Differences in innate immune responses (in vitro) to HeLa cells infected with nondisseminating serovar E and disseminating serovar L2 of Chlamydia trachomatis

Abstract

The inflammatory response associated with Chlamydia trachomatis genital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions between C. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants from C. trachomatis serovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-alpha) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1beta antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.

FIG. 1.

Protocol used for the coculture of HeLa cells with MdM prestimulated with supernatants from PMN incubated with C. trachomatis-infected HeLa cells. Briefly, 10⁶ PMN were added to the apical side of polarized HeLa cells infected with C. trachomatis for 36 h, and the cultures were reincubated for 12 h on a shaker (CULTURE 1). The apical medium was then collected and centrifuged to pellet the PMN. A portion of the supernatant was stored at −80°C (SUPERNATANT I), and the rest was diluted 1:1 in fresh RPMI before being added to MdM. After 48 h of stimulation, the macrophage culture supernatant was collected and stored at −80°C (SUPERNATANT II) and then replaced by 500 μl of fresh RPMI, and these stimulated MdM were next cocultivated with freshly inoculated cultures of polarized HeLa cells (CULTURE 2). At 48 hpi, basal supernatants from the cocultures were collected and stored at −80°C (SUPERNATANT III), and inclusion counts and progeny titration were performed on HeLa cells.

FIG. 2.

Semiquantitative analysis of IL-11 mRNA expression in C3H mice infected with C. trachomatis MoPn. The PCR products from murine IL-11 (413 bp) primers after 28 cycles of amplification (lanes 1 to 5) and from GAPDH (536 bp) primers after 18 cycles (lanes 6 to 10) were analyzed on a 2% agarose gel (A) and after Southern blotting with an internal probe (B). Then, 10-μl portions of sample were loaded onto the gels as follows: uninfected control mice (lanes 1 and 6), MoPn-infected mice at 48 hpi (lanes 2 and 7) and at 72 hpi (lanes 3 and 8), a negative control of amplification with no cDNA added to the reaction mix (lanes 4 and 9), and cDNA from uninfected McCoy cells used as positive control for the RT and PCRs (lanes 5 and 10).

FIG. 3.

Titration of C. trachomatis serovar E and L2 after 48 h of coculture of polarized infected HeLa cells with dTHP-1 cells. HeLa cells, seeded in inserts and infected with C. trachomatis, were cultivated in the presence of dTHP-1 cells. At 48 hpi, chlamydial inclusions were counted after fluorescent immunostaining (direct count, shaded bars), and the titer of the infectious progeny was determined after passage on fresh HeLa cells (subpassage, open bars). Since some differences in the absolute numbers of inclusions were found between independent experiments, inclusion counts for serovar E (A) and serovar L2 (B) were expressed as percentages of chlamydial growth compared to the control cultures, i.e., infected HeLa cells cultivated alone, that represent 100% of growth. In some experiments, the cocultures were performed in the presence of anti-TNF-α, anti-IL-1β, or anti-IL-8 antibodies; Trp (200 μg/ml); or SOD (300 U/ml). The lack of or minimal effect of the antibodies, Trp, or SOD on chlamydia growth was tested on control cultures of HeLa cells alone. This figure shows average (± the standard deviation) results from three independent experiments, and statistically significant recovery (P ≤ 0.05) in chlamydial growth or infectivity compared to the control cocultures is indicated by an asterisk.

FIG.4.

Morphology of chlamydial inclusions in infected HeLa cells cocultured with dTHP-1 cells. Fluorescence microscopy and transmission electron photomicrographs of polarized HeLa cells infected with C. trachomatis serovar E cultivated alone (A and C) or in the presence of dTHP-1 cells (B, D, E, and F). Serovar E inclusions, immunostained with an anti-major outer membrane protein fluorescein isothiocyanate-conjugated antibody, appeared much smaller and in reduced number in cocultures (B) compared to controls (A) (×400 magnification). (C) At 48 hpi, inclusions from control cultures contain mostly small, electron-dense EB and some RB. (D to F) A delay in chlamydial maturation was observed in inclusions from cocultures of HeLa and dTHP-1 cells, as well as the presence of damaged RB (arrows, E and F), many outer membrane blebs, and associated dark globular components (arrowheads, D and E). Magnifications: ×3,000 (C, E, and F); ×2,000 (D).

FIG. 5.

RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis-infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.

FIG. 6.

Titration of C. trachomatis serovar E and serovar L2 after a 48-h coculture of polarized infected HeLa cells with MdM prestimulated with different culture supernatants. HeLa cells, seeded in inserts and infected with C. trachomatis (culture 1, C1), were cultivated for 36 h, and then 10⁶ PMN were added to the apical side of these cultures for 12 h. Control culture 1, corresponding to HeLa cells incubated for 48 h without the addition of PMN, was also included, as well as a control wherein 10⁶ PMN were incubated in a tissue culture plate for 12 h in RPMI medium alone. The apical medium from HeLa cultures or PMN seeded in plates was then collected and centrifuged to pellet the cells. For convenience, the various supernatants are designated as follows: supernatants of PMN incubated in RPMI alone (RPMI/PMN), with uninfected HeLa cells (UC1/PMN), or with serovar E- or serovar L2-infected cells (EC1/PMN and L₂C1/PMN, respectively, and EC1 and L₂C1, respectively, corresponding to apical supernatants from E- or L2-infected HeLa cells). These supernatants were then diluted 1:1 in fresh RPMI before being added to MdM (EC1 or L₂C1, RPMI/PMN, UC1/PMN, EC1/PMN, or L₂C1/PMN + MdM). After 48 h of stimulation, the culture medium from macrophages was replaced by fresh RPMI, and these stimulated MdM were then cocultivated for 48 additional hours with freshly inoculated cultures of polarized HeLa cells (culture 2, EC2 and L₂C2, corresponding to serovar E- and serovar L2-infected cultures). Controls consisting of infected HeLa cells incubated with EC1 and EC1/PMN supernatants diluted 1:1 with fresh medium were also included in these experiments. Serovar E (A) and serovar L2 (B) inclusion counts (shaded bars) and progeny titration after subpassage (open bars) were expressed as percentages of chlamydial growth compared to the control cultures, i.e., infected HeLa cells cultivated alone, representing 100% of growth. These coculture experiments were repeated twice but, since the actual number of MdM per well is a difficult parameter to control, some variations in the level of chlamydial inhibition were found between independent experiments. However, the pattern of inhibition was similar in both experiments. This figure shows the results from a representative experiment.

FIG. 7.

Summary of in vitro TNF-α production and chlamydial survival during chronological appearance of PMN and MdM to chlamydia-infected HeLa cells. The left part of the chart presents a summary of the protocol used (for more details, see Fig. 1). Briefly, polarized HeLa cells infected with C. trachomatis serovar E or serovar L2 for 36 h were incubated in the presence of PMN. After 12 h of incubation, the supernatants from infected HeLa/PMN cocultures (SUPERNATANT I) were collected and used, after 1:1 dilution in fresh medium, to activate MdM. After 48 h of incubation, MdM supernatants (SUPERNATANT II) were collected, and these “stimulated” MdM, in fresh medium, were then cocultured with fresh HeLa cells infected with the respective serovars of C. trachomatis (culture 2). At 48 hpi, basal supernatants from cocultures were collected (SUPERNATANT III), and chlamydial inclusions and infectious progeny were counted. The right portion of the chart presents the progression in TNF-α production at each step of the two-stage coculture experiments by using serovar E (E) or serovar L2 (L2), with TNF-α concentrations in supernatants I, II (TNF-α released by the “stimulated” MdM only), and III represented by the shaded, open, and solid bars, respectively. The numbers shown on the graph above the open and solid bars represent the infectivity yields obtained when serovar E- or serovar L2-infected HeLa cells (culture 2) were cultured in the presence of the respective supernatant I or cocultured in the presence of PMN preactivated MdM, respectively. The infectious progeny titers were expressed as percentages of the control infected HeLa cells cultivated alone. The graph summarizes the data presented in Table 3 (TNF-α ELISA) and Fig. 6 (chlamydia titration).