Caspase-1 Contributes to Chlamydia trachomatis-Induced Upper Urogenital Tract Inflammatory Pathologies without Affecting the Course of Infection

Chlamydial infection. C. trachomatis serovar L2 was used to infect HeLa cells (human cervical carcinoma epithelial cells; ATCC catalog number CCL2), and C. muridarum was used to infect mice and mouse macrophages (Mφs). Both organisms were propagated, purified, aliquoted, and stored as described previously (6). To infect HeLa cells, HeLa cells grown in tissue flasks containing Dulbecco's modified Eagle's medium (GIBCO BRL, Rockville, MD) with 10% fetal calf serum (GIBCO BRL) at 37°C in an incubator supplied with 5% CO₂ were inoculated with serovar L2 cells as described previously (6). The infected cultures were harvested at different time points after infection for Western blot analyses as described below. To infect mice, female NOD mice with [NOD.129S2(B6)-Casp1^tm/Sesh/LtJ; stock number 004947; 13 mice] or without (NOD/Ltj; stock number 001976; 14 mice) caspase-1 gene knockout (KO) that were 5 to 6 weeks old were purchased from Jackson Laboratories (Bar Harbor, ME). Each mouse was inoculated intravaginally with 1 × 10⁴ inclusion-forming units (IFUs) of live C. muridarum in 20 μl of sucrose-phosphate-glutamate buffer (218 mM sucrose, 3.76 mM KH₂PO₄, 7.1 mM K₂HPO₄, 4.9 mM glutamate; pH 7.2). Five days prior to infection, each mouse was inoculated with 2.5 mg Depo-provera (Pharmacia Upjohn, Kalamazoo, MI) subcutaneously to synchronize the menstrual cycles and to increase mouse susceptibility to chlamydial infection. For some mice, a secondary infection was performed similarly on day 51 after the first infection. Depo-provera was also given to the mice 5 days prior to the secondary infection. For in vitro infection of Mφs, mouse Mφs were collected from the peritoneal cavity as described previously (56). Briefly, 4 to 5 ml of cold Hanks buffer (2.5 mM HEPES [pH 7.4], 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM Na₂HPO₄, 25 mM glucose, 0.05% bovine serum albumin) was inoculated into the mouse peritoneal cavity using a 27-gauge needle. After gentle massaging, the solution was slowly withdrawn from the mouse peritoneal cavity using a 20-gauge needle. After the total number of viable cells was determined, the peritoneal cavity-derived cells were resuspended in RPMI 1640 with 10% fetal calf serum and 10 μg/ml gentamicin, and 2 × 10⁵ cells were added to each well of 48-well plates. The plates were incubated at 37°C for 2 h in a CO₂ incubator to allow Mφs to adhere. After nonadherent cells were washed away, fresh medium was added to each well and incubation was continued. The adherent Mφs were cultured overnight prior to chlamydial inoculation. Chlamydiae diluted in cell growth medium were directly inoculated onto the cell monolayers at a multiplicity of infection of 5. The infected cultures were incubated for 24 h at 37°C in a CO₂ incubator before they were harvested for measurement of cytokines by an enzyme-linked immunosorbent assay (ELISA).

Western blot assay.The Western blot assay was carried out as described elsewhere (11, 12, 14, 42, 52). Briefly, the cell samples were solubilized in 2% sodium dodecyl sulfate sample buffer and loaded onto sodium dodecyl sulfate-polyacrylamide gel wells. After electrophoresis, the proteins were transferred to nitrocellulose membranes, and the blots were detected with primary antibodies, including rabbit anti-caspase-1 (catalog number 06-503; Upstate, Chicago, IL) and anti-IL-1β (catalog number SC-2022; Santa Cruz Biotech, Santa Cruz, CA) and mouse anti-chlamydial major outer membrane protein (clone MC22) (53) and anti-mammalian heat shock protein 70 (catalog number SC-24; clone w27; Santa Cruz Biotech). The primary antibody binding was probed with a horseradish peroxidase-conjugated secondary antibody (either goat anti-rabbit or anti-mouse immunoglobulin G; Jackson Immunologicals, Westgrove, PA) and visualized with an enhanced chemiluminescence kit (Santa Cruz Biotech).

ELISA.After the infected Mφs were cultured, the supernatants were collected in order to measure secreted cytokines, while after two washes with warm medium the remaining cell monolayers were collected and lysates were prepared by sonication in an equal amount of medium in order to measure cell-associated or intracellular cytokines using commercially available ELISA kits. The kits used for mouse IL-1β (catalog number DY400), IL-6 (catalog number DY406), tumor necrosis factor alpha (catalog number DY410), and macrophage inflammatory protein 2 (mouse homolog of human IL-8) (catalog number DY452) were all obtained from R&D Systems, Inc. (Minneapolis, MN). The ELISA was carried out by following the instructions provided by the manufacturer or instructions described elsewhere (41, 54, 55). Briefly, 96-well ELISA microplates (Nunc, Rochester, NY) were coated with a capture antibody, and after blocking, the cytokine samples or standards were added to the coated plates, followed by a biotin-conjugated detection antibody. The antibody binding was measured with horseradish peroxidase-conjugated Avidin plus a soluble colorimetric substrate [2,2′-azino-di-(3-ethylbenzthiazolinesulfonic acid) (ABTS)]. The absorbance at 405 nm was determined using a microplate reader (Molecular Devices Corporation, Sunnyvale, CA). The cytokine concentrations were calculated using absorbance values, cytokine standards, and sample dilution factors and were expressed in ng or pg per ml.

Monitoring mouse shedding of live chlamydiae.To monitor the shedding of live organisms, vaginal swabs were taken once every 3 to 4 days after inoculation for the first 30 days and once per week thereafter. Each swab was dissolved and sonicated in 500 μl of sucrose-phosphate-glutamate buffer and then titrated using HeLa cell monolayers in duplicate as described previously (27). Briefly, serially diluted swab samples were inoculated onto HeLa cell monolayers grown on coverslips in 24-well plates. After incubation for 24 h in the presence of 2 μg/ml cycloheximide, the cultures were fixed with 2% paraformaldehyde dissolved in phosphate-buffered saline for 30 min at room temperature, followed by permeabilization with 2% saponin for an additional 1 h. After washing and blocking, the cells were stained with Hoechst stain (Sigma, St. Louis, MO) (blue) to visualize nuclear DNA and a mouse anti-chlamydial lipopolysaccharide antibody (clone MB5H9) (unpublished observations) plus goat anti-mouse immunoglobulin G conjugated with Cy3 (red) (Jackson ImmunoResearch) to visualize chlamydial inclusions. The inclusions were counted using an Olympus AX-70 fluorescence microscope equipped with multiple filter sets (Olympus, Melville, NY). Five random fields were counted per coverslip. For coverslips containing less than 1 IFU per field, the entire coverslip was counted. Coverslips showing obvious cytotoxicity of HeLa cells were not taken into account. The total number of IFUs per swab was calculated based on the number of IFUs per field, the number of fields per coverslip, the dilution factor, and the inoculation and total sample volumes. An average was calculated using the serially diluted and duplicate samples for a given swab. The calculated total number of IFUs/swab was converted into a log₁₀ value, and the log₁₀ IFU values were used to calculate the mean and standard deviation for each group at each time point.

Evaluating mouse genital tract tissue pathologies and histological scoring.Eighty days after the primary infection, all mice were sacrificed, and the mouse urogenital tract tissues were isolated. Before the tissues were removed from a mouse body, an in situ gross examination was performed to obtain evidence of hydrosalpinx formation and any other related abnormalities. The excised tissues were then fixed in 10% neutral formalin, embedded in paraffin, and serially sectioned longitudinally (5-μm sections). A effort was made to include the cervix and both uterine horns and oviducts, as well as luminal structures of each tissue, in each section. The sections were stained with hematoxylin and eosin (H&E) as described elsewhere (40). The H&E-stained sections were assessed by a certified pathologist (I-T.Y) blinded to mouse treatment, and the severity of inflammation and pathologies was scored based on the modified schemes established previously (18, 27, 40). The uterine horns and fallopian tubes were scored separately. The scores for dilatation of the uterine horn or fallopian tube were as follows: 0, no significant dilatation; 1, mild dilatation of a single cross section; 2, one to three dilated cross sections; 3, more than three dilated cross sections; and 4, confluent pronounced dilation. The scores for inflammatory cell infiltrates (at the chronic stage of infection, the infiltrates mainly contained mononuclear cells) were as follows: 0, no significant infiltration; 1, infiltration at a single focus; 2, infiltration at two to four foci; 3, infiltration at more than four foci; and 4, confluent infiltration. Scores assigned to individual mice were used to calculate the mean ± standard error for each group of animals (n = 5 to 9).

Statistical analysis.The chi-square test (Microsoft Excel) was used to analyze qualitative (categorical incidence) data. An analysis of variance (http://www.physics.csbsju.edu/stats/anova.html ) was performed to analyze quantitative data from multiple groups, and a two-tailed Student t test (Microsoft Excel) was used to compare two groups.

Caspase-1 is activated during C. trachomatis infection.We monitored the processing of both caspase-1 and IL-1β during infection of human cervical carcinoma epithelial cells with C. trachomatis (Fig. 1A) or C. muridarum (Fig. 1B). The 45-kDa procaspase-1 was both up-regulated and cleaved into the active p20 fragment at 12 h, and the processing continued to increase up to 48 h for C. trachomatis and up to 36 h for C. muridarum after infection (Fig. 1, panels a). The caspase-1 cleavage was accompanied by processing of pro-IL-1β (panels b), indicating that the cleaved caspase-1 was active in processing its substrates. The chlamydial major outer membrane protein became detectable 24 h after infection (panels c), and the total amount of protein loaded into each lane was monitored by simultaneous detection of host heat shock protein 70 (panels d). The results described above confirm the previous observation that infection with either C. trachomatis or C. muridarum can lead to activation of caspase-1 and processing of IL-1β and IL-18 (21, 29, 49).

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FIG. 1.

Activation of caspase-1 by chlamydial infection. HeLa cells infected with C. trachomatis (A) or C. muridarum (MoPn) (B) were harvested at various time points after infection as indicated at the top in order to monitor caspase-1 (panels a) and IL-1β (panels b) processing in a Western blot. The anti-caspase-1 and IL-1β antibodies indicated on the left detected both the pro and mature forms of caspase-1 and IL-1β, as indicated on the right. A mouse anti-chlamydial major outer membrane protein (MOMP) monoclonal antibody was used to monitor chlamydial infection (panels c), and an anti-mammalian heat shock protein 70 (HSP70) antibody was used to monitor total protein loading (panels d). The sample harvested at zero time was normal HeLa cells not infected with chlamydiae (lane 1). Note that both caspase-1 processing and IL-1β processing were induced by chlamydial infection. MW, molecular mass; ns, not significant.

Caspase-1 is not required for host defense against chlamydial infection.After confirming that caspase-1 is activated during C. trachomatis infection, we next determined whether the caspase-1 activation could contribute to host defense against chlamydial challenge and infection. Groups of mice with or without a caspase-1 gene deficiency were intravaginally infected with C. muridarum, and the vaginal shedding of live organisms was monitored over the course of infection (Fig. 2). All mice, regardless of the genotype, were infected with C. muridarum and shed live organisms up to 2 weeks after infection. Although 3 and 7 of the 14 wild-type mice no longer shed live organisms at days 17 and 21 postinfection, respectively, the average numbers of live organisms shed from both caspase-1 KO and wild-type mice were not significantly different at these two time points. By day 24, no live organisms could be detected for any mouse. To test whether caspase-1 can contribute to the adaptive immunity against Chlamydia infection, six and five mice from the caspase-1 KO and wild-type mouse groups, respectively, were reinfected with C. muridarum on day 51. As expected, the time course following reinfection was greatly shortened and lasted only about 1 week. This was true for both groups of mice, and no significant difference in the shedding of live organisms between the caspase-1 KO and wild-type mice was observed. The observations described above suggest that caspase-1 is not required for either innate or adaptive immunity against chlamydial infection. When the expression and secretion of cytokines by mouse Mφs were measured, we found that a deficiency in caspase-1 did not alter the ability of mouse Mφs to produce and secrete other inflammatory cytokines in response to chlamydial infection, although caspase-1-deficient Mφs did not process IL-1β (Fig. 3).

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FIG. 2.

Effect of caspase-1 deficiency on live chlamydia shedding following chlamydial infection. Mice deficient (open bars) or competent (filled bars) for caspase-1 were infected intravaginally with C. muridarum, and vaginal swabs were taken during the course of infection as indicated on the x axis to determine the number of live organisms (expressed in IFUs) shed from the urogenital tract. The number of IFU obtained from each swab was converted into a log₁₀ value, and the log₁₀ IFU values were used to calculate the mean and standard deviation for each mouse group at each time point, as shown on the y axis. At the start of the experiment the caspase-1 KO group contained 13 mice, while the wild-type (wt) group contained 14 mice. The number of mice with detectable IFUs at each time point is indicated above the horizontal line at the top. On day 51 after the primary infection, six and five mice in the caspase-1 KO and wild-type groups, respectively, were reinfected with C. muridarum. All mice were sacrificed on day 80 after the primary infection. The log₁₀ IFU values for the KO and wild-type groups at each time point were analyzed using a two-tailed Student t test, and no statistically significant differences were found. Note that the course of infection was dramatically shortened following the secondary infection.

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FIG. 3.

Effect of caspase-1 deficiency on cytokine production and secretion by mouse Mφs after Chlamydia infection. Peritoneal Mφs harvested from either caspase-1 KO (open bars) or wild-type (filled bars) mice were infected with chlamydiae for 24 h in 48-well plates. The intracellular IL-1β (b), secreted IL-1β (a), IL-6 (c), macrophage inflammatory protein 2 (MIP-2) (mouse IL-8 homolog) (d), and tumor necrosis factor alpha (TNFα) (panel e) levels were measured using a commercially available ELISA kit. The data were obtained using seven to nine mice in each group and were expressed in ng or pg per ml of culture supernatant; the bars indicate means, and the error bars indicate standard deviations. The asterisk indicates a statistically significant difference (P < 0.01) in the level of secreted IL-1β between the caspase-1 KO and wild-type Mφs (both infected with chlamydiae).

Deficiency in caspase-1 protected mice from upper genital tract pathologies following C. muridarum infection.At 80 days after infection, all mice were sacrificed in order to evaluate the pathologies of mouse genital tract tissues. Inflammatory infiltrates and luminal dilation as determined with a microscope, together with hydrosalpinx formation as determined by gross appearance, are hallmarks of the urogenital tract pathologies caused by chlamydial vaginal infection in mice (40). We found that the wild-type NOD mice developed these typical pathological changes in their genital tracts upon chlamydial intravaginal infection (Fig. 4A). When the gross appearance of the isolated genital tracts was examined, we found that the incidence of hydrosalpinx formation was significantly lower in caspase-1 KO mice after reinfection (Table 1) (P < 0.05, as determined by a chi-square test). The H&E-stained histological sections were viewed with a microscope, and inflammation and luminal dilation were semiquantitatively scored. Reduced levels of inflammatory infiltrates and luminal dilation were observed in both the uterine horn and fallopian tube tissue sections from the caspase-1-deficient mice (compared to the sections from wild-type mice) after either primary or secondary infection (Table 2). It is worth noting that none of the NOD mice showed uterine horn luminal dilation despite reinfection. Due to the low number of samples in each score category, we reclassified the samples with a positive inflammation or dilation score (≥1) as inflammation or dilation positive and compared the rates of positivity for the wild-type and caspase-1 KO mouse groups. We found that the rates of positive inflammation in both uterine horn and fallopian tube tissue samples were significantly lower in caspase-1-deficient mice (P < 0.05 and P < 0.01, respectively, as determined by a chi-square test). Furthermore, the caspase-1-deficient mice also displayed a significantly lower rate of fallopian tube dilation after the secondary infection (P < 0.05). This observation is consistent with the significant difference in the incidence of hydrosalpinx formation shown in Table 1. The same mouse samples with hydrosalpinx formation as determined by gross appearance were also scored positive for fallopian tube luminal dilation using the microscope. When the means and standard errors of the inflammation scores for the wild-type and caspase-1-deficient mice were compared using the Student t test, we found that the capspase-1-deficient mice had significantly less inflammation in the fallopian tube tissues (Fig. 4C) (P < 0.01). However, the reduction in inflammation disappeared after reinfection. This was mainly due to the dramatic increase in inflammation induced by the secondary infection in the caspase-1-deficient mice (P < 0.05).

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FIG. 4.

Effect of caspase-1 deficiency on the development of inflammatory pathologies in the mouse urogenital tract following chlamydial infection. (A) When urogenital tract tissues from wild-type NOD mice with (panels b and d) or without (panels a and c) a chlamydial infection were examined at the level of gross appearance (panels a and b) and with a microscope (panels c and d), obvious inflammatory pathologies were noted in the samples from the infected mouse but not in the samples from the normal mouse, and these pathologies included hydrosalpinx formation (panel b, white arrow), extensive infiltration of mononuclear cells (panel d, arrows), and fallopian tube luminal dilation (panel d, arrows). Different types of tissues in a NOD mouse urogenital tract with a normal gross appearance are indicated in panel a (arrows). (B) Mouse genital tract tissues were sectioned and analyzed using a microscope after H&E staining. A representative image of a histological section from either the uterine horn (panels a, c, e, g, I, and k) or fallopian tube (panels b, d, f, h, j, and l) tissues is shown for each group of mice with or without a primary or secondary infection. The asterisks indicate inflammatory cell infiltration, the number sign indicates fibrosis, and the ampersand indicates luminal dilation. Scores based on the severity of inflammatory infiltration are indicated in some of the images. Note that chlamydial infection induced more severe inflammatory changes in wild-type mice. (C) Inflammation scores assigned to individual mice were used to calculate the means (bars) and standard errors (error bars) for different groups. The various tissue and mouse groups are indicated on the x axis. The open bars indicate tissue samples from caspase-1 KO mice, while the filled bars indicate tissue samples from wild-type mice. An analysis of variance test was used to analyze differences among different groups, and a two-tailed Student t test was used to analyze differences between the caspase-1 KO and wild-type groups. There was a highly significant difference in inflammation scores (P < 0.01, Student t test) between the caspase-1 KO and wild-type fallopian tissues, and there was a significant difference in the caspase-1 KO fallopian tube tissue inflammation (P < 0.05, Student t test) between the primary and secondary infections.