- Email: [email protected]
Lung Alveolar Repair: Not All Cells Are Equal
TRMOME 1273 No. of Pages 3
Spotlight
Lung Alveolar Repair: Not All Cells Are Equal Charlotte H. Dean1,* and Clare M. Lloyd1 The lungs are capable of repair but the extent to which this occurs varies widely. Recent data indicate that, following injury, different progenitor cell populations can arise, depending on the molecular environment. In turn, these result in either normal or aberrant alveolar repair. Thus, a key question in lung regenerative medicine is how to maintain a ‘Goldilocks zone’ of repair. Diseases involving the destruction of lung alveoli represent a huge global health burden [1]. These diseases can be broadly separated into two categories: (i) chronic disease [including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD)]; and (ii) acute disease [including infections, such as influenza or pneumonia, in addition to acute respiratory distress syndrome (ARDS)]. Moreover, congenital lung diseases involving aberrant or insufficient generation of tissue exist, such as congenital pulmonary airway malformations and bronchopulmonary dysplasia, among others. Currently, there are no curative treatments for most of these conditions and we have no way of restoring lost alveolar tissue. Therefore, the advent of regenerative medicine provides an exciting opportunity to fulfill this unmet clinical need. Adult lungs are capable of intrinsic repair [2] but the extent to which this occurs can differ vastly between different diseases, as well as between individuals. Loss of tissue is catastrophic for effective lung function, preventing proper gaseous exchange and, therefore, tissue repair is
critical to [105_TD$IF]restore pulmonary function. However, the process must be finely tuned to provide sufficient, but not excessive, repair; hence, the ‘Goldilocks’ concept of fine-tuning. At one end of the spectrum, excessive repair can potentially lead to fibrotic lung disease. For instance, IPF can result from an aberrant woundhealing response; and lung cancer can result from hyperproliferation [3]. At the other end of the spectrum, morbidities in COPD and influenza infection have been linked to a lack of lung tissue repair [3]. As we begin to work towards therapeutic lung regeneration, a comprehensive understanding of the intrinsic molecular mechanisms driving alveolar regeneration is critical. A recent article by Xi et al. [4] highlights our rapidly increasing knowledge of the complexity of lung alveolar regeneration. Previous work in mice identified distinct lineage-negative epithelial progenitor cell [106_TD$IF]populations (LNEPs) lacking the expression of mature lineage markers, [107_TD$IF]that expand in the lungs following influenzamediated injury in vivo [5,6]. Some LNEPs expressed the stem and/or basal epithelial cell marker P63, while others did not [5,6]. Influenza or bleomycin-induced injury stimulated a Notch-driven remodeling repair program where LNEPs activated P63 and cytokeratin 5 (KRT5pos[104_TD$IF]) progenitors, leading to dysplastic repair of the alveolar epithelium [5]. By contrast, subsequent depletion of Notch allowed the generation of surfactant protein C (SPC)-positive cells and the differentiation of alveolar epithelial cells, facilitating normal repair [5]. In the latest study by Xi and coworkers, the authors shed light on the mechanisms of alveolar regeneration in murine and human lungs; they identified hypoxia as a critical controller of pulmonary epithelial cell fate decisions [4]. Specifically, by using the mouse model of influenza-mediated lung injury, they demonstrated that expression of hypoxiainducible factor (Hif)-1a could drive Krt5pos progenitor cells to persist in the lung, resulting in aberrant alveolar repair.
By contrast, in LNEP, Hif1a deletion or upregulation of Wnt/ß-catenin activity (a key regenerative pathway) promoted differentiation into normal SPC-positive alveolar type II epithelial cells (ATII) and enhanced repair [4]. This study underlines the fact that not all types of cell repair are equal and demonstrates examples of signals that fine-tune tissue repair, such as Hif1a and Wnt/ ß-catenin signaling. Thus, the balance between ‘good’ (i.e., healing) and ‘bad’ (i.e., fibrotic repair) is a key consideration for the development of regenerative therapies; for this, further knowledge of the signals that can drive normal versus abnormal lung repair is required (Figure 1). The important role of Notch in cell fate decisions also extends to inflammatory cells where it [109_TD$IF]is required for immune signaling [7]. Therefore, it is possible that maintaining Notch signaling might diminish effective lung repair by prolonging inflammation, as well as by regulating progenitor identity, although this remains to be directly tested. The link between enhanced repair in an inflammation-free context is well known from comparisons of embryonic and adult wound repair; in the embryo, wound repair can occur in a highly reduced inflammatory environment, resulting in scar-free healing, unlike adult wound repair, where scarring occurs and repair is less efficient [8]. A question that remains is whether there are differences in the molecular mechanisms of alveolar lung repair when triggered by different types of stimulus. For instance, are there differences in alveolar repair following a sterile stimulus, such as a mechanical injury [ventilator associated lung injury (VALI[10_TD$IF])]; a nonsterile stimulus (influenza infection); or in chronic lung diseases, where injury can be exacerbated by pulmonary infection (e.g., COPD or IPF)? Based on RNA-Seq data comparing normal versus abnormal lungs in humans and mice, the authors suggested that, in humans, the progenitor cell populations
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
1
TRMOME 1273 No. of Pages 3
Figure 1. Maintaining the Balance between ‘Good’ and ‘Bad’ Lung Repair. In humans and mice, the alveolar epithelium comprises long, thin squamous alveolar type I (ATI) cells and cuboidal alveolar type II (AT II) cells. Following tissue injury, progenitor and/or stem cell populations are generated to repair lost or damaged alveoli. Different types of progenitor population can arise, depending on the molecular environment. P63pos, Krt5pos cells are generated in response to hypoxia or persistent high levels of Notch. These precursors give rise to squamous, dysplastic epithelial cells and result in aberrant alveolar repair. By contrast, P63neg, SPCpos progenitor cells are generated in response to Wnt signaling. These cells expand and eventually give rise to both ATI and ATII cells to fully restore normal alveolar epithelium. Maintaining the balance between ‘good’ (normal) alveolar repair and ‘bad’ (dysplastic) repair is essential for restoring lung health. Several factors may tip the balance towards ‘bad’ repair, including the type of injury trigger, aging, lifestyle, genetics, and environment.
triggered by influenza infection, or in ARDS or IPF, may be largely the same: but is this really the case? Xi et al. have demonstrated that different progenitor populations predominate depending on the precise signals received, and that, in turn, these can lead to normal or dysplastic repair [4]. Undoubtedly, further detailed analysis is needed to identify the subpopulations that are prevalent in specific diseases [1_TD$IF]in order to target efficient therapeutic strategies. For instance, we know that, in skin, epidermal wound
2
repair can occur in the absence of an immune response, but different molecular mechanisms are utilized compared with those triggered during inflammationinduced repair; this illustrates that the presence of inflammation can affect tissue repair mechanisms. Moreover, the influence of aging on tissue repair capacity is also an important consideration for lung regenerative medicine; indeed, it is well known that fibrotic lung diseases are more frequent in older people, while classic inflammatory diseases, such as
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
asthma, are more common in children. Consequently, the response to injury may differ over a lifetime. A key strength of the study by Vaughan’s laboratory is that data from both mice and humans are presented. As is clear from their results, this is imperative because, when examining lung responses to injury, key differences, as well as commonalities, in regenerative signatures exist between species [4]. For example, RNA-Seq data show that, in response to lung injury, 102
TRMOME 1273 No. of Pages 3
common genes and pathways (predominantly involved in cell migration and proliferation) are upregulated in [12_TD$IF]both activated LNEP murine cells [13_TD$IF]and hypoxic ATII human cells. However, [14_TD$IF]many additional genes and/or pathways appear to be upregulated in [15_TD$IF]only one of these cell types [4]. This is perhaps unsurprising given the known differences between human and mouse lung morphology. Accordingly, the proportion of different epithelial cell types differs; there are more basal cells in human lung than in mouse lung [2]. Moreover, the immune environment of laboratory-reared mice differs considerably from that of humans [9]. The findings presented also remind us that experimental design, as well as whether data are obtained in vivo or in vitro, are important considerations, potentially leading to varying results. Indeed, two examples of different results are highlighted: first, LNEP cultures immersed in media were reported to be hypoxic, showing a different transcriptomic signature compared with those cultured at the air–liquid interface, illustrating the influence of varying culture conditions [4]. Second, mouse LNEP cultures rapidly lost SPC mRNA expression in vitro but not in vivo, highlighting the potential environmental effects on transcriptomic outcomes [4].
destruction, some patients with ARDS recover lost alveolar tissue, while others do not [10]. The variability in alveolar tissue repair capacity strongly suggests a genetic component to this process. Indeed, a recent study identified a critical role for the noncanonical Wnt/Planar Cell Polarity (PCP) pathway in lung repair [11]. The authors used population data to reveal the presence of a particular single nucleotide polymorphism (SNP) in Vangl2 (a key gene in this pathway), which rendered individuals more susceptible to lung function decline in response to smoking, [16_TD$IF]compared to nonsmokers not receiving [17_TD$IF]this damaging injury [11]. Thus, subtle genetic differences between individuals might affect lung function only when an injuriousinsult is received. This could partially explain why some people [18_TD$IF]do not mount an efficient lung repair response while others [19_TD$IF]do. In the era of genomicmedicine, this isanimportantconsideration that we can begin to address. All in all, a greater understanding of the molecular mechanisms regulating cell fate decisions following pulmonary damage, be it inflammatory, infectious, or due to physical injury, may eventually facilitate our ability to ensure lung tissue repair is maintained in the ‘Goldilocks zone’, mediated by cues that either prevent aberrant repair or promote sufficient ‘normal’ alveolar repair.
Finally, why do some individuals effectively repair damaged lung tissue while others are Acknowledgments incapable of doing so? It is well known that The authors wish to acknowledge funding provided despite extensive alveolar tissue by the Leverhulme trust (C.H.D.) and a Wellcome
Senior Fellowship in basic biomedical sciences (C.M.L[120_TD$IF].). We thank Laura Yates for assistance with Figure 1. 1 Inflammation, Repair and Development Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ
*Correspondence: [email protected] (C.H. Dean). http://dx.doi.org/10.1016/j.molmed.2017.08.009 References 1. Ferkol, T. and Schraufnagel, D. (2014) The global burden of respiratory disease. Ann. Am. Thorac. Soc. 11, 404–406 2. Hogan, B.L. et al. (2014) Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 3. Spella, M. et al. (2017) Shared epithelial pathways to lung repair and disease. Eur. Respir. Rev. 26, 170048 4. Xi, Y. et al. (2017) Local lung hypoxia determines epithelial fate decisions during alveolar regeneration. Nat. Cell Biol. 19, 904–914 5. Vaughan, A.E. et al. (2015) Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 517, 621–625 6. Kumar, P.A. et al. (2011) Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell 147, 525–538 7. Shang, Y. et al. (2016) Role of Notch signaling in regulating innate immunity and inflammation in health and disease. Protein Cell 7, 159–174 8. Eming, S.A. et al. (2014) Wound repair and regeneration: mechanisms, signaling, and translation. Sci. Transl. Med. 6, 265sr6 9. Beura, L.K. et al. (2016) Normalizing the environment recapitulates adult human immune traits in laboratory mice. Nature 532, 512–516 10. Martin, C. et al. (1995) Pulmonary fibrosis correlates with outcome in adult respiratory distress syndrome. A study in mechanically ventilated patients. Chest 107, 196–200 11. Poobalasingam, T. et al. (2017) Heterozygous Vangl2Looptail mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair. Dis. Models Mech. 10, 409–423
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
3
Spotlight
Lung Alveolar Repair: Not All Cells Are Equal Charlotte H. Dean1,* and Clare M. Lloyd1 The lungs are capable of repair but the extent to which this occurs varies widely. Recent data indicate that, following injury, different progenitor cell populations can arise, depending on the molecular environment. In turn, these result in either normal or aberrant alveolar repair. Thus, a key question in lung regenerative medicine is how to maintain a ‘Goldilocks zone’ of repair. Diseases involving the destruction of lung alveoli represent a huge global health burden [1]. These diseases can be broadly separated into two categories: (i) chronic disease [including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD)]; and (ii) acute disease [including infections, such as influenza or pneumonia, in addition to acute respiratory distress syndrome (ARDS)]. Moreover, congenital lung diseases involving aberrant or insufficient generation of tissue exist, such as congenital pulmonary airway malformations and bronchopulmonary dysplasia, among others. Currently, there are no curative treatments for most of these conditions and we have no way of restoring lost alveolar tissue. Therefore, the advent of regenerative medicine provides an exciting opportunity to fulfill this unmet clinical need. Adult lungs are capable of intrinsic repair [2] but the extent to which this occurs can differ vastly between different diseases, as well as between individuals. Loss of tissue is catastrophic for effective lung function, preventing proper gaseous exchange and, therefore, tissue repair is
critical to [105_TD$IF]restore pulmonary function. However, the process must be finely tuned to provide sufficient, but not excessive, repair; hence, the ‘Goldilocks’ concept of fine-tuning. At one end of the spectrum, excessive repair can potentially lead to fibrotic lung disease. For instance, IPF can result from an aberrant woundhealing response; and lung cancer can result from hyperproliferation [3]. At the other end of the spectrum, morbidities in COPD and influenza infection have been linked to a lack of lung tissue repair [3]. As we begin to work towards therapeutic lung regeneration, a comprehensive understanding of the intrinsic molecular mechanisms driving alveolar regeneration is critical. A recent article by Xi et al. [4] highlights our rapidly increasing knowledge of the complexity of lung alveolar regeneration. Previous work in mice identified distinct lineage-negative epithelial progenitor cell [106_TD$IF]populations (LNEPs) lacking the expression of mature lineage markers, [107_TD$IF]that expand in the lungs following influenzamediated injury in vivo [5,6]. Some LNEPs expressed the stem and/or basal epithelial cell marker P63, while others did not [5,6]. Influenza or bleomycin-induced injury stimulated a Notch-driven remodeling repair program where LNEPs activated P63 and cytokeratin 5 (KRT5pos[104_TD$IF]) progenitors, leading to dysplastic repair of the alveolar epithelium [5]. By contrast, subsequent depletion of Notch allowed the generation of surfactant protein C (SPC)-positive cells and the differentiation of alveolar epithelial cells, facilitating normal repair [5]. In the latest study by Xi and coworkers, the authors shed light on the mechanisms of alveolar regeneration in murine and human lungs; they identified hypoxia as a critical controller of pulmonary epithelial cell fate decisions [4]. Specifically, by using the mouse model of influenza-mediated lung injury, they demonstrated that expression of hypoxiainducible factor (Hif)-1a could drive Krt5pos progenitor cells to persist in the lung, resulting in aberrant alveolar repair.
By contrast, in LNEP, Hif1a deletion or upregulation of Wnt/ß-catenin activity (a key regenerative pathway) promoted differentiation into normal SPC-positive alveolar type II epithelial cells (ATII) and enhanced repair [4]. This study underlines the fact that not all types of cell repair are equal and demonstrates examples of signals that fine-tune tissue repair, such as Hif1a and Wnt/ ß-catenin signaling. Thus, the balance between ‘good’ (i.e., healing) and ‘bad’ (i.e., fibrotic repair) is a key consideration for the development of regenerative therapies; for this, further knowledge of the signals that can drive normal versus abnormal lung repair is required (Figure 1). The important role of Notch in cell fate decisions also extends to inflammatory cells where it [109_TD$IF]is required for immune signaling [7]. Therefore, it is possible that maintaining Notch signaling might diminish effective lung repair by prolonging inflammation, as well as by regulating progenitor identity, although this remains to be directly tested. The link between enhanced repair in an inflammation-free context is well known from comparisons of embryonic and adult wound repair; in the embryo, wound repair can occur in a highly reduced inflammatory environment, resulting in scar-free healing, unlike adult wound repair, where scarring occurs and repair is less efficient [8]. A question that remains is whether there are differences in the molecular mechanisms of alveolar lung repair when triggered by different types of stimulus. For instance, are there differences in alveolar repair following a sterile stimulus, such as a mechanical injury [ventilator associated lung injury (VALI[10_TD$IF])]; a nonsterile stimulus (influenza infection); or in chronic lung diseases, where injury can be exacerbated by pulmonary infection (e.g., COPD or IPF)? Based on RNA-Seq data comparing normal versus abnormal lungs in humans and mice, the authors suggested that, in humans, the progenitor cell populations
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
1
TRMOME 1273 No. of Pages 3
Figure 1. Maintaining the Balance between ‘Good’ and ‘Bad’ Lung Repair. In humans and mice, the alveolar epithelium comprises long, thin squamous alveolar type I (ATI) cells and cuboidal alveolar type II (AT II) cells. Following tissue injury, progenitor and/or stem cell populations are generated to repair lost or damaged alveoli. Different types of progenitor population can arise, depending on the molecular environment. P63pos, Krt5pos cells are generated in response to hypoxia or persistent high levels of Notch. These precursors give rise to squamous, dysplastic epithelial cells and result in aberrant alveolar repair. By contrast, P63neg, SPCpos progenitor cells are generated in response to Wnt signaling. These cells expand and eventually give rise to both ATI and ATII cells to fully restore normal alveolar epithelium. Maintaining the balance between ‘good’ (normal) alveolar repair and ‘bad’ (dysplastic) repair is essential for restoring lung health. Several factors may tip the balance towards ‘bad’ repair, including the type of injury trigger, aging, lifestyle, genetics, and environment.
triggered by influenza infection, or in ARDS or IPF, may be largely the same: but is this really the case? Xi et al. have demonstrated that different progenitor populations predominate depending on the precise signals received, and that, in turn, these can lead to normal or dysplastic repair [4]. Undoubtedly, further detailed analysis is needed to identify the subpopulations that are prevalent in specific diseases [1_TD$IF]in order to target efficient therapeutic strategies. For instance, we know that, in skin, epidermal wound
2
repair can occur in the absence of an immune response, but different molecular mechanisms are utilized compared with those triggered during inflammationinduced repair; this illustrates that the presence of inflammation can affect tissue repair mechanisms. Moreover, the influence of aging on tissue repair capacity is also an important consideration for lung regenerative medicine; indeed, it is well known that fibrotic lung diseases are more frequent in older people, while classic inflammatory diseases, such as
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
asthma, are more common in children. Consequently, the response to injury may differ over a lifetime. A key strength of the study by Vaughan’s laboratory is that data from both mice and humans are presented. As is clear from their results, this is imperative because, when examining lung responses to injury, key differences, as well as commonalities, in regenerative signatures exist between species [4]. For example, RNA-Seq data show that, in response to lung injury, 102
TRMOME 1273 No. of Pages 3
common genes and pathways (predominantly involved in cell migration and proliferation) are upregulated in [12_TD$IF]both activated LNEP murine cells [13_TD$IF]and hypoxic ATII human cells. However, [14_TD$IF]many additional genes and/or pathways appear to be upregulated in [15_TD$IF]only one of these cell types [4]. This is perhaps unsurprising given the known differences between human and mouse lung morphology. Accordingly, the proportion of different epithelial cell types differs; there are more basal cells in human lung than in mouse lung [2]. Moreover, the immune environment of laboratory-reared mice differs considerably from that of humans [9]. The findings presented also remind us that experimental design, as well as whether data are obtained in vivo or in vitro, are important considerations, potentially leading to varying results. Indeed, two examples of different results are highlighted: first, LNEP cultures immersed in media were reported to be hypoxic, showing a different transcriptomic signature compared with those cultured at the air–liquid interface, illustrating the influence of varying culture conditions [4]. Second, mouse LNEP cultures rapidly lost SPC mRNA expression in vitro but not in vivo, highlighting the potential environmental effects on transcriptomic outcomes [4].
destruction, some patients with ARDS recover lost alveolar tissue, while others do not [10]. The variability in alveolar tissue repair capacity strongly suggests a genetic component to this process. Indeed, a recent study identified a critical role for the noncanonical Wnt/Planar Cell Polarity (PCP) pathway in lung repair [11]. The authors used population data to reveal the presence of a particular single nucleotide polymorphism (SNP) in Vangl2 (a key gene in this pathway), which rendered individuals more susceptible to lung function decline in response to smoking, [16_TD$IF]compared to nonsmokers not receiving [17_TD$IF]this damaging injury [11]. Thus, subtle genetic differences between individuals might affect lung function only when an injuriousinsult is received. This could partially explain why some people [18_TD$IF]do not mount an efficient lung repair response while others [19_TD$IF]do. In the era of genomicmedicine, this isanimportantconsideration that we can begin to address. All in all, a greater understanding of the molecular mechanisms regulating cell fate decisions following pulmonary damage, be it inflammatory, infectious, or due to physical injury, may eventually facilitate our ability to ensure lung tissue repair is maintained in the ‘Goldilocks zone’, mediated by cues that either prevent aberrant repair or promote sufficient ‘normal’ alveolar repair.
Finally, why do some individuals effectively repair damaged lung tissue while others are Acknowledgments incapable of doing so? It is well known that The authors wish to acknowledge funding provided despite extensive alveolar tissue by the Leverhulme trust (C.H.D.) and a Wellcome
Senior Fellowship in basic biomedical sciences (C.M.L[120_TD$IF].). We thank Laura Yates for assistance with Figure 1. 1 Inflammation, Repair and Development Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ
*Correspondence: [email protected] (C.H. Dean). http://dx.doi.org/10.1016/j.molmed.2017.08.009 References 1. Ferkol, T. and Schraufnagel, D. (2014) The global burden of respiratory disease. Ann. Am. Thorac. Soc. 11, 404–406 2. Hogan, B.L. et al. (2014) Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123–138 3. Spella, M. et al. (2017) Shared epithelial pathways to lung repair and disease. Eur. Respir. Rev. 26, 170048 4. Xi, Y. et al. (2017) Local lung hypoxia determines epithelial fate decisions during alveolar regeneration. Nat. Cell Biol. 19, 904–914 5. Vaughan, A.E. et al. (2015) Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 517, 621–625 6. Kumar, P.A. et al. (2011) Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell 147, 525–538 7. Shang, Y. et al. (2016) Role of Notch signaling in regulating innate immunity and inflammation in health and disease. Protein Cell 7, 159–174 8. Eming, S.A. et al. (2014) Wound repair and regeneration: mechanisms, signaling, and translation. Sci. Transl. Med. 6, 265sr6 9. Beura, L.K. et al. (2016) Normalizing the environment recapitulates adult human immune traits in laboratory mice. Nature 532, 512–516 10. Martin, C. et al. (1995) Pulmonary fibrosis correlates with outcome in adult respiratory distress syndrome. A study in mechanically ventilated patients. Chest 107, 196–200 11. Poobalasingam, T. et al. (2017) Heterozygous Vangl2Looptail mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair. Dis. Models Mech. 10, 409–423
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
3