Tumor necrosis factor alpha production from CD8+ T cells mediates oviduct pathological sequelae following primary genital Chlamydia muridarum infection

Abstract

The immunopathogenesis of Chlamydia trachomatis-induced oviduct pathological sequelae is not well understood. Mice genetically deficient in perforin (perforin(-/-) mice) or tumor necrosis factor alpha (TNF-α) production (TNF-α(-/-) mice) displayed comparable vaginal chlamydial clearance rates but significantly reduced oviduct pathology (hydrosalpinx) compared to that of wild-type mice. Since both perforin and TNF-α are effector mechanisms of CD8(+) T cells, we evaluated the role of CD8(+) T cells during genital Chlamydia muridarum infection and oviduct sequelae. Following vaginal chlamydial challenge, (i) mice deficient in TAP I (and therefore the major histocompatibility complex [MHC] I pathway and CD8(+) T cells), (ii) wild-type mice depleted of CD8(+) T cells, and (iii) mice genetically deficient in CD8 (CD8(-/-) mice) all displayed similar levels of vaginal chlamydial clearance but significantly reduced hydrosalpinx, compared to those of wild-type C57BL/6 mice, suggesting a role for CD8(+) T cells in chlamydial pathogenesis. Repletion of CD8(-/-) mice with wild-type or perforin(-/-), but not TNF-α(-/-), CD8(+) T cells at the time of challenge restored hydrosalpinx to levels observed in wild-type C57BL/6 mice, suggesting that TNF-α production from CD8(+) T cells is important for pathogenesis. Additionally, repletion of TNF-α(-/-) mice with TNF-α(+/+) CD8(+) T cells significantly enhanced the incidence of hydrosalpinx and oviduct dilatation compared to those of TNF-α(-/-) mice but not to the levels found in wild-type mice, suggesting that TNF-α production from CD8(+) T cells and non-CD8(+) cells cooperates to induce optimal oviduct pathology following genital chlamydial infection. These results provide compelling new evidence supporting the contribution of CD8(+) T cells and TNF-α production to Chlamydia-induced reproductive tract sequelae.

Fig. 1.

Role of perforin and TNF-α in genital chlamydial infection and pathology. (A and B) Groups (n = 33) of C57BL/6 mice were challenged i.vag. with C. muridarum, and upper genital tracts (n = 3) were collected on day 0 before challenge and at weekly intervals after challenge for 10 consecutive weeks. Tissue homogenates were examined for levels of granzyme B (A) and TNF-α (B). Results are expressed as mean ± SEM of cytokine level at each time point per group and are representative of two independent experiments. (C and D) Groups (n = 6) of perforin^−/−, TNF-α^−/−, and C57BL/6 mice were challenged i.vag. with C. muridarum. (C) The vaginal chlamydial shedding was measured over a period of 1 month following challenge. (D) On day 80 after challenge, the gross oviduct diameter was measured. Each individual marker represents one oviduct, and the mean ± SEM of oviduct diameter per group is also shown. The number of normal oviducts (numerator) and the total number of oviducts evaluated (denominator) per respective group of mice have been indicated in parentheses. *, significant (P ≤ 0.05) difference between indicated group and C57BL/6 mice in incidence of hydrosalpinx (chi-square test) and the oviduct diameter (ANOVA). Results are a composite from two independent experiments.

Fig. 2.

Role of CD8⁺ T cells in genital chlamydial infection. (A to C) Groups (n = 3) of mice (C57BL/6 and TAP I^−/− [A], control rat Ig and anti-CD8 antibody treated [B], and C57BL/6 and CD8^−/− [C]) were euthanized, and splenic populations of CD8⁺ T cells were analyzed by flow cytometry. Results are shown as mean ± SEM of the percentage of CD8⁺ T cells in total splenocytes and are representative of two independent experiments. (D to F) Groups (n = 6) of mice (C57BL/6 and TAP I^−/− [D], control rat Ig and anti-CD8 antibody treated [E], and C57BL/6 and CD8^−/− [F]) were challenged with 5 × 10⁴ IFU of C. muridarum on day 0, and chlamydial shedding was enumerated every third day for a period of 1 month. The mean ± SEM of vaginal chlamydial shedding per group at each time point is shown. Results are representative of three independent experiments.

Fig. 3.

Role of CD8⁺ T cells in oviduct sequelae after vaginal C. muridarum infection. Groups (n = 6) of mice (C57BL/6 and TAP I^−/− [A], control rat Ig and anti-CD8 antibody treated [B], and C57BL/6 and CD8^−/− [C]) were challenged with 5 × 10⁴ IFU of C. muridarum on day 0, and the macroscopic oviduct diameter was measured on day 80 after challenge. Each individual marker represents one oviduct, and the mean ± SEM of oviduct diameter per group of mice also is shown. The number of normal oviducts (numerator) and the total number of oviducts evaluated (denominator) per respective group of mice have been indicated in parentheses. *, significant (P ≤ 0.05, Student's t test) difference in oviduct diameter between the indicated group and C57BL/6 mice in panels A and C or control rat Ig-treated mice in panel B). In panels B and C, * indicates significant (P ≤ 0.05, chi-square test) difference in oviduct diameter between the indicated group and control rat Ig-treated mice or C57BL/6 mice, respectively. Results are representative of three independent experiments.

Fig. 4.

Role of TNF-α production from CD8⁺ T cells in oviduct pathological sequelae following vaginal C. muridarum infection. (A and B) Groups (n = 3) of C57BL/6 and CD8^−/− mice were challenged i.vag. with 5 × 10⁴ IFU of C. muridarum on day 0, and granzyme B (A) and TNF-α (B) production was evaluated in the upper genital tract homogenates on days 3, 8, and 13 following challenge. *, significant (P ≤ 0.05, Student's t test) difference between the CD8^−/− and C57BL/6 mice. Results are representative of two independent experiments. (C) Groups (n = 3 to 6) of mice as indicated were challenged i.vag. with 5 × 10⁴ IFU of C. muridarum on day 0, and macroscopic oviduct dilatation was measured on day 80 after challenge. Each individual marker represents one oviduct, and the mean ± SEM of oviduct diameter per group of mice also is shown. The number of normal oviducts (numerator) and the total number of oviducts evaluated (denominator) per respective group of mice have been indicated in parentheses. *, significant (P ≤ 0.05, chi-square test and ANOVA) difference in incidence and dilatation, respectively, of hydrosalpinx between CD8^−/− and C57BL/6 mice and between CD8^−/− mice repleted with TNF-α^−/− CD8⁺ T cells and CD8^−/− mice repleted with C57BL/6 CD8⁺ T cells. Results are a composite from two independent experiments.

Fig. 5.

Role of TNF-α production from CD8⁺ T cells in oviduct pathological sequelae following vaginal C. muridarum infection. Groups (n = 3) of mice as indicated were challenged with 5 × 10⁴ IFU of C. muridarum on day 0, and macroscopic oviduct dilatation was measured on day 80 after challenge. Each individual marker represents one oviduct, and the mean ± SEM of oviduct diameter per group of mice also is shown. The number of normal oviducts (numerator) and the total number of oviducts evaluated (denominator) per respective group of mice have been indicated in parentheses. *, significant (P ≤ 0.05, chi-square test and ANOVA) difference in incidence and dilatation, respectively, of hydrosalpinx between TNF-α^−/− and C57BL/6 mice and in incidence of hydrosalpinx between TNF-α^−/− and TNF-α^−/− receiving C57BL/6 CD8⁺ T cells. Results are a composite from two independent experiments.