Cervico-vaginal immunoglobulin g levels increase post-ovulation independently of neutrophils

The prevalence of sexually transmitted infections (STIs) is often higher in females than in males. Although the reproductive cycle profoundly modulates local immunity in the female reproductive tract (FRT) system, significant gaps in our knowledge of the immunobiology of the FRT still exist. An intriguing and frequently observed characteristic of the FRT is the predominant presence of immunoglobulin (Ig) G in cervico-vaginal secretions. We show here that in the mouse, IgG accumulation was enhanced approximately 5-fold post-ovulation, and was accompanied by an influx of neutrophils into the FRT. To determine whether these two events were causally related, we performed short-term neutrophil depletion experiments at individual stages throughout the estrous cycle. Our results demonstrate that neutrophils were not necessary for cycle-dependent tissue remodeling and cycle progression and that cycle-dependent IgG accumulation occurred independent of neutrophils. We thus conclude that neutrophil influx and IgG accumulation are independent events that occur in the FRT during the reproductive cycle

Influence of the estrous cycle on cervico-vaginal IgG levels.

<p>Vaginal washings were taken from naïve adult female C57BL/6 mice (8–12 weeks old) throughout the course of the estrous cycle. Samples were analyzed by mouse IgG-specific ELISA. Each graphic symbol represents one experimental animal. Data are derived from three independent experiments (n = 4) and were analyzed using one-way ANOVA with post-test (Tukey's). The mean±SEM is shown (*, p≤0.05; **, p≤0.01).</p

Tissue changes in the FRT during the estrous cycle of the mouse.

<p>Vaginal smears were taken from naïve virgin C57BL/6 female mice (8–12 weeks old) over the course of one estrous cycle and used for determination of cycle stage. Mice were killed at PE (<b>A</b>), E (<b>B</b>), ME (<b>C</b>) and DE (<b>D</b>) and tissue sections of vagina were cut. Vaginal smears (top panel) and tissue sections (bottom panel) were stained with H&E. The insets in the top panel show typical cell types found in vaginal smears of each cycle stage. The inset in the bottom panel shows neutrophils present in vaginal epithelium. Representative images from conventional cycle stage assessments are shown. Top panel, 100× magnification; bottom panel, 400× magnification.</p

Effects of Ly6G depletion on cervico-vaginal IgG.

<p>Naïve virgin C57BL/6 mice were treated with the Ly6G-depleting antibody 1A8 or the isotype control 2A3 at PE (<b>A</b>), E (<b>B</b>), ME (<b>C</b>) or DE (<b>D</b>). Cervico-vaginal washings were taken 24 and 48h after mAb administration according to the described protocol. Data are presented as ratios of genital IgG levels post-injection, measured by ELISA, relative to genital IgG levels pre-injection (baseline). Data are derived from one experiment (n≥4 mice) and were analyzed using unpaired t test. The mean±SEM is shown (n.s., non-significant).</p

Detection and quantification of neutrophils and monocytes in the total lower FRT during the estrous cycle by flow cytometry.

<p>Lower FRT was isolated from naïve adult virgin female C57BL/6 mice and prepared for flow cytometry. Cells were gated as CD45.2⁺CD11b⁺CD11c^lo (as shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114824#pone.0114824.s001" target="_blank">Figure S1</a></b>) prior to further analysis. (<b>A</b>) Percentage of CD45.2⁺CD11b⁺CD11c^loLy6C⁺Ly6G⁺ cells (neutrophils) and CD45.2⁺CD11b⁺CD11c^loLy6C⁺Ly6G⁻ (monocytes) at each cycle stage (estrus,E; proestrus, PE: metestrus, M; diestrus, DE). (<b>B–D</b>) Frequency (<b>B</b>), absolute number (<b>C</b>) and morphology (<b>D</b>) of neutrophils at each cycle stage. (<b>E–G</b>) Frequency (<b>E</b>), absolute number (<b>F</b>) and morphology (<b>G</b>) of monocytes at each cycle stage. Samples in <b>(D)</b> and <b>(G)</b> were Giemsa stained cytospins after sorting. Representative images shown in <b>(D)</b> and <b>(G)</b> were taken at 400× magnification. Data derived from three independent flow cytometry experiments (each run with tissue pooled from four mice per time point) were quantified and analyzed using one-way ANOVA with Tukey's post-test. The mean±SEM is shown (*, p≤0.05; **, p≤0.01; ***, p≤0.001).</p