Although EMT has been implicated in lung fibrosis, the mechanisms underlying EMT remain unclear. In this report, using an in vitro and in vivo model of MHV68 infection of lung epithelial cells, we show that γ-herpes virus infection induces Twist and expression of mesenchymal markers. Inhibition of Twist expression by shRNA in MLE15 cells infected with MHV68 resulted in persistent E-cadherin expression and lower expression of collagen compared to cells without Twist knockdown. Importantly, ectopic overexpression of Twist in MLE-15 was sufficient to reduce the expression of E-cadherin, a protein required in the cell-cell contact and usually downmodulated during EMT, and to increase the expression of FSP-1, fibronectin and collagen, mesenchymal cell markers. Furthermore, this report is the first to demonstrate upregulation of Twist protein expression in a murine model of virus-induced lung fibrosis and in IPF lung tissue. Twist was expressed in the nuclei of alveolar type II epithelial cells of MHV68 infected lungs and was not expressed in mock-infected lung tissue. The levels of Twist changed with time after lung infection starting with low levels in early infection and high levels at the time of chronic MHV68 infection. Expression of Twist in IPF lungs was upregulated significantly. Furthermore, expression of Twist in alveolar epithelial cells in IPF was associated with expression of pro-SP-C protein and the mesenchymal cell marker N-cadherin. In addition, Twist-positive lung epithelial cells in IPF tissues had cytoplasmic instead of membranous localization of E-cadherin.
Microarray analysis of IPF tissue identified an IPF-specific gene expression signature characterized by the up-regulation of genes involved in tissue remodeling and enriched with genes associated with lung development 
. Among the developmental genes discovered were members of the Sry-related high mobility group-box and forkhead box families, and genes related to the Wnt/β-catenin pathway. Twist has been shown to be up-regulated in response to Wnt1 expression in mouse mammary epithelial cell lines and tumors 
. Twist expression is also induced by NF-κB, insulin-like growth factor 1, HIF-1, and EGF/EGFR signaling pathways 
. Once expressed, Twist directly or indirectly represses E-cadherin expression via E-boxes that are also targeted by Snail and SIP1. However, the repression of E-cadherin is not sufficient to induce EMT and ectopic expression of E-cadherin does not restore the epithelial phenotype in Twist-overexpressing cells 
. Thus, EMT completion requires the acquisition of mesenchymal markers.
Twist regulates EMT, cell movement, and tissue reorganization during early embryogenesis. Similarly, Twist expression in tumor cells is associated with EMT and increased cell motility, suggesting that Twist may contribute to metastasis. Suppression of Twist expression in highly aggressive 4T1 mammary carcinoma cells specifically inhibits their ability to metastasize from the mammary gland to the lung 
. Twist has been also related to EMT in a model of renal fibrosis induced by unilateral ureteral obstruction 
. In that model, Twist expression was increased in tubular epithelia of the dilated tubules and the expanded interstitial areas where proliferating cells were frequently found in a time-dependent manner. The genes downstream of Twist that are essential to initiate and maintain EMT are still under investigation. In breast carcinoma cells, Twist causes transcriptional repression of E-cadherin, α, β and γ-catenins, and stimulates the expression of mesenchymal cell markers like fibronectin, vimentin, α-smooth muscle actin and N-cadherin as well as the angiogenic factor VEGF 
. Additionally, Twist is able to inhibit oncogene- and p53-dependent cell death and, therefore, considered as an anti-apoptotic factor 
Recently, several studies demonstrated EMT induction in the setting of viral infection. Li et al. showed positive expression of hepatitis C virus core protein associated with decreased expression of E-cadherin and α-catenin in conjunction with increased expression of N-cadherin, vimentin, and fibronectin in tissues from cholangiocarcinoma 
. In a study of cervical cancer, the early stages of keratinocyte transformation by HPV16 were characterized by cellular changes associated with EMT including reduction in expression of cytokeratin, formation of desmosomes, adherents junctions and focal adhesions 
. As we mentioned before, expression of the EBV protein LMP-1 in nasopharyngeal carcinoma cells was associated with Twist expression and EMT 
. Virus infection has also been shown to contribute to TGF-β activation and expression of extracellular matrix components in endothelial cells. Infection of endothelial cells with cytomegalovirus (CMV) upregulated integrin-β6 followed by activation of the TGF-β pathway and expression of collagen IV 
. Those observations are intriguing in view that EMT in alveolar epithelial cells is induced by linking TGF-β and β-catenin signaling through integrins 
Strong evidence for EMT in vivo
in the setting of epithelial injury/stress is difficult to obtain because of the isolated and possible transient nature of the process; such transitioning cells are difficult to track. However recent studies with bone marrow chimeras and transgenic reporter mice showed that during renal fibrogenesis fibroblasts are derived both from bone marrow and by local EMT with EMT being the dominant source of the fibroblasts 
. EMT has also been detected in IPF lung tissue and animal models of pulmonary fibrosis 
. Using a triple transgenic mouse reporter system, Kim et al. demonstrated that EMT plays an important role during lung fibrogenesis and may be more widespread than previously thought 
Our data suggest that Twist might contribute to the EMT process in pulmonary fibrosis. Furthermore, they suggest that γ-herpes virus might be, in some IPF cases, the source of injury precipitating EMT. We demonstrated that biopsies with relatively high Twist expression were positive for EBV genome. Several other studies suggest a potential role for EBV in viral epithelial injury and potential EMT development. In a viral cell line model, TGFβ1 was shown to be induced in epithelial cells following EBV lytic phase induction. TGFβ1 expression was promoted by the EBV early genes Rta and Zta 
. In human nasopharyngeal carcinoma tissue, expression of Twist and EBV LMP-1 was directly correlated. EBV LMP-1 caused morphologic and molecular changes of EMT in cultured epithelial cells and this EMT was reversed by suppressing Twist 
Although our studies suggest that virus-induced Twist activation might represent an important mechanism for EMT in IPF, further studies will be needed to test the true role of Twist in EMT in vivo and to determine the role of virus infection in the fibrogenesis pathways that characterize IPF and related lung fibrotic disorders.
In summary, our studies, suggest that EMT may be a cellular mechanism of fibrogenesis in the lung associated with virus-induced epithelial injury. Twist is a well known master and a transcriptional regulator of EMT during embryogenesis and metastasis. The abundant expression of Twist in alveolar epithelial cells is likely to contribute to EMT and an important source of fibroblasts in IPF lungs. The identification of the downstream effector pathways that are activated during EMT holds the promise of revealing new diagnostic markers of early stages of pulmonary fibrosis and, quite possibly, novel targets for anti-fibrotic therapeutics.