The duration of Chlamydia muridarum genital tract infection and associated chronic pathological changes are reduced in IL-17 knockout mice but protection is not increased further by immunization

Abstract

IL-17 is believed to be important for protection against extracellular pathogens, where clearance is dependent on neutrophil recruitment and local activation of epithelial cell defences. However, the role of IL-17 in protection against intracellular pathogens such as Chlamydia is less clear. We have compared (i) the course of natural genital tract C. muridarum infection, (ii) the development of oviduct pathology and (iii) the development of vaccine-induced immunity against infection in wild type (WT) BALB/c and IL-17 knockout mice (IL-17-/-) to determine if IL-17-mediated immunity is implicated in the development of infection-induced pathology and/or protection. Both the magnitude and duration of genital infection was significantly reduced in IL-17-/- mice compared to BALB/c. Similarly, hydrosalpinx was also greatly reduced in IL-17-/- mice and this correlated with reduced neutrophil and macrophage infiltration of oviduct tissues. Matrix metalloproteinase (MMP) 9 and MMP2 were increased in WT oviducts compared to IL-17-/- animals at day 7 post-infection. In contrast, oviducts from IL-17-/- mice contained higher MMP9 and MMP2 at day 21. Infection also elicited higher levels of Chlamydia-neutralizing antibody in serum of IL-17-/- mice than WT mice. Following intranasal immunization with C. muridarumMajor Outer Membrane Protein (MOMP) and cholera toxin plus CpG adjuvants, significantly higher levels of chlamydial MOMP-specific IgG and IgA were found in serum and vaginal washes of IL-17-/- mice. T cell proliferation and IFNγ production by splenocytes was greater in WT animals following in vitro re-stimulation, however vaccination was only effective at reducing infection in WT, not IL-17-/- mice. Intranasal or transcutaneous immunization protected WT but not IL-17-/- mice against hydrosalpinx development. Our data show that in the absence of IL-17, the severity of C. muridarum genital infection and associated oviduct pathology are significantly attenuated, however neither infection or pathology can be reduced further by vaccination protocols that effectively protect WT mice.

Figure 1. Clearance of vaginal C. muridarum infection in BALB/c and IL-17-/- mice.

Vaginal swabs were collected every 3 days following challenge of progesterone primed mice with 5 x 10⁴ C. muridarum. The number of IFU per swab was determined by culture on McCoy cells. The numbers of IFU per swab (A) and the area under the clearance curves (B) are presented. (*) or dotted bar represents p<0.05 significance.

Figure 2. Infection-induced occlusion of oviducts.

Hydrosalpinx was measured at 35 days post infection following challenge of WT BALB/c and IL-17-/- mice with 5 x 10⁴ C. muridarum. Dotted bar represents p<0.05 significance.

Figure 3. Neutrophil infiltration of oviducts following C. muridarum infection.

Oviducts were collected from WT BALB/c mice (A), IL-17-/- mice (B) 6 days post infection and stained for Ly-6G/Ly-6C as described in materials and methods. Staining of oviduct tissues was quantitated by image analysis (C) as described in methods. Scale bar = 100 µM. Dotted bar represents p<0.05 significance.

Figure 4. Macrophage infiltration of oviducts following C. muridarum infection.

Oviducts were collected from WT BALB/c mice (A), IL-17-/- mice (B) 6 days post infection and stained for F4/80 as described in materials and methods. Staining of oviduct tissues was quantitated by image analysis (C) as described in methods. Scale bar = 200 µM. Dotted bar represents p<0.05 significance.

Figure 5. Expression of matrix metalloproteinase MMP9 and MMP2 in oviducts and uterine horns of wild type BALB/c and IL-17-/- mice 7 days post infection with C. muridarum.

MMP expression was determined by both ELISA and gelatin zymography as described in materials and methods. Dotted bar represents p<0.05 significance.

Figure 6. MOMP-specific IgG and IgA in serum and vaginal wash fluids following intranasal (IN) immunization and resolution of a primary C. muridarum infection (LIC).

MOMP-specific IgG and IgA antibodies were measured by ELISA as described in materials and methods following intranasal immunization or resolution of a primary vaginal infection. Dotted bar represents p<0.05 significance.

Figure 7. MOMP-induced splenocyte proliferation and cytokine secretion following intranasal (IN) immunization or following resolution of infection (LIC).

Splenocytes were stimulated in vitro with MOMP and proliferation (A) determined by ³H-thymidine incorporation (CPM = counts per minute). Cytokines secreted by MOMP-stimulated cells (B) were measured by Bio-Plex assay as described in methods. Dotted bar represents p<0.05 significance.

Figure 8. The effect of immunization or a prior infection on chlamydial clearance and development of hydrosalpinx.

BALB/c or IL-17-/- mice were immunized by either the intranasal (IN) or transcutaneous (TC) routes or allowed to recover from a C. muridarum infection (LIC). Vaginal swabs were collected every 3 days following challenge. The number of IFU per swab was determined by culture on McCoy cells and plotted against time. From these plots, the area under the curve was calculated (A). Hydrosalpinx was measured at day 35 post-infection (B) as described in materials and methods. Dotted bar represents p<0.05 significance.