteristic hallmarks of M2 macrophages . In summary, these results indicate that IL-37 treatment ameliorates the lung damage by polarizing macrophages from an M1 to an M2 phenotype. Further research regarding the mechanisms of the inhibitory e ect of IL-37 is needed.
To further con rm that macrophages play an important role in the anti-in ammatory e ect of IL-37, RAW264.7 cells were infected with H1N1, and treated with oseltamivir in combination with/without IL-37. At the indicated intervals, macrophages were collected, and the expression of cytokine mRNA in the cells was detected. As shown in Figure 7A, compared with that in the oseltamivir group, the mRNA expression of IL-1β, IL-6, TNF-α and MIP-1 was obviously downregulated in the IL-37 treatment group. ese results are consistent with those of pulmonary studies, which further indicates that IL-37 treatment can ameliorate H1N1-induced in ammation by reducing macrophage cytokine production. e activity of IL-37 has been reported to largely depend upon IL-18Rα and SIGIRR for the extracellular activation of the anti-in ammatory pathway (Lunding et al., 2015;Zeng et al., 2017). Herein, we found that paralleling the augmented macrophages percentage, the IL-18Rα + -macrophages percentage was enhanced markedly in the lungs of IL-37-treated mice (Figure 5), indicating that IL-37 down-regulates the increased production of pro-in ammatory cytokines in an IL-18Rα activation-dependent manner. en, by using the RAW264.7 cell line, the underlying mechanisms of the IL-37 e ect in macrophages were further investigated. Studies have shown that MAPK-related signaling can be inhibited by IL-37 in activated mast cells (Gallenga et al., 2019). In addition, the intraperitoneal injection of IL-37 signi cantly decreases the expression of NLRP3 in the mouse lung aspergillosis model (Moretti et al., 2014;Jia and Liu, 2018). Indeed, our results showed that the increased phosphorylation of ERK1/2 and p38 MAPK was signi cantly downregulated in RAW264.7 cells treated with IL-37; furthermore, the ratio of NLRP3 in the IL-37-treated group was decreased in vitro (Figure 7). In contrast, treatment with IL-37 did not inhibit the production of JNK protein in in uenza A virus-infected RAW 264.7 cells (data not shown). ese results con rm that IL-37 ameliorates in uenza pneumonia by attenuating macrophage cytokine production in a MAPK pathway-dependent manner, especially the ERK1/2 and p38 pathways.
In conclusion, these data provide evidence that IL-37 inhibits the pathogenesis of in uenza pneumonia by decreasing the production of essential pro-in ammatory cytokines, indicating a new and promising therapeutic approach for excessively activated immune responses in in uenza A infection-induced pneumonia. However, the function of IL-37 in other viral infections, especially serious emergent and re-emerged infectious diseases, remains unclear. Further detailed research remains necessary to fully determine the possible functions of IL-37 in viral infections.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
e animal study was reviewed and approved by e Institute of Animal Use and Care Committee of the Institute of Laboratory Animal Science, Peking Union Medical College.

AUTHOR CONTRIBUTIONS
CQ, LB, and FQ conceived, designed, and supervised the experiments. FQ and ML performed most experiments, analyzed the data, and wrote the original dra . LB, FQ, and ML analyzed the data. FL, QL, GW, SG, SW, and YX conducted some experiments. FQ and ML edited the manuscript. All authors reviewed and approved the manuscript.

SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02482/ full#supplementary-material SUPPLEMENTARY FIGURE S1 | Percentages of lymphocytes were detected in IL-37-treated mice. (A) The percentages of different lymphocytes that were identi ed as CD3 + CD4 + or CD3 + CD8 + as well as IL-18Rα + lymphocytes in the lungs of IL-37 treated mice were determined by ow cytometry on day 6 during H1N1 infection. Data are representative of three independent experiments with three mice for each group. *Signi cant difference (p < 0.05), compared with oseltamivir-treated mice.