Cell Intrinsic Factors Modulate the Effects of IFNγ on the Development of Chlamydia trachomatis

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that cannot synthesize several amino acids, including tryptophan. Rather, C. trachomatis acquires these essential metabolites from its human host cell. Chlamydial dependence on host-provided tryptophan underlies a major host defense mechanism against the bacterium; namely, the induction of the host tryptophan-catabolizing enzyme, indoleamine 2,3- dioxygenase (IDO1) by interferon gamma (IFNγ), which leads to eradication of C. trachomatis by tryptophan starvation. For this reason, IFNγ is proposed to be the major host protective cytokine against genital C. trachomatis infections. The protective effect of IFNγ against C. trachomatis can be recapitulated in vitro using epithelial cell-lines such as the cervical carcinoma derived cell-line Hela, the Hela subclone HEp-2, and the cervical carcinoma derived cell-line ME180. Addition of IFNγ to these cells infected with C. trachomatis results in a strong bactericidal or bacteriostatic effect dependent on the concentration of IFNγ administered. Unlike Hela, HEp-2, and ME180, there are other human epithelial, or epithelial-like cell-lines where administration of IFNγ does not affect chlamydial replication, although they express the IFNγ receptor (IFNGR). In this report, we have characterized the mechanisms that underlie this dichotomy using the cell-lines C33A and 293. Akin to Hela, C33A is derived from a human cervical carcinoma, while 293 cells were produced by transfection of adenovirus type 5 DNA into embryonic kidney cells. We demonstrate that although IFNGR is expressed at high levels in C33A cells, its ligation by IFNγ does not result in STAT1 phosphorylation, an essential step for activation of the IDO1 promoter. Our results indicate that although the IFNγ-dependent signaling cascade is intact in 293 cells; the IDO1 promoter is not activated in these cells because it is epigenetically silenced, most likely by DNA methylation. Because polymorphisms in IFNγ, IFNGR, and the IDO1 promoter are known to affect other human infections or diseased states, our results indicate that the effect of allelic differences in these genes and the pathways they activate should be evaluated for their effect on C. trachomatis pathology.

Figure 1

The effect of IFNγ treatment on the development of Chlamydia trachomatis is cell-line dependent. Hela, A2EN, C33A and 293 cells were infected with C. trachomatis at an m.o.i. of 5 as described in the methods. After infection, cells were grown in RPMI media in the absence or presence (300 U/ml) of IFNγ. At 42 h.p.i. cells were fixed, permeabilized and stained using a FITC-conjugated antibody against chlamydial, and counter-stained with Hoechst dye. Infected cells were also harvested to measure IFU released by infection of Hela cells. A) Primary chlamydial inclusions (green) formed in Hela, A2EN, C33A and 293 cells 42 h.p.i. Hoechst dye (blue) shows cell nuclei and chlamydial DNA. Scale bar indicates 50 μm. B) IFU/ml released after 42 h.p.i. in Hela, A2EN, C33A and 293 cells as evaluated by infection of Hela cells. The mean and standard deviation from three independent experiments were represented as bars (** indicates p<0.01 by Wilcoxon Rank Sum Test).

Figure 2

IDO1 is induced in Hela and A2EN cells but not in C33A and 293 cells treated with IFNγ. Hela, A2EN, C33A and 293 cells were grown in the absence or presence of IFNγ. (600 U/ml) for 24 hours, after which, cells were harvested and lysates were used to evaluate expression of IDO1 by immunoblot using an antibody against IDO1. β-actin expression was evaluated as a loading control.

Figure 3

Surface IFNGR1 expression is observed in Hela, A2EN, C33A and 293 cells. Hela, A2EN, C33A and 293 cells were grown in the absence or presence of IFNγ (600 U/ml) After 24 hours, cells were lifted, counted, fixed but not permeabilized, and stained with antibody against IFNGR1. Surface expression of IFNGR1 was evaluated by flow cytometry (profile in green). Flow cytometry profiles obtained with the isotype control antibody are shown in grey.

Figure 4

IFNγ does not induce STAT1 phosphorylation in C33A cells. Hela, A2EN, C33A and 293 cells were plated in regular media, which was replaced with or without 600 U/ml of IFNγ 24 hours post-plating. After 24 hours of IFNγ treatment, cells were harvested and used to make extracts that were evaluated by immunoblot using antibodies against STAT1 and pSTAT1.

Figure 5

IFNγ induces the IDO1 promoter on an episomal reporter plasmid in 293 cells. A) A map of p616 reporter plasmid used to construct 293/p616 cells. p616 replicates as a stable episome in these cells. B) IFNγ (600 U/ml) was added to 293/p616 for 24 hours after which luciferase expression was evaluated. Expression in indicated as fold-over the expression level observed with untreated cells.

Figure 6

IDO1 promoter is epigenetically modified in 293 cells. 293 cells were grown in media containing 8 μM 5-azacytidine for 48 hours, after which cells were treated with 600 U/ml of IFNγ for 24 hours. Cells were harvested, counted and lysed and evaluated for expression of IDO1 by immunoblot. Immunoblots evaluating β-actin expression was used as a loading control. Lane 1: Untreated cells; Lane 2: Cells treated with 5-azacytidine alone; and Lane 3: Cells were treated with 5-azacytidine, followed by 600 U/ml of IFNγ.