Epithelial to Mesenchymal Transition in Microbial Pathogenesis

The first chapters of the volume "Cytokeratins - Tools in Oncology" discuss multiple functions of cytokeratins in organization of the intermediary filaments in normal intestine and liver as well as microfold L cells and the usability of cytokeratins 7, 8 and 20 in tumor diagnosis in detail. Epithelial to mesenchymal transition as a mechanism important in pathogenesis is touched in another chapter, followed by several articles dealing with the role of cytokeratins for detection of disseminated tumor cells and as response markers during chemotherapy. This book is therefore destined to all cancer researchers and therapists who want to understand the diagnostic application of cytokeratins in histology and, especially, the use of anti-cytokeratin antibodies to identify viable residual tumor cells accounting for a higher risk of tumor recurrence or cancer cells responding to chemotherapy, respectively.


Introduction
Epithelia are physical barriers that constitute a functional interface between distinct body compartments and the outside. Under healthy condition, cells that composed the epithelial sheets are tightly bound to neighboring cells and to underlying basement membranes by adherens junctions, tight junctions, desmosomes and hemi-desmosomes (Farquhar, M. G. & Palade, G. E., 1963). However, epithelial cells empower high degree of plasticity and under certain circumstances such as developmental processes, fibrogenesis or tumor progression, they loss their static phenotype and acquire migratory and invasive behavior (Grunert, S., et al., 2003). Epithelial plasticity could be limited to relocalization of junctional proteins or to a more drastic epithelial to mesenchymal transition (EMT) which is characterized by disruption of intercellular contacts, loss of epithelium-specific proteins, switch to a mesenchymal gene expression pattern, and gain of invasive properties (Thiery, J. P., 2002). It is to note that EMT is different than collective cell movement which occurs when two or more cells that retain their genetic and phenotypic feature move together across a twodimensional (layer of extracellular matrix) or through a three-dimensional interstitial tissue (Ilina, O. & Friedl, P., 2009).

Deciphering the EMT process 2.1 General concept
EMT has been extensively reviewed in the litterature (Nieto, M. A., 2011, Thiery, J. P., 2002) and we summarized in Figure 1 the key point steps of this cellular process. As stated above, epithelial cells are apico-basal polarized cells with lateral adherence to their neighbors under the control of E-cadherins. The adhesion sites to extracellular matrix (ECM) are focused to the basal lamina, and cytokeratins are the main intermediate filaments. In contrast, EMT can result from various extracellular (ligands for RTKs such as FGFs, PDFG; ligands for RS/TKs such as TGF or ligands for specific receptors such as Wnts) and cellular stimuli (extracellular matrix compounds, hypoxia, microRNAs, ROS) that through interactions with specific receptors or other mechanisms can stimulate intracellular signaling pathways, leading to activation of transcription factors that govern the transcription of EMT-related genes. The Figure highlights NF-κB and MAPK signaling as major pathways involved in EMT triggering. The most relevant criteria to detect EMT are loss of epithelial markers (Ecadherin, Cytokeratins, ZO-1 and etc.) gain of mesenchymal markers (vimentin, fibronectin, MMPs, proteins of the extracellular matrix, etc.) associated to appearance of the fibroblastoide phenotype and increased migratory and invasive properties.

EMT and transcription factors
EMT is controlled by a small group of transcription factors defined as the core EMT regulatory factors that comprises SNAI1/Snail1 (Twigg, S. R. & Wilkie, A. O., 1999), SNAI2/Slug/Snail2 (Cohen, M. E., et al., 1998), Sip1/ZEB2 (Verschueren, K., et al., 1999) and Twist (Wang, S. M., et al., 1997). Whereas these proteins share the same function that is a transcriptional repression of E-cadherin, they have different structures. The Snail family is composed of zinc finger proteins, the ZEB family has 2 zinc finger clusters and Twist proteins has a helix loop helix motif (Peinado, H., et al., 2007). Interestingly enough, it was recently shown that in neural crest cells all these factors are coordinately regulated by an E3 ubiquitin ligase named Partner of paired (Ppa) (Lander, R., et al., 2011). Ppa is a F-box containing protein that targets its bound substrates to the ubiquitin-proteasome system for degradation. Given the importance of EMT in physiological development the existence of a common regulatory protein that can be tightly controlled in a spatio-temporal manner makes sense. However, it remains to be defined whether Ppa is also involved in pathophysiogical EMT such as tumor progression and microbial pathogenesis.

EMT and intracellular signaling pathways
Multiple signaling pathways, including receptor tyrosine kinase-mediated signals, transforming growth factor (TGF)-/Smad, Wnt, Notch and hypoxia have been implicated as upstream initiators of the EMT process as highlighted in recent reviews (Moustakas, A. & Heldin, C. H., 2007, Peinado, H., et al., 2007, Said, N. A. & Williams, E. D., 2011. We will focus here on pathways that are activated by pathogen recognition receptors as detailed later in this review.

The MAPK module
MAPK signaling pathways are organized in modular cascades in which activation of upstream kinases by cell surface receptors leads to sequential activation of a MAPK module (MAPKKK → MAPKK → MAPK). This module comprises four different signaling pathways activated by mitogens, inflammation, stress and oxidative stress (Junttila, M. R., et al., 2008). These signaling pathways are interconnected.
The Ras>Raf>MAPK kinase cascade is activated by a large number of mitogen receptors including tyrosine kinase receptors, such as fibroblast growth factor receptor, epithelial growth factor receptor, hepatocyte growth factor, vascular endothelial growth factor and the G-protein coupled receptors, a family of seven trans-membrane domains proteins including cytokine and chemokine receptors. This signaling cascade, which is extremely well conserved from yeast to man, allows activation of a set of transcription factors which in turn control many cellular responses that are relevant for EMT (Keshet, Y. & Seger, R., 2010). Indeed, in addition to repress E-cadherin via activation of Snail/Slug, this pathway also controls upregulation of mesenchymal genes and cell motility via activation of SRE, AP1 and SP transcription factors (Grunert, S., et al., 2003) and references herein.
The p38 MAPK pathway can be activated in response to various cytokines, as well as pathogens and by environmental stress such as hypoxia. p38 MAPK was first described to down-regulate E-cadherin expression during mouse gastrulation (Zohn, I. E., et al., 2006). Further, p38 MAPK was described to participate in TNF- (Grund, E. M., et al., 2008) and TGF--induced EMT (Borthwick, L. A., et al., 2011). In addition a crosstalk between the Smad and NR-κB pathways accentuates TGF--induced EMT in presence of TNF-.
The c-Jun N-terminal kinase (JNK) pathway is mainly activated by cellular stress and by cytokines that act through several upstream kinases such as TAK1 and TRAF6. JNK pathway mediates TGF--induced EMT in keratinocytes (Santibanez, J. F., 2006). Further it was shown that activation of Smad3 by JNK is necessary to mediate TGF--induced EMT (Liu, Q., et al., 2008).

The Smad pathway
The best described inductor of the Smad pathway is the TGF-that is widely described as an EMT inductor; for a review see (Zavadil, J. & Bottinger, E. P., 2005). Briefly, TGF-binds to its receptor which then activates by phosphorylation two transcription factors, Smad-2 and Smad-3 (Massague, J., 1998). Phospho-Smad2/3 heterodimerize with Smad-4 and the Smad-complex translocate to the nucleus to regulate the transcription of genes that control cell proliferation, differentiation and cell migration (Wu, J. W., et al., 2001). Moreover, TGFactivates Smad-independent signaling cascade leading to the activation of the classical Ras-MAPK pathway (Said, N. A. & Williams, E. D., 2011).
In addition to its well-known function in tumor progression, the TGF-signaling plays an essential role in establishing immunological tolerance (Wan, Y. Y. & Flavell, R. A., 2007). Interestingly, reports indicate that microbe invasion lead to TGF-modulation (Reed, S. G., 1999). First, it was shown, in vitro and in vivo, that macrophages invasion by Trypanosoma cruzi led to production of TGF- (Silva, J. S., et al., 1991). This observation was then extended to bacterial infections with studies using Mycobacterium avium and Mycobacterium tuberculosis (Champsi, J., et al., 1995, Toossi, Z., et al., 1995. In the last decade it appears that many bacteria or viruses induce TGF-production via signaling pathways that require Toll like receptors (TLR) as described below.
Macrophages represent the first line of defense; indeed most of these studies were performed on immune cells. However, TGF-released by macrophages could activate TGF signaling on epithelial cells and then induce EMT. In agreement with this paracrine loop hypothesis, it has recently been demonstrated that increasing numbers of leukocytes (macrophages and T cells) infiltrating the kidney after acute unilateral ureteral obstruction in a mouse model correlate with increased EMT (Lange-Sperandio, B., et al., 2007).

The STAT pathway
The signal transducers and activators of transcription (STAT) family consist of seven proteins. STATs are activated by tyrosine phosphorylation of receptor tyrosine kinases, by the cytokine and chemokine receptor/Janus activated kinase (JAK) complexes or by nonwww.intechopen.com receptor tyrosine kinases (Reich, N. C. & Liu, L., 2006). In general, STAT proteins have important roles in the immune response (Ihle, J. N., 2001), however STAT3 has been more particularly involved in EMT. Invalidation of the stat3 gene in mice results in early embryonic lethality (Takeda, K., et al., 1997), therefore using small interference RNA technology to efficiently block STAT3 signaling, Huang and co-authors demonstrated in pancreatic cancer cells that silencing of STAT3 resulted in suppression of EMT (Huang, C., et al., 2011).

Hypoxia
Alteration in microenvironmental oxygen tension and activation of hypoxic signaling through hypoxia-inducible factor (HIF) are emerging as important triggers and modulators of EMT (Haase, V. H., 2009, Jiang, J., et al., 2011. In vivo, O 2 tension varies from 2.5% to 9% in most healthy tissues. However, inflamed or diseased tissues can be deprived of O 2 (hypoxia) due to vascular damage, intensive metabolic activity of bacteria and other pathogens, and large numbers of infiltrating cells, leading to O 2 levels of less than 1%. This phenomenon results in activation of the well-coordinated mechanism leading to regulation of HIF transcriptional pathways as described in many reviews such as (Imtiyaz, H. Z., et al., 2010).
Oxygen deprivation is not the only inducer of HIF. Indeed, inflammatory cytokines, growth factors and bacterial products under normoxic conditions also induce HIFs (Blouin, C. C., et al., 2004, Cane, G., et al., 2010, Peyssonnaux, C., et al., 2008, Zhou, J., et al., 2003. Once activated HIFs, and more particularly HIF-1, controls E-cadherin repression, loss of cell-cell adhesion and cell motility via regulation of the core EMT regulatory factors in various cell types, as described in (Haase, V. H., 2009). For example, HIF-1, regulates the expression of TWIST by binding directly to the hypoxia response element in the TWIST proximal promoter (Yang, M. H., et al., 2008).

NF-κB
The NF-κB family of transcription factors which is composed of five members -p65 (REL-A), REL-B, cytoplasmic (c) REL, p50 and p52 -is widely activated under cytokines and/or microbial challenge (Li, Q. & Verma, I. M., 2002, Min, C., et al., 2008. For example, the proinflammatory cytokines interleukin-1 and tumor necrosis factor (TNF)-both activate and are activated by NF-κB, thus creating a positive feedback loop that results in perpetual amplification of the response.
Together with SMADs and HIF-1 , NF-κB has been shown, in an integrative genomic analysis, to regulate ZEB2, an EMT regulator, (Katoh, M., 2009). In addition, NF-κB is involved in the up regulation of twist-1 and twist-2 expression in response to TNF-; this regulation is lost in fibroblasts lacking the p65 subunit of NF-κB (Sosic, D., et al., 2003). Moreover, the authors proposed a model in which TWIST orchestrates a negative feedback loop by repressing cytokine expression under cytokine challenge and therefore maintaining a controlled inflammatory response.
Interestingly enough, the classical NF-κB pathway is also responsible for the EMT process attributable to Von Hippel-Lindau (VHL) loss and subsequent HIF-1 activation since molecular and pharmacological approaches to inhibit NF-κB promote a partial reversion to an epithelial phenotype (Pantuck, A. J., et al., 2010).

MicroRNAs
MicroRNAs (miRs) are non-coding RNA of 18-24 bp that post transcriptionally regulate gene expression. The key region of miRs that governs their target specificity, named the seed sequence, encompasses bases 2-7 from their 5' end (Lewis, B. P., et al., 2005). More than 1200 miRNAs have been identified in humans, and each individual miR could regulate ten to hundreds of genes according to the presence of seed sequence matches in their 3'UTRs. The ability of a specific miR to modify gene expression is governed by its seed sequence but also its expression, which could be spatiotemporally regulated.
A microRNA microarray profiling performed on MDCK undergoing EMT allowed to characterize the implication of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205. Decrease in expression of each of these miRs correlates with decreased expression of E-cadherin and increases in mesenchymal markers mRNA such as vimentin and fibronectin. In addition, overexpression of these miRs in MDCK cells prevents EMT demonstrating that down-regulation of these miRs is an essential component of the EMT process. Finally, it was shown that the miR-200 family represses endogenous expression of ZEB1 and ZEB2 (Bracken, C. P., et al., 2008, Korpal, M., et al., 2008, Park, S. M., et al., 2008. As mentioned previously in this report, TGF is a powerful inductor of EMT. A combination of miRs and mRNA profiling was used to identify miRs that destabilize mRNAs in TGFdirected EMT. Such strategy allowed the characterization of eight miRs specific of a particular signature of EMT-like response (Zavadil, J., et al., 2007).

A general overview on PRR
Charles Janeway was the first to understand that recognition of pathogen-associated molecular patterns (PAMPs) by host pathogen-recognition receptors (PRRs) is the basis of immune immunity and represents the first defense against pathogens (Janeway, C. A., Jr., 1989). His discovery was further confirmed by the identification of Toll-like receptor (TLR)4 as the protein involved in the recognition of lipopolysaccharide (LPS), therefore making the link between a microbial motif, LPS, and a host receptor, TLR4 (Poltorak, A., et al., 1998). A new axe of researches was then opened and after more than a decade the TLRs family encounters 10 members in human and each TLR has a distinct function in terms of PAMP recognition (Kawai, T. & Akira, S., 2010).
TLRs are divided into two subgroups based on their cellular localization and respective PAMP ligands. The first group, expressed on cell surfaces which recognize mainly microbial membrane components such as lipids, lipoproteins and proteins, is composed of TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11; the second group, expressed exclusively in intracellular vesicles where the receptors recognize microbial nucleic acids, is composed of TLR3, TLR7, TLR8 and TLR9.

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In mammals, in addition to TLRs, an intra-cytoplasmic sensing system for microbial effector exists. This second family of receptors is named Nod (nucleotide-binding oligomerization domain)-like receptors (NLRs); NLRs sense the presence of intracellular muropeptides (Fritz, J. H., et al., 2006). As highlighted in Figure 2 both TLRs and NLRs activate intracellular signaling pathways that share common adaptors with receptors of growth factors, cytokines or chemokines.
Note that in addition to TLRs and NLRs other microbial sensors exist as reviewed in (Bouchon, A., et al., 2000, Crocker, P. R., 2005, Klesney-Tait, J., et al., 2006, Robinson, M. J., et al., 2006. IKK2 and nuclear factor-κB (NF-κB) essential modulator, NEMO, respectively -and MAPKs. Alternatively, TIRAP (TIR domain-containing adaptor protein), a second TIRdomain-containing adaptor protein, is involved in the MyD88-dependent signalling pathway through TLR2 and TLR4. A third TIR-domain-containing adaptor, TRIF (TIR domain-containing adaptor protein inducing IFN-), is essential for the MyD88independent pathway. Further, the non-typical IKKs IKK-ε and TBK1 (TRAF-familymember-associated NF-κB activator (TANK)-binding kinase 1) mediate activation of IRF3 downstream of TRIF. A fourth TIR-domain containing adaptor, TRAM (TRIF-related adaptor molecule), is specific to the TLR4-mediated, MyD88-independent/TRIF-dependent pathway. By contrast, activation of NLRs leads to the recruitment of the receptor-interacting protein 2 (RIP2) kinase, which is essential for the activation of the IKK complex. In addition, activation of NOD1 leads to JNK stimulation. Finally, double strand DNA has been linked to inflammasome activation. This protein complex which is composed of NLRs of the NALPfamily and adaptor-proteins apoptosis-associated speck-like protein (ASC), mediates the generation of IL-1 through cleavage of its precursor by caspase-1.

PRR and mediators of EMT
TLRs are type I trans-membrane proteins with extracellular domains containing leucine-rich repeats and mediating the recognition of PAMPs, trans-membrane domains and intracellular Toll-interleukin 1 (IL-1) receptor (TIR) domains which recruit TIR domaincontaining adaptor molecules to induce downstream signal transduction.
MyD88 was identified as the first member of the TIR family adaptors. Once bound to TLRs, MyD88 recruits the IL-1 receptor-associated kinases IRAK4, IRAK1, IRAK2 and IRAK-M. Mostly, direct or indirect activation of IRAK allows the activation of NF-κB and MAPK which in turn induces various transcription factors (Kawai, T. & Akira, S., 2010).
The TIR family also comprise TIRAP (Mal), TRAM and TRIF. TIRAP and TRAM function as additional sorting adaptors allowing the recruitment of MyD88 to TLR2 and TLR4. The final consequence of this signaling puzzle is the activation of NF-κB and MAPK signaling pathways. TRIF is used by TLR3 and TLR4 and induces alternative pathways that lead to activation of the transcription factors IRF3 and NF-κB Interestingly enough, host recognition of pathogens lead to activation of NF-κB and MAPK pathways. As mentioned earlier, these two pathways are particularly relevant for EMT since they control the activation of transcription factors that in turn regulate the expression of the EMT core genes.

EMT and bacterial pathogens
The microbes normally present in humans are collectively estimated to number tenfold that of human cells. Mainly located in the gut, the microbiota is crucial for human life by influencing human physiology and nutriment uptake (Ley, R. E., et al., 2006). In addition, the microbiota contributes to the shaping of healthy intestinal immune responses (Inagaki, H., et al., 1996). It has been proposed that an alteration in the development and/or composition of the microbiota may disturb the relationship between microbes and the immune system. In turn, immune defects may favor pathogenesis of various human inflammatory disorders (Round, J. L. & Mazmanian, S. K., 2009) and inflammatory disorders promote EMT.

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We can therefore speculate that most of microbes that persist in the body have the potential to indirectly favor an EMT behavior. In this review we will only focus on the few examples that describe a direct involvement of microbial pathogens in EMT induction.

Lipopolysaccharide
Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. LPS is an endotoxin which induces a strong response from normal animal immune systems; therefore it is widely used to study gram-negative bacteria-induced cellular responses. Intriguingly, we found in the literature only one report that studies LPS-induced EMT. Using a model of intrahepatic biliary epithelial cells, Zhao and co-authors have shown that in response to LPS stimulation a decrease in E-cadherin expression was observed whereas expression of the mesenchymal markers (S100A and -SMA) increased by more than 12-fold . In addition to EMT markers, they noticed that the messenger coding for TGF -1 was significantly increased. As indicated previously, TGF -1 is a well-known inductor of EMT that transmits its effect via Smad2/3. Indeed, silencing of Smad 2/3 in biliary epithelial cells resulted in a significant decrease of mesenchymal markers and an increase in E-cadherin expression. Therefore, the authors concluded that LPS induced the EMT probably through the TGF-1/Smad2/3 pathway.

Helicobacter pylori
Helicobacter pylori is a gram-negative bacteria which colonizes the human stomach of about 50% of the world's population. Although a large proportion of infected subjects can develop gastritis, 80% of these individuals remain asymptomatic. Severe H. pylori-mediated diseases are duodenal and gastric ulcer disease, gastric cancer and mucosa-associated lymphoid tissues (MALT) lymphomas affecting about 15%, 1% and 0.1% of infected people, respectively (Amieva, M. R. & El-Omar, E. M., 2008). Since 1994, H. pylori is classified as a class I carcinogen by the World Health Organization. More than 350 genetically different strains have been identified. To avoid mechanical clearance, H. pylori first adhere to the gastric epithelium due to adhesins. Among their numerous virulence factors, the two major virulence factors of H. pylori, the cytotoxin VacA and the cag pathogenicity island and its effector CagA, can co-opt epithelial cell function. Whereas VacA can disrupt the barrier function of tight junction, it does not perturb junction integrity (Papini, E., et al., 1998), CagA has major effects on the apical junctional complex allowing the deregulation of epithelial cell-cell adhesion and a loss in epithelial polarity (Amieva, M. R., et al., 2003, Murata-Kamiya, N., et al., 2007. Using the pathogenic H. Pylori strain 60190, Yin and co-authors observed expression of Snail and Slug in gastric epithelial cells (Yin, Y., et al., 2010). Further, they demonstrated that induction of EMT genes depends on H. pylori-induced signaling cascade pathways that involve gastrin, MMP7 and shedding of soluble heparin-binding epidermal growth factor. Interestingly, the increase of gastrin observed in response to H. pylori infection occurred via a Ras>Raf>Mek>Erk>NF-κb signaling pathway (Brandt, S., et al., 2005). Then, it appears that NF-κB is a central common effector that plays a key role in the EMT process.
As mentioned earlier, HIFs are also involved in EMT regulation. It is noteworthy that ROS stabilize HIF-1 (Park, J. H., et al., 2003) and H. pylori induce ROS (Bagchi, D., et al., 1996). Therefore, one could speculate that H. pylori-induced stabilization of HIF-1 acts in combination with NF-κB to maximally induce the EMT program.

Enterovirulent Escherichia coli strains
Escherichia coli which colonize the gastrointestinal tract of human infants within a few hours after birth normally coexist in harmony with its human hosts. However, there are several highly adapted E. coli clones that have acquired specific virulence factors, which confer an increased ability to adapt to new niches and allow them to cause a broad spectrum of diseases. Among the intestinal pathogens there are six well described classes: enteropathogenic-, enterohaemorrhagic-, enterotoxigenic-, enteroaggregative-, enteroinvasive-and diffusely adherent-E. coli. Enteropathogenic E. coli cause entero/diarrhoeal disease as a consequence of lack of intestinal barrier permeability (Kaper, J. B., et al., 2004). In most of the cases this epithelial plasticity is limited to relocalization of junctional proteins; however, depending on the bacterial strain used to infect epithelial cells, it could lead to a more drastic EMT.
Among the families of entero-pathogenic E. coli, diffusely adherent E. coli (DAEC) is a heterogeneous group with variable virulence factors promoting adherence to epithelial cells (Servin, A. L., 2005) The pathogenicity of such bacteria is still controversial; however, the presence of DAEC expressing Afa/Dr adhesins has been reported in epidemiological studies of various types of enterocolitis (Meraz, I. M., et al., 2007, Vargas, M., et al., 1998. Afa/Dr DAEC strains are a family of DAEC expressing the afimbrial Afa-I and Afa-III adhesins, Dr haemagglutinin and fimbrial F1845 adhesin. Afa/Dr adhesins interact with receptors such as the membrane-associated decay accelerating factor (DAF/ CD55), the carcino-embryonic-antigen (CEA/CD66e) and CEACAM-1, -3, -6 (Berger, C. N., et al., 2004), leading to cell signaling. Using the clinical isolate DAEC C1845, we have shown that infection of intestinal epithelial cells promotes EMT-like behavior. We have deciphered the molecular mechanisms leading to EMT and observed that F1845 adhesin binding to the DAF receptor promotes Ras>Raf>MAPK and PI3K pathways (Betis, F., et al., 2003a, 2003b, Cane, G., et al., 2007. Activation of these signaling pathways is required to induce an increase in HIF-1 protein expression but also Twist1 mRNA expression. We noticed that HIF-1 silencing significantly blocked the expression of Twist1 gene, revealing a role for HIF-1 in the transcriptional regulation of this gene. Furthermore, we observed that C1845-induced HIF-1 protein expression leads to a loss of E-cadherin and cytokeratin 18 and an increase in fibronectin expression, which are reversed in HIF-1 silenced cells (Cane, G., et al., 2010), therefore highlighting the critical role of HIF in DAEC-induced EMT.

EMT and viral pathogens
As for microbial pathogens, viral infection leads to activation of intracellular signaling pathways (Rathinam, V. A. & Fitzgerald, K. A., 2011); thus we can intuitively speculate that viruses can induce EMT. The major pathogenic viruses include cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus, Kaposi's sarcoma-associated herpes virus, polyoma virus, hepatitis B and C virus and human papilloma virus. Previous works indeed confirmed that at least two families of viruses (Epstein-barr and hepatitis B and C) induce EMT in epithelial cells. www.intechopen.com

Epstein-barr virus
Epstein-Barr virus (EBV) is a member of the herpes virus family which infects more than 90% of world population. EBV utilizes normal B cell biology to infect, persist, and replicate in B cells. Beyond immune cells, EBV also infects epithelial cells and it has been associated with neoplastic diseases such as nasopharyngeal carcinoma (Chen, M.-R., 2011); the link between EBV and EMT has been studied in this particular context. Latent EBV encodes for eight proteins, two of them, the latent membrane protein 1 and 2A (LMPs), which highjack cell host signaling (Caldwell, R. G., et al., 1998, Gires, O., et al., 1997, are particularly involved in EMT. Horikawa and coauthors were the first to describe that transformation of MDCK epithelial cells with LMP1 induces EMT, characterized by loss of epithelial markers, gain of mesenchymal markers and its associated increase in cell motility and invasiveness (Horikawa, T., et al., 2007). To go further, the authors have shown that Twist1-silencing in MDCK cells resulted in changes from scattered and fibroblast-like shapes to tightly packed cobblestone morphology, characteristics of mesenchymal-toepithelial transition, the reverse of EMT. Finally, the authors demonstrated that LMP1 induces Twist through NF-κB in nasopharyngeal epithelial cells. More recently the same group demonstrated that Snail1 acts in combination to twist1 to induce EMT in nasopharyngeal carcinoma cells (Horikawa, T., et al., 2011).
Using nasopharyngeal carcinoma tumor samples the group of Zeng has shown that 57.6% of tumors overexpressed LMP2A at the tumor invasive front (Kong, Q. L., et al., 2010). Interestingly enough, LMP2A increases the size of the stem-like cell population and the number of tumor initial cells; this effect being reversed by inhibitors of AKT.
In addition to a classical effect on intracellular signaling, EBV also down regulates expression of miR-200a and miR-200b, the down regulation of which induces EMT (Shinozaki, A., et al., 2010). First, the authors demonstrated an association between miR-200a and miR-200b down regulation and E-cadherin expression on resected gastric carcinoma tissue. Further, using in vitro established EBV-infected cell lines they confirmed that down regulation of these miRs correlates with up regulation the ZEB family of transcription factors and their associated loss of cell-to-cell adhesion. Finally they uncovered the ability of LMP2A, EBNA1 and BARF0 to down regulate the pri-miR-200 transcript.
EBV is found in alveolar epithelial cells where it is suspected to promote idiopathic pulmonary fibrosis. Indeed, active EVB infection regulates EMT in alveolar epithelial cells (Malizia, A. P., et al., 2009). In this report the authors highlighted the role of Wnt signaling, since Wnt5Bsilenced cells are resistant to EBV-induced EMT. Further, using an ex vivo cell system model the authors demonstrated that activation of non-canonical Wnt signaling pathway by EBV is dependent of CUX1 signaling. Therefore a link between EBV and fibrosis was demonstrated with EMT being the core of the process. This former observation was recently confirmed and extended. Indeed, the group of Lasky demonstrated that LMP1 induces pro-EMT signaling that occurs primarily through the nuclear factor-κB pathway and secondarily through the extracellular signal-regulated kinase (ERK) pathway (Sides, M. D., et al., 2011).

Hepatitis B and C viruses
At least seven different viruses cause hepatitis, hepatitis viruses A, B and C are the most known. Whereas hepatitis virus A (HAV) induces acute infection disease of the liver, HBV www.intechopen.com and HCV induce more chronic diseases that can lead to cirrhosis and hepatocellular carcinoma. Both HBV and HCV have been shown to induce EMT. Viral particles of mammalian HBV encode for a small regulatory protein, known as the X protein that modulates intracellular signaling pathways by directly or indirectly interacting with host factors. Therefore it was hypothesized that HBV X protein may induce EMT in hepatocytes. To test this hypothesis Yang and coauthors transfected hepatocytes with HBx gene and observed that cells underwent morphological changes from an epithelial morphology to spindle-like shape associated with an increase in invasive potential (Yang, S. Z., et al., 2009). When the authors treated the cells with PP2, a well-known inhibitor of the Src kinase family, they noticed that cells recovered their original epithelial morphology. Therefore, they claimed that activated c-Src played a critical role in the HBx-induced EMT of hepatocytes.
HCV core protein which interacts with various cellular proteins induces host cells responses (Delhem, N., et al., 2001, Lai, M. M. & Ware, C. F., 2000, Zhu, N., et al., 1998. Of particular interest, HCV core protein interacts with Smad3 and consequently inhibits TGF-induced Smad3 transcriptional activity (Pavio, N., et al., 2005). Since the TGF-/Smad3 pathway induces EMT, it was suspected that HCV core protein directly impacts on the EMT process. Using stably transfected cell lines and primary mouse hepatocytes, as well as primary human hepatocytes infected in vitro with lentiviruses encoding HCV core protein, Battaglia and coauthors demonstrated that core protein expression was sufficient to provoke EMT in primary hepatocytes. This effect was reverted by addition of a specific inhibitor of TGF-I receptor thus demonstrating a TGF-dependent effect of core on EMT development (Battaglia, S., et al., 2009).
HCV core protein has also been involved in the pathogenesis of cholangiocarcinoma. In agreement with this idea, HCV core protein expression in cholangiocarcinoma cells induces EMT through a mechanism dependent on LOXL2 pathway (Li, T., et al., 2010).

Perspective: EMT and microbial pathogenesis
The field of research encompassing EMT has been one of the most exciting areas in embryogenesis, organ development, wound repair and tissue remodeling over the past 10 years. This overview is by no means intended to provide a global view on EMT. Instead, as shown in Figure 3, we have attempted to depict the main lines which govern EMT in order to highlight similarities that exist between growth factor-and pathogens-induced signaling pathways allowing us to give a coherent picture of the place of microbial infection in EMT and subsequent human pathologies. However, it is important to note that in healthy individuals, infection is effectively controlled, and the inflammatory response is promptly resolved. Indeed, microbes-induced chronic inflammation is intimately linked to defective innate immunity correlating with microenvironment, genetic and epigenetic susceptibilities but also treatment access. For example, H. pylori colonize the human stomach of about 50% of the world's population, however less than 2% of this population will develop a stomack cancer, implying the existence of individual predispositon.
Interestingly, it appears that only pathogens associated to chronic pathologies (fibrinogenesis, cancer) (Hofman, P. M., 2010) have been described to induce EMT. Given that all pathogen recognition receptors induce NF-κB and MAPK module, one can speculate www.intechopen.com that each pathogen may have the potential to induce EMT as for as its attack remains unresolved by innate immunity. Keeping that in mind, we can assume that a large part of EMT knowledge can be moved to translational research in molecular medicine with potential future new therapeutics in treating diseases linked to infections.

Fig. 3. Microbe-induced chronic inflammation in predisposed individuals leads to EMT
Here we suggest a model in which microbe infection plays a critical role as an EMT promoter. In healthy individuals, microbe infection is contained by the innate immunity. By contrast in predisposed individuals the innate immunity is exceeded by microbe infection leading to chronic inflammation. Chronic inflammation, associated to chronic infection lead to sustained NF-κB and MAPK module activation: the basement of EMT. Finally, EMT plays a critical role in onset of various human pathologies such as fibrinogenesis, cancer progression and metastasis.

Acknowledgment
We thank L. Bianchini for her critical feedback and thoughtful suggestions. We apologize to those investigators whose experimental work has not been cited or cited indirectly owing to space limitations. Experimental work was supported by the «Institut National de la Santé et de la Recherche Médicale», the « Centre National de la Recherche Scientifique », the « Ministère de l'Education, de la Recherche et de la Technologie », and by grants from « Association pour la Recherche sur le Cancer » (ARC, subvention 1142), « Association François Aupetit » and « Fondation Infectiopole Sud ».