Initial Influenza Virus Replication Can Be Limited in Allergic Asthma Through Rapid Induction of Type III Interferons in Respiratory Epithelium

Although asthmatics has been considered to be highly susceptible to respiratory viral infection and most studies have focused on exacerbation of asthma by influenza A virus (IAV) infection, few experimental evidences exist to directly demonstrate that asthmatic mice are actually resistant to IAV infection. Here, we show that asthmatic mice are not highly susceptible to IAV in the early stage of infection and type III interferon (IFN) maintains antiviral immune response in the lung of IAV-infected asthmatic mouse resulting in inhibition of initial viral spread. C57BL/6 mice with allergic asthma were infected with IAV (WS/33: H1N1) and survival rate, body weight, viral titer, histopathological findings of lung and cytokine profiles including IFNs and Th2 cytokines were measured. Notably, asthmatic mice were significantly resistant to IAV and showed lower viral load until 7 days after infection. Furthermore, IAV-infected asthmatic mice exhibited decreased Th2-related inflammation in lung tissue until 7 days. These increased antiviral resistant mechanism and reduced Th2 inflammation were attributable to rapid induction of type III IFNs and blockade of type III IFNs in asthmatic lung led to aggravated IAV infection and to enhance the production of Th2 cytokines. Asthmatic mice showed bi-phasic responses against IAV-caused lung infection such as rapid production of type III IFNs and subsequent induction of type II IFNs. Actually, IAV-infected asthmatic mice become vulnerable to IAV infection after 7 days with noticeable morbidity and severe weight loss. However, intranasal administration of type III IFNs protects completely asthmatic mice from IAV-mediated immunopathology and lung infection until 14 days after infection. Taken together, our study indicates that the rapid induction of type III IFN might be distinctive immunological findings in the respiratory tract of IAV-infected asthmatic mice at the early stage of infection and crucial for suppression of initial viral spread in vivo asthma accompanying with restriction of Th2 cytokine productions.

inTrODUcTiOn Allergic asthma is caused by sensitization to innocuous allergens via airway exposure. This type of asthma is thought to arise from an imbalance in T helper type I (Th1)-Th2 immune regulation, resulting in increased levels of the Th2 cytokines interleukin (IL)-4, IL-5, and IL-13, which have been proven to be important drivers of allergic airway inflammation in asthma (1,2). Asthma exacerbations are acute attacks of asthma, accompanied by sudden decrease of lung function, most often precipitated by a respiratory viral infection and are responsible for the vast majority of the mortality associated with asthma. Although adequate control of asthma has been achieved, more appropriate controls for respiratory viral infection are needed to reduce acute exacerbation of respiratory symptoms in asthmatics (3,4). The innate immune system of the respiratory epithelium serves as the first line of antiviral defense against invading respiratory viruses including influenza A virus (IAV) (5). Traditionally, the antiviral innate immune response has been thought to be exclusively mediated by type I interferons (IFNs) followed by the adaptive immune response (5,6). However, emerging evidence indicates that type III IFN is likely to be mainly required for immune responses in respiratory tract. In particular, type III IFN has been shown to be dominant IFN which is produced in respiratory tract against respiratory viral infection and provide front-line protection against respiratory virus to suppress initial viral spread in respiratory epithelium (7,8). Moreover, type III IFN-mediated innate immune response is necessary to protect the lungs from IAV infection beyond antiviral properties of type I IFNs (9). Impaired innate immune responses have been reported to be potentially responsible for the increased susceptibility to infections in asthmatics. Moreover, dysregulation of antiviral immune responses related to Th2 cytokines has been suggested to explain the higher susceptibility of the asthmatic respiratory epithelium to viral infection (10)(11)(12). Strong links between low expression of type III IFNs, severity of allergic asthma, and asthma exacerbations have been described (13). In the absence of detectable viral infection, asthmatic patients with active disease still exhibit an inverse correlation between type III IFN level and the severity of allergic response in the airways (12). However, some studies have also investigated whether asthmatic mice are more resistant to respiratory viral infection and which bystander immune mechanisms are activated during the induction of asthma and contribute to protecting the asthmatics against respiratory viral infection (14,15).
To address the antiviral resistance of IAV-infected asthmatic mice, we sought to determine first if the asthmatic mice are more susceptible or resistant to IAV infection. We found that the asthmatic mice were not highly susceptible to IAV infection and type III IFNs were preferentially induced in the lung of IAV-infected asthmatic mouse at early stage of infection. Rapid induction of type III IFNs led to accelerated clearance of IAV, accompanied by increased type II IFN secretion. In addition, intranasal administration of type III IFNs provided more potent antiviral resistance to IAV-infected asthmatic mice. This study suggest that a better understanding about the role of type III IFNs in this asthmatic airway need to be achieved to prevent higher viral loads in the asthmatic airway and it can provide new insights into strategies for reducing asthmatic exacerbation from respiratory viral infection.

MaTerials anD MeThODs allergen sensitization and challenge Protocol
C57BL/6J (B6) mice (Orientalbio, Seoul, Korea) aged 7 weeks (19-23 g) were used for the development of non-asthmatic and asthmatic mice. Asthma was induced by first sensitizing male B6 mice intraperitoneally (i.p.) with OVA in aluminum hydroxide and then challenging intranasally (i.n.) with soluble OVA (OVA/ OVA). Phosphate-buffered saline (PBS)-challenged mice (OVA/ PBS) (hereafter referred to as non-asthmatic mice) were used as a negative control. B6 mice were sensitized with two intraperitoneal injections on days 0 and 14 of 7.5 μg OVA (Grade V; Sigma, MO, USA) complexed with aluminum hydroxide as adjuvant (Sigma, MO, USA). On days 21, 22, 23, and 24, mice were challenged intranasally with 7.5 g OVA mixed with PBS (OVA/OVA). Control mice received an intraperitoneal injection of OVA at the same concentration and were challenged with PBS alone by intranasal inoculation (OVA/PBS). Airway hyper-responsiveness (AHR) was measured in anesthetized mechanically ventilated B6 mice (Flexivent ventilator, SciReq, Montreal, QC, Canada) at 24 h after the last intranasal OVA exposure. AHR was measured invasively using a body plethysmograph (Buxco Electronics, Inc., Wilmington, NC, USA).

Mice and Virus inoculation
Influenza A virus (WS/33: H1N1, ATCC, Manassas, VA, USA) was used to induce acute viral lung infection. Virus stocks were grown in Madin-Darby canine kidney cells in virus growth medium according to a standard procedure (16). Briefly, after 48 h of incubation at 37°C, the supernatants were harvested and centrifuged at 5,000 rpm for 30 min to remove cellular debris. Virus stocks were titrated on MDCK cells using a tissue culture infectious dose assay and stored at −80°C. The B6 mice used in the study, like other commercially available strains of inbred mice, carry a dysfunctional Mx1 gene and are not congenic B6 mice with a functional Mx1 gene, which are derived from influenzaresistant mice.
For viral infections, IAV (WS/33, H1N1; 213 pfu in 30 µl PBS) were inoculated into WT mice by intranasal delivery and asthmatic mice (OVA/OVA) were also infected with IAV at 25 days after first sensitization. Mice were euthanized at the end of each experiment with overdose of tiletamine/zolazepam (5 mg) and xylazine (0.23 mg) and after euthanizing, bronchoalveolar lavage (BAL) fluid was obtained from the lungs by lavaging with 1,000 µl 0.5 mM ethylene diamine tetraacetic acid in PBS after cannulation of the trachea. The BAL fluid was used for enzyme-linked immunosorbent assay (ELISA) for measuring secreted protein levels and plaque assay to determine the viral titer. Mouse lung tissue was also harvested for real-time polymerase chain reaction (PCR), microarray, and immunohistochemistry analyses.

real-Time Pcr
Lung tissue was obtained from mice infected with WS/33 (H1N1) on 1, 3, 5, 7, 10, and 14 days postinfection, after which total RNA was isolated using TRIzol (Invitrogen). cDNA was synthesized from 3 µg of RNA with random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Perkin Elmer Life Sciences, Waltham, MA, USA and Roche Applied Science, Indianapolis, IN, USA). Amplification was performed using the TaqMan Universal PCR Master Mix (PE Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Briefly, amplification reactions had a total volume of 12 µl and contained 2 µl of cDNA (reverse transcription mixture), oligonucleotide primers (final concentration of 800 nM), and TaqMan hybridi zation probe (200 nM). Real-time PCR probes were labeled at the 5′ end with carboxyfluorescein (FAM) and at the 3′ end with the quencher carboxytetramethylrhodamine (TAMRA).
To quantify the intracellular levels of viral RNA and host gene expression levels, cellular RNA was used to generate cDNA. IAV level was monitored using quantitative PCR to amplify the PA gene (segment 3) with forward and reverse primers and probe 5′-ggccgactacactctcgatga-3′, 5′-tgtcttatggtgaatagcctggttt-3′, and 5′-agcagggctaggatc-3′, respectively. Primers for mouse IFN-α, IFN-β, IFN-λ2/3, and IFN-γ were purchased from Applied Biosystems (Foster City, CA, USA). Real-time PCR was performed using the PE Biosystems ABI PRISM ® 7700 Sequence Detection System. Thermocyling parameters were as follows: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min. Target mRNA levels were quantified using target-specific primer and probe sets for IAV WS/33 (H1N1), IFN-α, IFN-β, IFN-λ2/3, and IFN-γ. All PCR assays were quantitative and utilized plasmids containing the target gene sequences as standards. All reactions were performed in triplicate, and all real-time PCR data were normalized to the level of the housekeeping gene glyceraldehyde phosphate dehydrogenase (1 × 10 6 copies) to correct for variation between samples.
These antibodies were found to inhibit the secretion of IFN-λ2/3 and IFN-γ by more than 70% in BAL fluid. Neutralizing antibodies (10 μg/30 μl) were mixed with PBS and inoculated by intranasal delivery according to the manufacturer's instructions (R&D Systems Inc.), concurrent with IAV infection. This procedure did not affect mouse viability. ELISA analysis confirmed that the neutralizing antibodies partially inhibited IFN-λ2/3 and IFN-γ secretion.

immunohistochemistry and histological analysis
Lung tissue was fixed in 10% (vol/vol) neutral buffered formalin and embedded in paraffin. Paraffin-embedded tissue slices were stained with hematoxylin/eosin (H&E) or periodic acid Schiff (PAS) solution (Sigma, Deisenhofen, Germany). Histopathological analysis of inflammatory cells in H&E-stained lung sections was performed in a blinded fashion using a semi-quantitative scoring system as previously described (13). Lung sections from at least five mice were examined. Briefly, peribronchiolar inflammation was scored as follows: 0, normal; 1, a few cells; 2, a ring of inflammatory cells one layer deep; 3, a ring of inflammatory cells two to four cells deep; and 4, a ring of inflammatory cells more than four cells deep (maximum score = 8). The histological score for PBS/PBS control mouse lung tissue was 0. At least six separate areas from similar sections within a single mouse were assessed, and at least five mice were assessed. The five best sections were used for evaluation. PMNs were counted by an examiner who was blinded to the experimental group; results are expressed as the number of cells per high power field.

Plaque assay
Virus samples were serially diluted with PBS. Confluent monolayers of MDCK cells in six-well plates were washed twice with PBS and then infected in duplicate with 250 μl/well of each virus dilution. The plates were incubated at 37°C for 45 min to facilitate virus adsorption. Following adsorption, a 1% agarose overlay in complete MEM supplemented with TPCK trypsin (1 µg/ml) and 1% fetal bovine serum was applied. The plates were incubated at 37°C, after which cells were fixed with 10% formalin at 2 days postinfection.

intracellular cytokine staining
For intracellular cytokine staining, single-cell suspensions were incubated in RPMI medium containing 10% FBS with PMA (100 ng/ml), ionomycin (1 µg/ml), and GolgiStop (BD) at 37°C for 4 h. After surface staining, the cells were fixed and permeabilized with a Fixation/Permeabilization Kit (eBioscience). Finally, the cells were stained with PE-anti-T-bet and APC-anti-IFN-γ antibodies (Biolegend). The respective isotype-control antibody was also used for each experiment.

statistical analyses
Real-time PCR, plaque assay, and ELISA results are presented as median values (interquartile ranges for 25 and 75%). The statistical significance of differences between two groups was determined by the Mann-Whitney test. Histological scores were also evaluated by a non-parametric test (Wilcoxon rank sum test). All statistical analysis was performed with GraphPad Prism software (version 5; GraphPad Software, La Jolla, CA, USA). p Values <0.05 were considered to be statistically significant.

resUlTs
OVa-sensitized/challenged c57Bl/6 (B6) Mice exhibit a Typical asthmatic Phenotype To assess the influence of allergic airway inflammation on susceptibility to respiratory viral infection, we developed an OVA-sensitized/challenged allergic asthma mouse model on a B6 genetic background ( Figure 1A). Initially, AHR was measured in mice after inoculation of methacholine. Asthmatic mice (OVA/ OVA) were observed to have a methacholine-induced increase in total lung resistance (N = 3, Figure 1B). As a complementary approach, H&E-and PAS-stained micrographs of lung sections were obtained from non-asthmatic (OVA/PBS) and asthmatic mice. Histological analysis revealed that the lungs of asthmatic mice were severely inflamed, with extensive inflammatory cell infiltration at the peribronchial areas of the lung. This infiltration was accompanied by significantly increased goblet cell metaplasia ( Figure 1C). We found that the number of eosinophils was also increased in total lung tissue of asthmatic mice, and that the numbers of lymphocytes, neutrophils, and eosinophils were significantly increased in the BAL fluid of asthmatic mice (Figures 1D,E). Collectively, these findings demonstrate that an allergic asthma mouse model could be established with B6 mice, and that this model could be used to investigate the susceptibility of asthmatic mice to influenza virus infection.
asthmatic Mice are not highly susceptible to iaV infection Previously, we found that IAV-infected non-asthmatic mice exhibited a significant decrease in mean body weight and a decrease in body temperature, with the lowest drop at 7 dpi. They also showed 80% survival until 14 dpi (17). In this study, non-asthmatic mice (N = 5) and asthmatic mice (N = 5) were inoculated with WS/33 (H1N1) to determine the susceptibility of asthmatic mice to IAV lung infection. Then, the body weights and survival rates of non-asthmatic and asthmatic mice were compared until 7 dpi. Non-asthmatic mice showed significant weight loss from 5 dpi, and 20% of the mice died in the first 7 days after IAV infection. However, asthmatic mice did not exhibit significant weight loss or noticeable morbidity until 7 dpi, and all asthmatic mice survived the IAV infection (Figures 2A,B). The levels of IAV mRNA in non-asthmatic and asthmatic mice lung were next analyzed by real-time PCR, and the viral titers in the BAL fluid were measured by plaque assay at 7 dpi. The mean level of IAV mRNA and the mean viral titer were both significantly elevated at 7 dpi (mRNA level: 2.1 × 10 4 , viral titer: 3.3 × 10 5 pfu/ml) in non-asthmatic mice. By contrast, the levels of IAV mRNA and viral titer were much lower in IAV-infected asthmatic mice (mRNA level: 1.4 × 10 3 , viral titer: 8.1 × 10 4 pfu/ml, Figures 2C,D). As a complementary approach, lung sections were obtained from non-asthmatic and asthmatic mice at 7 dpi, and H&E-stained micrographs were generated. Histological analysis revealed severe subepithelial consolidation, peribronchial edema, and increased epithelium detachment in non-asthmatic mouse lung sections harvested at 7 dpi. The lungs of asthmatic mice were severely inflamed, with extensive inflammatory cell infiltration at the peribronchial areas without IAV infection. Notably, these histopathological findings were not detectable in the lung sections harvested from IAVinfected asthmatic mice at 7 dpi and the mean histological score was significantly lower in IAV-infected asthmatic mice (8.6 for the non-asthmatic mice vs. 1.8 for the asthmatic mice) ( Figure 2E). This finding was accompanied by significantly increased goblet cell metaplasia in asthmatic mice, whereas remarkable reduction of goblet cells was observed in the respiratory epithelium of IAVinfected asthmatic mice at 7 dpi (13.2 in asthmatic mice vs. 4.6 in IAV-infected asthmatic mice, Figure 2F). In addition, AHR was measured after inoculation of methacholine in IAV-infected asthmatic mice at 7 dpi and a methacholine-induced increase in total lung resistance was not observed in IAV-infected asthmatic mice ( Figure S1 in Supplementary Material).
A multiplex assay was next performed to quantify the levels of secreted Th2 cytokines, such as IL-4, IL-5, and IL-13, in the BAL fluid of asthmatic mice. While higher secretion of Th2 cytokines was observed at 7 dpi in asthmatic mice without IAV infection (IL-4: 532.3 ± 85.1 pg/ml, IL-5: 964.7 ± 98.5 ng/ml, IL-13: 2,487.5 ± 765.5 ng/ml), cytokine secretion was significantly attenuated in IAV-infected asthmatic mice (IL-4: 64.2 ± 11.5 pg/ml, IL-5: 121.7 ± 32.6 ng/ml, IL-13: 498.5 ± 104.8 ng/ml, Figure 2G). These results indicate that asthmatic mice were not highly susceptible to IAV infection. Moreover, IAV-infected asthmatic mice asthmatic Mice Maintain intact iFn-related innate immunity after iaV infection To assess whether the increased antiviral resistance of asthmatic mice was due to an increased IFN-related immune response, we investigated the influence of asthma status on induction of IFN from the respiratory tract after IAV infection. Real-time PCR analysis revealed that the mRNA levels of IFN-α4, IFN-β, and IFN-λ2/3 were higher at day 7 after IAV infection in lung tissue from non-asthmatic mice (IFN-α4: 1.2 × 10 4 ± 3.4 × 10 3 , IFN-β: 2.8 × 10 4 ± 4.3 × 10 3 , IFN-λ2/3:1.4 × 10 5 ± 5.2 × 10 4 , Figure 3A) but minimal induction of IFN mRNA, especially IFN-λ2/3, was observed in lung tissue from IAV-infected asthmatic mice (IFN-λ2/3:4.8 × 10 4 ± 1.4 × 10 4 ). Next, ELISA was performed to quantify the levels of secreted IFNs in the BAL fluid after IAV infection. The levels of secreted IFN-β and IFN-λ2/3 were all increased  . This finding suggests that the IFN-γ production in IAV-infected asthmatic mice observed at 7 dpi might be critically involved in resistance to IAV infection and decreased secretion of Th2 cytokines in the asthmatic respiratory tract. While neither IFN-β nor IFN-λ2/3 was induced in the respiratory tract of IAV-infected asthmatic mice at 7 days after infection, we next analyzed the levels of these IFNs in asthmatic mice until 7 days after infection to determine if rapid and transient alterations occurred. To this end, asthmatic mice were infected with IAV WS/33 (H1N1) via the intranasal route (213 pfu/30 μl), and the production of IFNs and Th2 cytokines was measured at 0, 1, 3, 5, and 7 dpi.
These findings show that IFN-λ2/3 and IFN-γ were both markedly induced, and that IFN-λ2/3 induction was driven rapidly after IAV infection in the lungs of IAV-infected asthmatic mice than IFN-γ. This induction appears to reduce Th2 cytokine secretion in asthmatic mice after infection, which might be required for the enhanced resistance of asthmatic mice to IAV replication.
Influenza A virus infection has been shown to incite a robust IFN-γ response in the lung, promoting the development of adaptive immunity (18). IFN-γ has been proposed to be predominantly produced by activated innate lymphoid cell type I (ILC1) and natural killer (NK) cells (19). Our current findings also showed production of IFN-γ peaked at 7 dpi in the lung tissue and BAL fluid of IAV-infected asthmatic mice and we investigated the target cells that produce IFN-γ in asthmatic mice at 7 dpi. Non-asthmatic and asthmatic mice were infected with IAV    Figure S3 in Supplementary Material). Then, we also investigate the correlation between IFN-λ and -γ in IAV-infected asthmatic mice with administration of IFN-λ2/3-and γ-neutralizing antibodies. The ELISA results showed that IAV-induced IFN-λ secretion was not altered in the BAL fluid of asthmatic mice with IFN-γ-neutralizing Ab, whereas IAV-induced IFN-γ secretion (1,724.5 ± 378.5 pg/ml) was significantly reduced in asthmatic mice that were administered IFN-λ2/3neutralizing antibodies before IAV infection (669.5 ± 214.6 pg/ ml) (Figures 4C,D). In addition, the decreased goblet cell metaplasia and airway mucus secretion observed after IAV infection were not seen until 7 dpi in the lung tissue of IAVinfected asthmatic mice inoculated with IFN-λ2/3-neutralizing antibodies ( Figure 4E). This finding was accompanied by higher viral titer until 7 dpi (PI 3d: 3.0 × 10 6 + 1.3 × 10 6 , PI 5d: 3.1 × 10 6 + 1.8 × 10 6 , PI 7d: 4.3 × 10 6 + 1.6 × 10 5 , Figure 4F). A multiplex assay was also performed to quantify the levels of the secreted Th2 cytokines IL-4, IL-5, IL-6, and IL-13, in the BAL fluid of IAV-infected asthmatic mice with functional blocking of IFN-λ2/3. As noted above, secretion of Th2 cytokines was significantly attenuated in IAV-infected asthmatic mice, whereas it was not attenuated in IAV-infected mice that were inoculated with IFN-λ2/3-neutralizing antibodies ( Figure 4G).
Overall, these results indicate that neither ILC1 nor NK cells contributed to the increased production of IFN-γ in IAV-infected asthmatic mice. They also indicate that IFN-γ is produced predominantly at the respiratory epithelium in the lungs of IAVinfected asthmatic mice. The rapid induction of IFN-λ2/3 could be related to the robust induction of IFN-γ at 7 days after IAV infection in respiratory epithelium of asthmatic mice; this induction seems to be central for expedite antiviral immune response and for limiting Th2 cytokine secretion after IAV infection.

DiscUssiOn
This study provides a different view of the antiviral innate immune system in the asthmatic respiratory tract. Specifically, it has been generally accepted that the asthmatic airway is highly vulnerable to respiratory viral infection due to its reduced production of IFNs, which are required for clearance of viral infection and lower induction of IFNs can be involved in robust production of Th2 cytokines (10)(11)(12). Here, we showed that IFN-λ2/3 is induced rapidly in asthmatic mice after IAV infection, and that this cytokine is important for maintaining the antiviral immune response against IAV lung infection and for restricting Th2 cytokine secretion in asthmatic mice. These effects were observed within 7 days. Our findings also imply that intranasal administration of IFN-λ2/3 is a potential strategy for controlling the enhanced susceptibility of asthmatic mice to IAV infection from 7 days postchallenge onward.
Asthmatics have been reported to have increased susceptibility to respiratory viral infection including increased severity, longer duration of respiratory symptoms, and acute exacerbation of asthma (20,21). This increased susceptibility to respiratory viral infection has been shown to be mediated by novel mechanisms including virus-induced secretion of epithelial-derived Th2 cytokines, deficient apoptosis of viral-infected cells, and lower production of IFNs after viral infection at the asthmatic airway epithelium (22,23). In particular, IFNs are the most important component of the innate immune response and have been shown to be involved in defective innate immune responses to respiratory viral infection. The asthmatic airway epithelium appears to be profoundly deficient in viral-induced production of IFNs; reduced induction of IFNs has been shown to dampen the adaptive immune response (24)(25)(26). Although in vivo asthma model does not show exactly same characteristics or phenotypes as human asthma, we thought B6-oriented asthmatic model exhibit the biomedical relevance with human asthma considering histological findings, increased AHR after methacholine inhalation and peribronchial infiltration of inflammatory cells. This model could be used to investigate the susceptibility of asthmatic mice to influenza virus infection. The experiments performed here showed that asthmatic mice exhibited a significantly lower survival rate and greater loss of body weight, accompanied by higher viral titer, from 7 days after IAV lung infection onward. In addition, induction of IFN-λ2/3 and -γ was significantly impaired in asthmatic mouse lung tissue at 14 days after IAV infection. To the best of our knowledge, deficient innate immune responses (including reduced induction of IFNs) are expected in the asthmatic respiratory tract. Additional research aiming to understand the mechanisms driving these deficient innate immune responses is urgently required to reduce the susceptibility to viral infection of asthmatics.
While most studies have focused on exacerbation of asthma by influenza viral lung infection and the higher susceptibility to influenza virus of asthmatics, studies using a novel mouse model of asthma and influenza infection have shown that asthmatic mice are highly susceptible to influenza viral infection compared with non-asthmatic mice. Such studies are necessary for identifying novel targets for the development of effective therapies against viral infection in asthmatics (11)(12)(13).
The different hypothesis has been introduced that asthmatic mice are more resistant to influenza virus as a result of a lower viral burden in the lungs than control mice, leading to improvement of their clinical condition (14). This prompt viral clearance in asthmatic mice has been shown to be mediated by increased production of antiviral cytokines, activation of NK cells, and enhanced antigen-specific CD8+ T cell activity after infection. Other immunological mechanisms explaining the viral resistance of asthmatic mice have also been suggested that the increased survival of asthmatic mice was due to increased TGF-β-mediated tolerance to influenza infection-mediated tissue damage, rather than enhanced antiviral immunity (15). We think that it will be of interest to determine whether asthmatic mice might be also resistant to lung infection by IAV, which causes an exacerbation of asthmatic symptoms and higher mortality.
In this study, we found that asthmatic mice were also resistant to IAV infection until 7 days. Specifically, all mice survived, the mean viral titer was significantly lower, and the IAV-induced lung pathologies were reduced. Interestingly, IAV infection led to reduced Th2-related immune responses, including decreased IL-4, IL-5, and IL-13 secretion. Moreover, resolution of extensive asthma-related lung pathologies such as goblet cell hyperplasia was observed consistently in the lungs of IAV-infected asthmatic mice at 7 dpi. Based on these results, we estimate that asthmatic mice were not completely vulnerable to influenza virus infection and particularly asthmatic mice exhibited distinct immune mechanisms for resisting respiratory viral infection and restricting Th2 immunopathology in the respiratory tract.
We focused on the phase at which IFN-λ2/3 is rapidly induced in the lungs of IAV-infected asthmatic mice and IFN-λ2/3 contributes to resistance to IAV infection in asthmatic mice. In allergic airway diseases, IFN-λ cytokines are critical for driving Th1 differentiation in vivo and limiting Th2 and Th17 responses in the airway (7,10). Our data also show that IFN-λ2/3 in particular was preferentially secreted in the lungs of IAV-infected asthmatic mice from 1 dpi and its level was maintained until 7 days. In addition, secretion of IFN-λ2/3 drives the transcription of IFN-γ, which was accompanied by increased viral clearance in respiratory epithelium and attenuation of Th2 cytokine secretion in IAV-infected asthmatic mice from 1 dpi onward. Interestingly, functional inhibition of IFN-λ2/3 in IAV-infected asthmatic mice aggravated IAV infection. Moreover, the level of Th2 cytokine and the degree of asthma-related lung histopathological findings in vivo were improved after IAV infection. Given these findings, the impairment of IFN-λ2/3 induction in response to viral infection in asthmatics has profound implications relating to the pathogenesis of virus-induced asthma exacerbation. Thus, rapid production of IFN-λ2/3 in the lungs of asthmatic mice in response to IAV infection could constitute the primary antiviral defense and could also restrict the secretion of Th2 cytokines. Previously, we found that the highest mRNA IFN-λ2/3 level and the highest level of secreted IFN-λ2/3 were observed at 10 dpi in the lungs of IAV-infected B6 mice (17,27). By contrast, IFN-λ2/3 was released in the lungs of asthmatic mice from 1 day after IAV infection and this rapid production and maintenance of IFN-λs until 5 days after infection was accompanied by increase of IFN-γ secretion at 7 dpi. The levels of secreted IFN-β was increased by 3 dpi and the highest level of IFN-β secretion was also observed at 7 dpi in the lung of IAV-infected non-asthmatic mice (17) but IFN-β was minimally induced in the lung of IAV-infected asthmatic mice until 7 dpi. We hypothesize that the pattern of IFN secretion in response to IAV infection was altered in asthmatic mice. Specifically, asthmatic mice exhibited more rapid release of IFN-λ2/3, which is a central mediator of the antiviral immune response. This mechanism could explain the high resistance of asthmatic mice to respiratory viral infection at the early stage of IAV infection.
Although IFN-λ2/3 production was rapidly driven after IAV infection in the respiratory tract of IAV-infected asthmatic mice, asthmatic mice became vulnerable to the effect of IAV infection after 7 dpi and the levels of secreted IFN-β, IFN-λ2/3, and IFN-γ were completely reduced at 14 dpi in the lung of IAV-infected asthmatic mice. However, intranasal delivery of IFN-λ2/3 was shown to almost completely inhibit viral replication in IAV-infected asthmatic mice, as demonstrated by the significantly decreased viral titer in the lungs of mice that received intranasal IFN-λ2/3 compared with that of non-infected mice. In addition, the lungs of mice that received intranasal IFN-λ2/3 exhibited similar histological results to those of noninfected mice. Therefore, strategies involving the compensation or maintenance of IFN-λ are new opportunities for invoking an effective antiviral defense against IAV infection. IFN-λ is thus a promising new target for reducing susceptibility to respiratory viral infection in asthmatics.
In summary, rapid induction of IFN-λ is a distinctive immunologic finding in IAV-infected asthmatic mice and this effect is accompanied by reduced initial viral spread in asthmatic lung. This rapid induction of IFN-λ is crucial for controlling viral load and for maintaining effective antiviral immune mechanisms in asthmatics at early stage of infection. Our study provides compelling evidence that IFN-λ could have therapeutic potential for treating IAV-related respiratory infection in asthmatics.     FigUre s3 | Immunohistochemistry for IFN-γ in non-asthmatic and asthmatic mice. Immunohistochemical analysis of IFN-γ using DAB chromogen was performed in lung sections from non-asthmatic (OVA/PBS) and asthmatic mice (OVA/OVA) before and after IAV infection. The number of IFN-γ-positive cells was significantly increased in the lung tissue of IAV-infected asthmatic mice and bronchial epithelial cells showed strong expression of IFN-γ (Original magnification x200).