Epithelial-mesenchymal transition: potential role in obliterative bronchiolitis?

Lung transplantation remains the only viable option for many patients suffering from a variety of progressive or intractable end-stage lung diseases. Despite significant advances in the prevention of early graft rejection, ischemia-reperfusion injury, and acute management of lung transplant recipients, significant challenges remain in the chronic management of patients after lung transplantation (1). In this issue of Thorax, Borthwick and colleagues provide intriguing new evidence that implicates the airway epithelium directly in the pathogenesis of bronchiolitis obliterans syndrome (BOS), the most significant factor in determination of long-term lung graft survival. As discussed in the study, the pathological lesion of BOS is obliterative bronchiolitis (OB), which recently has been postulated to be at least partially a disease of aberrant epithelial repair processes (2). Borthwick and colleagues provide evidence that epithelial-mesenchymal transition (EMT), a process whereby epithelial cells undergo a complete lineage transition to become fibroblasts and myofibroblasts, may underlie the dysfunctional airway repair processes that lead to OB. This study, and others like it, attempt to redefine traditional paradigms regarding normal airway epithelial biology and disease pathogenesis, and have the potential to lead to entirely new therapeutic avenues for previously untreatable disease processes such as BOS.

OB is characterized by initial inflammation of the small airways, followed by airway remodeling, aberrant epithelial regeneration and repair, proliferation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and eventual airway obstruction (3). The initial inflammatory response is the result of an allogeneic immune response initiated against donor antigens in the graft endothelial and airway epithelial cells. This response characteristically generates antigen-specific, graft-infiltrating destructive lymphocytes. The lymphocytes facilitate activation of macrophages and a variety of other inflammatory cells, with resultant epithelial damage (4). The critical role of this allogeneic immune response as the initial trigger leading to OB is supported by the fact that the primary risk factors for the development of BOS after lung transplantation are class I- and II- mismatches between donor and recipient, as well as the number and severity of rejection episodes (3). While it has been recognized for over a decade that the airway epithelium is a target of the initial immune response (5), the pathogenetic pathway that leads to disruption of normal epithelial repair processes, excessive fibroblastic responses and resultant excessive ECM deposition, and eventual obliteration of the small airways is still incompletely elucidated.

While bronchial epithelial cells (BECs) have been shown to directly present antigen (6), and are potentially the primary target of immunologic attack during the pathogenesis of OB (7), their precise link to the proliferation of fibroblasts and the propagation of fibrosis is not entirely clear. It is known that epithelial cell apoptosis and disruption of epithelial integrity likely contributes to sub-epithelial fibroblastic proliferation (8), but the primary source of the proliferating fibroblasts during airway fibrosis is still unknown. One possible source is direct conversion of proliferating BECs into pathogenetic fibroblasts and myofibroblasts through the process termed EMT. EMT is a process by which epithelial cells lose fundamental epithelial characteristics such as tight junctions, apical:basolateral polarity, and the expression of epithelial-specific markers, and assume a mesenchymal phenotype, expressing a variety of mesenchymal markers and acquiring functional characteristics of fibroblasts and myofibroblasts, such as ECM production, motility, and the ability to invade surrounding tissues (9). EMT is not a new concept, being critical during normal development and for the development of metastatic potential and increased invasiveness during cancer progression (10). However, the role of EMT in the pathogenesis of adult tissue fibrosis is a relatively new area of study. EMT has been most commonly investigated as a mechanism underlying fibrosis in renal and lens epithelium. In the kidney, ~ 40% of new fibroblasts are thought to arise via EMT during injury (11), while in the eye EMT has been demonstrated both in vitro and in vivo (12). Very recently, the role of EMT in the pathogenesis of pulmonary disease has begun to be investigated. The first demonstrations of EMT in pulmonary epithelium were in alveolar epithelial cells. In response to TGF-β in vitro, several groups have demonstrated conversion of alveolar epithelial cells to a myofibroblast-like phenotype, with a complete morphologic and functional change and acquisition of mesenchymal markers, concomitant with the loss of detectable epithelial characteristics (13–15). Further, EMT has been demonstrated in alveolar epithelium in vivo in mice. In one recent study, lineage-tagged alveolar epithelial cells were shown to contribute over 30% of the alveolar fibroblastic response during experimental lung injury (16). Finally, immunohistochemical evidence from lung biopsy samples from patients with idiopathic pulmonary fibrosis (IPF) suggests that up to 80% of the epithelium in areas of active fibrosis exhibit evidence of EMT (13). Given the growing body of evidence implicating the importance of EMT of alveolar epithelial cells in the pathogenesis of pulmonary fibrosis, the possibility that BECs may undergo EMT to contribute to airway remodeling and fibrosis is intriguing.

In this issue, Borthwick et al. cultured primary human bronchial epithelial cells in the presence and absence of TGF-β and TNF-α. Only TGF-β alone was able to induce a fibroblastoid morphology, but the combination of both TGF-β and TNF-α resulted in the most robust phenotypic change together with upregulation of mesenchymal markers (vimentin and fibronectin) and downregulation of the epithelial markers cytokeratin 19 and E-cadherin. Treated cells also acquired functional characteristics of myofibroblasts, demonstrating an increase in the ability to invade collagen and deposit ECM. The authors extended their study to examine normal lung and pathologic specimens from stable transplant patients and those with progressive BOS. They used quantitative immunohistochemistry to demonstrate a correlation between disease severity and both loss of epithelial markers and acquisition of mesenchymal markers in the bronchial epithelium. Finally, they utilized confocal microscopy to demonstrate extensive co-localization of epithelial and mesenchymal markers within the epithelium of transplant recipients with OB. Together, the findings of the current study provide strong support for the hypothesis that EMT may play a significant role in the pathogenesis of OB.

While few, there have been other suggestions of EMT as a potential pathogenetic mechanism in OB. An early report that included authors of the current study demonstrated that 15% of the epithelium in biopsy specimens from clinically stable transplant patients co-stained for the fibroblast marker S100A4 (2). Recently, mouse tracheal epithelial cells were shown to undergo at least partial EMT in response to TGF-β1, possibly through a JNK-1 mediated mechanism (17). Finally, peribronchial fibrosis during bleomycin-induced lung injury was found to contain a subset of BECs possibly derived from EMT (18). Clearly, the possibility that BECs can contribute to the airway fibrotic response in a variety of disease processes deserves further investigation.

An intriguing aspect of the current study is the requirement for both TGF-β and TNF-α together to induce a robust EMT response in BECs in vitro. A potential criticism of previous studies has been that the dose of TGF-β required to induce EMT in BECs has been higher than that required to induce alveolar EMT (5–50 ng/mL vs. 0.5–1 ng/mL) (17). Given that physiologic concentrations of TGF-β1 during lung injury only reach concentrations of approximately 1 ng/mL or lower (19), the possibility that bronchial epithelial-mesenchymal transition is a purely in vitro phenomenon has been raised. The combination of TGF-β and TNF-α is intriguing, in that EMT during airway fibrosis would likely occur in the presence of both of these cytokines, due to the ongoing inflammation during rejection. The addition of TNF-α resulted in the induction of EMT at doses of TGF-β as low as 1 ng/mL, within the physiologic range. This synergy between TGF-β and TNF-α confirms prior observations in EMT of alveolar epithelial cells (13), and suggests an interesting cross-talk between TGF-β and TNF-α-induced intracellular signaling pathways.

In summary, Borthwick and colleagues have provided strong evidence for the possibility of EMT as a pathogenetic contributor to airways fibrosis in OB and perhaps other diseases of the small airways. Studies like this one force us to re-examine long-held disease paradigms and hopefully embark on new investigations to find novel therapeutics. Given the historic lack of progress in the prevention and treatment of BOS, new insights and avenues of inquiry are critical to improving the outcomes and lives of patients after lung transplantation.