Inhibition of chlamydia trachomatis growth by human interferon-α : mechanisms and synergistic effect with interferon-γ and tumor necrosis factor-α

We have evaluated the effect of natural human interferon (IFN)-α on the growth of chlamydia trachomatis in human epithelial cells in vitro and revealed that IFN-α has reduced both growth and infectivity of C. trachomatis. The effect of IFN-α was reversed by the addition of exogenous L-tryptophan and iron to the culture medium, suggesting that antichlamydial effect of IFN-α was caused by depletion of intracellular tryptophan and iron, both of which are essential for chlamydial growth. When IFN-α was combined with another antichlamydial cytokines, IFN-γ and tumor necrosis factor (TNF)-α, the effect was synergistically enhanced. Therefore, IFN-α would act coordinately with other cytokines such as IFN-γ and TNF-α, and play an important role in host defense against infection and in the establishment of persistent chlamydial infection of host, in which the organism remains viable, but in a culture-negative state. Chlamydia trachomatis is an obligate intracellular bacterium, which causes a wide spectrum of human diseases and is one of the most common sexually transmitted pathogens in the world. Upper genital tract infections with C. trachomatis in woman tend to have very serious repercussions, including the onset of pelvic inflammatory disease. This syndrome often results in severe and irreversible sequelae, such as infertility or ectopic pregnancy (5, 13). Various cytokines have been demonstrated to restrict the growth of intracellular pathogens and are significant activators of host cell immune responses to infections. Interferon (IFN)-γ is required for resolution of chlamydial infections, and has been shown to inhibit the growth of chlamydia in cell culture (22). The mechanisms of the action of IFN-γ in cultured human cell lines have been elucidated and involve the induction of a cellular enzyme, indoleamine 2,3-dioxygenase (IDO), which causes a depletion of intracellular tryptophan. In addition to IFN-γ, two other cytokines such as tumor necrosis factor (TNF)-γ and interleukin (IL)-1β have been shown to play a role in host defense against chlamydial infection (3, 24). Furthermore, it has been reported that these cytokines exerts the inhibitory effect synergistically when combined with each other (26). IFN-α is a well recognized antiviral cytokine, in addition, with antiproliferative and immunomodulatory properties (1, 23). Beside its protective role against viral infection, IFN-α has been implicated in innate immunity against a variety of facultative and obligate intracellular non-viral pathogens such as Leishmania donovani and Txoplasma gondii (14, 16). Early works reported that IFN, reported to be IFN-α/β, showed to inhibit chlamydial growth (6, 20). However, the mechanisms of the action of IFN-α, and the interaction with other cytokines have not been intensively evaluated. In this study, we examined the antichlamydial activity of natural IFN-α and evaluated the mechanisms of its action. Furthermore, since IFN-α is known to act synergistically with other cytokines Address correspondence to: Dr. Miho Aga, Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama 702-8006, Japan Tel: +81-86-276-3141, Fax: +81-86-276-6885 E-mail: tariyasu@hayashibara.co.jp T. Ishihara et al. 180 popolysaccharide (LPS) antibody (1/50 dilution) (Denka Seiken Co., Ltd., Tokyo, Japan) was added to each well and the plates were incubated at room temperature for 2 h. After washing three times, 50 μL of β-galactosidase-conjugated anti-mouse IgG antibody (1/2000 dilution) (American Qualex International, Inc., San Clemente, CA) was added to each well and the plates were incubated at room temperature for 2 h. The wells were washed three times and 100 μL of 10 mM phosphate buffer (pH 7.8) containing 0.1 mg/mL 4-methyl-umbelliferyl β-D-galactopyranoside (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and 1 mM MgCl2 were added. After incubation at 37°C for 2 h, the fluorescence intensities (excition at 355 nm, emission at 460 nm) were measured. Assessment of chlamydia infectivity. HeLa cells were plated in 24 well culture plates at 1 × 10 cells/well and cultured for 24 h. Then, cells were treated with various doses of cytokines for 24 h and were infected with C. trachomatis to develop 5 × 10 inclusions per well. Forty-eight hours later, cells were washed once with medium, scraped into SPG and sonicated briefly. Serial dilutions of these suspensions were inoculated on fresh HeLa cell monolayer in 96 well microtiter plates as described above, and then incubated in the medium with 2 μg/mL cycloheximide (Wako Pure Chemical Industries) for 48 h. Cells were fixed with methanol and the number of inclusions was assessed by staining with FITC-labeled mouse anti-chlamydia antibody (Denka Seiken). Statistical analysis. Results were analyzed by Student’s unpaired t-test and a p value of < 0.05 was taken to be significant. The 50% inhibitory concentrations (IC50) were estimated by probit analysis. To assess the synergistic interaction, the combination index (CI) was calculated by median effect analysis using CalcuSyn Software (Biosoft Inc., Cambridge, U. K.), a computer software for automated dose-effect analysis (4). CalcuSyn analyzes the shape of the drug does response curves for each drug or combination of drugs and calculates the combination index (CI) by median effect analyze described by Chou. CI values of < 0.9, 0.9–1.1, and > 1.1 indicate synergism, additive effect and antagonism, respectively.