Clinical Science (2014) 126, 567–580 (Printed in Great Britain) doi: 10.1042/CS20130149
Development of a mouse model mimicking key aspects of a viral asthma exacerbation Deborah L. CLARKE∗ , Nicola H. E. DAVIS∗ , Jayesh B. MAJITHIYA∗ , Sian C. PIPER∗ , Arthur LEWIS∗ , Matthew A. SLEEMAN∗ , Dominic J. CORKILL∗ and Richard D. MAY∗
Clinical Science
www.clinsci.org
∗
Department of Respiratory, Inflammation and Autoimmunity, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
Abstract Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children. The current standard of care, inhaled CS (corticosteroids) and LABAs (long-acting β 2 -adrenoceptor agonists), fails to prevent the loss of control that manifests as an exacerbation. In order to better understand the mechanisms underlying viral asthma exacerbations we established an in vivo model using the clinically relevant aeroallergen HDM (house dust mite) and the viral mimetic/TLR3 (Toll-like receptor 3) agonist poly(I:C). Poly(I:C) alone induced a similar neutrophilic inflammatory profile in the BAL (bronchoalveolar lavage) to that of HRV1b (human rhinovirus 1b) alone, accompanied by both elevated BAL KC (keratinocyte-derived chemokine) and IL-1β (interleukin-1β). When mice allergic to HDM were also challenged with poly(I:C) the neutrophilic inflammatory profile was exacerbated. Increased CD8 + T-cell numbers, increased CD4 + and CD8 + cell activation and elevated KC and IL-1β were observed. No increases in Th2 cytokines or the eosinophil chemoattractant CCL11 [chemokine (C-C motif) ligand 11], above those induced by HDM alone, were observed. The poly(I:C)-exacerbated neutrophilia did not translate into changes in AHR (airways hyper-responsiveness), indicating that in this model inflammation and AHR are two mechanistically independent events. To test the clinical relevance of this model CS sensitivity was assessed using prednisone, a synthetic oral CS used to manage exacerbations in asthmatic patients already on maximal doses of inhaled CS. The increased neutrophils, and accompanying cytokines/chemokines KC and IL-1β induced by poly(I:C) challenge of HDM-sensitized and challenged mice were insensitive to oral prednisone therapy. In summary we have described a CS-resistant mouse model mimicking the key aspects of viral asthma exacerbation using the clinically relevant aeroallergen HDM and the viral mimic poly(I:C). This model may provide better understanding of disease mechanisms underlying viral exacerbations and could be used to build early confidence in novel therapeutic axes targeting viral asthma exacerbations in Th2 asthmatics. Key words: airway inflammation, asthma, disease exacerbation, mouse model, steroid resistance, viral infection
INTRODUCTION Asthma is a complex and persistent inflammatory disease characterized by AHR (airway hyper-responsiveness) and airway inflammation which affects up to 10 % of adults and 30 % of children in the Western world [1]. Treatment of moderate-to-severe asthmatic patients with inhaled CS (corticosteroids) and β 2 -adrenoceptor agonists generally provides adequate disease control and enable patients to live a ‘normal’ life not restricted by their disease [Global Initiative For Asthma (GINA) Guidelines 2010 (http://www.ginasthma.org/
local/uploads/files/GINA_Report_2010_1.pdf)]). However, despite advances in asthma management, inhaled CS often fail to provide disease control in patients with severe asthma [2,3] and are ineffective in managing asthma exacerbations in both adults [4,5] and children [6]. Exacerbations are an ongoing issue for many asthmatic people, imposing considerable morbidity and constituting a major burden on healthcare resources. It is estimated that severe asthma and asthma exacerbations across asthma severities contributes more than 50 % of the considerable social and economic burden on healthcare resources, estimated at $14.7 billion/annum in the U.S.A. and €17.7
Abbreviations: AHR, airway hyper-responsiveness; APC, allophycocyanin; BAL, bronchoalveolar lavage; CCL11, chemokine (C-C motif) ligand 11; COPD, chronic obstructive pulmonary disease; CRA, cockroach allergen; CS, corticosteroid(s); HDM, house dust mite; H&E, haematoxylin and eosin; HRV1b, human RV1b; ICAM-1, intercellular adhesion molecule 1; IFNγ , interferon γ ; IL, interleukin; KC, keratinocyte-derived chemokine; NBF, neutral-buffered formalin; NEAA, non-essential amino acid; ns, not significant; PE, phycoerythrin; PerCP, peridinin–chlorophyll protein complex; RSV, respiratory syncytial virus; RV, rhinovirus; TCID50 , median tissue culture infective dose; TLR3, Toll-like receptor 3. Correspondence: Dr Deborah Clarke (email [email protected]).
www.clinsci.org
567
D. Clarke and others
billion/annum in the European Union [7] [American Lung Association (http://www.lung.org/) and European Lung Foundation (http://www.europeanlung.org/en/)]. Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children [8,9]. Viruses are detected in approximately 80 % of wheezing episodes in school-aged children [10] and in approximately 50–75 % of acute wheezing episodes in adults [11–13]. With the exception of RSV (respiratory syncytial virus) in infants hospitalized with bronchiolitis, RVs (rhinoviruses) are the most frequently detected virus type in patients hospitalized with non-COPD (chronic obstructive pulmonary disease)-associated breathing difficulties [7]. As the major unmet medical need in asthma is the severe patient suffering recurrent exacerbations, a major focus in recent years has been on the development of models mimicking key aspects of CS-resistant asthma and virally induced exacerbations. As the Th2 paradigm is well-modelled in mice and seems to be analogous to the Th2-high subset of human asthma [14,15], several attempts have been made to combine lung allergy and lung infection models to further understand the mechanisms underlying disease. However attempts to develop small animal models of RV infection been largely unsuccessful due to the fact that 90 % of RV serotypes (major group RV and RV-1A) specifically require binding to human ICAM-1 (intercellular adhesion molecule-1) to enter cells. Previously a model of virus-induced exacerbations of asthma has emerged using transgenic mice overexpressing human ICAM-1 that allowed the mice to be infected by the major group serotypes [16]. Use of the minor group RV serotype RV1b, which infects via the low-density lipoprotein receptor [17], has also been successfully used in the context of the allergen ovalbumin to model aspects of clinical disease in na¨ıve BALB/c mice [16,18]. The sole similarity in these studies was the enhancement of neutrophilia observed at 24 h after HRV1b exposure in ovalbumin-challenged mice when compared with sham-exposed mice. Other changes in inflammatory cell recruitment, such as eosinophils, lymphocytes and mononuclear cells, differed in their kinetics, as did the accompanying cytokine and chemokine induction {IL (interleukin)-4, IL-13 and CCL11 [chemokine (C-C motif) ligand 11]} [16,18]. Similarly, Bartlett et al. [16] observed changes in lung resistance 24 h after HRV1b challenge; however, these changes were observed 4 days after viral exposure. A number of studies have also been performed to assess the effects of viral exposure prior to allergen challenge. dsRNA [poly(I:C)] administered to mice during ovalbumin sensitization enhanced airway eosinophilia and AHR and was associated with enhanced induction of IL-13-producing CD8 + T-cells [19]. In a rat model, administration of poly(I:C) before and throughout ovalbumin challenge increased the inflammatory cell burden (total cells and neutrophils) as well as enhancing structural changes in the airway [20]. Additionally exposure to low or high doses of poly(I:C) before allergen challenge enhanced airway inflammation in mice [21]. We sought to establish a model mimicking key aspects of viral exacerbation of asthma using the clinically relevant aeroallergen HDM (house dust mite) [22] and a virus. TLR3 (Toll-like receptor 3) recognizes dsRNA, a molecular pattern associated with viral infection, and as such can be used to mimic viral infection.
568
C The Authors Journal compilation C 2014 Biochemical Society
Therefore we compared the ability of the minor group RV HRV1b (due to its ability to infect mice [16]) and the synthetic TLR3 ligand poly(I:C) to affect the HDM-induced allergic lung phenotype in terms of inflammatory profile and response to current standard of care treatment.
MATERIALS AND METHODS HRV1b production Virus was propagated by infection of HeLa H1 cells (A.T.C.C., Manassas, VA, U.S.A.) with HRV1b (A.T.C.C.) for 48 h at 33 ◦ C in EMEM (Eagle’s minimum essential medium) containing 2 mM L-glutamine and 1 % NEAAs (non-essential amino acids). Virus-containing supernatant was collected, centrifuged and the virus precipitated with 7 % PEG 6000 and 0.5 M NaCl at 4 ◦ C overnight. Precipitated virus was centrifuged (1860 g for 5 min) and resuspended by overnight incubation in PBS. This sample was then centrifuged (1860 g for 5 min), filtered (0.22 μm Steriflip filter; Millipore) and concentrated [100 000 NMCO (nominal molecular-mass cut-off) centrifugal filter device; Millipore] to a final target of (6–10) × 108 TCID50 (median tissue culture infective dose)/ml. During the concentration step the buffer was exchanged with fresh PBS in order to remove any contaminating cytokines. The TCID50 of purified HRV1b was determined by incubating 3 × 104 HeLa Ohio cells [ECACC (European Collection of Cell Culture), Porton Down, U.K.] seeded in a 96-well plate at 24 h before infection with 10-fold serial dilutions of virus in MEM (minimal essential medium) containing 100 units/ml of both penicillin and streptomycin, 1 % NEAAs and 10 % FBS (Invitrogen). Cells and virus were incubated at 37 ◦ C and 5 % CO2 for 6 days before being fixed in 3.7 % formaldehyde and stained for 10 min in 0.1 % Crystal Violet. The mean absorbance of 100 scans at 600 nm was calculated per well. All wells with an average absorbance P < 0.001 poly(I:C) and prednisone compared with the corresponding HDM and prednisone group; ◦ P < 0.05, ◦◦ P < 0.01 and ◦◦◦ P < 0.001 HDM and prednisone compared with the corresponding HDM/poly(I:C) and prednisone group.
C The Authors Journal compilation C 2014 Biochemical Society
Steroid-resistant model of asthma exacerbations
Figure S3
Inflammatory mediator profile after HDM/poly(I:C) challenge BALB/c mice were intranasally administered 25 μg of HDM extract in 25 μl of PBS or PBS alone three times a week for 3 weeks. At 2 h after the final challenge mice were challenged with poly(I:C) (10 mg/kg of body mass in a 50 μl volume) or vehicle (PBS, 50 μl volume) and killed 24 h later. Mice were treated with vehicle or prednisone (Pred) orally twice daily (3–30 mg/kg of body mass) for the final 2 weeks. Lungs were removed and snap-frozen, after which they were homogenized and assayed for cytokine content by Meso Scale Discovery (A–E) or ELISA (F). Results are means + − S.E.M., n = 6–8 mice/group from one experiment. Results were analysed by one-way ANOVA followed by Bonferroni’s post-hoc ∗ ∗∗ ∗∗∗ ## test comparing all groups. P < 0.05, P < 0.01 and P < 0.001 compared with the PBS alone group; P < 0.01 and ### P < 0.001 compared with the poly(I:C) alone group; ∼∼∼ P < 0.001 compared with the HDM alone group; + P < 0.05, ++ P < 0.01 and + + + P < 0.001 prednisone-treated group compared with the relevant control; >>> P < 0.001 poly(I:C) and prednisone compared with the corresponding HDM and prednisone group; ◦◦◦ P < 0.001 HDM and prednisone compared with the corresponding HDM/poly(I:C) and prednisone group.
Received 14 March 2013/18 October 2013; accepted 24 October 2013 Published as Immediate Publication 24 October 2013, doi: 10.1042/CS20130149
www.clinsci.org
Copyright of Clinical Science is the property of Portland Press Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Development of a mouse model mimicking key aspects of a viral asthma exacerbation Deborah L. CLARKE∗ , Nicola H. E. DAVIS∗ , Jayesh B. MAJITHIYA∗ , Sian C. PIPER∗ , Arthur LEWIS∗ , Matthew A. SLEEMAN∗ , Dominic J. CORKILL∗ and Richard D. MAY∗
Clinical Science
www.clinsci.org
∗
Department of Respiratory, Inflammation and Autoimmunity, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.
Abstract Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children. The current standard of care, inhaled CS (corticosteroids) and LABAs (long-acting β 2 -adrenoceptor agonists), fails to prevent the loss of control that manifests as an exacerbation. In order to better understand the mechanisms underlying viral asthma exacerbations we established an in vivo model using the clinically relevant aeroallergen HDM (house dust mite) and the viral mimetic/TLR3 (Toll-like receptor 3) agonist poly(I:C). Poly(I:C) alone induced a similar neutrophilic inflammatory profile in the BAL (bronchoalveolar lavage) to that of HRV1b (human rhinovirus 1b) alone, accompanied by both elevated BAL KC (keratinocyte-derived chemokine) and IL-1β (interleukin-1β). When mice allergic to HDM were also challenged with poly(I:C) the neutrophilic inflammatory profile was exacerbated. Increased CD8 + T-cell numbers, increased CD4 + and CD8 + cell activation and elevated KC and IL-1β were observed. No increases in Th2 cytokines or the eosinophil chemoattractant CCL11 [chemokine (C-C motif) ligand 11], above those induced by HDM alone, were observed. The poly(I:C)-exacerbated neutrophilia did not translate into changes in AHR (airways hyper-responsiveness), indicating that in this model inflammation and AHR are two mechanistically independent events. To test the clinical relevance of this model CS sensitivity was assessed using prednisone, a synthetic oral CS used to manage exacerbations in asthmatic patients already on maximal doses of inhaled CS. The increased neutrophils, and accompanying cytokines/chemokines KC and IL-1β induced by poly(I:C) challenge of HDM-sensitized and challenged mice were insensitive to oral prednisone therapy. In summary we have described a CS-resistant mouse model mimicking the key aspects of viral asthma exacerbation using the clinically relevant aeroallergen HDM and the viral mimic poly(I:C). This model may provide better understanding of disease mechanisms underlying viral exacerbations and could be used to build early confidence in novel therapeutic axes targeting viral asthma exacerbations in Th2 asthmatics. Key words: airway inflammation, asthma, disease exacerbation, mouse model, steroid resistance, viral infection
INTRODUCTION Asthma is a complex and persistent inflammatory disease characterized by AHR (airway hyper-responsiveness) and airway inflammation which affects up to 10 % of adults and 30 % of children in the Western world [1]. Treatment of moderate-to-severe asthmatic patients with inhaled CS (corticosteroids) and β 2 -adrenoceptor agonists generally provides adequate disease control and enable patients to live a ‘normal’ life not restricted by their disease [Global Initiative For Asthma (GINA) Guidelines 2010 (http://www.ginasthma.org/
local/uploads/files/GINA_Report_2010_1.pdf)]). However, despite advances in asthma management, inhaled CS often fail to provide disease control in patients with severe asthma [2,3] and are ineffective in managing asthma exacerbations in both adults [4,5] and children [6]. Exacerbations are an ongoing issue for many asthmatic people, imposing considerable morbidity and constituting a major burden on healthcare resources. It is estimated that severe asthma and asthma exacerbations across asthma severities contributes more than 50 % of the considerable social and economic burden on healthcare resources, estimated at $14.7 billion/annum in the U.S.A. and €17.7
Abbreviations: AHR, airway hyper-responsiveness; APC, allophycocyanin; BAL, bronchoalveolar lavage; CCL11, chemokine (C-C motif) ligand 11; COPD, chronic obstructive pulmonary disease; CRA, cockroach allergen; CS, corticosteroid(s); HDM, house dust mite; H&E, haematoxylin and eosin; HRV1b, human RV1b; ICAM-1, intercellular adhesion molecule 1; IFNγ , interferon γ ; IL, interleukin; KC, keratinocyte-derived chemokine; NBF, neutral-buffered formalin; NEAA, non-essential amino acid; ns, not significant; PE, phycoerythrin; PerCP, peridinin–chlorophyll protein complex; RSV, respiratory syncytial virus; RV, rhinovirus; TCID50 , median tissue culture infective dose; TLR3, Toll-like receptor 3. Correspondence: Dr Deborah Clarke (email [email protected]).
www.clinsci.org
567
D. Clarke and others
billion/annum in the European Union [7] [American Lung Association (http://www.lung.org/) and European Lung Foundation (http://www.europeanlung.org/en/)]. Viral respiratory tract infections are known triggers of asthma exacerbations in both adults and children [8,9]. Viruses are detected in approximately 80 % of wheezing episodes in school-aged children [10] and in approximately 50–75 % of acute wheezing episodes in adults [11–13]. With the exception of RSV (respiratory syncytial virus) in infants hospitalized with bronchiolitis, RVs (rhinoviruses) are the most frequently detected virus type in patients hospitalized with non-COPD (chronic obstructive pulmonary disease)-associated breathing difficulties [7]. As the major unmet medical need in asthma is the severe patient suffering recurrent exacerbations, a major focus in recent years has been on the development of models mimicking key aspects of CS-resistant asthma and virally induced exacerbations. As the Th2 paradigm is well-modelled in mice and seems to be analogous to the Th2-high subset of human asthma [14,15], several attempts have been made to combine lung allergy and lung infection models to further understand the mechanisms underlying disease. However attempts to develop small animal models of RV infection been largely unsuccessful due to the fact that 90 % of RV serotypes (major group RV and RV-1A) specifically require binding to human ICAM-1 (intercellular adhesion molecule-1) to enter cells. Previously a model of virus-induced exacerbations of asthma has emerged using transgenic mice overexpressing human ICAM-1 that allowed the mice to be infected by the major group serotypes [16]. Use of the minor group RV serotype RV1b, which infects via the low-density lipoprotein receptor [17], has also been successfully used in the context of the allergen ovalbumin to model aspects of clinical disease in na¨ıve BALB/c mice [16,18]. The sole similarity in these studies was the enhancement of neutrophilia observed at 24 h after HRV1b exposure in ovalbumin-challenged mice when compared with sham-exposed mice. Other changes in inflammatory cell recruitment, such as eosinophils, lymphocytes and mononuclear cells, differed in their kinetics, as did the accompanying cytokine and chemokine induction {IL (interleukin)-4, IL-13 and CCL11 [chemokine (C-C motif) ligand 11]} [16,18]. Similarly, Bartlett et al. [16] observed changes in lung resistance 24 h after HRV1b challenge; however, these changes were observed 4 days after viral exposure. A number of studies have also been performed to assess the effects of viral exposure prior to allergen challenge. dsRNA [poly(I:C)] administered to mice during ovalbumin sensitization enhanced airway eosinophilia and AHR and was associated with enhanced induction of IL-13-producing CD8 + T-cells [19]. In a rat model, administration of poly(I:C) before and throughout ovalbumin challenge increased the inflammatory cell burden (total cells and neutrophils) as well as enhancing structural changes in the airway [20]. Additionally exposure to low or high doses of poly(I:C) before allergen challenge enhanced airway inflammation in mice [21]. We sought to establish a model mimicking key aspects of viral exacerbation of asthma using the clinically relevant aeroallergen HDM (house dust mite) [22] and a virus. TLR3 (Toll-like receptor 3) recognizes dsRNA, a molecular pattern associated with viral infection, and as such can be used to mimic viral infection.
568
C The Authors Journal compilation C 2014 Biochemical Society
Therefore we compared the ability of the minor group RV HRV1b (due to its ability to infect mice [16]) and the synthetic TLR3 ligand poly(I:C) to affect the HDM-induced allergic lung phenotype in terms of inflammatory profile and response to current standard of care treatment.
MATERIALS AND METHODS HRV1b production Virus was propagated by infection of HeLa H1 cells (A.T.C.C., Manassas, VA, U.S.A.) with HRV1b (A.T.C.C.) for 48 h at 33 ◦ C in EMEM (Eagle’s minimum essential medium) containing 2 mM L-glutamine and 1 % NEAAs (non-essential amino acids). Virus-containing supernatant was collected, centrifuged and the virus precipitated with 7 % PEG 6000 and 0.5 M NaCl at 4 ◦ C overnight. Precipitated virus was centrifuged (1860 g for 5 min) and resuspended by overnight incubation in PBS. This sample was then centrifuged (1860 g for 5 min), filtered (0.22 μm Steriflip filter; Millipore) and concentrated [100 000 NMCO (nominal molecular-mass cut-off) centrifugal filter device; Millipore] to a final target of (6–10) × 108 TCID50 (median tissue culture infective dose)/ml. During the concentration step the buffer was exchanged with fresh PBS in order to remove any contaminating cytokines. The TCID50 of purified HRV1b was determined by incubating 3 × 104 HeLa Ohio cells [ECACC (European Collection of Cell Culture), Porton Down, U.K.] seeded in a 96-well plate at 24 h before infection with 10-fold serial dilutions of virus in MEM (minimal essential medium) containing 100 units/ml of both penicillin and streptomycin, 1 % NEAAs and 10 % FBS (Invitrogen). Cells and virus were incubated at 37 ◦ C and 5 % CO2 for 6 days before being fixed in 3.7 % formaldehyde and stained for 10 min in 0.1 % Crystal Violet. The mean absorbance of 100 scans at 600 nm was calculated per well. All wells with an average absorbance P < 0.001 poly(I:C) and prednisone compared with the corresponding HDM and prednisone group; ◦ P < 0.05, ◦◦ P < 0.01 and ◦◦◦ P < 0.001 HDM and prednisone compared with the corresponding HDM/poly(I:C) and prednisone group.
C The Authors Journal compilation C 2014 Biochemical Society
Steroid-resistant model of asthma exacerbations
Figure S3
Inflammatory mediator profile after HDM/poly(I:C) challenge BALB/c mice were intranasally administered 25 μg of HDM extract in 25 μl of PBS or PBS alone three times a week for 3 weeks. At 2 h after the final challenge mice were challenged with poly(I:C) (10 mg/kg of body mass in a 50 μl volume) or vehicle (PBS, 50 μl volume) and killed 24 h later. Mice were treated with vehicle or prednisone (Pred) orally twice daily (3–30 mg/kg of body mass) for the final 2 weeks. Lungs were removed and snap-frozen, after which they were homogenized and assayed for cytokine content by Meso Scale Discovery (A–E) or ELISA (F). Results are means + − S.E.M., n = 6–8 mice/group from one experiment. Results were analysed by one-way ANOVA followed by Bonferroni’s post-hoc ∗ ∗∗ ∗∗∗ ## test comparing all groups. P < 0.05, P < 0.01 and P < 0.001 compared with the PBS alone group; P < 0.01 and ### P < 0.001 compared with the poly(I:C) alone group; ∼∼∼ P < 0.001 compared with the HDM alone group; + P < 0.05, ++ P < 0.01 and + + + P < 0.001 prednisone-treated group compared with the relevant control; >>> P < 0.001 poly(I:C) and prednisone compared with the corresponding HDM and prednisone group; ◦◦◦ P < 0.001 HDM and prednisone compared with the corresponding HDM/poly(I:C) and prednisone group.
Received 14 March 2013/18 October 2013; accepted 24 October 2013 Published as Immediate Publication 24 October 2013, doi: 10.1042/CS20130149
www.clinsci.org
Copyright of Clinical Science is the property of Portland Press Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
