Chlamydia trachomatis infection increases the expression of inflammatory tumorigenic cytokines and chemokines as well as components of the Toll-like receptor and NF-κB pathways in human prostate epithelial cells

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Abstract

Inflammation has been reported to play a major role in prostate carcinogenesis. Several bacterial infections can lead to prostate inflammation; however, until now, the precise molecular and cellular mechanisms linking inflammation to carcinogenesis have remained unclear. We therefore investigated the initiation of inflammation induced by Chlamydia trachomatis (C. trachomatis) infection in human prostate epithelial cells using an in vitro culture system in which human androgen-independent PC-3 prostate cancer epithelial cells were infected with C. trachomatis serovar L2. The expression levels of VEGF, ICAM-1, IL-6, IL-8, IL-1β, TNFα, CCL5, CCL2 and iNOS inflammation-related genes, as well as genes involved in the Toll-like receptor (TLR) pathway (TLR2, TLR4, CD14 and MyD88), were evaluated at the mRNA level in infected PC-3 cells 24 h after infection with C. trachomatis serovar L2. The expression levels of components of the NF-κB pathway (p65 and IκBα) were evaluated at the mRNA level in infected PC-3 cells at different time points (1, 6, 12 and 24 h) after infection. The expression levels of inflammation-related genes, components of the Toll-like receptor pathway and genes involved in NF-κB activation were analyzed in infected and uninfected cells using semi-quantitative RT-PCR. We detected a significant increase (p < 0.001) in inflammation-related cytokines in infected PC-3 cells. During infection, PC-3 cells elicited a proinflammatory response, as shown by NF-κB activation, TLR2 and TLR4 upregulation and the increased expression of inflammation-related genes. Furthermore, we observed significant upregulation of the adhesion molecules ICAM-1 and VEGF, which are two biomarkers correlated with tumor progression and immune system evasion. The present study suggests that human prostate cancer epithelial cells are susceptible to C. trachomatis infection and upregulate proinflammatory markers during infection.

Introduction

Prostate cancer (PCa) is the most common malignancy in older men. Worldwide, more than 670,000 men are diagnosed with prostate cancer each year [1]. In Tunisia, PCa is the fourth commonest cancer in men [2].

The pathogenesis of PCa, as well as other cancers, involves not only heritable but also environmental factors [3]. Among these environmental factors, chronic inflammation has been postulated to be an important driving force toward the development of prostate carcinoma [4]. Chronic inflammation is implicated in the development of a diverse range of human cancers, including cancer of the esophagus, lungs, pancreas and stomach [4]. Epidemiological studies have shown a significant association between infection/inflammation and prostate carcinoma [5]. Moreover, men with a previous history of particular sexually transmitted infections also have a higher incidence of PCa than age-matched controls [6]. Therefore, knowledge about the intrinsic interplay between microbes and urogenital cells is central our understanding of the involvement of microbial pathogens in the development of PCa.

Epithelial cells in the human genital tract can secrete different cytokines and chemokines in response to abnormal conditions, such as infections. However, some of these epithelial surfaces also secrete certain cytokines under normal conditions. It has been suggested that epithelial cells can act as an early alarm system by creating a network connected to local and distant cells in the host that mount a coordinated inflammatory response [7], [8]. C. trachomatis is the most common agent of bacterial sexually transmitted disease in the world [9]. C. trachomatis is an obligate intracellular gram-negative bacterium, which, similar to all chlamydial species, has a tendency to cause chronic persistent infections. Despite the similar prevalence of Chlamydia infection in human males and females and the accepted role for Chlamydia in the development of male urethritis, epididymitis, orchitis and prostatitis, research focusing on the pathogenic mechanisms involved in male genitourinary tract infections is still very limited [10], [11].

Chronic inflammation and inflammatory damage to infected tissues are a primary source of pathology and disease from infection by numerous pathogens such as Chlamydia [12]. Infection with C. trachomatis can induce inflammatory cytokine production in both in vivo and in vitro models [13], [14]. Proinflammatory cytokines and chemokines produced by infected epithelial cells has been suspected to initiate and potentiate chronic inflammation associated with chlamydial diseases [12].

The effect of Chlamydia muridarum, a pathogen closely related to C. trachomatis that is often used for murine infections, on a murine prostate epithelial cell line was previously studied. Murine prostate epithelial cells were susceptible to Chlamydia infection and responded by upregulating inflammatory mediators via NF-κB activation. TLR2 and TLR4 were recruited to the chlamydial inclusion, suggesting an active role for these receptors in recognition of the bacterium and activation of prostate epithelial cells [15], [16], [17].

Given the association between infection, inflammation and prostate carcinoma as well as the difference between mice and human infection models and Chlamydia serovars, there is a need for well characterized models to study the inflammatory response elicited by human prostate cancer epithelial cells during C. trachomatis infection and the effects of this inflammatory response on cell proliferation and carcinogenesis.

In this study, we present an in vitro model to elucidate the inflammatory response of human androgen-independent PC-3 prostate cancer epithelial cells during infection with C. trachomatis serovar L2.

Section snippets

Cell culture

PC-3, a human prostate cancer cell line (CRL-1435), was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and 50 mg/ml gentamicin at 37 °C in a humidified incubator containing 5% CO2 in air.

Chlamydial stocks

The C. trachomatis LGV/L2 serovar was grown in Mycoplasma-free McCoy cells. Elementary bodies (EBs) were harvested from infected

Susceptibility of PC-3 cells to C. trachomatis infection

Infection of PC-3 cells with C. trachomatis serovar L2 resulted in the formation of inclusions, as detected by staining with an anti-cLPS Chlamydia antibody followed by fluorescence microscopy 48 h after infection. The infection rates ranged from 60 to 80% at MOIs of 15:1 (Fig. 1), and the MOI was measured using McCoy cells.

Moreover, C. trachomatis was able to complete its biphasic developmental cycle, as supernatants from infected PC-3 cells could re-infect highly susceptible epithelial cell

Discussion

Previously, it was shown that C. muridarum could infect and survive in a rat prostate adenocarcinoma cell line [15]. However, the ability of C. trachomatis serovar L2 to infect human prostate adenocarcinomas and to induce inflammatory cytokines has not yet been examined. In the present study, we examined whether C. trachomatis serovar L2 can multiply in the human prostate adenocarcinoma cell line, PC-3. This lymphogranuloma strain is known to be very invasive and cause chronic inflammatory

Acknowledgments

This work is part of a doctoral thesis by Hanen SELLAMI. We would like to thank the Ministry of Higher Education and Scientific Research, Tunisia, and the University of California, Merced, USA, for their financial support.

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