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Paediatric Respiratory Reviews 12 (2011) 216222

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Paediatric Respiratory Reviews

Mini-Symposium: Interstitial lung diseases in childhood

Interstitial lung disease: Physiopathology in the context of lung growth

Nadia Nathan 1,2,3, Guillaume Thouvenin 1,2,3, Brigitte Fauroux 1,2,3, Harriet Corvol 1,2,3, Annick Clement 1,2,3,*
1 2

Inserm, UMR-S U938, Paris, F-75012 France Pierre et Marie Curie-Paris6, Paris, F-75012 France Universite 3 pital Trousseau, Pediatric Pulmonary Department, Paris, F-75012 France AP-HP, Ho



Keywords: Interstitial lung disease Alveolar epithelial cells Endoplasmic reticulum stress Transforming growth factor-b Surfactant

Interstitial lung diseases (ILDs) in children represent a heterogeneous group of respiratory disorders characterized by derangements of the alveolar walls. The key pathologic feature of ILDs is the altered repair of the alveolar surface after injury with a marked disruption in the integrity of the epithelium and, consequently, a dysregulated communication between epithelial and mesenchymal pulmonary components. Concomitant to the loss of cell-cell contact, epithelial cells undergo a process called epithelial to mesenchymal transition and acquire a mesenchymal identity. Among the factors involved in disease progression, transforming growth factor-b has been identied as a master switch in the induction of brosis. This article reviews recent advances in the understanding of the mechanisms involved in the pathogenesis of ILDs, and provides information on their adaptation in the context of lung growth. Crown Copyright 2011 Published by Elsevier Ltd. All rights reserved.

INTRODUCTION Interstitial lung diseases (ILDs) in children represent a heterogeneous group of respiratory disorders characterized by the presence of diffuse inltrates on chest radiographs, and abnormal pulmonary function tests with evidence of restrictive ventilatory defect and/or impaired gas exchange.15 The main pathologic changes are derangements of the alveolar walls. ILD develops after epithelial damage which triggers the accumulation and activation of immuno-inammatory cells, with subsequent migration and proliferation of broblasts and deposition of extra-cellular matrix. From numerous clinical studies, there is compelling evidence that expression and outcome of ILD in children differ from adult ILD, strongly suggesting that additional events related to lung growth and development play a critical role in disease progression.6 This article reviews recent advances in the understanding of the mechanisms involved in the pathogenesis of ILD in children.

ALTERED REPAIR PROCESS OF THE ALVEOLAR EPITHELIUM IN ILD The key pathologic feature of ILD is the altered repair of the alveolar surface with marked disruption in the integrity of the

pital Trousseau, Pediatric Pulmonary Depart* Corresponding author. AP-HP, Ho ment, Inserm, UMR-S U938, UPMC, Paris6, 26 Avenue du Docteur Arnold Netter, 75571 Paris cedex 12, France. E-mail address: (A. Clement).

epithelium.7,8 The alveolar epithelium consists of type I and type II alveolar epithelial cells (AECsI and AECsII). The alveolar surface is mainly covered by AECsI, which are membranous cells usually found overlying the capillaries. The AECsII are large cuboidal cells located in the alveolar corners.9 They have the ability to divide, and are believed to serve as the progenitors of the alveolar epithelium, being capable of both self-renewal and of giving rise to AECsI through a process of transdifferentiation. Consistent with a central role of the alveolar epithelium in disease pathogenesis, AECs in idiopathic pulmonary brosis (IPF) appear morphologically abnormal, with hyperplastic AECsII and reactive elongated cells displaying an intermediate phenotype between AECII and AECI, suggesting an impaired transdifferentiation of AECII to AECI, which may contribute to the abnormal epithelium repair.1012 An important feature of the pathologic process observed in ILD is linked to an altered communication between epithelial and mesenchymal pulmonary components. Prolonged denudation of the basement membrane after injury contributes to altered interactions between AECs and mesenchymal cells, resulting in profound modications of cell functions with imbalanced production of polypeptide mediators including cytokines, growth factors, oxidants, and proteases.1315 The population of broblasts and myobroblasts progressively increases due to stimulation of proliferation by local mitogenic factors and reduction of apoptosis. The resulting aberrant tissue remodeling is characterized by disorganization of extracellular matrix component deposition, including brillar collagen, elastic bers, bronectin and proteoglycans (Figure 1). This is associated with the formation of new blood vessels following the induction of angiogenic molecules,

1526-0542/$ see front matter . Crown Copyright 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.prrv.2011.04.003

N. Nathan et al. / Paediatric Respiratory Reviews 12 (2011) 216222


Figure 1. The repair process of the lung alveolar structure following injury, and the inuence of aging TGF-b = transforming growth factor-b.

which promote endothelial cell migration and neovascularization.16 Histopathological analyses of brotic lungs also have documented the presence of broblastic/myobroblastic foci.16 Myobroblasts may form through conversion of resident broblasts and differentiation of circulating bone marrow-derived progenitors; but in recent years, a new source has emerged with the contribution of AECs.17,18 Indeed, due to their high plasticity, AECs can convert to mesenchymal cells by epithelial-mesenchymal transition (EMT). During this process, epithelial cells lose polarity and epithelial-specic markers. Molecular changes include dissolution of epithelial tight junctions, reorganisation of the actin cytoskeleton, loss of apical-basal polarity, induction of a mesenchymal gene-expression program and migration through basement membrane and tissues. The process of alveolar surface regeneration is complex, involving spreading and migration of neighbouring AECsII followed by proliferation and differentiation.19 This program is governed by intrinsic and external environmental factors, which ultimately converge to regulate cellular signaling pathways.11,12 During development, this program allows the progressive constitution of the complex lung architecture. Of interest are the numerous observations in various pulmonary diseases showing that this complex architecture can reform precisely after extensive damage, supporting the view that the molecular developmental pathways can in part be recapitulated during postnatal life to restore a normal tissue. Therefore, it is strongly suggested that a dysregulation of these pathways can underlie the progression towards brosis.20 Supporting this hypothesis, an abnormal activation of the main tissue homeostasis pathway, Wnt (Wingless

iNTegration site) signaling pathway, has been observed in lung tissues of patients with pulmonary brosis.21,22 Wnt signaling has been shown to have an essential role in development and maintenance of multiple organs including the lung, with a number of target genes being expressed in developing and in adult lung. Wnt signaling, through the activation of the multifunctional protein b-catenin, is implicated in both opposing processes of cell proliferation and differentiation. In brosis, it is suggested that the abnormal activation of the Wnt pathway leads to an imbalance characterized by an increased cell proliferation and a decreased differentiation. Indeed, hyperplastic and proliferating AECsII are present in lung brotic tissues, with accumulation of nuclear bcatenin.23 TGF-B: AN IMPORTANT MEDIATOR OF DISEASE PROGRESSION Transforming-growth factor-b (TGF-b) is a pleiotropic growth factor that is synthesized by many cells, and has been identied as a master switch in the induction of brosis in many tissues.24,25 Among its numerous actions, TGF-b1 can increase the production of interstitial collagens, bronectin, and proteoglycans by myobroblasts and can trigger its own production by myobroblasts, thereby establishing an autocrine cycle of myobroblast differentiation and activation. A number of observations has strongly contributed to establish the key role of TGF-b in the ILD pathological process (Figure 2).26,27 Of importance are the data obtained in various organs indicating that tissue damage following injury was associated with increased TGF-b production before induction of extra-cellular matrix components, and that inhibitors


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Figure 2. The role of TGF-b in the remodeling process of the lung alveolar structure following injury TGF-b = transforming growth factor-b EMT = epithelial-mesenchymal transition

of TGF-b receptor binding could reduce or abolish brosis.28 Overexpression of active TGF-b in rat lung was shown to result in severe interstitial brosis with emergence of cells with a myobroblast phenotype. Recently, it has been reported that exposure of AECs to TGF-b resulted in increased expression of mesenchymal markers including smooth muscle actin, type 1 collagen, N-cadherin, and vimentin, and decreased expression of epithelial markers such as E-cadherin and cytokeratins.29,30 A key function of TGF-b in the context of brosis relies on its ability to induce EMT. The molecular mechanisms that mediate TGF-b induction of EMT are being elucidated. TGF-b initiates signaling in responsive cells by forming an activated heteromeric complex with specic transmembrane TGF-b type I and II serine/ threonine kinase receptors. The type II receptor kinase phosphorylates and activates the type I receptor, which in turn induces the activation of the canonical intracellular Smad signaling pathway. EMT comprises epithelial cell polarity and cell-cell adhesion due to changes in cytoskeletal architecture and cell junctional proteins such as the protein E-cadherin which exerts a central role in epithelial homeostasis. Reduction in E-cadherin levels at the sites of cell-cell attachments has been reported in situations of EMT, and its loss at the cell-cell junctions leads to reduced expression and/or organization of other epithelial markers. Interestingly, the Smad proteins have recently been reported to be required for the ability of TGF-b to induce EMT. These proteins can induce the expression of other transcription factors, including Snail and Zeb1 that are thought to repress the expression of the E-cadherin gene31 Another important function of TGF-b in the context of alveolar epithelium repair is linked to its role as modulator of apoptosis and maintenance of tissue homeostasis.32 In situations of brosis, increased cell loss in the epithelium is observed early in progression, while reduced broblast apoptosis has been associated with the formation of scar lesions.33 In addition, inam-

matory cells can modulate the apoptosis of the other cell types by ineffective removal of apoptotic cells and debris, and cytokine production. In ILD and lung brosis, increased apoptosis with upregulation of the Fas-FasL molecules has been demonstrated in AECsII overlying myobroblasts.12 In a model of bleomycininduced pulmonary brosis, the use of neutralization of FasL was shown to prevent the development of brosis. Of importance is the observation that TGF-b can directly induce AECs apoptosis by enhancing the FasL-Fas interaction. In contrast to AECsII, broblasts/myobroblasts isolated from the lungs of patients with ILD showed decreased apoptosis.34 The increased survival of the cells in brotic disorders supports the view that anti-apoptotic mediators and pathways are activated. Indeed, broblasts from brotic lungs have been reported to be resistant to Fas mediated apoptosis. This may be explained in part by decreased levels of surface Fas and increased levels of soluble Fas. Of interest is the observation that TGF-b could activate the anti-apoptotic pathways in broblasts, which include the focal adhesion kinase pathway and the phosphatidyl-inositol 3-kinase/protein-kinase B pathway.35,36 ROLE OF ENDOPLASMIC RETICULUM STRESS RESPONSE Recent reports strongly suggest that the endoplasmic reticulum (ER) stress may represent an important mechanism of the altered repair process observed in the alveolar epithelium of brotic lung, through its participation in cell death through both apoptosis and autophagic pathways.37,38 The ER is responsible for synthesis of polypeptides and posttranslational modications and folding of peptides to form functional proteins. Misfolded proteins can disrupt normal cellular function by inappropriate interactions. As part of routine housekeeping, the ER has control mechanisms that recognize misfolded proteins and remove them for degradation

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Figure 3. Endoplasmic reticulum (ER) stress response in the context of injury

(Figure 3). They include chaperones such as binding immunoglobulin protein (BiP) that belongs to the heat shock protein 70 family. BiP binds to the hydrophobic region of unfolded proteins and functions as intermediary to facilitate protein folding through conformational changes. If the client protein load is excessive compared with the reserve of ER chaperones, the cell is said to be experiencing an ER stress. If unchecked, ER stress could overwhelm the processing capacity of the ER. In such situations, the unfolded protein response (UPR) is activated to facilitate the folding, processing, export and degradation of the proteins. Three UPR signaling pathways exist in mammalian cells that include the activating transcription factors (ATF) 4 and 6. Through a coordinated process, these pathways expand the protein folding capacity of the cell in order to restore ER homeostasis.39 Unresolved ER stress leads to the accumulation of unfolded proteins and collapse of the secretory pathway, and to abnormal expression of selected genes of the UPR such as ATF6, with consequently increased cell susceptibility to programmed cell death. The alveolar epithelium is likely to be highly susceptible to the ER stress because of its environmental situation accounting for an important level of injury exposure, and of its sustained protein metabolism activity. Indeed, the consensus understanding is that the proportion of protein misfolding is directly linked to the level of molecule complexity, as a result of a stochastic process.40 In epithelial cells, proteins destined for secretion or insertion into the plasma membrane require modications, such as glycosylation and disulde bond formation. Generation of these modications, which are inappropriate for the cytosol, necessitates a specic compartment provided by the ER and its protein maturation machinery to allow synthetic and quality assurance processes to proceed efciently. Epithelial proteins are therefore highly susceptible to misfolding, which can be observed under several pathologic conditions.4143 Intra-cellular aggregations of misfolded toxic proteins are now reported in a growing family of human disorders involving

oxidative stress. From endogenous and exogenous sources, the production of oxidants is maintained at a high level in situations of ILD and brosis through a positive feedback mechanism involving many factors such as TGF-b. Other factors which can contribute to ER stress are viruses, especially in children who are particulary exposed to a viral environment in the rst years of life. Recently, it has been reported that Herpes virus infection, which can induce ER stress, was present in the lungs of patients with lung brosis and can participate to brosis progression.44 The viral protein was found in the AECsII from the patients and colocalized with UPR markers.45 ROLE OF AUTOPHAGY IN TISSUE REMODELING In the context of ILD, ER stress linked to protein dysfunction and misfolding is likely to participate in the altered repair process of the alveolar epithelium through several mechanisms that include an inappropriate autophagy.46,47 Autophagy plays a central role in cellular and tissue homeostasis by mediating the turnover of intracellular organelles and proteins through a lysosome-dependent degradative pathway.48 Autophagy operates by capturing cellular components (proteins, lipids, organelles) in double membrane vesicles that trafc to and fuse with lysosomes where the content of the autophagosomes is degraded. It occurs at basal levels in all cells to degrade long-lived proteins and to regulate organelle turnover. Autophagy is dramatically induced in situations of stress and is crucial in preventing the accumulation of toxic components including damaged proteins. Autophagy is a complex self-degradative process that involves several steps.49 The rst step is the isolation of a membrane, derived from the lipid bilayer, primarily from the ER, leading to the formation of a membrane known as phagophore. Progressively, the phagophore expands through interaction with its binding partners, including the autophagy-related gene (Atg) products and beclin-1, and multimerization. This structure allows the sequestration of


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intra-cellular cargo such as protein aggregates in a doublemembraned autophagosome, its fusion with the lysosome, and ultimately proteolytic degradation of engulfed molecules by lysosomal proteases. Recently, autophagy-decient mouse models and human diseases linked to defects in specic autophagy genes have been reported. As an example, loss of functional Atg16L in mice was associated with aberrant inammatory response of intestinal cells and block of autophagy.50 A growing body of evidence implicates autophagy as a protective response in genetic diseases associated with cytoplasmic aggregation-prone proteins. Excess misfolded proteins that escape this quality-control mechanism begin to aggregate.38 The presence of protein aggregates, in turn, overwhelms and inhibits proteasome activity, potentially disrupting other important proteasome functions. Autophagy can relieve proteasome inhibition by removing aggregates that have escaped proteasome clearance. In parallel, unprocessed protein aggregates are directed toward sequestration in the perinuclear aggregasome. As an example, recent studies indicate that the mutant alpha-1antitrypsin protein forms aggregates that can induce the ER to form autophagosome, leading to activation of autophagy.51 The participation of autophagy in the degradation of aggregated protein allows viewing autophagy as a protective cellular response in genetic diseases associated with aggregated forms of mutant proteins. In lung alveolar epithelium, autophagy is certainly an important regulatory process in AECII function linked to surfactant metabolism. Indeed, surfactant protein (SP)-C has a high tendency to misfold and aggregate into amyloid brils mediated by its polyvaline domain.5255 In the context of childhood and viral exposure, autophagy is crucial in cell homeostasis. Indeed, viruses such as Herpes virus have been associated to ER stress and SP-C accumulation in IPF.42 The C-terminal domain of pro-SP-C is thought to have a chaperone function by preventing the polyvaline fragment from promoting formation of amyloid-like deposits. Consequently, mutations in the SP-C gene may lead to AECII dysfunction and ILD via loss of the chaperone function of the Cterminal domain. Defective autophagy has recently been reported in several pathological conditions associated with tissue damage and inammation.38 In the context of lung diseases, an example is provided by new observations in Hermansky-Pudlak syndrome (HPS) pathology.43,56 HPS is a rare autosomal recessive disorder characterized by several organ dysfunctions including the lung with progressive development of interstitial pneumonia. Current understanding suggests the HPS pulmonary disease results from chronic AECII injury induced by impaired lysosomal trafcking. In lung tissue from HPS patients, an increased expression of pro-SP-C was documented by immuno histochemistry in AECII. In these cells, positive staining for cathepsin D and cleaved caspase-3 was also observed, providing evidence of ER stress and lysosomal pathway dysfunction. In murine models of HPS, abnormal lung structures were reported with development of pulmonary brosis and enlarged AECII with giant lamellar bodies. These features were associated with increased levels of phospholipids as well as SP-B and SP-C in lung homogenates, indicating accumulated storage and altered secretion of these surfactant components. Surfactant abnormalities were associated with activation of lysosomal stress markers such as cathepsin D as well as induction of ER stress molecules including ATF-4.57 The AECII dysfunction observed in HPS lung disease is not due to abnormal surfactant synthesis or uptake, but to alterations in the secretory machinery of lamellar bodies. Such alterations can occur in various situations of stress, and can induce an ER stress through increasing amounts of accumulating surfactant protein and phosphatidylcholine, as well as retrograde stacking of lysosomal surfactant components into the ER.54,55 The consequence is a chronic lysosomal/ER stress and the

occurrence of cell death through both apoptosis and autophagic pathways. ILD IN THE CONTEXT OF LUNG GROWTH AND DEVELOPMENT ILD and lung brotic disorders are less frequently diagnosed in children than in adults.4,6 In addition, paediatric ILD is more responsive to therapeutic strategies than adult ILD.2,58 These differences may be explained by the types of initial injury, which may be different due to changes in the host environment. In addition, modications of the immune function and wound healing process with age are certainly important contributors. From clinical and experimental studies, it is now well established that the innate and adaptative immune response become less effective with increasing age, explaining the concept of immunosenescence. Immunosenescence affects all the cell types involved in the immunomodulation process, including epithelial cells, neutrophils, monocytes and macrophages. With age, epithelial cells display increased susceptibility to oxidative stress and to other forms of stress-induced damage, leading to more sustained chronic inammation and remodeling due to alteration in the proliferative response and to abnormal control of apoptotic pathways (Figure 1). Profound modications in broblast metabolism and function are also observed.59 Skin wound healing in the foetus is characterized by complete regeneration of the skin and the absence of any scar formation. This capacity for scarless repair seems to be inherent to the foetal skin itself and its genetic program. With progressing age, the skin loses the capacity to regenerate the original tissue architecture with the result being scar formation that extends outside the wound bed. In a recent work, Coolen and coworkers compared adult and foetal skin and documented important differences in the composition and architecture of the extra-cellular matrix, including bronectin, elastin and cytokeratins.60 The process of healing involves the coordinated regulation of cell proliferation and migration, and tissue remodeling, predominantly by polypeptide growth factors. The slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory factors. In a study on the role of aging in the development of cardiac brosis in the rabbit, differences in the cascade of events leading to myocardial remodeling were observed, with mainly the presence of more myobroblasts synthesizing collagen in the old animals.61 A study of growth factors involved in skin wound healing in young and aged mice also showed age-dependent changes.62 Expression of all the broblast growth factors was diminished in aged mice, even in healthy skin. In addition, the post-wound regulation of expression of these factors and of TGF-b was less pronounced and slower than in the young mice. These ndings are in agreement with data observed in muscle that indicated signicant alterations in the TGF-b production with age.63 This is linked to the observation that injury in adult tissues does in certain circumstances stimulate tissue regeneration, depending on the presence of small subset of primitive stem cells. Other potential mechanisms of normal lung reconstruction following injury during childhood include stem cells. Stem cells are the self-renewing, primitive, undifferentiated, multipotent source of multiple cell lineages.64 While such cells are critical during development, poor residual pools of adult stem cells are hypothesized to be the source of the frequently limited tissue regeneration and repair that occurs in adults.65 In addition to stem cell niches located within the lung, a growing body of evidence in recent years supports the concept of bone marrow-derived stem cell (BMSC) plasticity with the capacity of these cells to differentiate into epithelial cells of multiple organs including the lung. Indeed, several groups have reported that BMSC can

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engraft into the lung and differentiate into lung epithelial cells.66 The underlying mechanisms for recruitment and phenotypic differentiation remain to be established, and may depend on the type of damage, the route of delivery, and the population of BMSC used. The importance of the stem cell process in lung repair is likely to be inuenced by age. Indeed, unlike embryonic stem cells, adult stem cells are not immortal, and show decreasing telomere length with increasing age.67,68 The naturally limited replacement capacity of such endogenous stem cell pools may occur via exhaustion of the stem cell pool or arise as a consequence of inherited or acquired mutations that alter proper stem cell function.32,69,70 The limited life span of cells may result from replicative senescence in response to various stresses including DNA damage, oxidants, and telomere erosion.71,72 Such mechanisms are likely to interact also in infant lungs, but to date, because of the difculty to biopsy lungs compared to skin, no study is available. However, all these forms of injury have been documented in the brotic lung from adult patients. Recently, Adler and coworkers studied telomere length in leukocytes from adult patients with lung brosis and from healthy age-matched controls.73 Shorter telomeres were found in individuals with lung disease. In addition, telomerase mutations were reported to be associated with familial IPF.74 CONCLUSION Much progress has been made recently in the identication of the pathological processes associated with ILD development and progression. This should help dening new therapeutic strategies including those capable of interfering with the pathways that lead to myobroblast expansion and AEC apoptosis. Such therapeutic interventions may be particularly promising in children, who usually experience a less devastating disease with a potential of signicant regeneration of the alveolar structure. Acknowledgments Pierre et Marie This work was supported by Inserm, Universite pitaux de Paris, Curie-Paris 6, Paris, Assistance Publique-Ho ` re de la Sante (Centre de Re fe rence des Maladies Ministe Respiratoires Rares), France. References
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