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Murine models of infectious exacerbations of airway inflammation
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Murine models of infectious exacerbations of airway inflammation Malcolm Ronald Starkey1, Andrew Gregory Jarnicki1, Ama-Tawiah Essilfie1, Shaan Lae Gellatly, Richard Yong Kim, Alexandra Cerelina Brown, Paul Stephen Foster, Jay Christopher Horvat1 and Philip Michael Hansbro1 Airway inflammation underpins the pathogenesis of the major human chronic respiratory diseases. It is now well recognized that respiratory infections with bacteria and viruses are important in the induction, progression and exacerbation of these diseases. There are no effective therapies that prevent or reverse these events. The development and use of mouse models are proving valuable in understanding the role of infection in disease pathogenesis. They have recently been used to show that infections in early life alter immune responses and lung structure to increase asthma severity, and alter immune responses in later life to induce steroid resistance. Infection following smoke exposure or in experimental chronic obstructive pulmonary disease exacerbates inflammation and remodeling, and worsens cystic fibrosis. Further exploration of these models will facilitate the identification of new therapeutic approaches and the testing of new preventions and treatments. Addresses Centre for Asthma and Respiratory Disease, The Hunter Medical Research Institute and The University of Newcastle, Newcastle, Australia Corresponding author: Hansbro, Philip Michael ([email protected]) 1
These authors contributed equally.
Current Opinion in Pharmacology 2013, 13:337–344 This review comes from a themed issue on Respiratory Edited by Alastair G Stewart For a complete overview see the Issue and the Editorial
Mouse models of asthma involve the induction of acute or chronic allergic airway disease with model allergens such as ovalbumin (Ova) or house dust mite (HDM) extract (Figures 1 and 2) [1]. Acute models involve systemic (Ova) or local (HDM) allergic sensitisation followed by airway challenge with cognate antigen that induces T helper type 2 lymphocyte (Th2 cell) and cytokine responses, airway inflammation with elevated numbers of eosinophils, mucus hypersecretion and airway hyperreactivity (AHR) [2–4]. Chronic models are used to examine airway remodelling [5]. Models of COPD have also been developed. The administration of elastase and/or LPS induces lung damage and emphysematous-like changes. However they do not replicate the effects of exposure to cigarette smoke, which is the predominant COPD risk factor in humans [6,7]. Models involving acute nose-only or whole body cigarette smoke-exposure for 4 days to 4 weeks have proven valuable in elucidating the early effects in the lung but do not induce emphysema or lung function changes that are important features of COPD [6]. Chronic exposure for 5–6 months induces airway remodelling, emphysema and mild lung function changes [8]. Recently we have developed a short-term model of COPD induced by cigarette smoke exposure for 8 weeks that has the major pathological features of disease; chronic airway/lung inflammation, mucus hypersecretion, emphysema-like changes and reduced lung function (Figure 3) [9].
Available online 6th April 2013 1471-4892/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2013.03.005
Introduction Airway inflammation underpins human respiratory diseases such as asthma, COPD, cystic fibrosis (CF) and bronchiectasis. These conditions may be induced or exacerbated by respiratory infections that drive the progression of disease. There are no effective therapies that reverse the tissue lesions or that modulate the inducing or exacerbating factors that drive disease progression. Human clinical and in vitro studies characterise epidemiological and clinical features and causes of respiratory diseases. Mouse models that accurately reflect these features are invaluable in determining the mechanisms of pathogenesis and for the identification of modifiable targets for the development of new therapeutics. www.sciencedirect.com
CF is a genetic disease that is caused by a wide variety of mutations of the CF transmembrane regulator (CFTR). Both wild-type and transgenic mouse models have been developed, with the latter differing in the deletion or the nature of the mutation introduced [10]. Transgenic mice develop intestinal disease and some have lung abnormalities that are features of early CF [11], including airway inflammation, impaired mucociliary clearance, lung hyperinflation and remodelling. There are no specific models of bronchiectasis, although we have recently developed a model that has the combination of chronic airway inflammation and persistent bacterial infection [12]. These models and the use of genetically modified (factordeficient and transgenic) mice, as well as chimera and adoptive transfer studies and the availability of molecular tools (monoclonal antibodies, recombinant proteins and Current Opinion in Pharmacology 2013, 13:337–344
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Figure 1
(a)
Ova-tolerized (1d-3wk)
Ova-induced AAD Endpoints (8wk)
RSV IN (3, 4 & 5wk old)
Ova+CT IN (6wk)
Ova aerosol (7-8wk)
HDM-induced AAD (7-25d p.i.) (b)
Endpoints (d36 & 57) Flu IN (8d old)
HDM IN (15-19d)
HDM IN HDM IN (22-26d) (29-33d) Ova-induced AAD (6 wks p.i.)
(c)
Endpoints (d16 of AAD) C. muridarum IN (0d or 3wks old)
Ova IP (0d)
Ova IN (12-15d)
Endpoints (5-9 wks p.i.)
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C. muridarum IN (0d, 1d, 1wk or 3wks old)
Early life ira RS to r Ch V, y in RV f l a ↑ m , F ect Th yd lu io ns 2 ia Im ; m un ity
Later life Allergic Sensitisation
Severe Asthma Altered immunity Altered lung function Abnormal lung structure
Re
sp
(e)
Balanced Immune responses
Tolerance to Allergens
Non asthmatic Normal immunity Normal lung function Normal lung structure Current Opinion in Pharmacology
Murine models of infection-induced, severe asthma in early life. Most models use allergic sensitisation to (a,c) ovalbumin (Ova) or (b) house dust mite (HDM). The impact of prior (a,b) viral (respiratory syncytial virus [RSV], influenza [50]) [19,22] or (c) bacterial (Chlamydia muridarum) [24,25] infection on the subsequent development of Ova-induced or HDM-induced allergic airway disease (AAD) can then be assessed to investigate their impact. (d) The effects of infection alone in driving features of AAD in later life can also be determined [13,24,25,26,27]. (e) These models have been used to show that infection in early life induces permanent alterations in immunity and lung structure that increases the severity of AAD. Other abbreviations: d: day; p.i.: post-inoculation; wk: week.
Current Opinion in Pharmacology 2013, 13:337–344
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Murine models of infection with airway inflammation Starkey et al. 339
Figure 2
(a)
Ova-induced AAD Endpoints (d16 of AAD) Ova IN Ova IP (12-15d) (0d) Ova-induced AAD
C. muridarum IN (-7d)
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Before, -10d
During, 0d
H. influenzae IT (-10d, 0d or 10d)
(c)
After, 10d
Endpoints (d16 of AAD)
Ova IN (10d) (12-15d)
Ova IP (0d)
Adult Asthmatic (Stable)
Adult Asthmatic (Exacerbation)
ira Ch tor y in lam inf flu yd ec en ia tio ns za ; e
Allergen Exposure
e
H.
↑
7 h1
m
Severe Asthma ↑ Neutrophilic inflammation Steroid-resistant
T
1/
sp Re
ated responses di
Th
Mild-moderate asthma Eosinophilic inflammation Steroid-sensitive
Th2-mediated responses
Allergen Exposure Current Opinion in Pharmacology
Murine models of infection-induced, steroid-resistant severe asthma in adulthood. (a,b) Models of infection have been combined with ovalbumin (Ova)-induced models of allergic airway disease (AAD) in later life to assess their impact [12,32,33]. (c) Infection drives a more neutrophilic form of disease that is resistant to steroid treatment. Other abbreviations: Chlamydia muridarum; d: day; H. influenzae: Haemophilus influenzae.
small molecule and RNA inhibitors) have proved valuable in assessing the role of specific cells and factors, and potential therapies [9,13,14,15]. Importantly each of these diseases are frequently induced or exacerbated by viral and bacterial infections. The approach of combining the models described above with representative models of infection is being used to elucidate the interactions and develop new preventions and treatments, which is the focus of this review. Such studies are either not possible and/or unsafe to conduct in a clinical setting. However, care must be taken when interpreting results from these models to ensure that they represent the human condition, or features of disease, as closely as possible, and when translating them into human studies.
Models of infection-induced, severe asthma in early life Viral and bacterial respiratory infections in both early and later life are associated with the development and www.sciencedirect.com
increasing the severity of asthma [16,17]. Some bacteria may be protective but this aspect of infection will not be considered further as it is reviewed elsewhere [2,4,18]. Respiratory syncytial virus (RSV), rhinovirus (RV) and influenza are important childhood pathogens and are strongly associated with the subsequent development of asthma [16]. The mechanisms involved are poorly understood but are increasingly being investigated using mouse models. A recent study demonstrated that repeated RSV infection of infant mice reduced maternally transferred tolerance to Ova and increased the severity of allergic airways disease (AAD) by increasing IgE, airway inflammation and AHR (Figure 1a). This was driven by reductions in the suppressive function of FoxP3+ regulatory T cells and increased GATA3 expression and Th2 cytokine production by these cells, which were dependent on signalling through the IL-4Ra [19]. This conclusion was also Current Opinion in Pharmacology 2013, 13:337–344
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Figure 3
(a)
Elastase/LPSinduced COPD
Elastase* & LPS** (d1* & d4**/wk, 4wks)
Endpoints (d1, 3, 5 & 15 p.i.)
H.Influenzae IN (4wks)
Cigarette smokeinduced COPD
(b)
Smoke exposure (1-5d/wk, 8wks)
(c) Smoker (Pre-COPD)
Endpoints (d1-10 p.i.)
S.pneumoniae IT H.Influenzae IN or Flu IN (8wks)
Respiratory infections; H. influenzae, S. pneumoniae, S. aureus, Flu
Smoker (COPD)
nate
R/In
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↑ inflammation ↓ protective responses
signalling
Exacerbation of COPD and infection Current Opinion in Pharmacology
Murine models of infection-induced exacerbations of COPD. (a) Elastase/lipopolysaccharide (LPS) [37] and (b) acute or chronic smoke exposure [38] or experimental chronic obstructive pulmonary disease (COPD) [9] have been used to assess the impact of infection. (c) These models have shown that infections increases inflammation, remodelling and emphysema and that smoke exposure reduces but experimental COPD increases susceptibility to respiratory infection. Other abbreviations: d: day; Flu: influenza; H. influenzae: Haemophilus influenzae; p.i.: post-inoculation; S. aureus: Staphylococcus aureus; S. pneumoniae: Streptococcus pneumoniae; wk: week.
supported by a previous study using pneumonia virus of mice (PVM), which is a natural mouse pneumovirus and infection in mice replicates severe RSV disease in human infants. Neonatal mice infected with PVM had increased IgE, AHR and Th2 responses during AAD in later-life, which was also dependent on IL-4Ra [5]. RV infection of neonatal mice also worsened the severity of subsequent AAD in another study by inducing mucus secreting cell (MSC) metaplasia, pulmonary inflammation and AHR that persisted into later life [20]. The effects were again IL-4Ra-dependent and IL-13-dependent. You et al. used influenza infection in mice to demonstrate that neonatal infection caused permanent pulmonary dysfunction characterised by chronic inflammation, emphysema-like alveolar enlargement and AHR [21]. This occurred as a result of insufficient CD8+ T cell responses and could be prevented by the adoptive transfer of adult CD8+ T cells before infection [21]. More Current Opinion in Pharmacology 2013, 13:337–344
recently, it was demonstrated that influenza in early life enhanced responses to allergens. Neonatal hypo-responsiveness to HDM was overcome when exposure occurred concurrently with acute influenza infection leading to increased allergic inflammation and lung remodelling. Remodelling persisted into adulthood and remained even after cessation of HDM exposure and was associated with significantly impaired lung function (Figure 1b) [22]. Early-life infections with certain bacterial pathogens such as Chlamydia also promote permanent pulmonary dysfunction and are associated with more severe asthma [17,23]. To study this we used the natural mouse pathogen C. muridarum, which induces a similar condition to that observed in humans with respiratory Chlamydia infection [23]. We first showed that neonatal, but not adult infection increased the severity of Ova-induced AAD in later-life by increasing the number of airway MSCs and AHR (Figure 1c) [24]. Interestingly, neonatal www.sciencedirect.com
Murine models of infection with airway inflammation Starkey et al. 341
infection alone, even in the absence of antigen sensitisation and challenge induced persistent AHR (Figure 1d). We then showed that both neonatal and infant infection enhanced AAD in later life by increasing MSCs, IL-13 expression, DC-induced IL-13 release from CD4+ T cells and AHR [25]. Importantly, neonatal but not infant or adult infection induced emphysema-like alveolar enlargement in the absence of AAD, which has since been confirmed by others [26]. The mechanisms underpinning these observations are now being explored. TLR2 expression in neutrophils is critical for protection against bacterial infection [27]. Reconstitution of irradiated naı¨ve adult mice with bone marrow from mice infected as infants increased the severity of AAD by increasing IL5 and IL-13 release from local lymph nodes and IL-13 levels in the lung, mucus hyper-secretion and AHR [14]. In contrast, reconstitution with bone marrow from infected adult mice had no effects. Therefore, early-life infection-induced alterations in hematopoietic cells may play a role in predisposing to severe AAD in later-life. The role of IL-13 in promoting early-life Chlamydia respiratory infection-induced persistent AHR and severe AAD has also been assessed [13]. Infection-induced persistent AHR was IL-13-dependent as it was totally ablated in IL-13-deficient mice, but was restored by reconstitution of constitutive levels of IL-13. Mice deficient in STAT6 (a critical downstream signalling molecule of IL-13) were also protected. Finally, neutralization with anti-IL-13 monoclonal antibody during earlylife infection prevented infection-induced severe AAD in later-life. Since Th2 and IL-13 responses are often elevated in early life, these studies support therapeutic targeting of IL-13 for the prevention or treatment of infection-induced asthma in childhood.
AAD suppressed Ova-specific Th2-mediated, eosinophilic inflammation but induced Th1 and Th17 responses and neutrophilic inflammation (Figure 2a) [32]. This model, therefore, has the hallmark features of neutrophilic asthma. Blocking neutrophil recruitment into the lungs by depleting CXCL1 and CXCL2 using monoclonal antibodies only during infection suppressed infection-induced Th1-dominated, neutrophilic inflammatory responses during subsequent AAD. This identified a potential role for infection-induced, neutrophilmediated responses in the modulation of immunological phenotype and the induction of severe disease. H. influenzae infection also induces neutrophilic AAD that is mediated by IL-17 responses (Figure 2b) [12,33]. Infection reduced both Th1 and Th2 responses, while enhancing innate IL-17 production from neutrophils and macrophages, and adaptive IL-17 production from Th17 cells. Significantly depleting IL-17 abrogated infectioninduced neutrophilic inflammation. Importantly, these models of infection-induced AAD are resistant to dexamethasone treatment, highlighting that infectioninduced Th1 and/or Th17 responses may be critical in the development of severe, non-eosinophilic asthma subtypes that are refractory to steroids.
Models of infection-induced, steroid-resistant severe asthma in adulthood
These findings are supported by recent studies showing that adoptive transfer of Ova-specific Th1 cells in combination with LPS induced neutrophilic inflammation and steroid-resistant AHR in mice [34]. It was the novel cooperation between Th1-induced IFN-g responses and TLR4/MyD88 signalling that resulted in the production of IL-27 by macrophages. The combination of IL-27 and IFN-g induced steroid-resistant AHR through MyD88dependent inhibition of the translocation of the glucocorticoid receptor to the nucleus of macrophages. In other studies, adoptive transfer of allergen specific Th17 cells resulted in neutrophilic inflammation and AHR during AAD that was resistant to dexamethasone treatment [35]. This was in contrast to the transfer of Th2 cells, which induced eosinophilic inflammation and AHR that was steroid sensitive.
Clinically, severe asthma in adults is associated with increased Th1 and/or Th17 and neutrophilic inflammatory responses and is refractory to steroid treatment [28,29]. Around 10% of asthmatics are resistant to treatment but because of limited treatment options these patients account for >50% of asthma heathcare costs. Respiratory infections are increasingly being associated with more severe asthma. Asthmatic patients with Chlamydia and Haemophilus influenzae infection have increased airway neutrophilic inflammation, have more severe disease and are resistant to steroid treatment [30,31].
We have also used murine models to demonstrate that the induction of AAD during H. influenzae infection promotes chronic bacterial infection in neutrophilic AAD by decreasing the phagocytic function of neutrophils and macrophages (Figure 2b) [12]. Furthermore, increased IL-13 responses (which dominate in the asthmatic lung) increase susceptibility to Chlamydia respiratory infection by reducing the anti-bacterial function of macrophages and increasing susceptibility of pulmonary epithelial cells to infection [36].
Animal models of severe steroid-resistant asthma have been developed only recently and are being used to investigate the association with infection. We have shown that Chlamydia respiratory infection during Ova-induced
Together, these findings suggest that infection may take advantage of Th2-dominant responses in the asthmatic lung in order to infect. They then induce Th1 and/or Th17 immune responses that drive the induction of
Collectively these studies show that early life infections may induce more severe asthma by inducing permanent alterations in immunity and lung structure (Figure 1e).
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steroid-resistant, neutrophil-dominated inflammatory responses and severe asthma (Figure 2c).
Models of infection-induced exacerbations of COPD COPD patients are more susceptible to bacterial and viral respiratory infections that cause exacerbations of their disease. Patients often do not fully recover from exacerbations, which drive the progressive deterioration of their condition. The mechanisms that underpin infectioninduced exacerbations are being unravelled by combining models of infections that are common causes of exacerbations, with elastase/LPS or smoke exposure models. When elastase/LPS-exposed mice were inoculated with H. influenzae the infection became more chronic and persisted for at least 5 days compared to 3 days with elastase, LPS or PBS alone (Figure 3a) [37]. Elastase/LPS-exposed mice also had sustained neutrophilic inflammation in the lung, goblet cell metaplasia, emphysema and AHR at 15 days after infection. However, the mechanisms of the induction of pathology are not well understood, and are likely to be different in smoke-exposure models. H. influenzae-induced exacerbations have also been studied following whole body cigarette smoke exposure for 8 weeks [38]. Infection increased inflammation and damage in the lung, and smoke exposure altered the type of inflammatory mediators induced in response to the bacteria. Bacterial clearance was enhanced by smoke exposure, which was associated with and dependent on, bacterial-specific IgA [39]. Steroid treatment suppressed infection-induced inflammation in the presence or absence of cigarette smoke, but increased the bacterial burden. In support of this, Huvanne et al. used a model of whole body cigarette-smoke exposure for 4 weeks, with nasal administration of Staphylococcus aureus enterotoxin B (SEB), after 2 weeks. Exposure to both cigarette smoke and SEB also resulted in increases in the influx of inflammatory cells in the lung, compared to either exposure alone [40]. Other recent studies have used Poly I:C as a surrogate of viral infection following acute (2 week) exposure to cigarette smoke. This combination increased airway inflammation and induced emphysema-like changes, which did not occur upon exposure to each stimulus alone [41]. These effects were associated with changes in innate immune responses and TLR3-dependent and TLR3independent pathways and could be replicated with influenza infection. In a subsequent study influenza infection of mice following short-term (4 days) smoke exposure resulted in an increase in chemokine responses compared to either insult alone [42]. We have infected mice after 8 weeks of cigarette smoke exposure in our short-term model of COPD (Figure 3b) Current Opinion in Pharmacology 2013, 13:337–344
and showed that they suffer more severe Streptococcus pneumoniae (10-fold), H. influenzae and influenza (2.5-fold) infections [9]. This was associated with increases in the influx of inflammatory and CD8+ T cells into the airways but these cells had reduced expression of the activation marker CD98, which may contribute to impaired pathogen clearance. Thus, infection was more severe and inflammation is increased but immune function was suppressed in experimental COPD (Figure 3c).
Models of Pseudomonas aeruginosa infection-induced exacerbations of CF and COPD In CF, P. aeruginosa is the dominant microorganism in the lungs, and this bacterium is also important in COPD. Patients with CF usually acquire P. aeruginosa by adolescence, and over time, despite multiple antibiotic treatments, this bacterium becomes the dominant species in the lung. Mouse models of acute and chronic infection with this and other bacteria have been developed and combined with models of CF and COPD, which have recently been reviewed [43]. They mimic the infectioninduced exacerbations of disease that are typical in CF patients. Acute models typically use inoculation with bacterial cultures whereas chronic models of P. aeruginosa infection use an immobilizing agent, typically agar or agarose. Recent studies have utilised these models to show that infection is age-dependent and can be aggravated by estrogen and that inhibition of the high-mobility group Box 1 protein, and therapy with the long pentraxin PTX3 protect against infection and pneumonia in CF [44–47]. Furthermore the CFTR in innate immune cells may be important in clearance of P. aeruginosa [48]. Six to eight weeks of cigarette smoke exposure reduced P. aeruginosa clearance and resulted in increased inflammatory responses to the infection [49].
Conclusions Murine models are valuable tools for investigating the underlying mechanisms of the induction, progression and exacerbation of diseases that are underpinned by airway inflammation and currently have no effective treatments or cures. They enable the investigation of the precise interplay between infection and chronic respiratory disease since the timing and level of infection and other parameters can be controlled. The availability and use of factor-deficient and transgenic mouse strains, and molecular tools allow the depletion/enhancement of individual factors to establish their roles in disease pathogenesis. Elucidating the mechanisms of disease may identify novel targets for therapeutic intervention and models can be utilised to test the efficacy of new therapies. In this regard murine models have recently been used to show that infection in early life alters immune responses and lung structure that increase asthma severity, whereas www.sciencedirect.com
Murine models of infection with airway inflammation Starkey et al. 343
in later life the immune phenotype is modified to induce severe steroid resistant disease. Infection following cigarette smoke exposure or experimental COPD exaggerates airway and alveolar inflammatory and remodelling responses, and smoke exposure improves whereas the development of COPD reduces the clearance of infection. Infection worsens disease in CF and numerous new therapeutics are being investigated. These studies and further exploration of novel mouse models are likely to identify new therapies for the treatment and prevention of infection-induced exacerbations of airway inflammation.
Acknowledgements Our work is supported by grants from the National Health and Medical Research Council of Australia, The Australian Research Council, the Asthma Foundation of NSW and the Hunter Medical Research Institute.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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2.
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3.
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8.
Huvenne W, Pe´rez-Novo CA, Derycke L, De Ruyck N, Krysko O, Maes T, Pauwels N, Robays L, Bracke KR, Joos G et al.: Different regulation of cigarette smoke induced inflammation in upper versus lower airways. Respir Res 2010, 11:100.
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Beckett EL, Stevens RL, Jarnicki AG, Kim RY, Hanish I, Hansbro NG, Deane A, Keely S, Horvat JC, Yang M et al.: A shortterm model of COPD identifies a role for mast cell tryptase. J Allergy Clin Immunol 2013, 131:752-762 e7. An important study that describes the development of a new, short-term mouse model of experimental COPD that develops the hallmarks features of the human condition in just 8 weeks. The use of this model in identifying a novel role for mast cell tryptase in pathogenesis is also described.
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24. Horvat JC, Beagley KW, Wade MA, Preston JA, Hansbro NG, Hickey DK, Kaiko GE, Gibson PG, Foster PS, Hansbro PM: Neonatal chlamydial infection induces mixed T-cell responses that drive allergic airway disease. Am J Respir Crit Care Med 2007, 176:556-564.
36. Asquith KL, Horvat JC, Kaiko GE, Carey AJ, Beagley KW, Hansbro PM, Foster PS: Interleukin-13 promotes susceptibility to chlamydial infection of the respiratory and genital tracts. PLoS Pathog 2011, 7:e1001339. See Ref. [13].
25. Horvat JC, Starkey MR, Kim RY, Phipps S, Gibson PG, Beagley KW, Foster PS, Hansbro PM: Early-life chlamydial lung infection enhances allergic airways disease through agedependent differences in immunopathology. J Allergy Clin Immunol 2010, 125:617-625 e611–e625, e616. Demonstrates for the first time that Chlamydia respiratory infection in early life permanently alters immune function and lung structure to increase the severity of AAD in later life.
37. Ganesan S, Faris AN, Comstock AT, Sonstein J, Curtis JL, Sajjan US: Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge. Am J Pathol 2012, 180:61-72.
26. Jupelli M, Murthy AK, Chaganty BK, Guentzel MN, Selby DM, Vasquez MM, Mustafa SB, Henson BM, Seidner SR, Zhong G et al.: Neonatal chlamydial pneumonia induces altered respiratory structure and function lasting into adult life. Lab Invest 2011, 91:1530-1539. 27. Beckett EL, Phipps S, Starkey MR, Kim RY, Horvat JC, Foster PS, Hansbro PM: TLR2, but not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life. PLoS ONE 2012, 7:e39460. TLR2 agonists may be beneficial in the treatment of early life bacterial infections and their sequelae. 28. Hansbro PM, Kaiko GE, Foster PS: Cytokine/anti-cytokine therapy — novel treatments for asthma? Br J Pharmacol 2011, 163:81-95. 29. Wang W, Li JJ, Foster PS, Hansbro PM, Yang M: Potential therapeutic targets for steroid-resistant asthma. Curr Drug Targets 2010, 11:957-970. 30. Cho YS, Kim TB, Lee TH, Moon KA, Lee J, Kim YK, Lee KY, Moon HB: Chlamydia pneumoniae infection enhances cellular proliferation and reduces steroid responsiveness of human peripheral blood mononuclear cells via a tumor necrosis factor-a-dependent pathway. Clin Exp Allergy 2005, 35:1625-1631. 31. Wood LG, Simpson JL, Hansbro PM, Gibson PG: Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia. Free Radic Res 2010, 44:146-154. 32. Horvat JC, Starkey MR, Kim RY, Beagley KW, Preston JA, Gibson PG, Foster PS, Hansbro PM: Chlamydial respiratory infection during allergen sensitization drives neutrophilic allergic airways disease. J Immunol 2010, 184:4159-4169. This and Ref. [33] were the first to demonstrate that the combination of bacterial infections and AAD could drive a more severe disease phenotype that is dominated by neutophilic inflammation. 33. Essilfie AT, Simpson JL, Horvat JC, Preston JA, Dunkley ML, Foster PS, Gibson PG, Hansbro PM: Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease. PLoS Pathog 2011, 7:e1002244. See Ref. [32]. 34. Li JJ, Wang W, Baines KJ, Bowden NA, Hansbro PM, Gibson PG, Kumar RK, Foster PS, Yang M: IL-27/IFN-g induce myd88dependent steroid-resistant airway hyperresponsiveness by inhibiting glucocorticoid signaling in macrophages. J Immunol 2010, 185:4401-4409. 35. McKinley L, Alcorn JF, Peterson A, Dupont RB, Kapadia S, Logar A, Henry A, Irvin CG, Piganelli JD, Ray A et al.: Th17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice. J Immunol 2008, 181:4089-4097.
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38. Gaschler GJ, Skrtic M, Zavitz CCJ, Lindahl M, Onnervik PO, Murphy TF, Sethi S, Stampfli MR: Bacteria challenge in smokeexposed mice exacerbates inflammation and skews the inflammatory profile. Am J Respir Crit Care Med 2009, 179:666-675. 39. Gaschler GJ, Zavitz CCJ, Bauer CMT, Stampfli MR: Mechanisms of clearance of nontypeable Haemophilus influenzae from cigarette smoke-exposed mouse lungs. Eur Respir J 2010, 36:1131-1142. 40. Huvenne W, Lanckacker EA, Krysko O, Bracke KR, Demoor T, Hellings PW, Brusselle GG, Joos GF, Bachert C, Maes T: Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus enterotoxin B in mice. Respir Res 2011, 12:69. 41. Kang MJ, Lee CG, Lee JY, Dela Cruz CS, Chen ZJ, Enelow R, Elias JA: Cigarette smoke selectively enhances viral PAMPand virus-induced pulmonary innate immune and remodeling responses in mice. J Clin Invest 2008, 118:2284-2771. 42. Bauer CMT, Zavitz CCJ, Botelho FM, Lambert KN, Brown EG, Mossman KL, Taylor JD, Sta¨mpfli MR: Treating viral exacerbations of chronic obstructive pulmonary disease: insights from a mouse model of cigarette smoke and H1N1 influenza infection. PLoS ONE 2010, 5:e13251. 43. Bragonzi A: Murine models of acute and chronic lung infection with cystic fibrosis pathogens. Int J Med Microbiol 2010, 300:584-593. A contemporary review of the animal models of infection in cystic fibrosis. 44. Munder A, Wo¨lbeling F, Kerber-Momot T, Wedekind D, Baumann U, Gulbins E, Tu¨mmler B: Acute intratracheal Pseudomonas aeruginosa infection in cystic fibrosis mice is age-independent. Respir Res 2011, 12:148. 45. Wang Y, Cela E, Gagnon S, Sweezey NB: Estrogen aggravates inflammation in Pseudomonas aeruginosa pneumonia in cystic fibrosis mice. Respir Res 2010, 11:166. 46. Entezari M, Weiss D, Sitapara R, Whittaker L, Wargo MJ, Li JH, Wang H, Yang H, Sharma L, Phan BD et al.: Inhibition of highmobility group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas aeruginosa pneumonia in cystic fibrosis. Mol Med 2012, 18:477-485. 47. Paroni M, Moalli F, Nebuloni M, Pasqualini F, Bonfield T, Nonis A, Mantovani A, Garlanda C, Bragonzi A: Response of CFTRdeficient mice to long-term chronic Pseudomonas aeruginosa infection and PTX3 therapy. J Infect Dis 2012. 48. Bonfield TL, Hodges CA, Cotton CU, Drumm ML: Absence of the cystic fibrosis transmembrane regulator (CFTR) from myeloidderived cells slows resolution of inflammation and infection. J Leukoc Biol 2012, 92:1111-1122. 49. Drannik AG, Pouladi MA, Robbins CS, Goncharova SI, Kianpour S, Stampfli MR: Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection. Am J Respir Crit Care Med 2004, 170:1164-1171.
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Murine models of infectious exacerbations of airway inflammation Malcolm Ronald Starkey1, Andrew Gregory Jarnicki1, Ama-Tawiah Essilfie1, Shaan Lae Gellatly, Richard Yong Kim, Alexandra Cerelina Brown, Paul Stephen Foster, Jay Christopher Horvat1 and Philip Michael Hansbro1 Airway inflammation underpins the pathogenesis of the major human chronic respiratory diseases. It is now well recognized that respiratory infections with bacteria and viruses are important in the induction, progression and exacerbation of these diseases. There are no effective therapies that prevent or reverse these events. The development and use of mouse models are proving valuable in understanding the role of infection in disease pathogenesis. They have recently been used to show that infections in early life alter immune responses and lung structure to increase asthma severity, and alter immune responses in later life to induce steroid resistance. Infection following smoke exposure or in experimental chronic obstructive pulmonary disease exacerbates inflammation and remodeling, and worsens cystic fibrosis. Further exploration of these models will facilitate the identification of new therapeutic approaches and the testing of new preventions and treatments. Addresses Centre for Asthma and Respiratory Disease, The Hunter Medical Research Institute and The University of Newcastle, Newcastle, Australia Corresponding author: Hansbro, Philip Michael ([email protected]) 1
These authors contributed equally.
Current Opinion in Pharmacology 2013, 13:337–344 This review comes from a themed issue on Respiratory Edited by Alastair G Stewart For a complete overview see the Issue and the Editorial
Mouse models of asthma involve the induction of acute or chronic allergic airway disease with model allergens such as ovalbumin (Ova) or house dust mite (HDM) extract (Figures 1 and 2) [1]. Acute models involve systemic (Ova) or local (HDM) allergic sensitisation followed by airway challenge with cognate antigen that induces T helper type 2 lymphocyte (Th2 cell) and cytokine responses, airway inflammation with elevated numbers of eosinophils, mucus hypersecretion and airway hyperreactivity (AHR) [2–4]. Chronic models are used to examine airway remodelling [5]. Models of COPD have also been developed. The administration of elastase and/or LPS induces lung damage and emphysematous-like changes. However they do not replicate the effects of exposure to cigarette smoke, which is the predominant COPD risk factor in humans [6,7]. Models involving acute nose-only or whole body cigarette smoke-exposure for 4 days to 4 weeks have proven valuable in elucidating the early effects in the lung but do not induce emphysema or lung function changes that are important features of COPD [6]. Chronic exposure for 5–6 months induces airway remodelling, emphysema and mild lung function changes [8]. Recently we have developed a short-term model of COPD induced by cigarette smoke exposure for 8 weeks that has the major pathological features of disease; chronic airway/lung inflammation, mucus hypersecretion, emphysema-like changes and reduced lung function (Figure 3) [9].
Available online 6th April 2013 1471-4892/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2013.03.005
Introduction Airway inflammation underpins human respiratory diseases such as asthma, COPD, cystic fibrosis (CF) and bronchiectasis. These conditions may be induced or exacerbated by respiratory infections that drive the progression of disease. There are no effective therapies that reverse the tissue lesions or that modulate the inducing or exacerbating factors that drive disease progression. Human clinical and in vitro studies characterise epidemiological and clinical features and causes of respiratory diseases. Mouse models that accurately reflect these features are invaluable in determining the mechanisms of pathogenesis and for the identification of modifiable targets for the development of new therapeutics. www.sciencedirect.com
CF is a genetic disease that is caused by a wide variety of mutations of the CF transmembrane regulator (CFTR). Both wild-type and transgenic mouse models have been developed, with the latter differing in the deletion or the nature of the mutation introduced [10]. Transgenic mice develop intestinal disease and some have lung abnormalities that are features of early CF [11], including airway inflammation, impaired mucociliary clearance, lung hyperinflation and remodelling. There are no specific models of bronchiectasis, although we have recently developed a model that has the combination of chronic airway inflammation and persistent bacterial infection [12]. These models and the use of genetically modified (factordeficient and transgenic) mice, as well as chimera and adoptive transfer studies and the availability of molecular tools (monoclonal antibodies, recombinant proteins and Current Opinion in Pharmacology 2013, 13:337–344
338 Respiratory
Figure 1
(a)
Ova-tolerized (1d-3wk)
Ova-induced AAD Endpoints (8wk)
RSV IN (3, 4 & 5wk old)
Ova+CT IN (6wk)
Ova aerosol (7-8wk)
HDM-induced AAD (7-25d p.i.) (b)
Endpoints (d36 & 57) Flu IN (8d old)
HDM IN (15-19d)
HDM IN HDM IN (22-26d) (29-33d) Ova-induced AAD (6 wks p.i.)
(c)
Endpoints (d16 of AAD) C. muridarum IN (0d or 3wks old)
Ova IP (0d)
Ova IN (12-15d)
Endpoints (5-9 wks p.i.)
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C. muridarum IN (0d, 1d, 1wk or 3wks old)
Early life ira RS to r Ch V, y in RV f l a ↑ m , F ect Th yd lu io ns 2 ia Im ; m un ity
Later life Allergic Sensitisation
Severe Asthma Altered immunity Altered lung function Abnormal lung structure
Re
sp
(e)
Balanced Immune responses
Tolerance to Allergens
Non asthmatic Normal immunity Normal lung function Normal lung structure Current Opinion in Pharmacology
Murine models of infection-induced, severe asthma in early life. Most models use allergic sensitisation to (a,c) ovalbumin (Ova) or (b) house dust mite (HDM). The impact of prior (a,b) viral (respiratory syncytial virus [RSV], influenza [50]) [19,22] or (c) bacterial (Chlamydia muridarum) [24,25] infection on the subsequent development of Ova-induced or HDM-induced allergic airway disease (AAD) can then be assessed to investigate their impact. (d) The effects of infection alone in driving features of AAD in later life can also be determined [13,24,25,26,27]. (e) These models have been used to show that infection in early life induces permanent alterations in immunity and lung structure that increases the severity of AAD. Other abbreviations: d: day; p.i.: post-inoculation; wk: week.
Current Opinion in Pharmacology 2013, 13:337–344
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Murine models of infection with airway inflammation Starkey et al. 339
Figure 2
(a)
Ova-induced AAD Endpoints (d16 of AAD) Ova IN Ova IP (12-15d) (0d) Ova-induced AAD
C. muridarum IN (-7d)
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Before, -10d
During, 0d
H. influenzae IT (-10d, 0d or 10d)
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Endpoints (d16 of AAD)
Ova IN (10d) (12-15d)
Ova IP (0d)
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Adult Asthmatic (Exacerbation)
ira Ch tor y in lam inf flu yd ec en ia tio ns za ; e
Allergen Exposure
e
H.
↑
7 h1
m
Severe Asthma ↑ Neutrophilic inflammation Steroid-resistant
T
1/
sp Re
ated responses di
Th
Mild-moderate asthma Eosinophilic inflammation Steroid-sensitive
Th2-mediated responses
Allergen Exposure Current Opinion in Pharmacology
Murine models of infection-induced, steroid-resistant severe asthma in adulthood. (a,b) Models of infection have been combined with ovalbumin (Ova)-induced models of allergic airway disease (AAD) in later life to assess their impact [12,32,33]. (c) Infection drives a more neutrophilic form of disease that is resistant to steroid treatment. Other abbreviations: Chlamydia muridarum; d: day; H. influenzae: Haemophilus influenzae.
small molecule and RNA inhibitors) have proved valuable in assessing the role of specific cells and factors, and potential therapies [9,13,14,15]. Importantly each of these diseases are frequently induced or exacerbated by viral and bacterial infections. The approach of combining the models described above with representative models of infection is being used to elucidate the interactions and develop new preventions and treatments, which is the focus of this review. Such studies are either not possible and/or unsafe to conduct in a clinical setting. However, care must be taken when interpreting results from these models to ensure that they represent the human condition, or features of disease, as closely as possible, and when translating them into human studies.
Models of infection-induced, severe asthma in early life Viral and bacterial respiratory infections in both early and later life are associated with the development and www.sciencedirect.com
increasing the severity of asthma [16,17]. Some bacteria may be protective but this aspect of infection will not be considered further as it is reviewed elsewhere [2,4,18]. Respiratory syncytial virus (RSV), rhinovirus (RV) and influenza are important childhood pathogens and are strongly associated with the subsequent development of asthma [16]. The mechanisms involved are poorly understood but are increasingly being investigated using mouse models. A recent study demonstrated that repeated RSV infection of infant mice reduced maternally transferred tolerance to Ova and increased the severity of allergic airways disease (AAD) by increasing IgE, airway inflammation and AHR (Figure 1a). This was driven by reductions in the suppressive function of FoxP3+ regulatory T cells and increased GATA3 expression and Th2 cytokine production by these cells, which were dependent on signalling through the IL-4Ra [19]. This conclusion was also Current Opinion in Pharmacology 2013, 13:337–344
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Figure 3
(a)
Elastase/LPSinduced COPD
Elastase* & LPS** (d1* & d4**/wk, 4wks)
Endpoints (d1, 3, 5 & 15 p.i.)
H.Influenzae IN (4wks)
Cigarette smokeinduced COPD
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Smoke exposure (1-5d/wk, 8wks)
(c) Smoker (Pre-COPD)
Endpoints (d1-10 p.i.)
S.pneumoniae IT H.Influenzae IN or Flu IN (8wks)
Respiratory infections; H. influenzae, S. pneumoniae, S. aureus, Flu
Smoker (COPD)
nate
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↑ inflammation ↓ protective responses
signalling
Exacerbation of COPD and infection Current Opinion in Pharmacology
Murine models of infection-induced exacerbations of COPD. (a) Elastase/lipopolysaccharide (LPS) [37] and (b) acute or chronic smoke exposure [38] or experimental chronic obstructive pulmonary disease (COPD) [9] have been used to assess the impact of infection. (c) These models have shown that infections increases inflammation, remodelling and emphysema and that smoke exposure reduces but experimental COPD increases susceptibility to respiratory infection. Other abbreviations: d: day; Flu: influenza; H. influenzae: Haemophilus influenzae; p.i.: post-inoculation; S. aureus: Staphylococcus aureus; S. pneumoniae: Streptococcus pneumoniae; wk: week.
supported by a previous study using pneumonia virus of mice (PVM), which is a natural mouse pneumovirus and infection in mice replicates severe RSV disease in human infants. Neonatal mice infected with PVM had increased IgE, AHR and Th2 responses during AAD in later-life, which was also dependent on IL-4Ra [5]. RV infection of neonatal mice also worsened the severity of subsequent AAD in another study by inducing mucus secreting cell (MSC) metaplasia, pulmonary inflammation and AHR that persisted into later life [20]. The effects were again IL-4Ra-dependent and IL-13-dependent. You et al. used influenza infection in mice to demonstrate that neonatal infection caused permanent pulmonary dysfunction characterised by chronic inflammation, emphysema-like alveolar enlargement and AHR [21]. This occurred as a result of insufficient CD8+ T cell responses and could be prevented by the adoptive transfer of adult CD8+ T cells before infection [21]. More Current Opinion in Pharmacology 2013, 13:337–344
recently, it was demonstrated that influenza in early life enhanced responses to allergens. Neonatal hypo-responsiveness to HDM was overcome when exposure occurred concurrently with acute influenza infection leading to increased allergic inflammation and lung remodelling. Remodelling persisted into adulthood and remained even after cessation of HDM exposure and was associated with significantly impaired lung function (Figure 1b) [22]. Early-life infections with certain bacterial pathogens such as Chlamydia also promote permanent pulmonary dysfunction and are associated with more severe asthma [17,23]. To study this we used the natural mouse pathogen C. muridarum, which induces a similar condition to that observed in humans with respiratory Chlamydia infection [23]. We first showed that neonatal, but not adult infection increased the severity of Ova-induced AAD in later-life by increasing the number of airway MSCs and AHR (Figure 1c) [24]. Interestingly, neonatal www.sciencedirect.com
Murine models of infection with airway inflammation Starkey et al. 341
infection alone, even in the absence of antigen sensitisation and challenge induced persistent AHR (Figure 1d). We then showed that both neonatal and infant infection enhanced AAD in later life by increasing MSCs, IL-13 expression, DC-induced IL-13 release from CD4+ T cells and AHR [25]. Importantly, neonatal but not infant or adult infection induced emphysema-like alveolar enlargement in the absence of AAD, which has since been confirmed by others [26]. The mechanisms underpinning these observations are now being explored. TLR2 expression in neutrophils is critical for protection against bacterial infection [27]. Reconstitution of irradiated naı¨ve adult mice with bone marrow from mice infected as infants increased the severity of AAD by increasing IL5 and IL-13 release from local lymph nodes and IL-13 levels in the lung, mucus hyper-secretion and AHR [14]. In contrast, reconstitution with bone marrow from infected adult mice had no effects. Therefore, early-life infection-induced alterations in hematopoietic cells may play a role in predisposing to severe AAD in later-life. The role of IL-13 in promoting early-life Chlamydia respiratory infection-induced persistent AHR and severe AAD has also been assessed [13]. Infection-induced persistent AHR was IL-13-dependent as it was totally ablated in IL-13-deficient mice, but was restored by reconstitution of constitutive levels of IL-13. Mice deficient in STAT6 (a critical downstream signalling molecule of IL-13) were also protected. Finally, neutralization with anti-IL-13 monoclonal antibody during earlylife infection prevented infection-induced severe AAD in later-life. Since Th2 and IL-13 responses are often elevated in early life, these studies support therapeutic targeting of IL-13 for the prevention or treatment of infection-induced asthma in childhood.
AAD suppressed Ova-specific Th2-mediated, eosinophilic inflammation but induced Th1 and Th17 responses and neutrophilic inflammation (Figure 2a) [32]. This model, therefore, has the hallmark features of neutrophilic asthma. Blocking neutrophil recruitment into the lungs by depleting CXCL1 and CXCL2 using monoclonal antibodies only during infection suppressed infection-induced Th1-dominated, neutrophilic inflammatory responses during subsequent AAD. This identified a potential role for infection-induced, neutrophilmediated responses in the modulation of immunological phenotype and the induction of severe disease. H. influenzae infection also induces neutrophilic AAD that is mediated by IL-17 responses (Figure 2b) [12,33]. Infection reduced both Th1 and Th2 responses, while enhancing innate IL-17 production from neutrophils and macrophages, and adaptive IL-17 production from Th17 cells. Significantly depleting IL-17 abrogated infectioninduced neutrophilic inflammation. Importantly, these models of infection-induced AAD are resistant to dexamethasone treatment, highlighting that infectioninduced Th1 and/or Th17 responses may be critical in the development of severe, non-eosinophilic asthma subtypes that are refractory to steroids.
Models of infection-induced, steroid-resistant severe asthma in adulthood
These findings are supported by recent studies showing that adoptive transfer of Ova-specific Th1 cells in combination with LPS induced neutrophilic inflammation and steroid-resistant AHR in mice [34]. It was the novel cooperation between Th1-induced IFN-g responses and TLR4/MyD88 signalling that resulted in the production of IL-27 by macrophages. The combination of IL-27 and IFN-g induced steroid-resistant AHR through MyD88dependent inhibition of the translocation of the glucocorticoid receptor to the nucleus of macrophages. In other studies, adoptive transfer of allergen specific Th17 cells resulted in neutrophilic inflammation and AHR during AAD that was resistant to dexamethasone treatment [35]. This was in contrast to the transfer of Th2 cells, which induced eosinophilic inflammation and AHR that was steroid sensitive.
Clinically, severe asthma in adults is associated with increased Th1 and/or Th17 and neutrophilic inflammatory responses and is refractory to steroid treatment [28,29]. Around 10% of asthmatics are resistant to treatment but because of limited treatment options these patients account for >50% of asthma heathcare costs. Respiratory infections are increasingly being associated with more severe asthma. Asthmatic patients with Chlamydia and Haemophilus influenzae infection have increased airway neutrophilic inflammation, have more severe disease and are resistant to steroid treatment [30,31].
We have also used murine models to demonstrate that the induction of AAD during H. influenzae infection promotes chronic bacterial infection in neutrophilic AAD by decreasing the phagocytic function of neutrophils and macrophages (Figure 2b) [12]. Furthermore, increased IL-13 responses (which dominate in the asthmatic lung) increase susceptibility to Chlamydia respiratory infection by reducing the anti-bacterial function of macrophages and increasing susceptibility of pulmonary epithelial cells to infection [36].
Animal models of severe steroid-resistant asthma have been developed only recently and are being used to investigate the association with infection. We have shown that Chlamydia respiratory infection during Ova-induced
Together, these findings suggest that infection may take advantage of Th2-dominant responses in the asthmatic lung in order to infect. They then induce Th1 and/or Th17 immune responses that drive the induction of
Collectively these studies show that early life infections may induce more severe asthma by inducing permanent alterations in immunity and lung structure (Figure 1e).
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steroid-resistant, neutrophil-dominated inflammatory responses and severe asthma (Figure 2c).
Models of infection-induced exacerbations of COPD COPD patients are more susceptible to bacterial and viral respiratory infections that cause exacerbations of their disease. Patients often do not fully recover from exacerbations, which drive the progressive deterioration of their condition. The mechanisms that underpin infectioninduced exacerbations are being unravelled by combining models of infections that are common causes of exacerbations, with elastase/LPS or smoke exposure models. When elastase/LPS-exposed mice were inoculated with H. influenzae the infection became more chronic and persisted for at least 5 days compared to 3 days with elastase, LPS or PBS alone (Figure 3a) [37]. Elastase/LPS-exposed mice also had sustained neutrophilic inflammation in the lung, goblet cell metaplasia, emphysema and AHR at 15 days after infection. However, the mechanisms of the induction of pathology are not well understood, and are likely to be different in smoke-exposure models. H. influenzae-induced exacerbations have also been studied following whole body cigarette smoke exposure for 8 weeks [38]. Infection increased inflammation and damage in the lung, and smoke exposure altered the type of inflammatory mediators induced in response to the bacteria. Bacterial clearance was enhanced by smoke exposure, which was associated with and dependent on, bacterial-specific IgA [39]. Steroid treatment suppressed infection-induced inflammation in the presence or absence of cigarette smoke, but increased the bacterial burden. In support of this, Huvanne et al. used a model of whole body cigarette-smoke exposure for 4 weeks, with nasal administration of Staphylococcus aureus enterotoxin B (SEB), after 2 weeks. Exposure to both cigarette smoke and SEB also resulted in increases in the influx of inflammatory cells in the lung, compared to either exposure alone [40]. Other recent studies have used Poly I:C as a surrogate of viral infection following acute (2 week) exposure to cigarette smoke. This combination increased airway inflammation and induced emphysema-like changes, which did not occur upon exposure to each stimulus alone [41]. These effects were associated with changes in innate immune responses and TLR3-dependent and TLR3independent pathways and could be replicated with influenza infection. In a subsequent study influenza infection of mice following short-term (4 days) smoke exposure resulted in an increase in chemokine responses compared to either insult alone [42]. We have infected mice after 8 weeks of cigarette smoke exposure in our short-term model of COPD (Figure 3b) Current Opinion in Pharmacology 2013, 13:337–344
and showed that they suffer more severe Streptococcus pneumoniae (10-fold), H. influenzae and influenza (2.5-fold) infections [9]. This was associated with increases in the influx of inflammatory and CD8+ T cells into the airways but these cells had reduced expression of the activation marker CD98, which may contribute to impaired pathogen clearance. Thus, infection was more severe and inflammation is increased but immune function was suppressed in experimental COPD (Figure 3c).
Models of Pseudomonas aeruginosa infection-induced exacerbations of CF and COPD In CF, P. aeruginosa is the dominant microorganism in the lungs, and this bacterium is also important in COPD. Patients with CF usually acquire P. aeruginosa by adolescence, and over time, despite multiple antibiotic treatments, this bacterium becomes the dominant species in the lung. Mouse models of acute and chronic infection with this and other bacteria have been developed and combined with models of CF and COPD, which have recently been reviewed [43]. They mimic the infectioninduced exacerbations of disease that are typical in CF patients. Acute models typically use inoculation with bacterial cultures whereas chronic models of P. aeruginosa infection use an immobilizing agent, typically agar or agarose. Recent studies have utilised these models to show that infection is age-dependent and can be aggravated by estrogen and that inhibition of the high-mobility group Box 1 protein, and therapy with the long pentraxin PTX3 protect against infection and pneumonia in CF [44–47]. Furthermore the CFTR in innate immune cells may be important in clearance of P. aeruginosa [48]. Six to eight weeks of cigarette smoke exposure reduced P. aeruginosa clearance and resulted in increased inflammatory responses to the infection [49].
Conclusions Murine models are valuable tools for investigating the underlying mechanisms of the induction, progression and exacerbation of diseases that are underpinned by airway inflammation and currently have no effective treatments or cures. They enable the investigation of the precise interplay between infection and chronic respiratory disease since the timing and level of infection and other parameters can be controlled. The availability and use of factor-deficient and transgenic mouse strains, and molecular tools allow the depletion/enhancement of individual factors to establish their roles in disease pathogenesis. Elucidating the mechanisms of disease may identify novel targets for therapeutic intervention and models can be utilised to test the efficacy of new therapies. In this regard murine models have recently been used to show that infection in early life alters immune responses and lung structure that increase asthma severity, whereas www.sciencedirect.com
Murine models of infection with airway inflammation Starkey et al. 343
in later life the immune phenotype is modified to induce severe steroid resistant disease. Infection following cigarette smoke exposure or experimental COPD exaggerates airway and alveolar inflammatory and remodelling responses, and smoke exposure improves whereas the development of COPD reduces the clearance of infection. Infection worsens disease in CF and numerous new therapeutics are being investigated. These studies and further exploration of novel mouse models are likely to identify new therapies for the treatment and prevention of infection-induced exacerbations of airway inflammation.
Acknowledgements Our work is supported by grants from the National Health and Medical Research Council of Australia, The Australian Research Council, the Asthma Foundation of NSW and the Hunter Medical Research Institute.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Hansbro PM, Scott GV, Essilfie AT, Kim RY, Starkey MR, Nguyen DH, Allen PD, Kaiko GE, Yang M, Horvat JC et al.: Th2 cytokine antagonists: potential treatments for severe asthma. Expert Opin Investig Drugs 2013, 22:49-69.
2.
Thorburn AN, Foster PS, Gibson PG, Hansbro PM: Components of Streptococcus pneumoniae suppress allergic airways disease and natural killer T cells by inducing regulatory T cells. J Immunol 2012, 188:4611-4620.
3.
Thorburn AN, O’Sullivan BJ, Thomas R, Kumar RK, Foster PS, Gibson PG, Hansbro PM: Pneumococcal conjugate vaccineinduced T regulatory cells suppress the development of allergic airways disease. Thorax 2010, 65:1053-1060.
4.
Preston JA, Essilfie AT, Horvat JC, Wade MA, Beagley KW, Gibson PG, Foster PS, Hansbro PM: Inhibition of allergic airways disease by immunomodulatory therapy with whole killed Streptococcus pneumoniae. Vaccine 2007, 25:8154-8162.
5.
Siegle JS, Hansbro N, Dong C, Angkasekwinai P, Foster PS, Kumar RK: Blocking induction of T helper type 2 responses prevents development of disease in a model of childhood asthma. Clin Exp Immunol 2011, 165:19-28. Viral infections in early life interact with Th2 responses to increase the severity of subsequent chronic AAD and indicate that targeting Th2 inducing responses can inhibit this process.
6.
Vlahos R, Bozinovski S, Gualano RC, Ernst M, Anderson GP: Modelling COPD in mice. Pulm Pharmacol Ther 2006, 19:12-17.
7.
Keely S, Talley NJ, Hansbro PM: Pulmonary-intestinal cross-talk in mucosal inflammatory disease. Mucosal Immunol 2012, 5:718. A current review of the known mechanisms involved in COPD pathogenesis and links with the gut.
8.
Huvenne W, Pe´rez-Novo CA, Derycke L, De Ruyck N, Krysko O, Maes T, Pauwels N, Robays L, Bracke KR, Joos G et al.: Different regulation of cigarette smoke induced inflammation in upper versus lower airways. Respir Res 2010, 11:100.
9.
Beckett EL, Stevens RL, Jarnicki AG, Kim RY, Hanish I, Hansbro NG, Deane A, Keely S, Horvat JC, Yang M et al.: A shortterm model of COPD identifies a role for mast cell tryptase. J Allergy Clin Immunol 2013, 131:752-762 e7. An important study that describes the development of a new, short-term mouse model of experimental COPD that develops the hallmarks features of the human condition in just 8 weeks. The use of this model in identifying a novel role for mast cell tryptase in pathogenesis is also described.
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10. Guilbault C, Saeed Z, Downey GP, Radzloch D: Cystic fibrosis mouse models. Am J Respir Cell Mol Biol 2007, 36:1-7. 11. Kukavica-Ibrulj I, Levesque RC: Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies. Lab Anim 2008, 42:389-412. 12. Essilfie AT, Simpson JL, Dunkley ML, Morgan LC, Oliver BG, Gibson PG, Foster PS, Hansbro PM: Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma. Thorax 2012, 67:588-599. Another important study that describes the development of a mouse model of chronic bacterial infection in combination with AAD that is underpinned by neutrophilic inflammation and is steroid resistant. This model accurately reflects the features of severe steroid-resistant asthma in humans. This model was used to show that although the numbers of phagocytes in the lung were increased they had reduced phagocytic capacity. 13. Starkey MR, Essilfie AT, Horvat JC, Kim RY, Nguyen DH, Beagley KW, Mattes J, Foster PS, Hansbro PM: Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease. Mucosal Immunol 2013. November 7 (Epub ahead of print). This study and Ref. [36] demonstrate for the first time that the Th2 cytokine IL-13 promotes bacterial infection and may predispose to more severe infection-induced asthma. It shows that IL-13 may be a novel therapeutic target for the prevention/treatment of bacterial infections in early life. 14. Starkey MR, Kim RY, Beckett EL, Schilter HC, Shim D, Essilfie AT, Nguyen DH, Beagley KW, Mattes J, Mackay CR et al.: Chlamydia muridarum lung infection in infants alters hematopoietic cells to promote allergic airway disease in mice. PLoS ONE 2012, 7:e42588. Chlamydia infection in early life may drive the development of more severe asthma by affecting the activity of haematopoietic cells. 15. Kaiko GE, Phipps S, Hickey DK, Lam E, Hansbro PM, Foster PS, Beagley KW: Chlamydia muridarum infection subverts dendritic cell function to promote Th2 immunity and airways hyperreactivity. J Immunol 2008, 180:2225-2232. 16. Hansbro NG, Horvat JC, Wark PA, Hansbro PM: Understanding the mechanisms of viral induced asthma: new therapeutic directions. Pharmacol Ther 2008, 117:313-353. 17. Hansbro PM, Starkey MR, Kim RY, Stevens RL, Foster PS, Horvat JC: Programming of the lung by early life infection. J Dev Orig Health Dis 2012, 3:153-158. 18. Thorburn AN, Hansbro PM: Harnessing regulatory T cells to suppress asthma: from potential to therapy. Am J Respir Cell Mol Biol 2010, 43:511-519. 19. Krishnamoorthy N, Khare A, Oriss TB, Raundhal M, Morse C, Yarlagadda M, Wenzel SE, Moore ML, Peebles RS, Ray A et al.: Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma. Nat Med 2012, 18:1525-1530. This and Refs [20] and [22] show how early life viral infections may promote the development of asthma. 20. Schneider D, Hong JY, Popova AP, Bowman ER, Linn MJ, McLean AM, Zhao Y, Sonstein J, Bentley JK, Weinberg JB et al.: Neonatal rhinovirus infection induces mucous metaplasia and airways hyperresponsiveness. J Immunol 2012, 188:2894-2904. See Ref. [19]. 21. You D, Ripple M, Balakrishna S, Troxclair D, Sandquist D, Ding L, Ahlert TA, Cormier SA: Inchoate CD8+ T cell responses in neonatal mice permit influenza-induced persistent pulmonary dysfunction. J Immunol 2008, 181:3486-3494. 22. Al-Garawi A, Fattouh R, Botelho F, Walker TD, Goncharova S, Moore CL, Mori M, Erjefalt JS, Chu DK, Humbles AA et al.: Influenza A facilitates sensitization to house dust mite in infant mice leading to an asthma phenotype in adulthood. Mucosal Immunol 2011, 4:682-694. See Ref. [19]. 23. Hansbro PM, Beagley KW, Horvat JC, Gibson PG: Role of atypical bacterial infection of the lung in predisposition/ protection of asthma. Pharmacol Ther 2004, 101:193-210. Current Opinion in Pharmacology 2013, 13:337–344
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24. Horvat JC, Beagley KW, Wade MA, Preston JA, Hansbro NG, Hickey DK, Kaiko GE, Gibson PG, Foster PS, Hansbro PM: Neonatal chlamydial infection induces mixed T-cell responses that drive allergic airway disease. Am J Respir Crit Care Med 2007, 176:556-564.
36. Asquith KL, Horvat JC, Kaiko GE, Carey AJ, Beagley KW, Hansbro PM, Foster PS: Interleukin-13 promotes susceptibility to chlamydial infection of the respiratory and genital tracts. PLoS Pathog 2011, 7:e1001339. See Ref. [13].
25. Horvat JC, Starkey MR, Kim RY, Phipps S, Gibson PG, Beagley KW, Foster PS, Hansbro PM: Early-life chlamydial lung infection enhances allergic airways disease through agedependent differences in immunopathology. J Allergy Clin Immunol 2010, 125:617-625 e611–e625, e616. Demonstrates for the first time that Chlamydia respiratory infection in early life permanently alters immune function and lung structure to increase the severity of AAD in later life.
37. Ganesan S, Faris AN, Comstock AT, Sonstein J, Curtis JL, Sajjan US: Elastase/LPS-exposed mice exhibit impaired innate immune responses to bacterial challenge. Am J Pathol 2012, 180:61-72.
26. Jupelli M, Murthy AK, Chaganty BK, Guentzel MN, Selby DM, Vasquez MM, Mustafa SB, Henson BM, Seidner SR, Zhong G et al.: Neonatal chlamydial pneumonia induces altered respiratory structure and function lasting into adult life. Lab Invest 2011, 91:1530-1539. 27. Beckett EL, Phipps S, Starkey MR, Kim RY, Horvat JC, Foster PS, Hansbro PM: TLR2, but not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life. PLoS ONE 2012, 7:e39460. TLR2 agonists may be beneficial in the treatment of early life bacterial infections and their sequelae. 28. Hansbro PM, Kaiko GE, Foster PS: Cytokine/anti-cytokine therapy — novel treatments for asthma? Br J Pharmacol 2011, 163:81-95. 29. Wang W, Li JJ, Foster PS, Hansbro PM, Yang M: Potential therapeutic targets for steroid-resistant asthma. Curr Drug Targets 2010, 11:957-970. 30. Cho YS, Kim TB, Lee TH, Moon KA, Lee J, Kim YK, Lee KY, Moon HB: Chlamydia pneumoniae infection enhances cellular proliferation and reduces steroid responsiveness of human peripheral blood mononuclear cells via a tumor necrosis factor-a-dependent pathway. Clin Exp Allergy 2005, 35:1625-1631. 31. Wood LG, Simpson JL, Hansbro PM, Gibson PG: Potentially pathogenic bacteria cultured from the sputum of stable asthmatics are associated with increased 8-isoprostane and airway neutrophilia. Free Radic Res 2010, 44:146-154. 32. Horvat JC, Starkey MR, Kim RY, Beagley KW, Preston JA, Gibson PG, Foster PS, Hansbro PM: Chlamydial respiratory infection during allergen sensitization drives neutrophilic allergic airways disease. J Immunol 2010, 184:4159-4169. This and Ref. [33] were the first to demonstrate that the combination of bacterial infections and AAD could drive a more severe disease phenotype that is dominated by neutophilic inflammation. 33. Essilfie AT, Simpson JL, Horvat JC, Preston JA, Dunkley ML, Foster PS, Gibson PG, Hansbro PM: Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease. PLoS Pathog 2011, 7:e1002244. See Ref. [32]. 34. Li JJ, Wang W, Baines KJ, Bowden NA, Hansbro PM, Gibson PG, Kumar RK, Foster PS, Yang M: IL-27/IFN-g induce myd88dependent steroid-resistant airway hyperresponsiveness by inhibiting glucocorticoid signaling in macrophages. J Immunol 2010, 185:4401-4409. 35. McKinley L, Alcorn JF, Peterson A, Dupont RB, Kapadia S, Logar A, Henry A, Irvin CG, Piganelli JD, Ray A et al.: Th17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice. J Immunol 2008, 181:4089-4097.
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38. Gaschler GJ, Skrtic M, Zavitz CCJ, Lindahl M, Onnervik PO, Murphy TF, Sethi S, Stampfli MR: Bacteria challenge in smokeexposed mice exacerbates inflammation and skews the inflammatory profile. Am J Respir Crit Care Med 2009, 179:666-675. 39. Gaschler GJ, Zavitz CCJ, Bauer CMT, Stampfli MR: Mechanisms of clearance of nontypeable Haemophilus influenzae from cigarette smoke-exposed mouse lungs. Eur Respir J 2010, 36:1131-1142. 40. Huvenne W, Lanckacker EA, Krysko O, Bracke KR, Demoor T, Hellings PW, Brusselle GG, Joos GF, Bachert C, Maes T: Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus enterotoxin B in mice. Respir Res 2011, 12:69. 41. Kang MJ, Lee CG, Lee JY, Dela Cruz CS, Chen ZJ, Enelow R, Elias JA: Cigarette smoke selectively enhances viral PAMPand virus-induced pulmonary innate immune and remodeling responses in mice. J Clin Invest 2008, 118:2284-2771. 42. Bauer CMT, Zavitz CCJ, Botelho FM, Lambert KN, Brown EG, Mossman KL, Taylor JD, Sta¨mpfli MR: Treating viral exacerbations of chronic obstructive pulmonary disease: insights from a mouse model of cigarette smoke and H1N1 influenza infection. PLoS ONE 2010, 5:e13251. 43. Bragonzi A: Murine models of acute and chronic lung infection with cystic fibrosis pathogens. Int J Med Microbiol 2010, 300:584-593. A contemporary review of the animal models of infection in cystic fibrosis. 44. Munder A, Wo¨lbeling F, Kerber-Momot T, Wedekind D, Baumann U, Gulbins E, Tu¨mmler B: Acute intratracheal Pseudomonas aeruginosa infection in cystic fibrosis mice is age-independent. Respir Res 2011, 12:148. 45. Wang Y, Cela E, Gagnon S, Sweezey NB: Estrogen aggravates inflammation in Pseudomonas aeruginosa pneumonia in cystic fibrosis mice. Respir Res 2010, 11:166. 46. Entezari M, Weiss D, Sitapara R, Whittaker L, Wargo MJ, Li JH, Wang H, Yang H, Sharma L, Phan BD et al.: Inhibition of highmobility group box 1 protein (HMGB1) enhances bacterial clearance and protects against Pseudomonas aeruginosa pneumonia in cystic fibrosis. Mol Med 2012, 18:477-485. 47. Paroni M, Moalli F, Nebuloni M, Pasqualini F, Bonfield T, Nonis A, Mantovani A, Garlanda C, Bragonzi A: Response of CFTRdeficient mice to long-term chronic Pseudomonas aeruginosa infection and PTX3 therapy. J Infect Dis 2012. 48. Bonfield TL, Hodges CA, Cotton CU, Drumm ML: Absence of the cystic fibrosis transmembrane regulator (CFTR) from myeloidderived cells slows resolution of inflammation and infection. J Leukoc Biol 2012, 92:1111-1122. 49. Drannik AG, Pouladi MA, Robbins CS, Goncharova SI, Kianpour S, Stampfli MR: Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection. Am J Respir Crit Care Med 2004, 170:1164-1171.
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