A significant number of clinical asthma exacerbations are triggered by viral infection. We aimed to characterize the effect of virus infection in an HDM (house dust mite) mouse model of asthma and assess the effect of oral corticosteroids. HDM alone significantly increased eosinophils, lymphocytes, neutrophils, macrophages and a number of cytokines in BAL (bronchoalveolar lavage), all of which were sensitive to treatment with prednisolone (with the exception of neutrophils). Virus infection also induced cell infiltration and cytokines. RSV (respiratory syncytial virus) infection in HDM-treated animals further increased all cell types in BAL (except eosinophils, which declined), but induced no further increase in HDM-elicited cytokines. However, while HDM-elicited TNF-α (tumour necrosis factor-α), IFN-γ (interferon-γ), IL (interleukin)-2, IL-5 and IL-10 were sensitive to prednisolone treatment, concomitant infection with RSV blocked the sensitivity towards steroid. In contrast, influenza infection in HDM- challenged animals resulted in increased BAL lymphocytes, neutrophils, IFN-γ, IL-1β, IL-4, IL-5, IL-10 and IL-12, but all were attenuated by prednisolone treatment. HDM also increased eNO (exhaled NO), which was further increased by concomitant virus infection. This increase was only partially attenuated by prednisolone. RSV infection alone increased BAL mucin. However, BAL mucin was increased in HDM animals with virus infection. Chronic HDM challenge in mice elicits a broad inflammatory response that shares many characteristics with clinical asthma. Concomitant influenza or RSV infection elicits differing inflammatory profiles that differ in their sensitivity towards steroids. This model may be suitable for the assessment of novel pharmacological interventions for asthmatic exacerbation.
- house dust mite
- mouse model
- respiratory syncytial virus (RSV)
• Asthma exacerbations are known to be triggered by viral infection and account for a high percentage of the overall healthcare costs.
• In the present study HDM significantly increased inflammatory cell influx and cytokines in BAL. Concomitant influenza or RSV infection elicited differing inflammatory profiles that differed in sensitivity towards steroids. RSV infection increased all cell types in BAL, but did not increase cytokines profile. Influenza infection increased BAL lymphocytes, neutrophils, IFN-γ, IL-1β, IL-4, IL-5, IL-10 and IL-12.
• This work characterizes the effect of two different virus infections in a mouse model of asthma and provides an insight into the drivers of the exacerbation phenotype and the alteration of drug efficacy.
Asthma is a chronic inflammatory disease of the airways characterized by pulmonary eosinophilia, AHR (airway hyper-responsiveness) and mucus hypersecretion, which ultimately results in airway remodelling . The airflow limitation induced by asthma is generally regarded as reversible and can usually be managed by steroid treatment. However, exacerbations, which cannot be effectively treated at present, are common and associated with increased health care costs, morbidity and mortality . It is therefore important to develop models of asthma exacerbation in which the efficacy of potential therapeutic interventions can be tested.
HDM (house dust mite) extract is often considered a more disease relevant challenge than OVA (ovalbumin) as it contains naturally occurring clinical allergens. HDM (Dermatophagoides farinae) has been used instead of OVA, to acutely sensitize and challenge mice . More recently, a chronic HDM (D. pteronyssinus) model has been developed which exposes mice to the allergen only via the intranasal route without the need of sensitization [4,5]. In this chronic model, allergen exposure is continual, which mirrors clinical asthma more closely. This chronic model displays AHR, eosinophilic infiltration, and increased Th2 (type 2 helper T-cell) cytokine production together with features of airway remodelling including goblet cell hyperplasia and collagen deposition [4,5], which other acute inflammation models cannot replicate.
It is now known that the majority of asthma exacerbations are triggered by respiratory viral infections, most notably rhinoviruses, RSV (respiratory syncytial virus) and parainfluenza viruses [6,7]. As a consequence, a number of viruses have been added to established models of inflammation in order to mimic an exacerbation. For example, Bartlett et al.  have developed a mouse model of asthma exacerbation by infecting BALB/c mice, sensitized and challenged with OVA, with rhinovirus-1B. These mice had significantly enhanced AHR, exacerbated neutrophilic, eosinophilic and lymphocytic airway inflammation and increased production of Th1 (type 1 helper T-cell) and Th2 cytokines. However, the technical challenges associated with establishing a robust rhinovirus infection in mice, together with the requirement for humanized ICAM-1 mice, limit the ability to establish a robust model capable of being benchmarked across several laboratories.
RSV is an enveloped RNA virus of the Paramyxoviridae family. It is a leading cause of bronchiolitis in children and has also been found to increase susceptibility to developing asthma in later life . RSV can cause repeated infections throughout life, usually associated with moderate cold-like symptoms. However, in addition, RSV has been implicated in the exacerbation of asthma and chronic obstructive pulmonary disease  due to its ability to lead to a more severe lower respiratory tract disease in those with compromised lung function. A number of studies have preclinically investigated the link between RSV infection and asthma exacerbation and/or predisposition to asthma [10–13] by combining RSV infection with a simple OVA sensitization-challenge model of inflammation.
Influenza is an enveloped RNA virus that comprises three of the five genera of the Orthomyxoviridae family (influenza virus-A, -B and -C). Influenza spreads around the world in seasonal epidemics, resulting in approximately 3–5 million yearly cases of severe illness and about 250000–500000 yearly deaths, rising to millions in some pandemic years . Influenza-attributable hospitalization rates and health care costs are generally higher in children with asthma . Dahl et al.  showed influenza-A infection induces an IFN-γ (interferon-γ) response in the lung, promoting the development of Th1-polarizing dendritic cells that enhanced both Th1 and Th2 cytokines following subsequent sensitization and challenge with keyhole limpet haemocyanin protein. More recently, reports have shown that in mice, neonatal infection with influenza A facilitates sensitization to HDM in infant mice leading to an asthma phenotype  and that acute, but not resolved, influenza-A infection enhances Th2 polarization towards HDM challenge .
The aim of these studies was to compare the effects of RSV or influenza infection in mice chronically challenged with HDM extract, to identify end points that were synergistically enhanced by co-challenge, and to investigate the therapeutic benefit of oral corticosteroids on these end points.
MATERIALS AND METHODS
Influenza H1N1 virus (strain A/PR/8/34) and RSV (A2 strain) were purchased from Advanced Biotechnologies. HDM extract (D. pteronyssinus) was purchased from Greer Laboratories. All other regents were purchased from Sigma-Aldrich unless otherwise stated.
Female BALB/c mice aged 8 weeks and weighing about 20 g were purchased from Charles River. Animals were housed and allowed food and water ad libitum, and handled according to Home Office legislation and local ethical regulations, in strict accordance with the Animals (Scientific Procedures) Act 1986.
HDM and virus administration
Mice were transiently anaesthetized with 2.5% (v/v) isoflurane in 100% (v/v) O2. HDM extract was administered by intranasal instillation (25 μg; 10 μl of sterile saline) directly into the nares as the animal was held in a vertical position. Control animals received sterile saline alone. Exposures were carried out for 5 days/week for 6 weeks. After 5 weeks of HDM exposure, mice were inoculated with influenza [15×103 FFU (fluorescent focus units); 20 μl] or RSV (2×106 FFU; 20 μl) by intranasal instillation.
Prednisolone was dissolved in water to a concentration of 1 mg/ml and administered in a 0.2 ml volume per os twice daily commencing 1 h prior to virus inoculation.
eNO (exhaled NO) determination
Mice were placed in modified whole body plethysmograph chambers (Buxco) for 20 min. Previous studies demonstrated that environmental NO (nitric oxide) levels increase for 15 min and then plateau (results not shown), indicating that the 20 min time point represents ‘steady state’ concentrations. Air from inside the plethysmograph chamber was sampled using a 280i NO analyser (Sievers, GEC) calibrated with NO-free air and commercial NO gas at 100 ppm (BOC Healthcare).
BAL (bronchoalveolar lavage), cell count and lung homogenization
Mice were killed with 0.1 ml intraperitoneal PentoJect® 7 days after viral inoculation. The thoracic cavity was opened and blood withdrawn by cardiac puncture. The trachea was cannulated with a 20 GA Insyte I.V. catheter (Becton Dickinson) and the lungs lavaged with 4×0.5 ml of DPBS (Dulbecco's PBS) containing 10 mM EDTA. The four lavages from single animals were pooled and yielded a consistent return of approximately 1.9 ml that consistently contained 90% of cells resident in the airways (results not shown). An aliquot of BAL was diluted 1:1 in Trypan Blue and cell density counted in a haemocytometer. Cytospins were prepared on microscope slides (Thermo Shandon), air dried and stained with DiffQuik (Dade Behring). Differential cell counts were performed, following staining with anti-CD45-PE and fixation in 1% (v/v) formaldehyde, using an FACS array (Becton Dickinson). Gating was validated in the same model versus manual cell counts from cytospins and CD45 gating was utilized to remove non-leukocytic events, followed by intrinsic cellular autofluorescence and side/forward scatter properties to discriminate the key cell populations.
Lungs were excised and homgenized on ice in 4 ml of PBS containing protease inhibitor cocktail (Sigma–Aldrich) using an Omni TH hand-held homogenizer (Omni International) until a uniform consistency was achieved. Samples were subsequently centrifuged and the supernatants removed and stored at −20°C prior to analysis.
Virus titre determination
Confluent Madin–Darby canine kidney cells (A.T.C.C. number: CCL34) plated in 96-well plate were inoculated with 50 μl of BAL samples for 90 min. Inoculum was removed and the cells were further incubated for 16 h to allow the virus to replicate. After fixing the cells with 75% (v/v) acetone in PBS, cells were immunostained by HistoMark TrueBlue™ (KPL) following the manufacturer's instructions. Stained spots were counted to determine the virus titre.
RNA was extracted from isolated lung tissue using an RNeasy midi kit (Qiagen) following the manufacturer's instructions. First-strand cDNA was then synthesized using reverse transcription reagents (Applied Biosystems), RSV strand-specific primers (positive sense 5′-CGGTCATGGTGGCGAATAATCCTGCAAAAATCCCTTCAACT-3′; negative sense 5′-CGGTCATGGTGGCGAATAAACTTTATAGATGTTTTTGTTCA-3′) and a GeneAmp PCR 9700 PCR system (Applied Biosystems) and an Applied Biosystems 7900HT. RSV copy number was normalized to the housekeeping gene β-actin (cDNA synthesized using the Applied Biosystems High Capacity cDNA reverse-transcription kit).
Cytokine and Muc5AC analysis
Lung homogenate concentrations of cytokines were measured using a Proinflammatory 9-plex Ultra-sensitive Kit (Meso Scale Discovery) in accordance with manufacturer's instructions. The mucin ELLA (enzyme-linked lectin assay) was performed as described previously .
All analyses were performed using a one-way ANOVA (using the non-parametric Kruskal–Wallis test) in GraphPad Prism version 5.0.
The weight in groups without HDM or virus showed no change during the period (Figure 1), whereas HDM treatment significantly reduced body weight. All mice treated with prednisolone, irrespective of concurrent HDM treatment, showed further weight decrease, consistent with known effects of oral corticosteroids.
RSV infection was not associated with loss of body weight in the presence or absence of HDM, unless animals were treated with prednisolone. In contrast, for influenza inoculated groups, the body weight of all groups decreased dramatically, regardless of the combination of treatments.
Virus titres in lung tissue
To determine the infection and replication of influenza and RSV virus, titre and copy number were measured, respectively (Table 1). Both viruses appeared to successfully infect and replicate in the mouse lung, with the viral load increasing by about 10-fold at day 3. Influenza titre further increased at day 7 by 46-fold compared with day 0.
BAL cellular composition
HDM alone significantly increased macrophages, lymphocytes, eosinophils and neutrophils in BAL, of which lymphocytes and eosinophils were sensitive to prednisolone treatment, whereas the other two were not (Figure 2).
Mice infected with virus had marked increases in BAL macrophages, lymphocytes and neutrophils (which were higher in the influenza-treated group compared with RSV-treatment), and slightly increased number of eosinophils. These effects were further enhanced with concomitant HDM treatment, particularly with respect to eosinophils. Increased lymphocytes and eosinophils in the groups of HDM treatment with virus infection were sensitive to prednisolone treatment. However, macrophages and neutrophils of these groups were insensitive to prednisolone treatment. Interestingly, prednisolone treatment in influenza-infected mice led to an increase in eosinophils and neutrophils compared with influenza infection only, whereas macrophages were reduced.
In preliminary experiments, histology, tissue cell content and structural changes associated with airway remodelling [collagen, SMC (smooth muscle cell) actin, mucus] were studied by image analysis (as described elsewhere ). Tissue cellular content consistently reflected that observed in BAL fluid (results not shown). Neither RSV nor influenza alone resulted in any differences in airway remodelling parameters compared with control animals (results not shown), although disruption to the epithelium was observed in the influenza-treated animals (results not shown). Airway remodelling as measured by increases in airway collagen, SMC actin and mucus in HDM-treated animals was similar to that reported previously . Neither RSV nor influenza significantly altered the airway remodelling caused by HDM-treatment alone (results not shown).
Lung homogenate cytokine composition
Infection with influenza and RSV both broadly induced production of various cytokines (Table 2). Although influenza induced all the cytokines examined, RSV had little effect on TNF-α (tumour necrosis factor-α) and IL (interleukin)-10 production. HDM treatment also significantly increased all the cytokines measured. When HDM-treated mice were concomitantly infected with influenza, further increases in the cytokines IL-12 (2.5-fold), IL-2 (2.1-fold), IL-4 (1.9-fold) and IL-10 (1.5-fold) were observed. IFN-γ production was reduced (2-fold) with concomitant influenza infection. Concomitant infection with RSV in HDM-treated groups also altered cytokine production, with TNF-α (4-fold), KC (keratonocyte chemoattractant; 3-fold), IL-2 (2.5-fold), IL-10 (2.5-fold), IL-1β (2-fold) and IL-5 (2-fold) increasing, and IL-4 (2-fold) decreasing.
Prednisolone treatment lowered TNF-α (16-fold), IFN-γ (5-fold), IL-2 (5-fold), IL-4 (2-fold), IL-5 (4-fold), IL-10 (5-fold) and IL-12 (6-fold) in HDM-treated animals compared with the corresponding vehicle group. Prednisolone treatment in HDM-treated animals concomitantly infected with influenza also reduced cytokine levels of TNF-α (5-fold), IFN-γ (2-fold), IL-2 (3-fold), IL-4 (2-fold), IL-5 (5-fold), IL-10 (2-fold) and IL-12 (3-fold). In HDM-treated animals concomitantly infected with RSV, cytokines levels were reduced by prednisolone treatment, IL-5 (3-fold) and IL-12 (2-fold). Interestingly, IL-10 was increased by prednisolone treatment (6-fold).
Mucin production in BAL
Mucin concentration was measured in groups with various combinations of HDM and virus treatments (Table 3). The influenza group had little effect on mucin production, whereas the RSV group had approximately 2-fold increase in mucin. HDM treatment only did not increase mucin, but in combination with virus, significant increases in mucin were observed especially in influenza infection.
The groups treated with no virus had low concentration of eNO (Figure 3). Influenza treatment induced eNO to some extent in all groups regardless of HDM and/or prednisolone treatment. However, although eNO concentration induced by RSV infection was fairly small, HDM further increased eNO produced by RSV, but was sensitive to prednisolone treatment. This effect of HDM was not observed in combination with influenza.
In allergic asthma, sensitization and subsequent exposure to allergens is believed to be key in inducing clinical symptoms including cellular infiltration in the lung. HDM is one of the well-known clinical allergens, and in the present study the HDM treatment of mice led to a significant increase in inflammatory cells infiltrating into the lung. Notably the increase in eosinophils was the most significant, which is consistent with the general consensus that eosinophils are the sentinel inflammatory cells in clinical asthmatic condition . Analyses of sputum, BAL and endobronchial biopsy specimens from living asthmatics and post-mortem samples from fatal asthma found that the majority of asthmatics have elevated eosinophils . Infection with virus as well as HDM treatment also increased various types of cells in the BAL samples compared with respective non-HDM treatment groups. Concomitant influenza infection in HDM-treated mice showed much less eosinophil infiltration than the HDM treatment only group. One possible explanation would be that the influenza virus induced Th1 immune responses in an IFN-γ-dependent manner while keeping existing Th2 responses induced by HDM, consistent with the data Dahl et al. reported . Indeed the concentrations of IFN-γ and IL-12 that induce IFN-γ production was much higher in the HDM/influenza group than either those in HDM/RSV or HDM only treatment groups. Certainly the blunting of the HDM-elicited eosinophilic response was less pronounced in the RSV/HDM group (compared with influenza/HDM), which further supports this notion. Moreover, Th2-type cytokines, i.e. IL-4, IL-5 and IL-10, were also much higher in the influenza/HDM groups compared with both RSV/influenza and HDM only groups, which also suggest a reinforced and concomitant Th2 response in these animals. These results appear to conflict with the well-known Th1/Th2 paradigm where Th1 and Th2 cells antagonize the effects of each other, but are in agreement with the study of Hansen et al. , in which Th1 cells induced airway inflammation and reduced the number of airway eosinophils induced by Th2 response, but increased Th1 response was ineffective in reducing airway hyper-reactivity caused by Th2. For example, the treatment of HDM with influenza infection induced IL-5 in BAL fluid more than other groups without pharmacological intervention, and this result is consistent with data reported previously . It may look contradictory since IL-5 is a Th2 type cytokine and it plays a critical role in Th2 immune response. Recent study demonstrated that the influenza infection clearly increases IL-33 mRNA and protein in the lung as well as the BAL fluid recovered . IL-33 is expressed and positioned to act on infiltrating inflammatory cells, and it is thought to be able to affect Th2 as well as Th1 immune responses under certain conditions [25,26]. Administration of IL-33 in mice leads to an increase in mRNA levels of Th2 type cytokines, i.e. IL-4, IL-5 and IL-13, and an increase in protein levels of IL-5 in the serum as well [25,27]. Therefore, although IL-5 is said to be a Th2 cytokine, it can be speculated that influenza virus may well be capable of indirectly inducing IL-5 via IL-33 production, while the virus can also induce Th1 immune response. This may be a reason why HDM treatment with influenza infection had high IL-5 concentration.
IL-5 is a key cytokine for eosinophil maturation, activation and recruitment. However, once eosinophils have migrated to the lung from circulation, mIL-5Rα (membrane IL-5 receptor α) on the surface of eosinophils is significantly reduced and sIL-5Rα (soluble IL-5 receptor α) is significantly increased instead [28,29]. It has been suggested that sIL-5Rα may work as an IL-5 antagonist, binding to IL-5 with high affinity , and the decrease of mIL-5Rα expression on BAL eosinophils and the lack of responsiveness to IL-5 may mean a switch to IL-5-independent cell function . This is considered an important mechanism of limiting IL-5-mediated eosinophils function. In the present study, the HDM treatment group had the highest eosinophilia with relatively low concentration of IL-5. This may therefore be due to these mechanisms: (1) excessive IL-5 in BAL fluid may have bound to sIL-5Rα, thereby making it difficult to detect by the assay targeting IL-5; (2) enough eosinophils had already migrated to the lung, so high concentration of IL-5 may have been of less importance, leading to the reduction in IL-5 release. And vice versa – a higher concentration of IL-5 with fewer eosinophils in the lung in the HDM treatment group and influenza infection can be explained by the mechanisms above: (i) sIL-5Rα levels may have been lower in the lung due to the fact that eosinophils were present at lower levels in BAL fluid; and (ii) keeping the concentration of IL-5 high enough to recruit more eosinophils in the lung, together with the notion mentioned above that influenza is capable of inducing IL-5.
It is very difficult to draw any definite conclusions about the cytokine regulation in the lung based on data from a single-point measurement. However, we have previously demonstrated that cytokines levels in the lung in response to HDM challenge are relatively stable after 3 weeks of challenge (results not shown). Furthermore, our preliminary experiments demonstrated that the time point chosen to measure both cells and cytokines post-viral infection coincided with the maximal inflammatory response to virus alone (results not shown). We therefore believe that the time point chosen is the best reflection of this dynamic process. The use of lung homogenates for cytokine measurements was chosen because cytokine levels in homogenates have in previous experiments closely paralleled those in BAL fluid (results not shown), but are present in greater quantities, therefore increasing the detection sensitivity.
An interesting question is how the differential effects of these viruses we observed in this study relate to humans. It has been reported that lower respiratory tract RSV and influenza infection in children is associated with higher cytokine secretion in influenza infection compared with infection by RSV . We also observed higher levels of cytokines following influenza infection compared with RSV for TNF-α, IFN-γ, IL-1β, IL-5, KC, IL-10 and IL-12, but not for IL-2 or IL-4. Matsuse et al.  recently demonstrated that TNF-α and IFN-γ were significantly higher in asthmatic exacerbations triggered by influenza, compared with RSV. This is also consistent with our data where both cytokines were elevated in the influenza-only group (compared with RSV) and influenza-HDM group (compared with HDM-RSV). Finally, a study in human epithelial cells showed that RSV, but not influenza, was able to increase transcription of Mucin 1 , which is consistent with our findings that RSV infection increased airway mucin content. Taken together, these data suggest that the differential effects observed between influenza and RSV infection in our study reflect the limited clinical observations that directly compare the two viruses in the same study.
Prednisolone generally reduced the number of lymphocytes and eosinophils significantly in all groups, while the number of macrophages and neutrophils remained unchanged. Corticosteroids are capable of inducing apoptosis of eosinophils  and lymphocytes . This hypothesis is supported by clinical data in which the number of eosinophils in the sputum was reduced by a tenth and the apoptotic eosinophils ratio was approximately 5-fold after corticosteroid administration for the treatment of asthma exacerbation . These results indicate that the cellular infiltration in this model would mimic clinical asthmatic conditions including exacerbation by virus infection, and may be used for evaluation of candidate compounds for the treatment of asthma.
The mucus layer that coats the airway epithelium provides a protective barrier against pathogenic and noxious agents and participates in the mucosal response to inflammation and infection . Mucins are the major components of mucus. They are sialylated, secreted glycoproteins present in mucus that may contribute to the homoeostasis of airway surface liquid and to the clearance of pathogens through diverse mechanisms . Of the various types of mucins, the MUC5AC product is thought to be the main component produced in respiratory tract  and the most frequently expressed mucin gene amongst others in both healthy and asthmatic subjects . Higher expression of MUC5AC mRNA is seen in asthmatic patients and the up-regulation of MUC5AC, assumed to be mediated by Th2 type cytokines, may account for increased mucin in this disease [41–43]. In the present study, mucin production was not altered by influenza infection, whereas the infection and replication were confirmed by measuring virus titre, and this result does not appear consistent with a previously reported study  in which mRNA expression and protein production of MUC5AC was increased in a mouse model of influenza infection. It would be possible to reason that this contrary observation could be attributed to experimental conditions such as mouse strains and sex, influenza subtypes/strains and preparation as well as the doses used. On the other hand, mucin production in the group infected with RSV was significantly increased and this increase is consistent with a previous report  in which the authors argue that leukocytes are responsible for RSV-induced airway hyper-reactivity and mucus overproduction, and that studies have shown that the inflammatory response that occurs after RSV infection is similar to that observed in asthmatics, including an increase in Th2-type cytokines, mucus production, and eosinophil infiltration that were all demonstrated in the present study. In the groups with virus infection in addition to HDM treatment, mucin production showed a clear synergistic increase compared with respective groups without HDM treatment. This is consistent with other reports that RSV infection increases mucus production and discharge from the goblet cells in the respiratory trachea in murine allergen challenge models causing asthmatic conditions and allergic airway inflammation [45,46]. A more synergistic effect between HDM and virus infection in the production of mucin was seen in influenza infected groups than in RSV infected groups. This synergistic effect is consistent with the cytokine profile, in which the Th1 type cytokines were much higher in the influenza infected HDM treatment group. There are reports that both TNF-α and IL-1β, both of which were elevated in the influenza/HDM group, activate transcription of MUC5AC [47,48].
It has been widely investigated and proposed that the eNO would be an alternative biomarker since it provides a non-invasive way of measuring airway inflammation  and it has been shown to correlate particularly with asthma [50,51]. In asthmatic patients, eNO level is increased and the source of NO in exhaled air is mainly derived from the lower respiratory tract rather than from the alveoli . NO is constitutively produced by a variety of cell types by the action of NOS (NO synthase) on l-arginine; for example, eNOS (endothelial NOS) in endothelial cells and nNOS (neuronal NOS) in neuronal cells , and the elevated eNO in asthmatic patients is thought to be attributed to increased transcription and production of iNOS (inducible NOS) in the airway epithelium as well as in inflammatory cells including macrophages, neutrophils and eosinophils . However, iNOS is believed to be up-regulated by a combination of TNF-α, IFN-γ and IL-1β. Although these cytokines were elevated in influenza and influenza/HDM groups in line with the increases in eNO observed, the largest increase in eNO was in the RSV/HDM group, in which these cytokines were present at lower levels. This perhaps suggests that either there is an additional unknown contributing factor present during RSV infection, or that the kinetics of cytokine production, iNOS transcription and eNO elaboration are not well described by a single time point measurement of all three parameters. Nevertheless, during severe exacerbation, the eNO level in asthmatic patients is significantly higher than that in symptom-free subjects . Furthermore, elevated eNO is decreased when treated with corticosteroids in patients with asthma, whereas the eNO level is unchanged in normal subjects [55,56]. eNO is increased in patients who remain symptomatic despite oral steroids and who have a relative steroid resistance, and may therefore be useful to quantify steroid resistance in asthma . In this respect, the eNO profile described in the present study closely resembles that reported for clinical asthma/asthma exacerbation. Moreover, prednisolone treatment successfully decreased the eNO levels compared to the corresponding groups, suggesting that measuring eNO in this animal model could be beneficial in estimating the effects of pharmacological agents including steroids in asthmatic exacerbations in the clinic.
In summary, the present study demonstrates that chronic challenge with HDM followed by acute infection with influenza or RSV in mice elicits a wide range of inflammatory responses that shares many characteristics with clinical exacerbation of asthmatic conditions. Virus infection shows a differing inflammatory profile coupled with an altered sensitivity towards steroids, depending on the virus used. This preclinical model may therefore be beneficial, as a translational model, for the evaluation of pharmacological candidates for the treatment of asthma as well as asthmatic exacerbations. Moreover, it could be useful to further characterize novel pharmacological interventions by selecting a virus of interest playing a role in asthma exacerbation, thereby mimicking the clinical condition.
Hiroki Mori performed the laboratory work and wrote the paper; Nicole Parker performed the laboratory work and approved the paper. Deborah Rodrigues performed the viral laboratory work and approved the paper. Kathryn Hulland, Deborah Chappell, Jennifer Hincks and Helen Bright performed the laboratory work and approved the paper. Steven Evans is the UBIOPRED Pfizer lead for severe asthma studies and wrote the paper. David Lamb is a UBIOPRED member for severe asthma and lead biologist for this in vivo work, and wrote the paper.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Abbreviations: AHR, airway hyper-responsiveness; BAL, bronchoalveolar lavage; ELLA, enzyme-linked lectin assay; eNO, exhaled NO; FFU, fluorescent focus units; HDM, house dust mite; IFN-γ, interferon-γ; IL, interleukin; iNOS, inducible NO synthase; KC, keratinocyte chemoattractant; mIL-5Rα, membrane IL-5 receptor α; NOS, NO synthase; OVA, ovalbumin; RSV, respiratory syncytial virus; sIL-5Rα, soluble IL-5 receptor α; Th1, type 1 helper T-cell; Th2, type 2 helper T-cell; TNF-α, tumour necrosis factor-α
- © The Authors Journal compilation © 2013 Biochemical Society