Article Figures & Data
Figures
- FIG. 1.
Effect of CD28, CD80/CD86, CD40, or CD40L deficiency on live organism shedding following chlamydial infection. Wild-type mice (black bar) or mice deficient in CD28 (CD28 KO, gray bar), CD80/CD86 (open bar), CD40 (CD40 KO, heavily hatched bar), or CD40L (CD40L KO, lightly hatched bar) were infected intravaginally with C. muridarum organisms, and vaginal swabs were obtained along the infection course as indicated along the x axis for measuring the number of live organisms (in IFU). The IFU count from each swab was converted into log10, and the log10 IFU values were used to calculate the means and standard deviations for each mouse group at each time point as presented along the y axis. The CD28 KO group started with a total of 20 mice and the CD80/CD86 KO group started with 10 mice while the wild-type, CD40 KO, and CD40L KO groups each started with 18. The number of mice with detectable IFU (IFU+) at each time point is listed in the bottom table. On day 73 after the primary infection, five to nine mice from each group were reinfected with C. muridarum organisms. The log10 IFU values along the time course were first analyzed by ANOVA and reanalyzed between the wild-type and KO groups (that displayed significant differences under ANOVA) by using a two-tailed Student t test (**, P < 0.01; *, P < 0.05). The wild-type and CD28 KO mice resolved their infections within 30 days after primary infection and within 7 days after secondary infection. However, mice deficient in CD40 or CD40L maintained statistically significant higher levels of live organism shedding and displayed extended infection courses (to 50 days after primary infection and 20 days after secondary infection).
- FIG. 2.
Effect of CD28, CD80/CD86, CD40, or CD40L deficiency on the development of inflammatory pathologies in the mouse urogenital tract after chlamydial infection. (A) When urogenital tract tissues from wild-type (a, f, and k), CD28 KO (b, g, and l), CD80/CD86 KO (c, h, and m), CD40 KO (d, i, and n), and CD40L KO (e, j, and o) mice were examined for gross appearance (a to e), on slides (f to j), and under a microscope (k to o), obvious inflammatory pathologies were noted in the representative images from wild-type, CD40 KO, and CD40L KO mice but not CD28 or CD80/CD86 KO mice. The pathologies were recorded as hydrosalpinx and uterine horn (UT) dilatation observable with the naked eye (a, d, and e, white arrows, see Table 1 for quantitative data), as well as extensive infiltration of mononuclear cells and oviduct (OV) luminal dilation observed under microscope after H&E staining (k, n, and o, white arrows). The representative images were from mice sacrificed on day 80 after primary infection. (B) Inflammation and lumen dilatation of both uterine horns and oviducts were semiquantitatively scored under a microscope, and the scores were used to calculate the means and standard errors for each group as shown along the y axis. The various tissue and mouse groups were indicated along the x axis. The solid bar represents tissue samples from wild-type mice, the gray bar represents tissue samples from C28 KO mice, the open bar represents tissue samples from CD80/CD86 KO mice, the heavily hatched bar represents tissue samples from CD40 KO mice, and the lightly hatched bar represents tissue samples from CD40L KO mice. The number of mice in each group is listed Table 1. Statistically significant differences in inflammation scores (*, P < 0.05) of uterine horn tissues and dilatation scores of both uterine horn and oviduct tissues were found between wild-type and CD28 or CD80/CD86 KO mice after primary but not after secondary infection.
- FIG. 3.
Effect of CD28, CD80/CD86, CD40, or CD40L deficiency on mouse serum IgG and IgM antibody production in response to chlamydial infection. The various mouse groups were infected with chlamydial organisms as described in the Fig. 1 legend, and the mice were bled on different days after primary infection, as indicated along the x axis. Serum samples displayed on the right side of the figure were from the reinfected mice only. The Chlamydia-specific IgG (a) and IgM (b) antibodies in the mouse sera were measured using C. muridarum-infected HeLa cells as antigens in an immunofluorescence assay. The highest dilution at which a given mouse serum still positively stained the C. muridarum inclusions was determined as the titer of that serum. The serum dilutions were converted into log10 titers for calculating the means and standard deviations, as displayed along the y axis. The wild-type mice (solid diamond, n = 10) developed a robust serum IgG response, while the CD28 (n = 12) or CD80/CD86 (n = 10) KO mice (solid or open square) developed a reduced level of IgG antibodies. However, no Chlamydia-specific IgG antibodies were detected in mice deficient in either CD40 (solid triangle, n = 10) or CD40L (open triangle, n = 10), although all mice developed Chlamydia-specific serum IgM antibodies.
- FIG. 4.
Effect of CD28, CD80/CD86, CD40, or CD40L deficiency on chlamydial antigen-specific T-cell responses. Splenocytes were harvested from wild-type (solid bar), CD28 KO (gray bar), CD80/CD86 KO (open bar), CD40 KO (heavily hatched bar), or CD40L KO (lightly hatched bar) mice and restimulated with chlamydial organism antigens for 72 h. The culture supernatants were measured for IFN-γ using ELISA, and the results were expressed as pg/ml as shown along the y axis. There were five mice for each group after primary infection and five for the CD80/CD86 KO mice and three for the remaining groups after secondary infection. Spleen cells from CD40 or CD40L KO mice failed to produce significantly high levels of IFN-γ (P < 0.01, Student t test).
- FIG. 5.
Effect of CD28, CD80/CD86, CD40, or CD40L deficiency on cytokine production in the mouse genital tract after chlamydial infection. On different days after intravaginal infection, vaginal swabs were collected from various groups of mice including wild-type (solid bar), CD28 KO (gray bar), CD40 KO (heavily hatched bar), or CD40L KO (lightly hatched bar) mice, as described in Fig. 1 legend and as displayed along the x axis. A portion of each vaginal swab sample was used to measure IL-1α (a), IL-6 (b), MIP-2 (a mouse homologue of IL-8 [c]), TNF-α (d), and IFN-γ (e) using ELISA, and the results were expressed as pg/ml along the y axis. The data came from 8 to 12 mice per group after primary infection and 3 to 6 mice per group after secondary infection. The varied number of mouse samples in each group was largely caused by the limited amount of each vaginal swab sample. No single sample had sufficient amount for measuring all five cytokines. The cytokine levels from CD80/CD86 KO mice were similar to those from CD28 KO mice and were not included into the figure. Please note that most cytokines peaked on day 7 after primary infection. The CD28 KO mice produced significantly lower levels of IL-1α, IL-6, MIP-2, and TNF-α (*, P < 0.05 [Student t test]), but not IFN-γ, whereas CD40 or CD40L KO mice failed to produce a significant level of IFN-γ (**, P < 0.01). Upon secondary infection, the cytokine peaks in most mice moved to day 3 and extended to day 6 or beyond.
Tables
- TABLE 1.
Incidence of gross pathologiesa
Mouse group No. of miceb sacrificed after: Primary infection Secondary infection Uterine horn dilation Hydrosalpinx Uterine horn dilation Hydrosalpinx None Single Both None Single Both None Single Both None Single Both Wild type 0 0 10 0 0 10 0 0 8 0 0 8 CD28 KO 5* 2 4 0 5 6 0 0 9 0 0 9 CD80/86 KO 2** 2 1 0 2 3 0 0 5 0 0 5 CD40L KO 0 0 10 0 2 8 0 0 8 0 0 8 CD40 KO 0 1 9 0 0 10 0 0 8 0 0 8 ↵ a Mice were sacrificed after primary infection alone or after secondary infection, and the reproductive tissues were isolated and inspected for gross pathologies, including uterine horn dilatation and oviduct hydrosalpinx (see Fig. 2A for images).
↵ b The number of mice showing the pathologies on single or both sides of the reproductive tissues were recorded and tabulated. The total uterine horn dilation incidence (both single and bilateral) of CD28 KO mice (*, P = 0.02) or CD80/86 KO mice (**, P = 0.09) was compared to that of wil-type mice using the Fisher exact test.
