Intracellular interleukin-1alpha mediates interleukin-8 production induced by Chlamydia trachomatis infection via a mechanism independent of type I interleukin-1 receptor

Abstract

Chlamydia trachomatis infection induces a wide array of inflammatory cytokines and chemokines, which may contribute to chlamydia-induced pathologies. However, the precise mechanisms by which Chlamydia induces cytokines remain unclear. Here we demonstrate that the proinflammatory cytokine interleukin-1alpha (IL-1alpha) plays an essential role in chlamydial induction of the chemokine IL-8. Cells deficient in IL-1alpha expression or IL-1alpha-competent cells treated with IL-1alpha-specific small interfering RNA failed to produce IL-8 in response to chlamydial infection. However, neutralization of extracellular IL-1alpha or blockade of or deficiency in type I IL-1 receptor (IL-1RI) signaling did not affect chlamydial induction of IL-8 in cells capable of producing IL-1alpha. These results suggest that IL-1alpha can mediate the chlamydial induction of IL-8 via an intracellular mechanism independent of IL-1RI, especially during the early stage of the infection cycle. This conclusion is further supported by the observations that expression of a transgene-encoded full-length IL-1alpha fusion protein in the nuclei enhanced IL-8 production and that nuclear localization of chlamydia-induced precursor IL-1alpha correlated with chlamydial induction of IL-8. Thus, we have identified a novel mechanism for chlamydial induction of the chemokine IL-8.

FIG. 1.

Inflammatory cytokine production during chlamydial infection. HeLa cells, with or without C. trachomatis L2 serovar infection for various periods, as indicated in the figure, were processed for the following measurements. (A) RNAs were extracted from whole-cell samples and reversely transcribed into cDNAs by use of random hexamer primers. The cDNAs were amplified by PCRs with primers for human IL-1α (a), IL-6 (b), IL-8 (c), and β-tubulin (d), and the PCR products were loaded into an agarose gel for visualization after ethidium bromide staining. MW, molecular weight in base pairs (bp), as indicated along the left side of the figure. The corresponding DNA bands are indicated along the right side of the figure. (B) Either the culture supernatants (for measuring secreted cytokines) (a, c, and e) or whole-cell lysates (for measuring intracellular cytokines) (b, d, and f) were analyzed for IL-1α (a and b), IL-6 (c and d), and IL-8 (e and f) by using a sandwich ELISA. The cytokine levels were determined based on absorbance readings, sample dilution factors, and corresponding cytokine standards and were expressed in nanograms (ng) per 1 million cells, in the format of means ± standard deviations. Please note the difference in scale between the y axes in the left and right panels. Each data point comes from four to six independent experiments done in duplicate. Asterisks indicate statistically significant differences (P < 0.05) between the infected (hatched bars) and uninfected (open bars) samples from the same time points, using a two-tailed Student t test. (C) HeLa cells grown on coverslips were fixed and permeabilized 48 h after infection. A combination of a goat anti-human IL-8 antibody (red), Hoechst DNA dye (blue; for chlamydial and host nuclear DNA), and a rabbit anti-CT395 antibody (green; for visualizing chlamydial organisms) (a to h) or anti-IL-8, the DNA dye, and a mouse anti-Golgi GM130 antibody (green) (i to l) was used to visualize the corresponding antigens or organelle under a fluorescence microscope. The images were taken one color at a time (a to c, e to g, and i to k), and the single-color images were finally overlaid as tricolor images (d, h, and l). White arrows point to IL-8 staining, and solid white triangles in panels i to l indicate chlamydial inclusions.

FIG. 2.

Role of IL-1α in chlamydial induction of IL-8. Five different cell lines, as indicated in the figure, with or without C. trachomatis L2 serovar infection for 48 h, were processed for either RT-PCR detection of IL-1α and β-tubulin transcripts (A) or ELISA detection of IL-1α, IL-6, and IL-8 proteins (B). The ELISA results were expressed in ng per well, in the format of means ± standard deviations, and each data point comes from three independent experiments done in duplicate. Asterisks indicate statistically significant differences (P < 0.05) between the infected (hatched bars) and uninfected (open bars) samples from the same cell lines, using a two-tailed Student t test. (C) HeLa cells grown in 24-well plates, with or without Lipofectamine-mediated siRNA transfection or chlamydial infection for 48 h, were measured for intracellular IL-1α (a) and secreted IL-8 (b) by ELISA. The cytokine concentrations were expressed in ng per well, in the format of means ± standard deviations. The experiments were repeated three times each, with duplicates. Asterisks indicate statistically significant differences (P < 0.05) between the IL-1α-specific siRNA-treated (column 7) and scrambled siRNA-treated (column 5) samples (uninfected cell samples; open bars) or between the IL-1α-specific siRNA-treated samples (column 8) and each of the three control groups, including no transfection (column 2), Lipofectamine-only treatment (column 4), and scrambled siRNA treatment (column 6) (infected samples; hatched bars). A two-tailed Student t test was used for statistical analysis. (D) Peritoneal macrophages harvested from either wt or IL-1α KO mice were infected with C. trachomatis serovar L2 for 48 h in 48-well plates. Both the intracellular IL-1α (a) and secreted MIP-2 (mouse IL-8 homolog) (b) were measured using a commercially available ELISA kit. The results came from six mice per group and were expressed in ng per well, in the format of means ± standard deviations. Asterisks indicate statistically significant differences (P < 0.05) between the wt and IL-1α KO macrophages (infected samples; hatched bars).

FIG. 3.

IL-1α-mediated chlamydial induction of IL-8 is independent of extracellular IL-1α and IL-1R. (A) HeLa cells, with or without C. trachomatis serovar L2 infection, were treated with 5 ng/ml of IL-1α, 2 μg/ml of an anti-IL-1α neutralizing antibody, or 500 ng/ml of IL-1RA. The treatments commenced at the time of infection and were maintained throughout the infection course by changing the medium with medium containing fresh cytokines every 24 h. Secreted IL-6 and IL-8 were measured 48 (a and c) or 72 (b and d) h after infection by ELISA. The experiments were repeated three or four times each, with duplicates. The results were expressed in ng per well. Asterisks indicate statistically significant differences (P < 0.05) between samples treated with IL-1α (column 2 or 11) and with neutralizing antibody (column 3 or 12) or IL-1RA (column 4 or 13) (uninfected samples; open bars) or between the 72-h infected sample (column 14) and the neutralizing antibody (column 15)- or IL-1RA (column 16)-treated samples (hatched bars). A two-tailed Student t test was used for the statistical analysis. (B) Peritoneal macrophages harvested from either wt or IL-1RI KO mice were infected with C. trachomatis serovar L2 for 48 h. Both the intracellular IL-1α and secreted MIP-2 (mouse homolog of IL-8) were measured using an ELISA. The results came from six mice per group and were expressed in ng per well, in the format of means ± standard deviations. Asterisks indicate statistically significant differences (P < 0.05) between the infected (hatched bars) and uninfected (open bars) samples from the wt macrophages.

FIG. 4.

Correlation of IL-1α nuclear expression with chlamydial induction of IL-8. 293T cells were transfected with a pDsRed plasmid encoding precursor full-length human IL-1α with RFP as an N-terminal fusion tag (RFP/IL-1α) or with vector alone (vector) or were treated with Lipofectamine alone (Lipo). The culture supernatants were harvested 48 h after infection for measurement of IL-8 by ELISA (B), and the corresponding cells grown on coverslips were processed for visualization of RFP (red) and nuclei (blue) after DNA dye staining (A). Note that the full-length IL-1α-RFP fusion protein was expressed in the nucleus and that a significant level of IL-8 was detected in the sample expressing IL-1α-RFP. (C) HeLa cells, with or without chlamydial infection for 48 h, were processed for immunofluorescence labeling of IL-8 (red), IL-1α (green), and host cell and chlamydial DNA (blue). The filled white triangles indicate chlamydial inclusions. Note that IL-1α was concentrated in the nuclei of the Chlamydia-infected cells, whereas IL-8 accumulated in the Golgi apparatuses of the same cells.