PLoS One. 2015 Dec 17;10(12):e0145198. doi: 10.1371/journal.pone.0145198.  

Role of Epithelial-Mesenchyme Transition in Chlamydia Pathogenesis.
 

Igietseme JU, Omosun Y, Stuchlik O, Reed MS, Partin J, He Q, Joseph K, Ellerson D, Bollweg B, George Z, Eko FO, Bandea C, Liu H, Yang G, Shieh WJ, Pohl J, Karem K, Black CM.

1National Center for Emerging Zoonotic and Infectious Diseases, Centers for Disease Control & Prevention (CDC) Atlanta, GA 30333 USA. 2Department of Microbiology, Biochemistry & Immunology, Morehouse School of Medicine, Atlanta, GA 30310 USA.

 

Abstract

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.

PMID: 26681200. PMCID: PMC4683008.

 

Supplement

Genital infection by Chlamydia trachomatis is a major public health concern because it is the most common bacterial sexually transmitted disease (STD) in the United States and several other industrialized nations. An estimated $2.5 billion is spent annually on more than 4 million reported cases of the genial infections in the United States alone. In women, the complications of genital chlamydial infection include pelvic inflammatory disease (PID), fibrosis, ectopic pregnancy and tubal factor infertility. Genital C. trachomatis infection is also a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma [1] (Figure 1). There are increasing threats of resistant chlamydial strains emerging globally, although there are still some effective antibiotics to treat diagnosed cases. However, the rampant asymptomatic infections in women (over 60%) often make the manifestation of complications the first evidence of infection. Therefore, since there is no human vaccine, a better understanding of the molecular pathogenesis of chlamydial complications in women and its role as a co-factor in HPV-related cervical carcinoma is crucial for developing new therapies and design efficacious vaccines to control chlamydia in the human population.

 

fig1Figure 1. Reproductive cancer complications of genital chlamydial infection 

 

Recent reports showed that chlamydial genital infection caused significant alterations in host regulatory micro-RNA (miRNA) expression profiles in the female reproductive tract. The alteration in miRNA profiles involved the activation of specific caspases that target Dicer [2], a ribonuclease III critical in the biogenesis of miRNAs and siRNAs [3]. Interestingly, the miRNAs altered during chlamydial infection regulate epithelial functional integrity required for secretory, barrier and cellular homeostasis [4]. This suggested that chlamydia might induce epithelial-mesenchyme transition (EMT) [4, 5] that altered epithelial state and adversely affected fertility-related reproductive tract epithelial functions and might also promote epithelial cell transformation and/or tumor progression. EMT is an important miRNA-regulated biological process that converts normal polarized, cobble-stone-like epithelial cells into fibroblastic (elongated) mesenchyme cells and consequently alters epithelial integrity and functions. Mesenchymal cells exhibit enhanced motility and migratory capacity, invasive capability, higher resistance to senescence and apoptosis, and increased production of extracellular matrix (ECM) components. EMT represents an important phase in development, differentiation, inflammation, fibrogenesis and tumorigenesis. It is developmental EMT when it drives cellular differentiation for specialized tissue formation and organogenesis during embryogenesis and development; however, it is pathologic EMT when it promotes the production of excess extracellular matrix (ECM)-producing myofibroblasts from epithelial cells during fibrosis that exceeds tissue repair or wound healing after injury; when it facilitates the secretion of excess pro-inflammatory molecules by epithelial cells during inflammation; and when it promotes the initiation of the invasive and metastatic behavior of epithelial cancers. For example, EMT drives several organ and tissue fibrotic diseases, including pulmonary, renal, and hepatic fibrosis [5]; it converts normal epithelial parenchymal tissue architecture into the non-functional scar tissues of fibrosis; and it alters the normal differentiated functions of epithelial cells (i.e., secretory, barrier and transport) that compromise epithelial physiology. EMT inducers include growth factors, hormones (e.g., estrogen) and proinflammatory cytokines (TNF-alpha and TGF-beta) and their receptors; these EMT inducers and receptors activate intracellular signaling pathways (e.g., the Smad, PI3K/ERK and the Wnt/beta-catenin pathways) that disrupt the fine miRNA regulatory processes maintaining the balance of E-cadherin on epithelial cells through key transcription factors (TFs) [4, 5]. The three families of TFs that are the principal mediators of EMT are: SNAIL1/2 (i.e., SNAIL1 and SLUG), TWIST, and zinc-finger E-box-binding, ZEB1; these TFs target the repression of epithelial cadherin and other normal epithelial cell markers such as beta-catenin and connexin proteins that are components of adherens, tight and gap junctions, causing loss of adherens junctions and cell-cell adhesion. Loss of E-cadherin and expression of T-/N-cadherin is the hallmark of EMT. Mesenchymal cell markers control epithelial cell-cell adhesion (N- and/or T-cadherin), regulate cell motility (vimentin, an intermediate filament protein), cell growth and migration (fibronectin 1) and attachment to ECM (matrix metalloproteinases)(Figure 2). Thus, mesenchymal state is caused by the reorganization of cellular cytoskeletal architecture leading to loss of epithelial tight junctions and apical-basolateral polarity, acquisition of a front-rear polarity, changes in cell shape with increased protrusions, and greater cellular motility for an invasive capability [4, 5]. The key miRNA signature profile of EMT is the down-regulation of members of the miR-200 family, especially miR-429, which normally exerts suppressive function on negative regulators of E-cadherin (e.g., ZEB1, TWIST and SNAIL1/2). In addition, there is the down-regulation of tumor suppressors, such as p53, and upregulation of a number of tumor promoters that involved in tumorigenesis and metastasis.

 

 

fig2Figure 2. Epithelial-mesenchyme transition 

 

Certain EMT-inducers such as proinflammatory cytokines are also key mediators of endometritis, salpingitis, tubal fibrosis and other inflammatory sequelae of genital chlamydial infection [6]; however, the involvement of miRNA-driven EMT in the pathologic processes that culminates in reproductive fertility-related complications was previously unknown. Besides, the contribution of infection-induced EMT to fibrosis or invasive and metastatic carcinoma had not been studied; however, chlamydia induces fibrosis, and is a strong co-factor for HPV-related cervical carcinoma [1], although the molecular basis has been an enigma. Our study investigated the role of EMT in the pathogenesis of reproductive epithelial dysfunction and tubal fibrosis associated with genital chlamydial disease, as well as the co-factor function in promoting HPV-related cervical carcinoma.

We tested the hypothesis that the molecular basis of chlamydia-induced infertility and chlamydia as a co-factor for HPV-induced cervical epithelial carcinoma is the induction of EMT. The result indicated that Chlamydia induced pathologic EMT, which was characterized by caspase activation, Dicer cleavage inactivation, and alterations in expression profiles of key miRNAs and transcription factors that regulate EMT, epithelial functional integrity, fibrosis and tumor promotion. EMT-induced fibrosis and compromised reproductive tract epithelium led to infertility possibly due to a dysfunction fallopian tube that could not convey the embryo to the uterus, unable to support the viability of the embryo or implantation abnormalities (a major cause of ectopic pregnancy); at the same time, we proposed that the co-factor role of Chlamydia in cervical carcinoma is the ability of EMT to convert quiescent HPV-transformed epithelial cells into invasive and metastatic tumor-forming stem cells that are responsible for cervical carcinomas (Figure 3). Clinico-pathologic studies indicate that the acquisition of invasive and metastatic characteristics by transformed cells involves the induction of EMT, the down-regulation of tumor suppressors such as p53, and the maintenance and expansion of cancer stem cells, CSCs [5].  Thus, EMT-inducing agents are co-factors in the development and progression of several cancers. Our working hypothesis is that EMT-inducing agents, including cytokines, estrogen and chlamydia, are co-factors for HPV-related invasive cervical carcinoma.

Cervical cancer is the most common malignancy in females worldwide and a leading cause of mortality among gynecological cancers. Oncogenic types of HPV (e.g., types 16 and 18) are important in the development of precursors of cervical cancer [5] but the fact that only a fraction of HPV-infected females develop the cervical carcinoma has prompted the search for co-factors in the progression to invasive cervical cancer [1]. Our results are consistent with the hypothesis that chlamydial induction of EMT during co-infection with HPV provides the pathophysiological basis for the cofactor role in HPV-induced cervical carcinoma. An ongoing study is using the HPV 16 E6/7 transgenic mouse model that requires estrogen as co-factor for accelerated development of invasive cervical carcinoma [7, 8], to determine whether chronic genital chlamydial infection can replace estrogen in disease pathogenesis.

Our hypothesis is further validated by our results showing that EMT inhibitors that target caspases also protected against Chlamydia-inducted complications, specifically tubal inflammation, fibrosis and infertility. Thus, the results implicate EMT as a central pathogenic event that drives the complications of genital chlamydial infection. Figure 3 illustrates the pathway and molecular events by which chlamydial infection may lead to infertility or tumor promotion via EMT induction.

The importance of this study

Our results demonstrated that the molecular pathogenesis of the complications of genital chlamydial infection and as a co-factor in HPV-associated cervical epithelial carcinoma is the induction of pathologic EMT that alters fertility-related epithelial functions, promotes fibrosis and tumorigenesis. Thus, EMT induction by chlamydia and the consequent alteration of epithelial functional integrity may provide a molecular basis for reproductive infertility associated with genital chlamydial infection, and the co-factor role of chlamydia in HPV-induced cervical epithelial neoplasia and invasive carcinoma. Besides, while evidence of EMT has been observed in the induction or progression of several tumors, our finding may represent the first evidence of a direct relationship between a co-factor and EMT. Thus, these findings provide a novel understanding of the molecular pathogenesis of chlamydia-induced infertility and its co-factor role in cervical carcinoma, which may guide to a rational strategy to prevent complications.

These findings open the possibility of using human compatible safe EMT inhibitors to prevent the complications of genital chlamydial infection and to control carcinomas progression, tumor expansion and metastasis in HPV-exposed individuals who are at risk for cervical carcinoma.  Thus, the results may opened up avenues to target specific molecules and pathways to control chlamydial complications in exposed unprotected individuals. Targeting EMT and especially the components of the signaling pathways of inducers with small molecule inhibitors such as antagomirs, siRNA- or anti-sense oligonucleotide, miRNA sponges, or antibodies and chemical inhibitors, represents an important therapeutic strategy for the clinical management of fibrosis and cancers [5]. Conversely, synthetic miRNAs administration can be used as a replacement therapy for EMT-/tumor-suppressor miRNAs that are commonly down-regulated in fibrosis and cancers [9]. Our present study may add a therapeutic option of targeting the caspases to control EMT and the pathophysiologic consequences. In addition, our findings may lead to the emergence of a diagnostic approach to using EMT molecules as biomarkers of progressing cervical carcinoma in HPV-positive individuals.

 

 

fig3

Figure 3. Summary pathway of the cellular and molecular events by which chlamydial infection may lead to infertility or tumor promotion via EMT induction

 

Acknowledgements: This study was supported by the National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention and the National Institutes of Health Grant #s: AI41231, GM 08248, RR03034, 1SC1GM098197 and 1SC2HD086066-01A1.

Contact: Dr Joseph U. Igietseme, Professor of Microbiology, Biochemistry and Immunology, Chief, Molecular Pathogenesis Laboratory, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), 1600 Clifton Road, MailStop G-36, Atlanta, GA 30333 USA. Tel: 404-639-3352. Fax: 404-718-4011. Email: jigietseme@cdc.gov

 

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