Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 22 Dec 2018 14:51:28 Chlamydia trachomatis induces an upregulation of molecular biomarkers podoplanin, Wilms’ tumour gene 1, osteopontin and inflammatory cytokines in human mesothelial cells Anna De Filippis, 1 Elisabetta Buommino, 1 Marina Di Domenico, 2 Antonia Feola, 2 Raffaella Brunetti-Pierri 3 and Antonietta Rizzo 1, * Abstract Chlamydia trachomatis is the most prevalent infection of the genital tract in women worldwide. C. trachomatis has a tendency to cause persistent infection and induce a state of chronic inflammation, which has been reported to play a role in carcinogenesis. We report that persistent C. trachomatis infection increases the expression of inflammatory tumour cytokines and upregulates molecular biomarkers such as podoplanin, Wilms’ tumour gene 1 and osteopontin in primary cultures of mesothelial cells (Mes1) and human mesothelioma cells (NCI). Infection experiments showed that Mes1 and NCI supported the growth of C. trachomatis in vitro, and at an m.o.i. of 4, the inclusion-forming units/cell showed many intracellular inclusion bodies after 3 days of infection. However, after 7 days of incubation, increased proliferative and invasive activity was also observed in Mes1 cells, which was more evident after 14 days of incubation. ELISA analysis revealed an increase in vascular endothelial growth factor, IL-6, IL-8, and TNF-a release in Mes1 cells infected for a longer period (14 days). Finally, real-time PCR analysis revealed a strong induction of podoplanin, Wilms’ tumour gene 1 and osteopontin gene expression in infected Mes1 cells. The aim of the present study was to investigate the inflammatory response elicited by C. trachomatis persistent infection and the role played by inflammation in cell proliferation, secretion of proinflammatory cytokines and molecular biomarkers of cancer. The results of this study suggest that increased molecular biomarkers of cancer by persistent inflammation from C. trachomatis infection might support cellular transformation, thus increasing the risk of cancer. INTRODUCTION Cancer continues to be the leading cause of death in developed countries. The role of bacteria as mediators of oncogenesis is an intriguing area of research, but how they influence disease development in the human host is poorly elucidated. Specific bacteria species can cause cancer through different and com- plex mechanisms. For some bacterial species this relationship is well established, whereas for other pathogens it is somewhat weak. An important common characteristic is the time inter- val between bacterial infection and cancer development [1]. Several bacteria can cause chronic infections or produce toxins that disturb the cell cycle and result in altered cell growth or DNA damage. Deficiencies in DNA-damage signalling and repair pathways lead to genetic instability, which in turn might enhance oncogenesis [2]. Chlamydia trachomatis, an obligate intracellular Gram-neg- ative bacterium, exhibits a biphasic life cycle: metabolically inactive elementary bodies (EBs) infect host cells and differ- entiate into metabolically active, replicating reticulate bodies (RBs) within a membrane-bound vacuole, which is called inclusion. C. trachomatis has a tendency to cause persistent infections that are usually asymptomatic but can lead to genital infections in women and generate cervicitis, cervical erosion, pelvic inflammatory disease, fallopian tube scarring and infertility [3]. In recent epidemiological studies, Chla- mydia infection has been linked to an increased risk of cer- vical cancer [4]. It has been suggested that cells infected by Chlamydia have a much higher risk of neoplastic transfor- mation because the pathogen is able to cause persistent inflammation and tissue damage and inhibit apoptosis in Received 30 August 2016; Accepted 22 March 2017 Author affiliations: 1 Department of Experimental Medicine, Section of Microbiology and Clinical Microbiology, Faculty of Medicine and Surgery - Second University of Naples, Via Santa Maria di Costantinopoli, 16 - 80138 Naples, Italy; 2 Department of Biochemistry, Biophysics and General Pathology - Second University of Naples, Via Santa Maria di Costantinopoli, 16 - 80138 Naples, Italy; 3 Multidisciplinary Department of Medical- Surgical and Dental Specialties - Second University of Naples, Via Pansini, 5 - 80131 Naples, Italy. *Correspondence: Antonietta Rizzo, antonietta.rizzo@unina2.it Keywords: cytokines; podoplanin; Wilms’ tumour gene 1; osteopontin. Abbreviations: EB, elementary body; ELISA, enzyme linked immunosorbent assay; IFU, inclusion-forming unit; MTT, methyltetrazolium; OPN, osteo- pontin; PL, podoplanin; RB, reticulate body; VEGF, vascular endothelial growth factor. RESEARCH ARTICLE Filippis et al., Microbiology 2017;163:654–663 DOI 10.1099/mic.0.000465 000465 ã 2017 The Authors 654