New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis

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Interstitial Lung Disease 1 New cellular and molecular mechanisms of lung injury and fibrosis in idiopathic pulmonary fibrosis Isis E Fernandez, Oliver Eickelberg Lancet 2012; 380: 680–88 This is the first in a Series of two papers about interstitial lung disease Comprehensive Pneumology Centre, University Hospital of the Ludwig-Maximilians University Munich, Munich, Germany (I E Fernandez MD, Prof O Eickelberg MD); and Helmholtz Centre Munich, German Centre for Lung Research, Munich, Germany (I E Fernandez, Prof O Eickelberg) Correspondence to: Prof Oliver Eickelberg, Comprehensive Pneumology Centre and Helmholtz Munich Centre, Munich D-81377, Germany oliver.eickelberg@ helmholtz-muenchen.de

Idiopathic pulmonary fibrosis is a serious and progressive chronic lung disease that is characterised by altered cellular composition and homoeostasis in the peripheral lung, leading to excessive accumulation of extracellular matrix and, ultimately, loss of lung function. It is the interstitial pneumonia with the worst prognosis—mortality 3–5 years after diagnosis is 50%. During the past decade, researchers have described several novel cellular and molecular mechanisms and signalling pathways implicated in the pathogenesis of idiopathic pulmonary fibrosis, resulting in the identification of new therapeutic targets. These advances will hopefully result in increased survival rates and improved quality of life for patients with this disorder in future.

Introduction Idiopathic pulmonary fibrosis is the interstitial pneumonia with the worst prognosis1—mortality 3–5 years after diagnosis is 50%.2 Although genetic determinants, environmental exposures, and common insults, such as smoking,3 pollutants, gastro-oesophageal reflux,4,5 occupational exposures,6 viral infections,7 and ageing8 have been identified as risk factors for this disorder, its origin and onset are not fully understood. Onset is thought to involve perpetuated microinjuries to the alveolar epithelium, leading to dysregulation of cellular homoeostasis in the alveolar epithelial– mesenchymal unit, reactivation of developmental signalling pathways (eg, TGFβ [transforming growth factor β],9 Wnt,10 SHH [sonic hedgehog],11 Notch12), induction of cell dysfunction and death, formation of scar tissue, and, consequently, distortion of lung homoeostasis and lung structures. Whereas much attention has focused on the general pathogenetic principles in idiopathic pulmonary fibrosis,13–15 we discuss novel concepts of distorted cellular homoeostasis and plasticity. We focus on newly described signalling pathways that are related to initiation, mediation, or perpetuation of lung fibrosis, drawing attention to those developments with potential to translate into clinical settings.

Pathognomonic hallmarks Diagnostic criteria for idiopathic pulmonary fibrosis have changed several times during the past decade, and the definition of this disorder has been refined.2,16 Although an open lung biopsy sample with a usual interstitial pneumonia pattern is the most convincing evidence of the altered structures in idiopathic pulmonary fibrosis, for many patients diagnosis of this disorder can nowadays be accurately made by clinicians and radiologists during multidisciplinary discussions. These newly defined clinical and radiological diagnostic criteria partly obviate the need to obtain a surgical lung biopsy sample, as stated in the 2011 American 680

Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin American Thoracic Association consensus statement on idiopathic pulmonary fibrosis.2 Lungs from patients with idiopathic pulmonary fibrosis are characterised by heterogeneous distribution of normal or mildly affected regions, alternating with regions of alveolar epithelial cell injury and hyperplasia, basal membrane disruption and capillary leakage, fibrin formation, subsequent aberrant wound healing, and interstitial fibrosis. Fibrotic areas contain septal thickening, honeycombing, and fibroblastic foci, predominantly in paraseptal or subpleural areas17 (figure 1). Honeycombing refers to cyst-like, dilated spaces surrounded by fibrotic tissue—changes that can be detected with high-resolution CT analysis, predominantly in lower lobes.18 Fibroblastic foci are regions of highly proliferative myofibroblast accumulations with positive staining for α smooth muscle actin (αSMA, encoded by ACTA2), which are located immediately adjacent to regions of alveolar epithelial cells that are hyperplastic or apoptotic, or both. These histological characteristics have led to the notion that epithelial microinjuries (of an as yet unknown cause) trigger abnormal epithelial–mesenchymal interactions and pathogenesis of idiopathic pulmonary fibrosis. Although no animal model has all of the histological characteristics of idiopathic pulmonary fibrosis, at least

Search strategy and selection criteria We searched PubMed and ClinicalTrials.gov with the terms “idiopathic pulmonary fibrosis”, “lung fibrosis”, “epithelial injury fibrosis”, “fibroblast fibrosis”, “pericytes fibrosis”, and “mesothelium fibrosis”. We mainly selected work published after January, 2007, although older articles that we deemed very relevant to this topic were included. The search was restricted to reports published in English and the last search was done in June, 2012.

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Figure 1: A healthy lung and a lung with idiopathic pulmonary fibrosis Whereas the healthy lung shows a normal macroscopic and microscopic lung architecture, the lung with idiopathic pulmonary fibrosis shows a heterogeneous pattern, with normal and fibrotic regions alongside areas of honeycombing with subpleural thickening, mostly in the lower lobes. (A) Magnification of the alveolar structure, with conserved alveolar walls and an intact bronchial and alveolar epithelial cell layer. (B) Injured epithelium with hyperplastic and apoptotic changes and epithelial-mesenchymal transition, contributing to subepithelial accumulation of activated myofibroblasts. Pleural thickening entails increased proliferation and migration of mesothelial cells, and pericytes from the surrounding of vessels, contributing to fibroblast foci.

two frequently used mouse models mimic many of the injurious and fibrogenic pathways that occur in this disorder. Bleomycin-induced fibrosis is the best-characterised and most prevalent model so far,19 in which reproducible lung fibrosis develops 10–14 days after exposure to bleomycin. However, this interstitial fibrosis almost fully resolves about 28 days after exposure. In another model, intratracheal application of fluorescein isothiocyanate induces non-reversible fibrosis20 after about 21 days, extending up to 6 months. www.thelancet.com Vol 380 August 18, 2012

Early injury response Some researchers have suggested that a series of repetitive microinjuries to the alveolar epithelium drives a pathogenetic cascade that initiates the histological changes that occur in idiopathic pulmonary fibrosis. Although not proven, the scientific literature largely supports the notion that these microinjuries change the homoeostatic microenvironment of the alveolar epithelial–mesenchymal unit and lead to its initial damage and subsequent aseptic inflammation. Under normal circumstances, such initial 681

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injuries will be repaired and the tissue will heal to a healthy physiological state (restitutio ad integrum), but in idiopathic pulmonary fibrosis healing does not occur. Continuous denudation and disruption of the alveolar epithelial cell layer promotes destruction of the basal membrane, subsequent activation of an intra-alveolar coagulation cascade, imbalance of proteases (matrix metalloproteinases) and antiproteases (tissue inhibitors of metalloproteinases), and activation of myofibroblasts. These changes ultimately result in the increased deposition of extracellular matrix that is characteristic of idiopathic pulmonary fibrosis. During the initial injury phase, activated alveolar epithelial cells and recruited inflammatory cells (eg, macrophages, neutrophils) release potent fibrogenic growth factors (eg, TGFβ, tumour necrosis factor α, PDFG [platelet-derived growth factor], or Wnt) that perpetuate the cycle of injury, failed repair, and fibrosis. These growth factors, particularly TGFβ, are involved in injury and apoptosis of alveolar epithelial cells, and in activation, invasion, or apoptosis resistance of fibroblasts and myofibroblasts. Most importantly, fibroblast foci, which form three-dimensional networks in lungs with usual interstitial pneumonia,21 predominantly occur in subepithelial regions of apoptotic or hyperplastic alveolar epithelial cells, suggesting that an initial injury to these cells promotes subsequent remodelling of the alveolar epithelial–mesenchymal unit. In the past decade substantial progress has been made in identification of potential cellular sources of the increased number of myofibroblasts that occur specifically in fibroblast foci in idiopathic pulmonary fibrosis. The origin of activated myofibroblasts, in particular, is at the centre of a vibrant scientific debate, because few other chronic lung diseases have fibroblast foci.

Cellular plasticity in pathogenesis The lung contains more than 40 different cell types, yet most of the increased extracellular matrix that is deposited in idiopathic pulmonary fibrosis is ascribed to activated myofibroblasts in fibroblast foci. These lesions do not arise in healthy lungs, yet are frequently identified in idiopathic pulmonary fibrosis biopsy samples and their number correlates with survival.22,23 Potential sources for activated myofibroblasts include alveolar epithelial cells or mesothelial cells (via epithelial-mesenchymal or mesothelialmesenchymal transition); local mesenchymal cells, such as fibroblasts or pericytes (via proliferation and invasion); or circulating progenitor cells (such as fibrocytes, via recruitment, invasion, and activation, figure 2).

Alveolar epithelial cells Any epithelial cell injury, depending on its severity or frequency, can induce cellular activation, dysfunction, necrosis, or apoptosis.24 These changes induce a local inflammatory response, including profibrotic cytokine release from resident or non-resident cells (paracrine 682

action) or hyperplastic changes with de-novo expression of mesenchymal markers in alveolar epithelial cells themselves (autocrine action). The direction of this response is largely dictated by the cell’s microenvironment, in particular the composition of extracellular matrix immediately surrounding alveolar epithelial cells. Several factors, secreted by alveolar epithelial cells, immune cells, or adjacent fibroblasts, induce activation and hyperplasia of alveolar epithelial cells. TGFβ is the prototypic growth factor for induction of epithelial-mesenchymal transition-like changes in alveolar epithelial cells. When stimulated with TGFβ in vitro, human,25,26 mouse,27,28 and rat29 alveolar epithelial cells showed mesenchymal gene expression patterns with the involvement of transcription factors including SNAI27 and TWIST.28 In-vivo cell-fate mapping strategies provide further evidence for epithelial-mesenchymal transition in fibrotic mouse lungs.25,30 Studies of mice with β-galactosidelabelled SFTPC-positive epithelial cells showed that, 2 weeks after bleomycin treatment, a third of S100A4 positively sorted fibroblasts were derived from these labelled epithelial cells.30 Furthermore, a recently described population of alveolar epithelial cells that do not express SFTPC but that do express ITGA6 and ITGB4 (8–10% of all alveolar epithelial cells) and that have high progenitor potential have been shown to repopulate injured alveolar epithelium after bleomycin treatment.31 However, Rock and colleagues32 did not identify evidence for epithelial cells (SFTPC-labeled type II alveolar epithelial cells) as a source of myofibroblasts in bleomycin-induced fibrosis when using novel fate mapping reporters. By contrast, they recorded evidence for the transition of pericytes and other mesenchymal cell populations to myofibroblasts in fibrosis (although other type II AEC populations, such as ITGA6 and ITGB4, were not assessed). Additional studies are needed to determine distinct subpopulations of alveolar epithelial cells with different progenitor and plasticity potential, not only in mice but also in tissues from patients with idiopathic pulmonary fibrosis. Among the recently identified novel regulators, WISP1, a target gene from the Wnt signalling pathway that is upregulated in patients with idiopathic pulmonary fibrosis and bleomycin-treated mice, decreases expression of TJP1, CDH1 (cadherin 1), and OCLN (occludin) in alveolar epithelial cells, while increasing expression of ACTA2, S100A4, and VIM (vimentin).33 Similarly, sphingosine-1phosphate, a bioactive lipid increased in serum, bronchoalveolar lavage, and lungs from patients with idiopathic pulmonary fibrosis, activates alveolar epithelial cells by increasing TGFβ secretion and induction of ACTA2, COL1A1 (collagen), and VIM expression.34 Importantly, although an overwhelming amount of experimental data suggests that complete epithelialmesenchymal transition occurs in vitro in alveolar epithelial cells, less evidence suggests that this process occurs in vivo. In vivo, alveolar epithelial cells with mesenchymal gene expression patterns are most probably www.thelancet.com Vol 380 August 18, 2012

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partly activated and hyperplastic epithelial cells, which contribute to and perpetuate fibrosis by increased release of profibrotic and injurious mediators in the absence of acquisition of a full mesenchymal phenotype.

Myofibroblasts Activated fibroblasts and myofibroblasts are regarded as the key cell types that bring about increased deposition of extracellular matrix in lungs of patients with idiopathic pulmonary fibrosis. Although this theory has not been proven experimentally, several investigators have identified mediators that cause fibroblast activation and increased synthesis of extracellular matrix. Fibroblast invasion into three-dimensional networks has attracted much attention as an important cellular effector function of fibroblasts, triggering pathogenesis of idiopathic pulmonary fibrosis. Hyaluronan is a non-sulphated glycosaminoglycan that is mainly produced by mesenchymal cells via the activity of hyaluronan synthases, three isoforms of which have been described (HAS1–3). Increased hyaluronan synthesis contributes to tumour invasiveness and metastasis,35 and promotes the inflammatory response after bleomycin treatment.36 Li and colleagues37 showed that increased synthesis of hyaluronan by myofibroblasts (via transgenic overexpression of HAS2) produces a more aggressive and invasive myofibroblast phenotype, thus promoting the fibrotic response after bleomycin treatment, leading to increased mortality. Conversely, conditional deletion of HAS2 or blocking of its biological effects with CD44blocking antibodies counteracted the invasive phenotype and reduced fibrosis. Importantly, fibroblasts isolated from patients with idiopathic pulmonary fibrosis showed increased migration and invasion compared with controls, which coincided with increased HAS expression in these cells.37 Thus, increased synthesis and deposition of glycosaminoglycan, which has also been identified in clinical samples from patients with idiopathic pulmonary fibrosis,38 might be an important regulatory mechanism for fibroblast activity and invasion.

Pericytes and mesothelial cells Although alveolar epithelial cells and fibroblasts have been the main candidates in the search for novel mechanisms and targets for idiopathic pulmonary fibrosis in the past two decades, several potential additional cellular sources for myofibroblasts have emerged, including mesothelial cells and pericytes. Lung pericytes are mesenchymalderived cells39 that are localised within basal membranes or perivascular linings40 and are involved in wound healing41 and collagen production.42 Studies of experimental kidney injury have identified a role for pericytes as collagenproducing cells during the development of fibrosis.43,44 Additionally, cell-fate mapping studies have shown that pericytes are one source of activated myofibroblasts in a pathway mediated by TGFβ or SHH.45,46 In the lung, such studies have identified pericytes as the predominant www.thelancet.com Vol 380 August 18, 2012

source of activated myofibroblasts in bleomycin-induced fibrosis.32 Investigations of spinal-cord injury also provided evidence for pericytes as direct effector cells in fibrosis.47 Most of these investigations relate to a study in which Cool and colleagues21 used reconstruction imaging to show that fibroblast foci form a highly complex, threedimensional, interconnected reticulum that extends from the pleural surface into the lung parenchyma. The pleura is largely formed by mesothelium, which originates from the embryonic mesoderm and consists of flattened, squamous-like epithelial cells that are surrounded by dense connective tissue. In mice, transgenic studies using the WT1 (a mesothelial-exclusive gene) promoter with three different reporters showed that mesothelial cells are able to move into the lung parenchyma and acquire ACTA2 and PDGFRB expression, further migrating to the walls of pulmonary blood vessels.39 Pleural mesothelial cells have also been associated with fibrotic disorders in the peritoneum,48 where PDGF-induced or TGFβ-induced mesothelial-mesenchymal transition occurs, contributing to the fibroblast pool.49 In-vitro stimulation of pleural mesothelial cells with TGFβ induces epithelial-tomesenchymal transition-like changes.50 Pleural mesothelial cells also seem to migrate into the lung parenchyma in people, because lung sections from patients with idiopathic pulmonary fibrosis show several mesothelial cells in the thickened pleura and fibrotic areas.51 In mice treated with TGFβ, pleural mesothelial cells labelled with green fluorescent protein were also identified within lung parenchyma coexpressing ACTA2.51 Taken together, these data strongly suggest a novel role for pericytes and pleural mesothelial cells as possible contributors to pathogenesis of idiopathic pulmonary fibrosis, which could also clarify the unexplained predominance of usual interstitial pneumonia lesions in subpleural spaces. Currently available data from various organs and cell-fate mapping studies clearly support a role for pericyte-to-myofibroblast transition, but further mapping studies are required for mesothelial cell plasticity to confirm present data suggesting that these cells could be targets in treatment of idiopathic pulmonary fibrosis. The extraordinary plasticity of resident lung cell populations (figure 2) requires a much more detailed analysis of their roles in fibrotic transformation as possible sources of activated myofibroblasts. In future, data obtained from genetically altered mice should be investigated in human disease samples, and novel approaches and strategies should be designed to examine cellular plasticity in lungs of patients with idiopathic pulmonary fibrosis.

Reactivation of developmental programmes Whereas some of the recently discovered pathways act in a very cell-specific fashion, others exert pleiotropic effects on several cell types during the pathogenesis of idiopathic pulmonary fibrosis. In this context, aberrant reactivation of developmental signalling pathways has been identified 683

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also induces perivascular perycite proliferation, further promoting kidney fibrosis.46 In the lung, activation of SHH in early injury stages is most probably an attempted reparative mechanism of the lung epithelium, as shown by upregulation of SHH and GLI1 during lung injury in much the same pattern as in early development.56 Fitch and colleagues57 reported that lung epithelial SHH is increased by oxidative stress and decreased by TGFβ in patients with idiopathic pulmonary fibrosis. Thus, further analysis of pathways that control cell–cell activation and crosstalk in a pleiotropic fashion, such as Notch and SHH, might enhance therapeutic options in early and late stages of the disease.

Soluble mediators of fibrotic injury Figure 2: Possible cellular components of lung injury in idiopathic pulmonary fibrosis Many different cell types have been suggested to contribute to the activated fibroblast and myofibroblast pool in fibroblast foci. These include (from left to right): type I and II alveolar epithelial cells, endothelial cells, fibrocytes, resident fibroblasts, pericytes, or pleural mesothelial cells.

as a key mechanism. These pathways include, among others, Wnt, SHH, Notch, and BMP (bone morphogenic protein) signalling, and they determine cell-cell communication, survival, and stem-cell differentiation, proliferation, and apoptosis. These pathways are thought to be initially activated as a normal healing response to lung injury, but their perpetuated, dysregulated, and non-coordinated activation leads to scarring and further enhancement of the disease process in idiopathic pulmonary fibrosis.52 Notch signalling is a complex pathway that determines lung morphogenesis, branching, and alveolarisation in a tightly controlled spatiotemporal manner during development. In the context of pulmonary fibrosis, RETNLB, which is exclusively expressed in the alveolar epithelium, promotes ACTA2 and COL1A1 expression and subsequent myofibroblast differentiation53 in co-culture systems or when artificially overexpressed in fibroblasts. Treatment with Fizz1 activates Notch signalling by overexpression of NIC, NOTCH1, JAG1, and HES1.12,54 Activation of Notch signalling in alveolar epithelial cells not only induces ACTA2 expression in fibroblasts (both correlate with each other in lungs with idiopathic pulmonary fibrosis), but also mediates epithelialmesenchymal transition in alveolar epithelial cells. Importantly, when Notch signalling is activated, mice show a more pronounced fibrotic response to bleomycin than when Notch is inactive,12 showing that Notch signalling as a profibrotic mediator. SHH is a developmental signalling pathway implicated in embryo differentiation, which also mediates epithelial– mesenchymal communication in lung and kidney fibrosis. The ligand SHH is mainly expressed in epithelial cells, with fibroblasts acting as responder cells.45,46 SHH induces GLI1, ACTA2, FN1 (fibronectin), COL1A1, and DES expression in fibroblasts. Importantly, GLI1-deficient animals are protected from fibrosis.55 SHH stimulation 684

Serotonin transporters have been identified as a novel signalling pathway that controls fibroblast activation. Increased expression of HTR1A, HTR1B, HTR2A, and HTR2B was detected in patients with idiopathic pulmonary fibrosis or non-specific interstitial pneumonia, with HTR2A expression being specific for idiopathic pulmonary fibrosis. Lung fibroblasts abundantly express HTR2A, whereas epithelial cells largely express HTR2B. Treatment with HTR2A and HTR2B inhibitors in bleomycin-injured mice resulted in an improvement in lung function, fibrotic score by histological examination, and a decrease in collagen content.58,59 In vitro, inhibition of HTR2A and HTR2B blocked TGFβ-induced and WNT3A-induced collagen production in fibroblasts.58 Importantly, HTR2B also regulates hepatic fibrosis by activation of TGFB expression and signalling through MAPK1 and JUND. Direct antagonism of HTR2B suppresses the development of fibrosis and enables tissue regeneration,60 lending further support to the idea that blockade of HTR2A and HTR2B might be a promising yet underexplored therapy for idiopathic pulmonary fibrosis. LPA (lysophosphatidic acid) is a newly identified mediator of early injury in fibrosis.61 It exerts positive effects on fibroblast accumulation after bleomycin injury;62 conversely, inhibition of LPA reduces lung fibrosis. LPA signalling through the LPA receptor increases epithelial apoptosis, yet leads to apoptosis resistance in fibroblasts.62,63 Similarly, the eicosanoid prostaglandin dinoprostone64 has also been associated with fibroblast resistance to apoptosis in patients with idiopathic pulmonary fibrosis. Administration of dinoprostone protected alveolar epithelial cells against FASL-induced apoptosis and increased apoptotic responses in fibroblasts.65 In human lung-resident mesenchymal stem cells, dinoprostone inhibits proliferation, collagen secretion, and ACTA2 expression.66 Further, treatment of bleomycin-induced fibrotic mice with diprostone or iloprost ameliorated fibrosis and improved lung function.67 Some novel soluble mediators in pathogenesis of idiopathic pulmonary fibrosis are being investigated as potent therapeutic targets by the biotechnology and pharmaceutical industries; for www.thelancet.com Vol 380 August 18, 2012

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example, an LPA1 antagonist is currently in phase 2 trials for the treatment of idiopathic pulmonary fibrosis and scleroderma.68 Another important novel soluble mediator with therapeutic potential is LOXL2 (lysyl oxidase-like 2). Lysyl oxidases are copper-dependent, secreted amine oxidases that initiate the first step of collagen and elastin crosslinking. Levental and colleagues69 described how crosslinking of extracellular matrix and tissue stiffness enhances malignant features of breast cancer and promotes fibrotic lesions in regions surrounding tumours. Invivo inhibition of LOXL2 modified tumour progression by decreasing fibrosis via regulation of collagen crosslinking and stiffness of extracellular matrix, decreasing focal adhesions, and preventing fibroblast activation.70 Rodriguez and collaegues71 developed a monoclonal antibody (AB0023) to induce allosteric inhibition of LOLX2 and showed that AB0023 administration reversed lung fibrosis, inhibited liver fibrosis, and reduced metastatic tumour burden in animals.71 Similarly, TGM2 (transglutaminase 2, also known as tissue transglutaminase) has a key role in matrix stabilisation and crosslinking of proteins by transamidation. This process requires high calcium concentrations, which occur in the extracellular environment during cell injury and loss of calcium ion homoeostasis.72 Olsen and colleagues73 showed that, on bleomycin treatment, Tgm2 knockout mice developed reduced tissue collagen accumulation and fibrosis, compared with wild-type animals. In biopsy samples from patients with idiopathic pulmonary fibrosis, TGM2 expression was increased compared with controls, and localised preferentially to interstitial fibrotic areas and fibroblastic foci. Artificial overexpression of TGM2 in primary human lung fibroblasts increased fibronectin deposition and matrix organisation. Consequently, these data collectively suggest that the extracellular matrix has a key role in mediation of fibrosis, and that enzymes regulating the stiffness or composition, or both, of the extracellular matrix are putative therapeutic targets in idiopathic pulmonary fibrosis. Importantly, increased crosslinking of extracellular matrix affects fibrogenesis and the pathogenesis of idiopathic pulmonary fibrosis in several ways. First, enzymes that regulate extracellular matrix composition and crosslinking biochemically modify fibrotic responses by controlling growth factor sequestration and availability. Second, extracellualr matrix stiffness directly affects cellular phenotypes via tensile forces, because matrix stiffness, independent of its composition, directly induces myofibroblast74 and TGFβ75 activation. Reactive oxygen species are well-known injurious agents for lung epithelial cells and fibroblasts. Generation of reactive oxygen species in fibroblasts, via NOX4 (NADPH oxidase 4), can induce and mediate fibrosis. NOX4 expression is increased in fibrotic regions from lung sections of patients with idiopathic pulmonary fibrosis.76 Hecker and colleagues77 showed that TGFβ induced NOX4 www.thelancet.com Vol 380 August 18, 2012

expression via canonical SMAD signalling. TGFβ also required production of reactive oxygen species to induce myofibroblast activation, extracellular matrix production, and contractility. In both fluorescein isothiocyanateinduced fibrosis and bleomycin-induced fibrosis, inhibition of NOX4 by small interfering RNA or chemical inhibition ameliorated fibrosis.77 Much the same results have been described in kidney fibrosis,78 where NOX4 also mediated TGFβ-induced myofibroblast activation. To investigate the idea that decreasing the oxidative load in the lung could be therapeutically beneficial, acetylcysteine, a precursor of the major antioxidant glutathione, has been clinically used to try to prevent injury perpetuation in lung epithelial cells. However, early results from the PANTHER trial79 showed that combination therapy (with prednisone, azathioprine, and acetylcysteine) increased the risk of death and admission to hospital, compared with placebo. Safety issues were not identified in the group of patients treated with acetylcysteine alone and the next data report is expected in 2013.

microRNAs Although several genetic, epigenetic, and proteomic studies have been done so far, studies investigating micro RNA (miRNA) regulatory networks in idiopathic pulmonary fibrosis have only recently gained much attention. miRNAs are small non-coding RNAs that specifically repress or induce the expression of a set of related target genes. Emerging evidence shows that miRNAs are fibrotic modulators in several organs, including the lung.80–82 Rather than working via one pathway, miRNA networks regulate the expression of entire sets of fibrosis-relevant genes by turning pathways on or off. Additionally, many profibrotic mediators, such as TGβ, regulate the expression of miRNAs. Let-7d was the first miRNA shown to be downregulated in idiopathic pulmonary fibrosis, in an unbiased miRNA array with whole lung RNA samples from patients with the disorder.83 Let-7d was negatively regulated by TGFβ inhibition, specifically SMAD3. Inhibition of let-7d induces the mesenchymal markers CDH1, VIM, ACTA2, and HMGA2 in alveolar epithelial cells. In vivo, let-7d inhibition leads to increased expression of ACTA2 and S100A4, increased collagen production in alveolar epithelial cells, and ultimately, alveolar septal thickening with lung injury and fibrosis, which functionally validates its role in idiopathic pulmonary fibrosis.83 Additionally, expression of the MIR200 family is downregulated in patients with idiopathic pulmonary fibrosis. Overexpression of MIR200 inhibited epithelial-mesenchymal transition-like changes induced by TGFβ in alveolar epithelial cells and mice treated with MIR200 showed an attenuation of pulmonary fibrosis.84 MIR31 is a negative regulator of fibrosis, because its expression was decreased in bleomycin-induced fibrosis and in fibroblasts from patients with idiopathic pulmonary fibrosis. Artificial overexpression of MIR31 in vivo diminished experimental lung fibrosis induced by 685

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bleomycin by preventing TGFβ1-induced profibrotic gene induction and fibroblast migration, contractility, and fibrogenesis. Most miRNAs that have been investigated so far are decreased in tissues with idiopathic pulmonary fibrosis. Restoration of their expression largely normalises cellular activities and gene expression, suggesting that systemic inhibition of miRNA patterns is an early event driving pathological gene expression patterns in idiopathic pulmonary fibrosis. Understanding of the signalling networks that synergistically link miRNA and gene expression of profibrotic mediators and effectors will be essential to intervene in fibrogenesis efficiently.

Autophagy Autophagy is a newly recognised regulatory mechanism of cellular homoeostasis and survival in lung diseases.85 It is a highly conserved catalytic pathway controlling protein and organelle transport and degradation via lysosomes in health and disease.86 Noxious environmental stimuli, such as pathogens, cigarette smoke, allergens, reactive oxygen species, or hyperoxia activate autophagic processes as an initial defence mechanism and in homoeostatic regulation of the lung microenvironment. In-vitro exposure of alveolar or bronchial epithelial cells to extracts from cigarette smoke activated autophagy and induced apoptosis via accumulation of MAP1LC3B and LC3B2 (its active form) in a time-dependent and dose-dependent manner.87 Further, increases in LC3B2 protein concentrations in patients with chronic obstructive pulmonary disease positively correlated with disease severity, suggesting pathogenetic relevance.88 Hypoxia also activated autophagy,89 specifically mitochondrial autophagy; this activation required high concentrations of intracellular reactive oxygen species and hypoxia-inducible factor 1α. Yang and colleagues90 linked activation of autophagy with proresolution and antifibrotic properties in acute and chronic models of pulmonary fibrosis, showing that activation of TLR4 (toll-like receptor 4) is needed to induce autophagy. Activation of autophagy on sirolimus treatment decreased hydroxyproline content and collagen deposition and improved survival rates. Importantly, Patel and colleagues91 showed that the autophagy-related proteins MAP1LC3B and NUP62, and the number of autophagosomes, are decreased in patients with idiopathic pulmonary fibrosis. In vitro, TGFβ1 prevented the activation of autophagy via upregulation of C12orf5 (an autophagy inhibitory protein) in fibroblasts.

Future perspectives The past two decades have seen an unprecedented expansion of our understanding of the cellular origins of activated myofibroblasts, soluble mediators of lung injury, and novel regulatory mechanisms of idiopathic pulmonary fibrosis. Currently, interest in the research community is focused on endogeneous and exogeneous repair mechanisms of distorted lung architecture and novel approaches 686

for lung regeneration. The studies we have discussed, and stem-cell approaches that have not been included in this report, have given hope to the idea that a diseased lung with idiopathic pulmonary fibrosis, which was previously regarded as a terminal and irreversible state, could be at least partly restored in future.92 Distinct cell populations seem to contribute to the restoration process, including tissue-specific stem cells and basal progenitor cells, along with resident lung cells with high plasticity potential (eg, alveolar epithelial cells, fibroblasts, mesothelial cells, and pericytes). Many of these populations have shown high degrees of plasticity in response to a multitude of stimuli, which might help to repopulate and restore regions of scarring in idiopathic pulmonary fibrosis.93,94 A spatiotemporally restricted yet closely coordinated interference with aberrantly activated developmental signalling pathways (eg, Wnt,95 SHH, and Notch96) might affect differentiation and restoration of lung architecture. We hope that in the future, in-vivo modulation of profibrotic and developmental pathways combined with manipulation of cellular plasticity will bring novel effective and safe therapeutic regimens to idiopathic pulmonary fibrosis in particular and chronic lung diseases in general. Contributors Both authors contributed equally to this Review. Conflicts of interest Both authors have sponsored grants for fibrosis research from Roche. OE has received speaker’s fees from Roche, Bayer, and InterMune. Acknowledgments We thank the Helmholtz Association for funding. References 1 Eickelberg O, Selman M. Update in diffuse parenchymal lung disease 2009. Am J Respir Crit Care Med 2010; 181: 883–88. 2 Raghu G, Collard HR, Egan JJ, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med 2011; 183: 788–824. 3 Oh CK, Murray LA, Molfino NA. Smoking and idiopathic pulmonary fibrosis. Pulm Med 2012; 2012: 808260. 4 Raghu G, Freudenberger TD, Yang S, et al. High prevalence of abnormal acid gastro-oesophageal reflux in idiopathic pulmonary fibrosis. Eur Respir J 2006; 27: 136–42. 5 Lee JS, Ryu JH, Elicker BM, et al. Gastroesophageal reflux therapy is associated with longer survival in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2011; 184: 1390–94. 6 Taskar VS, Coultas DB. Is idiopathic pulmonary fibrosis an environmental disease? Proc Am Thorac Soc 2006; 3: 293–98. 7 Lasithiotaki I, Antoniou KM, Vlahava VM, et al. Detection of herpes simplex virus type-1 in patients with fibrotic lung diseases. PLoS One 2011; 6: e27800. 8 Faner R, Rojas M, Macnee W, Agusti A. Abnormal lung aging in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2012; published online May 10. DOI:10.1164/rccm.201202-0282PP. 9 Wynn TA. Integrating mechanisms of pulmonary fibrosis. J Exp Med 2011; 208: 1339–50. 10 Konigshoff M, Eickelberg O. WNT signaling in lung disease: a failure or a regeneration signal? Am J Respir Cell Mol Biol 2010; 42: 21–31. 11 Crosby LM, Waters CM. Epithelial repair mechanisms in the lung. Am J Physiol Lung Cell Mol Physiol 2010; 298: L715–31. 12 Aoyagi-Ikeda K, Maeno T, Matsui H, et al. Notch induces myofibroblast differentiation of alveolar epithelial cells via transforming growth factor-β-Smad3 pathway. Am J Respir Cell Mol Biol 2011; 45: 136–44.

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