Elimination of Chlamydia muridarum from the female reproductive tract is IL-12p40 dependent, but independent of Th1 and Th2 cells

Abstract

Chlamydia vaccine approaches aspire to induce Th1 cells for optimal protection, despite the fact that there is no direct evidence demonstrating Th1-mediated Chlamydia clearance from the female reproductive tract (FRT). We recently reported that T-bet-deficient mice can resolve primary Chlamydia infection normally, undermining the potentially protective role of Th1 cells in Chlamydia immunity. Here, we show that T-bet-deficient mice develop robust Th17 responses and that mice deficient in Th17 cells exhibit delayed bacterial clearance, demonstrating that Chlamydia-specific Th17 cells represent an underappreciated protective population. Additionally, Th2-deficient mice competently clear cervicovaginal infection. Furthermore, we show that sensing of IFN-γ by non-hematopoietic cells is essential for Chlamydia immunity, yet bacterial clearance in the FRT does not require IFN-γ secretion by CD4 T cells. Despite the fact that Th1 cells are not necessary for Chlamydia clearance, protective immunity to Chlamydia is still dependent on MHC class-II-restricted CD4 T cells and IL-12p40. Together, these data point to IL-12p40-dependent CD4 effector maturation as essential for Chlamydia immunity, and Th17 cells to a lesser extent, yet neither Th1 nor Th2 cell development is critical. Future Chlamydia vaccination efforts will be more effective if they focus on induction of this protective CD4 T cell population.

Fig 1. STAT6-deficient mice do not show any deficiency in Chlamydia clearance.

(A) IFUs isolated from vaginal swabs at various time points after infection. For wild-type mice n = 10, STAT6-deficient mice n = 10, and MHC class II-deficient mice n = 5. Data is combined from two experiments, with the MHC class II-deficient group included in one experiment. Graph is displayed as mean ± SEM. Wild-type versus STAT6-deficient groups are not significantly different. For wild-type versus MHC class II-deficient groups p<0.05 on days 9–45. For STAT6-deficient versus MHC class II-deficient groups p<0.05 on days 7–45 (mixed-effects analysis). Dashed line represents limit of detection. (B) Data from A expressed as the percent of mice with culture positive vaginal swabs.

Fig 2. T-bet deficient mice display a significant shift towards Th17 responses.

Lymphocytes isolated from the FRT and ILN were stimulated with PMA and ionomycin with Brefeldin A before staining for flow cytometry. Results are gated on CD4+ CD44hi cells. (A) Percentage and number of total activated CD4 T cells isolated from FRT and ILN. (B) Expression of Th1 markers T-bet and IFN-γ. (C) Summary graphs from B. (D) Flow cytometry plots showing the expression of Th17 markers RORγt and IL-17A. (E) Summary graphs from D. All graphs are displayed as mean ± SD. Data is representative of two experiments.

Fig 3. IL-12p40-deficient mice exhibit a severe delay in Chlamydia clearance in the FRT.

(A) IFUs isolated from vaginal swabs at various time points after infection. n = 4 for all groups. Graph is displayed as mean ± SEM. For wild-type versus IL-12p40-/- groups, p<0.05 on days 3 and 15–36. For wild-type versus MHC class II-deficient groups, p<0.05 on days 9 and 15–80. For IL12p40-/- versus MHC class II-deficient groups, p<0.05 on days 27–80 (mixed-effects model). (B) Data from A expressed as the percent of mice with culture positive vaginal swabs. Data is representative of two experiments.

Fig 4. RORγt mutant mice have a delay in Chlamydia clearance.

(A) IFUs isolated from vaginal swabs at various time points after infection. For wild-type mice n = 10, RORγt mutant (RORγt^M) mice n = 10, and MHC class II-/- mice n = 5. Data is combined from two experiments, MHC class II-/- group was included in one experiment. Graph is displayed as mean ± SEM. For wild-type versus RORγt^M groups, p<0.05 on days 9 and 16–25. For wild-type versus MHC class II-deficient groups, p<0.05 on days 7–11 and 16–39. For RORγt^M versus MHC class II-deficient groups, p<0.05 on days 11, 19, and 25–39 (2-way ANOVA). (B) Data from A expressed as the percent of mice with culture positive vaginal swabs.

Fig 5. CD4 T cells from RORγt mutant mice have a similar expression pattern of Th1 and Th17 markers as wild-type mice.

Lymphocytes isolated from the FRT and ILN were stimulated with PMA and ionomycin with Brefeldin A before staining for flow cytometry. Results are gated on CD4+ CD44hi cells. (A) Percentage and number of total activated CD4 T cells isolated from FRT and ILN. (B) Expression of Th1 markers T-bet and IFN-γ. (C) Summary graphs from B. (D) Flow cytometry of expression of Th17 markers RORγt and IL-17A. (E) Summary graphs from D. All graphs are displayed as mean ± SD. Data is representative of two experiments.

Fig 6. Depleting Th1 and Th17 cells does not impact FRT clearance of Chlamydia.

(A and C) IFUs isolated from vaginal swabs at various time points after infection. Graphs are displayed as mean ± SEM. (B and D) Data from A and C expressed as the percent of mice with culture positive vaginal swabs. (A and B) Wild-type or T-bet-/- mice were given isotype or depleting antibodies for IL-6 and TGF-β. (C and D) T-bet-/- mice were bred with RORγt mutant mice and infected with Chlamydia. (A) n = 3 for isotype treated groups, n = 4 for antibody treated groups. All time points and comparisons are not significantly different except day 18 for wild-type isotype versus T-bet-/- isotype and day 18 for T-bet-/- isotype versus T-bet-/- anti-IL-6 anti-TGF-β (2-way ANOVA). (B) Data is combined from two experiments. n = 7 for wild-type, n = 6 for T-bet-/- x RORγt^M. p<0.05 on days 9–12 and 24 (mixed-effects analysis).

Fig 7. Bone marrow chimera mice require expression of IFN-γ receptor by the recipient host tissues, but not donor bone marrow cells to control systemic Chlamydia infection.

Four groups of bone marrow chimera mice were generated as follows: CD45.1 wild-type bone marrow was transferred into CD45.2 wild-type recipients (WT→WT), CD45.1 wild-type bone marrow was transferred into CD45.2 IFNγR1-/- recipients (WT→IFNγ R1-/-), CD45.2 IFNγR1-/- bone marrow was transferred into CD45.1 wild-type recipients (IFNγ R1-/-→WT), and CD45.2 IFNγR1-/- bone marrow was transferred into CD45.2 IFNγR1-/- recipients (IFNγ R1-/-→IFNγ R1-/-). These groups were synchronized and infected alongside an additional, unmanipulated group of IFNγ-/- mice. The weight of each mouse was monitored over the course of the experiment and mice were euthanized before day 21 post infection if their weight was below 80% of the starting value or their condition otherwise became too severe. All remaining mice were euthanized at day 21. Upon euthanasia, spleen, lung, and kidneys were harvested for counting Chlamydia burdens. (A) Graphs show the percentage of starting weight of each mouse over time ± SD. Controls and experimental groups are shown in separate plots for readability. WT→WT versus IFNγ R1-/-→WT is not significantly different. For WT→WT versus WT→IFNγ R1-/-, p<0.05 on days 11 and 14–20. For WT→WT versus IFNγ R1-/-→IFNγ R1-/-, p<0.05 on days 6, 10–11, and 16–21. For WT→WT versus IFNγ-/-, p<0.05 on days 12–19 and 21. For WT→IFNγ R1-/- versus IFNγ R1-/-→WT, p<0.05 on days 11, 14, 17, and 20–21 (mixed-effects analysis). (B) Survival curve. With Bonferroni correction for multiple comparisons, WT→WT versus IFNγ R1-/-→WT is not significantly different while all other groups compared to WT→WT are significant. (C) Bacterial load measured in each organ at the time of euthanasia ± SD (1-way ANOVA). (D) IFUs isolated from vaginal swabs over the course of infection ± SEM. For WT→WT versus IFNγ R1-/-→WT, p<0.05 on day 15. For WT→WT versus WT→IFNγ R1-/-, p<0.05 on days 9–21. For WT→WT versus IFNγ R1-/-→IFNγ R1-/-, p<0.05 on days 9–18. For WT→WT versus IFNγ-/-, p<0.05 on days 12–21 (mixed-effects analysis). Data is combined from two experiments, total n are 9 for WT→WT, 7 for WT→IFNγ R1-/-, 5 for IFNγ R1-/-→WT, 10 for IFNγ R1-/-→IFNγ R1-/-, and 8 for IFNγ-/-.

Fig 8. CD4 T cell derived IFN-γ is not required for bacterial clearance in the FRT.

Two groups of bone marrow chimeric mice were generated: CD4-Cre+ IFN-γ floxed bone marrow transferred into CD45.1 recipients (Cre+, n = 11) and IFN-γ floxed bone marrow transferred into CD45.1 recipients (Cre-, n = 10). Mice were synchronized and weighed before infection. Five mice from each group were euthanized at day 21 and spleen, lung, and kidneys were harvested for counting Chlamydia burdens, and the FRT was harvested to assess IFN-γ expression in CD4 T cells. Vaginal swabs were taken periodically from all mice up to day 21 and in remaining mice after day 21. Four Cre+ mice died between day 21 and end of experiment (days 24, 27, 31, and 40). (A) Expression of IFN-γ in CD4+ T cells from the FRT. Results are gated on CD4+ CD44hi cells. (B) The percentage of starting weight of each mouse over time ± SD. Groups are significantly different (p<0.05) on days 19–21 (mixed-effects analysis). (C) IFUs isolated from vaginal swabs over the course of infection ± SEM. Groups are not significantly different (mixed-effects analysis).