Chlamydia trachomatis infection results in a modest pro-inflammatory cytokine response and a decrease in T cell chemokine secretion in human polarized endocervical epithelial cells

Highlights

• A polarized endocervical epithelial cell model was infected with Chlamydia trachomatis.

• C. trachomatis infection failed to yield a robust pro-inflammatory cytokine response.

• Constitutive RANTES and IP10 were decreased by C. trachomatis infection.

• C. trachomatis infected primary epithelial cells yielded similar cytokine responses.

• Results suggest C. trachomatis uses evasive strategies to survive in the endocervix.

Abstract

The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72 h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120 h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion strategies, together with the inherent immune repertoire of endocervical epithelial cells, may aid chlamydiae in establishing, and possibly sustaining, an intracellular niche in microenvironments of the endocervix in vivo.

Introduction

Genital serovariants (D-K) of the obligate intracellular bacterium Chlamydia trachomatis are the world’s most common sexually transmitted bacterial pathogens, accounting for an estimated 90 million new cases reported annually [1]. C. trachomatis exhibits a tropism for the columnar epithelial cells of the genital mucosae, with the endocervix being the most commonly infected site in women. In a proportion of infected women, organisms also ascend into the endometrium and Fallopian tubes where chronic infection can lead to devastating reproductive consequences, including pelvic inflammatory disease (PID), tubal infertility, and ectopic pregnancy, all of which result from immune mediated damage [1]. The reason why C. trachomatis can cause extended infections, lasting months to years in the face of an immune response [2], [3], [4], [5], [6], is not well understood, but does suggest the organism can adapt to, or evade, elements of the local host immune response.

Chlamydiae have a biphasic developmental cycle that begins when non-metabolically active, infectious, elementary bodies (EBs) encounter the apical surface of polarized epithelial cells. Following entry into the host cell, EBs escape lysosomal fusion, and endosomes containing EBs fuse to form the membrane bound vacuole termed an inclusion. EBs differentiate into metabolically active, non-infectious reticulate bodies (RBs) that undergo DNA replication and binary fission. RBs then re-differentiate into EBs that may then escape the host cell through lysis or extrusion mechanisms [7], [8].

Traditional methods for culturing C. trachomatis in vitro utilize either murine fibroblast cell lines or the ectocervix derived cervical carcinoma cell line (HeLa). Recent studies, however, have highlighted the importance of the cell type in which chlamydiae are grown, as cell lines derived from different anatomical sites yield different growth rates and infectious yields [9], [10]. Neither HeLa cells nor murine fibroblast cells accurately represent the target cells in vivo, the polarized columnar epithelial cells, with respect to morphology, expression of innate immune mediators or responsiveness to TLR agonists [11], [12]. Furthermore, innate immune signaling pathways have been altered over the years during the time that HeLa cells have been grown in vitro [13], [14]. Alterations in innate immune pathways are significant, as it has been hypothesized that the epithelial cells, generally believed to be the only sites of chlamydial replication in the female reproductive tract (FRT), are responsible for eliciting and sustaining the inflammatory immune cascade associated with chlamydial disease via the release of intracellular IL1α upon chlamydiae induced host cell lysis [15], [16]. Therefore, traditional cell lines may not be the optimal model systems in which to investigate the innate immune cascade elicited by C. trachomatis infected epithelial cells.

In recent years the orientation of the cells used to culture chlamydiae has been shown to influence chlamydial biology. Columnar epithelial cells, the target cells for chlamydial infection, maintain functionally distinct apical and basolateral membrane domains that are separated by tight junctions. Epithelial cells grown in a polarized orientation contain greater nutrient pools that are important for chlamydial growth, such as tryptophan, than their traditionally-grown submerged cell counterparts grown on plastic surfaces [17]. The use of polarized epithelial cell culture models for chlamydial studies, pioneered by Wyrick, has also revealed differences in chlamydial entry and exit mechanisms, infectious progeny, duration of the developmental cycle, infectivity, duration of the persistent growth state, reactivity to antibiotics, responsiveness to female sex steroid hormones, and innate inflammatory responses (reviewed in [18]). All of these parameters of chlamydial biology may also influence the subsequent innate epithelial immune response to the bacteria as well, although this has not yet been investigated in more primary-like genital epithelial cells.

We recently developed an epithelial cell model derived from human endocervical tissue (A2EN cells). A2EN cells polarize and appropriately express many of the functional proteins of the endocervical epithelium in vivo such as hormone receptors, mucins, anti-microbial peptides, and cytokines [11]. The aim of this study was to determine the characteristics of, and the cytokine response to, C. trachomatis infection in polarized A2EN (polA2EN) cells in order to examine the potential role of the endocervical epithelium in initiating, sustaining and amplifying a proinflammatory immune response to this organism.