EDITORIALS ARDS lungs. Bellani and colleagues (4) used positron emission tomography and CT scans in 13 patients with ARDS to colocalize the site of increased CT density and areas of increased metabolic activity due to neutrophils, which are implicated in the inflammation of VILI. They found that throughout the ventilated lung, there was a significant positive correlation between the end-inspiratory plateau pressure, a known risk factor for stress-induced VILI, and the degree of metabolic activity. They also found no difference in the metabolic activity in regions that were normally aerated and those that were undergoing cyclic recruitment and derecruitment. They concluded that these findings did not support the importance of “atelectrauma” caused by cyclic recruitment and derecruitment of alveoli (5). Implicit in their observations is the absence of any indication that there is greater inflammatory activity at the borders of aerated and nonaerated lung. Although resolution of positron emission tomography is too limited to test the stress amplification hypothesis at present, this line of evidence seems not to support stress-induced lung inflammation bordering regions high CT density. We await further studies in this area. The study by Cressoni and colleagues makes a significant contribution to the literature by quantifying inhomogeneity in the lungs of patients with ARDS and by associating the degree of this inhomogeneity with disease severity and outcomes. The debate over the interpretation of CT density in ARDS, whether this represents areas of atelectasis subject to stress amplification or not, will need to be resolved by further studies. n

Author disclosures are available with the text of this article at www.atsjournals.org. Stephen H. Loring, M.D. Daniel Talmor, M.D., M.P.H. Department of Anesthesia, Critical Care and Pain Medicine Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts

References 1. Cressoni M, Cadringher P, Chiurazzi C, Amini M, Gallazzi E, Marino A, Brioni M, Carlesso E, Chiumello D, Quintel M, et al. Lung inhomogeneity in patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 2014;189:149–158. 2. Mead J, Takishima T, Leith D. Stress distribution in lungs: a model of pulmonary elasticity. J Appl Physiol 1970;28:596–608. 3. Hubmayr RD. Perspective on lung injury and recruitment: a skeptical look at the opening and collapse story. Am J Respir Crit Care Med 2002;165:1647–1653. 4. Bellani G, Messa C, Guerra L, Spagnolli E, Foti G, Patroniti N, Fumagalli R, Musch G, Fazio F, Pesenti A. Lungs of patients with acute respiratory distress syndrome show diffuse inflammation in normally aerated regions: a [18F]-fluoro-2-deoxy-D-glucose PET/CT study. Crit Care Med 2009;37:2216–2222. 5. Slutsky AS, Ranieri VM. Ventilator-induced lung injury. N Engl J Med 2013;369:2126–2136.

Copyright © 2014 by the American Thoracic Society

Modeling Pulmonary Alveolar Proteinosis with Induced Pluripotent Stem Cells In 2006, Takahashi and Yamanaka published their discovery that the transfer of just four transcription factors (Oct4, Sox2, Klf4, and cMyc) into mouse fibroblasts could reset the entire epigenetic landscape of somatic cells, reprogramming them into an embryonic-like pluripotent state virtually identical to embryonic stem (ES) cells (1). In the short 7 years since this discovery, there has been an exponential growth in the literature on induced pluripotent stem (iPS) cells (reviewed in Reference 2). iPS cells have been derived from a variety of human adult cells (3, 4), including dermal fibroblasts, keratinocytes, and even peripheral blood cells. Yamanaka’s Nobel Prize–winning discovery was a seminal event, because it made possible the derivation of an inexhaustible supply of patient-specific stem cells by reprogramming somatic cells taken from individuals suffering from any disease, including lung diseases (5). There is a shortage of model systems for understanding the underlying mechanisms of lung disease: smallanimal models often fail to replicate the human disease phenotypes, and large-animal models, though more similar to humans, are expensive and not widely available. Patient-specific iPS cells provide unprecedented opportunities to replay the emergence of human disease in vitro, provided that the appropriate cell types and cell–cell interactions can be generated. In 2010, the first 100 iPS cell lines were generated from individuals with genetic lung diseases, including those suffering from the two most common inherited 124

monogenic lung diseases, cystic fibrosis and a1-antitrypsin deficiency–related emphysema (6). Shortly thereafter, patientspecific and disease-specific lines were used to model the intracellular protein misfolding of mutant a1-antitrypsin protein (7, 8) or the aberrant intracellular trafficking of mutant cystic fibrosis transmembrane conductance regulator (9) in differentiated epithelial lineages derived from patient-specific iPS cells. Generating lung lineages from patient-specific iPS cells might also predict the personto-person differences in disease severity and drug responses observed in patients, raising the exciting possibility of using these cells to develop personalized therapeutic approaches in the years ahead. Two publications in this issue of the Journal by Suzuki and colleagues (pp. 183–193) and Lachmann and colleagues (pp. 167–182) represent an important milestone in the application of iPS cells to advance lung disease research (10, 11). The authors convincingly demonstrate the capacity of patient-derived iPS cells to model a rare pediatric lung disease in vitro that is otherwise difficult to study in vivo. The laboratory of Trapnell and colleagues has a long history of publications (12–16) on the basic biology of pulmonary alveolar proteinosis (PAP). The more common adult idiopathic form is associated with the presence of anti–granulocyte–macrophage colony–stimulating factor (GMCSF) autoantibodies, whereas the rare form studied in the current articles, PAP of childhood, results from homozygous inherited

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EDITORIALS mutations in genes encoding components of the receptor for GMCSF, such as CSF2RA or CSF2RB (14). Mutations in these genes are known to cause functional defects in the GM-CSF signaling pathway resulting in abnormal alveolar macrophage maturation and defective macrophage function, including impaired clearance of lung surfactant and resultant buildup of surfactant-laden proteinaceous material in lung alveoli (i.e., PAP). It is thought that defective function of alveolar macrophages underlies both the genetic and the idiopathic form of PAP, but in neither case is the mechanism of action entirely clear (17). Mechanistic studies using patient-derived macrophages are difficult, as these cells have limited proliferative potential in vitro, are notoriously resistant to conventional transfection and gene transfer techniques, and quickly change their phenotype in cell cultures. Generation of iPS cells from patients with the genetic form of PAP provides an inexhaustible source of patient-specific cells that can be genetically modified and can be differentiated into different cell types, including macrophages, for mechanistic studies on how genetic defects in GM-CSF signaling can lead to disease. Both groups were able to take iPS cells from patients with PAP and differentiate them into macrophages in vitro, using protocols that did not depend on GM-CSF. The PAP-derived macrophages showed predicted defects in GM-CSF signaling and in functional properties such as latex bead uptake. Intriguingly, Suzuki and colleagues went further and incubated the cells with surfactant material from patient lungs and showed that the mutant macrophages ingested the material and became morphologically similar to the foamy macrophages seen in patient lungs. Mutant cells were slow to clear the ingested material, suggesting that the defect in surfactant clearance in PAP is likely cellautonomous to the macrophage. Both groups then used lentiviruses expressing CSF2RA to rescue the genetic defect in the mutant iPS cells and showed that several properties of the macrophages, including surfactant clearance, were recovered. This genetic repair method relied on a “conventional” approach of viral vector–based gene transfer with random integration in the genome. Although it is appropriate as a proof of principle, this kind of approach is not ideal for future therapeutic intervention. Exciting new genome-editing technologies are now available that allow efficient correction of the genetic lesion in situ in the endogenous locus responsible for disease (8, 18, 19). Given that macrophages can be successfully engrafted in lung alveoli in vivo in rodent studies (20), in the years ahead it may be possible to treat patients with inherited forms of PAP by engrafting autologous genetically repaired macrophages into their lungs. This could provide the first clinical application of iPS cell technologies for lung disease. Engraftment of epithelial and endothelial cell lineages in the lung, in contrast, has not been convincingly or robustly demonstrated yet, a hurdle that is likely to continue to challenge researchers focused on iPS cell–derived reconstitution approaches for other monogenic and complex lung diseases in the years ahead. What other lessons for lung researchers interested in iPS cells can be learned from the emerging publications in this field? An unexpected lesson learned from the current work is the remarkably robust capacity to model postnatal disease in vitro even when the cell types derived in culture are not fully mature or organ specific. A perplexing issue in PAP has been why the overt clinical disease phenotype is lung localized, characterized by dysfunction of alveolar macrophages, rather than multisystemic disease affecting other organs that also have resident macrophage populations. The Editorials

macrophages generated by Suzuki and colleagues and Lachmann and colleagues were able to recapitulate features of PAP disease even though these macrophages were presumably not lung specific and had not been exposed to the complex lung microenvironment or to the multicellular alveolar niches that are assumed to contribute to development of the unique alveolar macrophage phenotype. In the future, it should be possible to gain further insights into the mechanism of disease by combining macrophages with matching patient-derived lung epithelial cells, endothelial cells, and other components of the alveolar niche, all derived from the same iPS cell source. There are still several challenges to the use of iPS cells in modeling lung disease that will need to be addressed in the years ahead. Although protocols for deriving iPS cells are becoming more and more robust, there is still variation between lines that needs to be accounted for in any study. Genetically correcting mutations in iPS lines and thus generating matched control lines, as performed here, is the ideal control experiment. However, this will not be possible with more complex disease models, and careful assessment of appropriate controls will be needed. In addition, the ongoing challenge with all iPS studies is developing the best protocols for differentiation of the appropriate cell types to model disease or, in the long run, for replacement therapy. In the lung field, there is a need for improved differentiation protocols for all lung cell types, in vitro reconstruction of the lung gas exchange system using bioengineering, and extension of studies of monogenic diseases to the most common diseases affecting pulmonary patients. Emphysema, pulmonary fibrosis, pulmonary vascular disease, and asthma, for example, continue to present an enormous healthcare burden. Because these diseases are seldom monogenic and result from complex multicellular and environmental interactions, it remains unclear how and if these more common lung diseases can be modeled in vitro using iPS cells. This high hurdle remains a key challenge facing this nascent field, and exciting research in a number of labs is increasingly dedicated to solving this challenge. n Author disclosures are available with the text of this article at www.atsjournals.org. Darrell N. Kotton, M.D. Center for Regenerative Medicine of Boston University and Boston Medical Center Boston University School of Medicine Boston, Massachusetts and Department of Medicine and The Pulmonary Center Boston University School of Medicine Boston, Massachusetts Janet Rossant, Ph.D. Program in Developmental and Stem Cell Biology Hospital for Sick Children Toronto, Ontario, Canada and Department of Molecular Genetics University of Toronto Toronto, Ontario, Canada

References 1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676.

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EDITORIALS 2. Stadtfeld M, Hochedlinger K. Induced pluripotency: history, mechanisms, and applications. Genes Dev 2010;24:2239–2263. 3. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131: 861–872. 4. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007;318:1917–1920. 5. Kotton DN. Next-generation regeneration: the hope and hype of lung stem cell research. Am J Respir Crit Care Med 2012;185: 1255–1260. 6. Somers A, Jean JC, Sommer CA, Omari A, Ford CC, Mills JA, Ying L, Sommer AG, Jean JM, Smith BW, et al. Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 2010;28:1728–1740. 7. Rashid ST, Corbineau S, Hannan N, Marciniak SJ, Miranda E, Alexander G, Huang-Doran I, Griffin J, Ahrlund-Richter L, Skepper J, et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest 2010;120: 3127–3136. 8. Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordoñez ´ A, Hannan NR, Rouhani FJ, et al. Targeted gene correction of a1-antitrypsin deficiency in induced pluripotent stem cells. Nature 2011;478:391–394. 9. Wong AP, Bear CE, Chin S, Pasceri P, Thompson TO, Huan LJ, Ratjen F, Ellis J, Rossant J. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol 2012;30:876–882. 10. Suzuki T, Mayhew C, Sallese A, Chalk C, Carey BC, Malik P, Wood RE, Trapnell BC. Use of induced pluripotent stem cells to recapitulate pulmonary alveolar proteinosis pathogenesis. Am J Respir Crit Care Med 2014;189:183–193.

11. Lachmann N, Happle C, Ackermann M, Luttge D, Wetzke M, Merkert S, Hetzel M, Kensah G, Jara-Avaca M, Mucci A, et al. Gene correction of human induced pluripotent stem cells repairs the cellular phenotype in pulmonary alveolar proteinosis. Am J Respir Crit Care Med 2014;189:167–182. 12. Trapnell BC, Whitsett JA. Gm-CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense. Annu Rev Physiol 2002;64:775–802. 13. Shibata Y, Berclaz PY, Chroneos ZC, Yoshida M, Whitsett JA, Trapnell BC. GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1. Immunity 2001;15: 557–567. 14. Suzuki T, Sakagami T, Rubin BK, Nogee LM, Wood RE, Zimmerman SL, Smolarek T, Dishop MK, Wert SE, Whitsett JA, et al. Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA. J Exp Med 2008;205:2703–2710. 15. Sakagami T, Uchida K, Suzuki T, Carey BC, Wood RE, Wert SE, Whitsett JA, Trapnell BC, Luisetti M. Human GM-CSF autoantibodies and reproduction of pulmonary alveolar proteinosis. N Engl J Med 2009;361:2679–2681. 16. Trapnell BC, Whitsett JA, Nakata K. Pulmonary alveolar proteinosis. N Engl J Med 2003;349:2527–2539. 17. Greenhill SR, Kotton DN. Pulmonary alveolar proteinosis: a bench-tobedside story of granulocyte-macrophage colony-stimulating factor dysfunction. Chest 2009;136:571–577. 18. Hockemeyer D, Jaenisch R. Gene targeting in human pluripotent cells. Cold Spring Harb Symp Quant Biol 2010;75:201–209. 19. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8:2281–2308. 20. Careau E, Bissonnette EY. Adoptive transfer of alveolar macrophages abrogates bronchial hyperresponsiveness. Am J Respir Cell Mol Biol 2004;31:22–27. Copyright © 2014 by the American Thoracic Society

Prior Exposure to Bacteria Attenuates Viral Disease of the Respiratory Tract: A Role for IL-17 and Innate Immune Memory? It has been recognized for some time that the incidence of asthma and autoimmune diseases is lower in rural areas of developing countries with higher incidence of infections, especially with helminth parasites (1). This “hygiene hypothesis” has been explained by infectioninduced regulatory T cells suppressing pathogenic T cells that mediate autoimmunity and allergy/asthma (1). Furthermore, there is growing evidence that the microbiota of the gut or respiratory tract can modulate innate and adaptive immune responses in the mucosa, and may also shape systemic immunity (2, 3). There is also evidence from experiments in mice that infection with one pathogen can suppress protective immune responses against another (4). Studies in humans have shown that infection with respiratory viruses, such as influenza and respiratory syncytial virus (RSV), can increase susceptibility to bacterial pathogens (5). A report from Schnoeller and colleagues (pp. 194–202) in this issue of the Journal provides a new twist to this story by showing that respiratory infection of neonatal mice with an attenuated Bordetella pertussis can protect against RSV-induced disease in adult life (6). The protective effect of the bacteria was associated with enhanced IL-10 and K.H.G.M. is supported by grants from Science Foundation Ireland and the Irish Health Research Board.

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IL-17 production and enhanced neutrophil and macrophage recruitments to the lungs and interestingly was reversed after neutralization of IL-17. IL-17 is a proinflammatory cytokine produced by a subtype of CD4 T cells called Th17 cells, but also by cells of the innate immune system, including gd T cells, natural killer T cells, and innate lymphoid cells (7, 8). IL-17 and Th17 cells play critical roles in many T-cell–mediated autoimmune diseases and have recently been implicated in allergic inflammation associated with asthma, and are now a major drug target for many immune-mediated diseases (9). Antibodies that block IL-17 or cytokines that promote induction of Th17 cells are highly effective against psoriasis and are showing promise in clinical trials with other autoimmune diseases (9). However, blocking IL-17 in patients with Crohn’s disease has been associated with enhanced inflammation and more frequent fungal infections (10). An increased incidence of respiratory tract infection has also been reported after blocking of the Th1/Th17 pathways (11). This is consistent with the role of IL-17–producing Th17 cells and gd T cells in protective immunity to fungal and extracellular bacterial infection, where they promote recruitment and activation of neutrophils (7). Th17 cells are also induced during infection with intracellular bacteria and viruses,

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 2 | January 15 2014