6 research outputs found
Th17 Cells and IL-17 in Protective Immunity to Vaginal Candidiasis
Background: Th17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC). Methods: To monitor the course of infection we exploited a new in vivo imaging technique. Results: i) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of bdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment. Conclusions: These results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis
Effect of halofuginone on vaginal infection.
<p>Mice, under pseudo-estrus conditions, were twice infected with 10<sup>7 </sup><i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl or 10 µg/100 µl of halofuginone solution or diluent of halofuginone and, in selected experiments, were treated intravaginally with 10 pg of mouse rIL-17. (A) Evaluation of IL-17 concentration by ELISA in supernatants of vaginal fluids obtained at different days after vaginal <i>Candida</i> infection and halofuginone treatment. Results are expressed as mean±SD (n = 9 mice, 3 mice for each of three separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice). At day 4, 14 and 25 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC) and treated with halofuginone or diluent. Images are representative of 5 out of 10 mice in two different experiments. (C) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes of mice (n = 10) treated with halofuginone or diluent. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. * <i>p</i><0.05, ** <i>p</i><0.01 (infected halofuginone treated mice vs infected diluent treated mice).</p
Effect of <i>Candida albicans</i> infection on draining lumbar lymph nodes.
<p>Lymph nodes, at different times after <i>Candida</i> infection, were aseptically recovered and mechanically homogenized. Cells were cultivated untreated or in presence of heat inactivated <i>C. albicans</i> for 72 hours. In the supernatant fluids of lymph node cell culture IL-17 (A) and IL-23 (B) were analyzed by ELISA. Results are expressed as mean±SD (n = 16 mice, 4 mice for each of four separate experiments). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (Lymphocytes from infected mice vs Lymphocytes from non infected mice). # <i>p</i><0.05, ## <i>p</i><0.01, (Lymphocytes re-stimulated from infected mice vs Lymphocytes re-stimulated from non infected mice).</p
Effect of halofuginone treatment on vaginal β defensin production.
<p>Mice, under pseudo-estrus conditions, were twice infected with 10<sup>7 </sup><i>Candida albicans</i> in vagina. Two days before and every two days after infection, mice were injected intraperitoneally with 5 µg/100 µl of halofuginone solution or diluent of halofuginone and were treated intravaginally with 10 pg of mouse rIL-17. At day 4, 8 and 14 after infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anesthesia using 2.5% isoflurane. (A) Dot plot of total photon emission from the infected regions and dot plot of CFU in vaginal washes. The statistical analysis was performed using non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one out of two independent experiments with similar results. After 4, 8, 14, 21, 28 days from challenge, the vaginal lumen was washed with 150 µl of saline and vaginal cells were recovered for β-defensin analysis. (B) Mean of fluorescence MIF of β-defensin 1, β-defensin 2 and β-defensin 3 cells evaluated by FACS analysis. The vaginal cells recovered by vaginal washes were stained with rabbit anti-mouse BD1, goat anti-mouse BD2 or goat anti-mouse BD3 and goat anti-rabbit TRIC conjugate or rabbit anti-goat PE conjugate respectively. (C) Percentage of epithelial cells producing d β-defensin 2. Data are the mean±SD (n = 8 mice, 4 mice for each of two separate experiments). Cells of vaginal washes were stained with FITC anti mouse pan-cytokeratin and goat anti-mouse BD2 and rabbit anti-goat PE conjugate. Data are representative of one out of two independent experiments (total 8 mice). The statistical analysis was performed using Mann-Whitney U test. * <i>p</i><0.05, (infected halofuginone+IL-17 treated mice vs infected halofuginone treated mice).</p
IL-17 and IL-23 concentration in murine vaginal washes of mice infected with <i>Candida albicans</i>.
<p>Evaluation of IL-17 (A–C) and IL-23 (D) concentration by ELISA test on supernatants of vaginal fluids obtained at different times after vaginal infection with different doses of <i>Candida albicans</i> gLUC59 (A–B) or CA1399 (D). Results are expressed as mean±SD (n = 12 mice, 4 mice for each of three separate experiments). * <i>p</i><0.05, (infected mice vs non infected mice).</p
Model of murine vaginal infection and monitoring.
<p>(A) Timeline of vaginal infection model. CD1 mice are resistant to <i>Candida</i> vaginal infection unless they are treated with estradiol valerate. CD1 mice were treated subcutaneously with estradiol valerate and infected for two consecutive days with 10 µl of 10<sup>9</sup>/ml suspension of <i>Candida albicans</i> cells (gLUC) into vaginal lumen. Two days before and every two days after challenge mice were treated intraperitoneally with halofuginone or diluent of halofuginone and, in selected experiments, intravaginally with 10 pg of recombinant mouse IL-17. After 4, 8, 12, 14, 18, 20, 25, 30 days post infection, mice were treated intravaginally with 10 µg of coelenterazine and imaged in the IVIS-200™ imaging system under anaesthesia using 2.5% isoflurane and the vaginal lumen was washed with 150 µl of saline using mechanical pipette. The fungal burden of vaginal fluids was evaluated by colony forming units (CFU) assay. (B) In vivo imaging of mice vaginally infected with <i>Candida albicans</i> cells (gLUC). Images are representative of 5 out of 10 mice for each experiment. C) Dot plots of total photon emission from the infected vaginal regions and corresponding CFU in vaginal washes of infected mice (n = 10). The statistical analysis was performed by non-parametric Mann-Whitney U test. The median is indicated by a straight line. Data are representative of one of two independent experiments with similar results. D) The correlation between the Total Photons emitted and CFU count in the vaginal wash was assessed using the Pearson's correlation statistics, and the correlation coefficients are shown for each time point. * <i>p</i><0.05, ** <i>p</i><0.01, (Log Photons Total Flux or Log CFU/ml of mice infected after 8, 12,14,18,20,25,30 days vs Log Photons Total Flux or Log CFU/ml of mice infected after 4 days).</p