- Email: [email protected]
The use of transgenic mice for modeling airways disease
Animal Models of Asthma and COPD
ARTICLE IN PRESS
Pulmonary Pharmacology & Therapeutics 21 (2008) 699–701 www.elsevier.com/locate/ypupt
The use of transgenic mice for modeling airways disease Steven D. Shapiro Jack D. Myers Professor, University of Pittsburgh, Department of Medicine, 1218 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA
Abstract Genetic manipulation in mice often combined with models of human disease has allowed investigators to dissect specific disease pathways. These tools have taught us much about the pathogenesis of chronic obstructive pulmonary disease (COPD). In particular, we have defined a complex inflammatory-protease network leading to emphysema. Translation of these findings to humans requires careful interpretation of the strengths and limitations of the models as well as similarities and differences between mouse and human biology. r 2008 Published by Elsevier Ltd. Keywords: COPD; Gene targeted mice; Transgenic mice; Cigarette smoking
1. Genetic engineering in mice Most genetic models of over- and underexpression in mammals involves transgenic and gene-targeted mice [1,2]. These techniques allow one to specifically increase or delete expression of one protein allowing for the design of a highly controlled experiment in vivo. Other species may be used, particularly for overexpression, however this discussion will be limited to the mouse. The main advantages of mice include the ability to isolate and manipulate their genome, an abundance of knowledge regarding mouse biology and probes available for the mouse, short breeding times, and large litters. 2. Generation of genetically engineered mice There are many reviews discussing techniques to generate transgenic and gene-targeted mice. Important practical points can be summarized as follows: Gene-targeted mice: Genomic DNA is altered (usually with a selectable marker) and incorporated into the germline of embryonic stem (ES) cells by homologous recombination. ES cells are placed in blastocysts and embryos selected for the expression of the mutant allele. Tel.: +412 648 9636; fax: +412 648 2117.
E-mail address: [email protected] 1094-5539/$ - see front matter r 2008 Published by Elsevier Ltd. doi:10.1016/j.pupt.2008.01.006
Traditionally, this leads to selective ‘‘knock-out’’ or inactivation of a gene product, however, more recently it has been used to ‘‘knock-in’’ gene products or specific mutations into the native promoter of the endogenous gene. Transgenic mice: Generally, the cDNA of interest is linked to a promoter (and polyA tail), often lungspecific, and the DNA or ‘‘transgene’’ is microinjected into pronuclei of fertilized eggs to randomly integrate genetic material into the genome. Upon placement of eggs into the oviduct, offspring then may express the transgene. Newer developments allow conditional (cellspecific), inducible expression, usually to avoid developmental expression of the transgene. One can also use color lineage specific promoters (or use knock-in technology) linked to color markers to tag particular lineages of cells. These techniques will become very useful as we try to identify specific stem cells in the future.
3. Modeling disease in mice The purpose of disease models is usually to determine pathogenetic mechanisms of disease. The utility of a given model depends upon the degree to which the animal model reflects the human disease. Two general ways in which models may be produced are: (1) overexpress
ARTICLE IN PRESS 700
S.D. Shapiro / Pulmonary Pharmacology & Therapeutics 21 (2008) 699–701
(or underexpress) a gene product and determine if the resulting phenotype reflects the disease of interest, or (2) natural model—apply the true (or surragate) etiologic agent resulting in the disease phenotype. One can apply genetargeted mice to the latter disease model to determine the contribution of that gene to the disease pathway. Of note, if transgenic overexpression of X results in the disease, one can conclude that X is ‘‘capable of’’ causing the phenotype, but cannot conclude that it is involved in the actual disease process. If however, it is known that X is up-regulated in the disease, then this is pretty strong evidence that its overexpression contributes to disease pathogenesis. The natural model approach is limited by our understanding of true disease etiology. For example, in chronic obstructive pulmonary disease (COPD) we have the advantage that we know that cigarette smoke (in susceptible individuals or strains of mice) causes the disease. However, we do not fully appreciate the cause of asthma limiting our ability to model it. Yet, we can induce an allergic state resulting in a characteristic inflammatory state with consequent hyperreactivity both phenotypes characteristic of asthma. More specifically asthma is difficult to model since we do not understand the cause. However, upon exposure to allergens, most commonly involving systemic sensitization and subsequent airway challenge with ovalbumin, one obtains a phenotype similar to aspects of asthma including inflammation (predominantly) eosinophilia and hyperreactivity. Applying gene-targeted mice to this model of allergic hyperreactivity, many different proteins have been shown to contribute to this phenotype, most of them involving Th-2 responses including IL-4, IL-5, IL-13, eotaxin, and RANTES. The important clinical question is whether this proof of concept studies will predict targets for human asthma. Our current therapy for asthma including beta-agonists, steroids, leukotriene receptor antagonists have been developed without gene targeting evidence. Newer therapy such as IgE directed therapy is supported by gene targeting, but clearly would have been predicted long before the advent of genetic engineering. Anti-IL-5 therapy represents a target with significant proof from IL-5 deficient (IL-5 / ) mice applied to the OVA model. IL-5 / mice were protected both from acute allergic inflammation, airway hyperresponsiveness [3], and chronic airway remodeling [4]. However, clinical trials utilizing IL-5 antagonists in both mild acute asthma [5] and chronic severe asthma [6] failed to provide a clinical benefit. The eosinophil community, however, has not accepted defeat. The number of patients in the studies were few and analysis of lung tissue following IL-5 antagonism caused less than a 50% depletion of tissue eosinophils [7]. Thus, the importance of IL-5 and eosinophils in asthma remains controversial, as does the utility of the murine models of asthma [8]. 4. Chronic obstructive pulmonary disease (COPD) In contrast to asthma, we do know that cigarette smoke, in susceptible individuals is the cause of COPD. Irrever-
sible airway obstruction that defines COPD can be caused both by airway obstruction and alveolar destruction with loss of recoil [9]. Upon exposure to long-term cigarette smoke exposure several mouse strains develop airspace enlargement, and evidence of airway remodeling with increased collagen, inflammation, and mucus hypersecretion [10]. Nevertheless, mice have a much simpler pattern of airway branching and few submucosal glands. Hence, the emphysematous component predominates mouse pathology making it easier to study this component in mice. Application of gene-targeted mice to this model has been largely restricted to proteinase ‘‘knockouts’’ at present. These studies have uncovered a dominant role for macrophage elastases, particularly MMP-12 [11], while also demonstrating a siginificant role for neutrophil elastase (NE) [12], with many interactions between MMP-12 and NE, each enhancing the others activity. Transgenic models have also shown that overexpression of a variety of proteins can lead to airspace enlargement and hence has the capacity to cause emphysema. One caveat is that overexpression during lung development can interfere with septation resulting in developmental (not destructive) airspace enlargement. Inducible and conditional overexpression can be used to avoid this caveat. Using this technology, it was found that overexpression of IL-13 [13] and IFNg [14], both led to inflammation and airspace enlargement. IFNg also has a marked element of structural cell apoptosis. 5. Limitations of genetic engineering in predicting drug targets Limitations relate mainly to the ability of mouse biology to replicate man and the limitations of the model used to assess a disease pathway. With respect to mouse and human biology, differences in both lung structure and function come into play. As mentioned mice have subtle differences in airway and alveolar structure such as fewer submucosal glands that make study of mucus hypersecretion difficult. Mice also have differences in protein expression. For example, mice do not have MMP-1, but do have the two other human interstitial collagenases (MMP-8 and MMP-13). Hence, functions of specific proteins may not translate directly from mouse to man. Nevertheless, the proteins classes, cell types, and general pathophysiologic pathways are well conserved between species. With respect to models, we discussed the major models of asthma and COPD and their strengths and weaknesses. We also discussed that overexpression of a specific protein may result in a disease phenotype, indicating that that protein has the potential to be a causative factor in the disease. However, asthma and COPD are complex diseases that result from multiple environmental and genetic factors. Nevertheless, genetic manipulation in the mouse as well as comparison of disease phenotypes in multiple strains of mice can be used to dissect these genetic components as well as disease pathways. Only time will
ARTICLE IN PRESS S.D. Shapiro / Pulmonary Pharmacology & Therapeutics 21 (2008) 699–701
tell how useful this line of research will be in helping understand and treat human airway disease, but the prediction here is that it will be invaluable if models are used and interpreted with care and caution. References [1] Shapiro SD. Mighty mice: transgenic technology ‘‘knocks out’’ questions of matrix metalloproteinase function. Matrix Biol 1997; 15:527–33. [2] Shapiro SD. Application of transgenic and gene-targeted mice to dissect mechanisms of lung disease. In: Stockley RA, editor. Molecular biology of the lung. Switzerland: Birkhauser Press; 1998. p. 1–15. [3] Foster P, Hogan S, Ramsay A, Matthaei K, YOung I. Interleukin-5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 1996;183:195–201. [4] Cho Y, Miller M, Baek K, et al. Inhibition of airway remodeling in IL-5-deficient mice. J Clin Invest 2004;113(4):551–60. [5] Leckie M, Brinke At, Khan J, et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000;356:2144–8. [6] Kips J, O’Connor B, Langley S, et al. Effect of SCH55700, a humanized anti-human interleukin-5 antibody, in severe persistent
[7]
[8] [9]
[10] [11]
[12]
[13]
[14]
701
asthma: a pilot study. Am J Respir Crit Care Med 2003;167(12): 1655–9. Flood-Page P, Menzies-Gow A, Kay A, Robinson D. Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003;167: 199–204. Williams T. The eosinophil enigma. J Clin Invest 2004;113:507–9. Pauwels R, Buist A, Calverley P, Jenkins C, Hurd S. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO global initiative for chronic obstructive lung disease (GOLD) workshop summary. Am J Respir Crit Care Med 2001;163:1256–76. Shapiro SD. Animal models for COPD. Chest 2000;117:223S–7S. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Macrophage elastase is required for cigarette smoke-induced emphysema in mice. Science 1997;277:2002–4. Shapiro S, Goldstein N, Kobayashi D, Kelley D, Houghton A, Belaaouaj A. Neutrophil elastase contributes to cigarette smokeinduced emphysema in mice. Am J Pathol 2004;163:2329–35. Zheng T, Zhu Z, Wang Z, et al. Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J Clin Invest 2000;106(9):1081–93. Wang Z, Zheng T, Zhu Z, et al. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J Exp Med 2000; 192(11):1587–600.
ARTICLE IN PRESS
Pulmonary Pharmacology & Therapeutics 21 (2008) 699–701 www.elsevier.com/locate/ypupt
The use of transgenic mice for modeling airways disease Steven D. Shapiro Jack D. Myers Professor, University of Pittsburgh, Department of Medicine, 1218 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA
Abstract Genetic manipulation in mice often combined with models of human disease has allowed investigators to dissect specific disease pathways. These tools have taught us much about the pathogenesis of chronic obstructive pulmonary disease (COPD). In particular, we have defined a complex inflammatory-protease network leading to emphysema. Translation of these findings to humans requires careful interpretation of the strengths and limitations of the models as well as similarities and differences between mouse and human biology. r 2008 Published by Elsevier Ltd. Keywords: COPD; Gene targeted mice; Transgenic mice; Cigarette smoking
1. Genetic engineering in mice Most genetic models of over- and underexpression in mammals involves transgenic and gene-targeted mice [1,2]. These techniques allow one to specifically increase or delete expression of one protein allowing for the design of a highly controlled experiment in vivo. Other species may be used, particularly for overexpression, however this discussion will be limited to the mouse. The main advantages of mice include the ability to isolate and manipulate their genome, an abundance of knowledge regarding mouse biology and probes available for the mouse, short breeding times, and large litters. 2. Generation of genetically engineered mice There are many reviews discussing techniques to generate transgenic and gene-targeted mice. Important practical points can be summarized as follows: Gene-targeted mice: Genomic DNA is altered (usually with a selectable marker) and incorporated into the germline of embryonic stem (ES) cells by homologous recombination. ES cells are placed in blastocysts and embryos selected for the expression of the mutant allele. Tel.: +412 648 9636; fax: +412 648 2117.
E-mail address: [email protected] 1094-5539/$ - see front matter r 2008 Published by Elsevier Ltd. doi:10.1016/j.pupt.2008.01.006
Traditionally, this leads to selective ‘‘knock-out’’ or inactivation of a gene product, however, more recently it has been used to ‘‘knock-in’’ gene products or specific mutations into the native promoter of the endogenous gene. Transgenic mice: Generally, the cDNA of interest is linked to a promoter (and polyA tail), often lungspecific, and the DNA or ‘‘transgene’’ is microinjected into pronuclei of fertilized eggs to randomly integrate genetic material into the genome. Upon placement of eggs into the oviduct, offspring then may express the transgene. Newer developments allow conditional (cellspecific), inducible expression, usually to avoid developmental expression of the transgene. One can also use color lineage specific promoters (or use knock-in technology) linked to color markers to tag particular lineages of cells. These techniques will become very useful as we try to identify specific stem cells in the future.
3. Modeling disease in mice The purpose of disease models is usually to determine pathogenetic mechanisms of disease. The utility of a given model depends upon the degree to which the animal model reflects the human disease. Two general ways in which models may be produced are: (1) overexpress
ARTICLE IN PRESS 700
S.D. Shapiro / Pulmonary Pharmacology & Therapeutics 21 (2008) 699–701
(or underexpress) a gene product and determine if the resulting phenotype reflects the disease of interest, or (2) natural model—apply the true (or surragate) etiologic agent resulting in the disease phenotype. One can apply genetargeted mice to the latter disease model to determine the contribution of that gene to the disease pathway. Of note, if transgenic overexpression of X results in the disease, one can conclude that X is ‘‘capable of’’ causing the phenotype, but cannot conclude that it is involved in the actual disease process. If however, it is known that X is up-regulated in the disease, then this is pretty strong evidence that its overexpression contributes to disease pathogenesis. The natural model approach is limited by our understanding of true disease etiology. For example, in chronic obstructive pulmonary disease (COPD) we have the advantage that we know that cigarette smoke (in susceptible individuals or strains of mice) causes the disease. However, we do not fully appreciate the cause of asthma limiting our ability to model it. Yet, we can induce an allergic state resulting in a characteristic inflammatory state with consequent hyperreactivity both phenotypes characteristic of asthma. More specifically asthma is difficult to model since we do not understand the cause. However, upon exposure to allergens, most commonly involving systemic sensitization and subsequent airway challenge with ovalbumin, one obtains a phenotype similar to aspects of asthma including inflammation (predominantly) eosinophilia and hyperreactivity. Applying gene-targeted mice to this model of allergic hyperreactivity, many different proteins have been shown to contribute to this phenotype, most of them involving Th-2 responses including IL-4, IL-5, IL-13, eotaxin, and RANTES. The important clinical question is whether this proof of concept studies will predict targets for human asthma. Our current therapy for asthma including beta-agonists, steroids, leukotriene receptor antagonists have been developed without gene targeting evidence. Newer therapy such as IgE directed therapy is supported by gene targeting, but clearly would have been predicted long before the advent of genetic engineering. Anti-IL-5 therapy represents a target with significant proof from IL-5 deficient (IL-5 / ) mice applied to the OVA model. IL-5 / mice were protected both from acute allergic inflammation, airway hyperresponsiveness [3], and chronic airway remodeling [4]. However, clinical trials utilizing IL-5 antagonists in both mild acute asthma [5] and chronic severe asthma [6] failed to provide a clinical benefit. The eosinophil community, however, has not accepted defeat. The number of patients in the studies were few and analysis of lung tissue following IL-5 antagonism caused less than a 50% depletion of tissue eosinophils [7]. Thus, the importance of IL-5 and eosinophils in asthma remains controversial, as does the utility of the murine models of asthma [8]. 4. Chronic obstructive pulmonary disease (COPD) In contrast to asthma, we do know that cigarette smoke, in susceptible individuals is the cause of COPD. Irrever-
sible airway obstruction that defines COPD can be caused both by airway obstruction and alveolar destruction with loss of recoil [9]. Upon exposure to long-term cigarette smoke exposure several mouse strains develop airspace enlargement, and evidence of airway remodeling with increased collagen, inflammation, and mucus hypersecretion [10]. Nevertheless, mice have a much simpler pattern of airway branching and few submucosal glands. Hence, the emphysematous component predominates mouse pathology making it easier to study this component in mice. Application of gene-targeted mice to this model has been largely restricted to proteinase ‘‘knockouts’’ at present. These studies have uncovered a dominant role for macrophage elastases, particularly MMP-12 [11], while also demonstrating a siginificant role for neutrophil elastase (NE) [12], with many interactions between MMP-12 and NE, each enhancing the others activity. Transgenic models have also shown that overexpression of a variety of proteins can lead to airspace enlargement and hence has the capacity to cause emphysema. One caveat is that overexpression during lung development can interfere with septation resulting in developmental (not destructive) airspace enlargement. Inducible and conditional overexpression can be used to avoid this caveat. Using this technology, it was found that overexpression of IL-13 [13] and IFNg [14], both led to inflammation and airspace enlargement. IFNg also has a marked element of structural cell apoptosis. 5. Limitations of genetic engineering in predicting drug targets Limitations relate mainly to the ability of mouse biology to replicate man and the limitations of the model used to assess a disease pathway. With respect to mouse and human biology, differences in both lung structure and function come into play. As mentioned mice have subtle differences in airway and alveolar structure such as fewer submucosal glands that make study of mucus hypersecretion difficult. Mice also have differences in protein expression. For example, mice do not have MMP-1, but do have the two other human interstitial collagenases (MMP-8 and MMP-13). Hence, functions of specific proteins may not translate directly from mouse to man. Nevertheless, the proteins classes, cell types, and general pathophysiologic pathways are well conserved between species. With respect to models, we discussed the major models of asthma and COPD and their strengths and weaknesses. We also discussed that overexpression of a specific protein may result in a disease phenotype, indicating that that protein has the potential to be a causative factor in the disease. However, asthma and COPD are complex diseases that result from multiple environmental and genetic factors. Nevertheless, genetic manipulation in the mouse as well as comparison of disease phenotypes in multiple strains of mice can be used to dissect these genetic components as well as disease pathways. Only time will
ARTICLE IN PRESS S.D. Shapiro / Pulmonary Pharmacology & Therapeutics 21 (2008) 699–701
tell how useful this line of research will be in helping understand and treat human airway disease, but the prediction here is that it will be invaluable if models are used and interpreted with care and caution. References [1] Shapiro SD. Mighty mice: transgenic technology ‘‘knocks out’’ questions of matrix metalloproteinase function. Matrix Biol 1997; 15:527–33. [2] Shapiro SD. Application of transgenic and gene-targeted mice to dissect mechanisms of lung disease. In: Stockley RA, editor. Molecular biology of the lung. Switzerland: Birkhauser Press; 1998. p. 1–15. [3] Foster P, Hogan S, Ramsay A, Matthaei K, YOung I. Interleukin-5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 1996;183:195–201. [4] Cho Y, Miller M, Baek K, et al. Inhibition of airway remodeling in IL-5-deficient mice. J Clin Invest 2004;113(4):551–60. [5] Leckie M, Brinke At, Khan J, et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000;356:2144–8. [6] Kips J, O’Connor B, Langley S, et al. Effect of SCH55700, a humanized anti-human interleukin-5 antibody, in severe persistent
[7]
[8] [9]
[10] [11]
[12]
[13]
[14]
701
asthma: a pilot study. Am J Respir Crit Care Med 2003;167(12): 1655–9. Flood-Page P, Menzies-Gow A, Kay A, Robinson D. Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003;167: 199–204. Williams T. The eosinophil enigma. J Clin Invest 2004;113:507–9. Pauwels R, Buist A, Calverley P, Jenkins C, Hurd S. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO global initiative for chronic obstructive lung disease (GOLD) workshop summary. Am J Respir Crit Care Med 2001;163:1256–76. Shapiro SD. Animal models for COPD. Chest 2000;117:223S–7S. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Macrophage elastase is required for cigarette smoke-induced emphysema in mice. Science 1997;277:2002–4. Shapiro S, Goldstein N, Kobayashi D, Kelley D, Houghton A, Belaaouaj A. Neutrophil elastase contributes to cigarette smokeinduced emphysema in mice. Am J Pathol 2004;163:2329–35. Zheng T, Zhu Z, Wang Z, et al. Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsin-dependent emphysema. J Clin Invest 2000;106(9):1081–93. Wang Z, Zheng T, Zhu Z, et al. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J Exp Med 2000; 192(11):1587–600.