Reversible deficiency of antimicrobial polypeptides in bacterial vaginosis

Abstract

Bacterial vaginosis is a common condition associated with increased risk of sexually transmitted diseases, including human immunodeficiency virus infections. In contrast, vulvovaginal candidiasis has a much weaker association with sexually transmitted diseases. We found that vaginal lavage fluid from women with bacterial vaginosis is deficient in antimicrobial polypeptides and antimicrobial activity compared to fluid from healthy women or women with vulvovaginal candidiasis. Effective treatment normalized the concentrations of antimicrobial polypeptides in both bacterial vaginosis and in vulvovaginal candidiasis, suggesting that the abnormalities were a result of the diseases. Unlike in vulvovaginal candidiasis, the neutrophil attractant chemokine interleukin-8 (IL-8) was not increased in bacterial vaginosis, accounting for low concentrations of neutrophil-derived defensins in vaginal fluid. In organotypic cultures of human vaginal epithelium containing dendritic cells, treatment with Lactobacillus jensenii, a typical vaginal resident, induced the synthesis of IL-8 mRNA and the epithelial human beta-defensin-2 mRNA, but a typical bacterial vaginosis pathogen, Gardnerella vaginalis, had no effect. When the two bacteria were combined, Gardnerella vaginalis did not interfere with the immunostimulatory effect of Lactobacillus jensenii. The loss of normal immunostimulatory flora in bacterial vaginosis is thus associated with a local deficiency of multiple innate immune factors, and this deficiency could predispose individuals to sexually transmitted diseases.

FIG. 1.

AMP in vaginal infections. VLF samples from healthy women (H) and those with BV or VVC before and after (-R) treatment were analyzed by immunoassay. Total protein was analyzed by the bicinchoninic acid assay. The box-whisker plot shows median values and the 25 to 75% interquartile range as lines within the boxes, the 10 to 90% range as whiskers, and values outside this range as dots. The Mann-Whitney rank-sum test was used to compare BV to H and BV to VVC, and P values of ≤0.05 are indicated in boldface. BV, n = 19; BVR, n = 16; BVR, n = 12; VVC, n = 24; VVCR, n = 20. The values after treatment (BVR and VVCR) are shown here for reference. The analysis of the effects of treatment was performed separately using paired before-and-after samples for each donor (Fig. 2).

FIG. 2.

Effects of treatment on vaginal AMP. For each protein, we calculated the ratio of the concentration in VLF after treatment to that before treatment. The individual ratios and group median and interquartile range are shown. BV, n = 12; VVC, n = 20. The Wilcoxon signed-rank statistic was used to compare the values before and after treatment, and the P values are shown above the horizontal axis, with P values of <0.05 indicated in boldface.

FIG. 3.

Differences in the protein composition of VLF in healthy women (H) and women with vaginal infection (BV and VVC) before and after treatment. The top panel shows acid-urea-PAGE in which the mobility of each protein species depends on its cationic charge and inversely on its size. The bottom panel is an SDS-Tricine-PAGE, which separates the proteins by size. The migration of lysozyme and HBD-2 standards is indicated in the upper panel, whereas molecular mass markers are indicated in the lower panel (in kilodaltons). For patients with BV or VVC, paired samples are shown before and after (r) treatment. Areas of marked differences are indicated by bars on the left side of each gel.

FIG. 4.

Acid-urea-PAGE of the cellular (c) and fluid (f) components of VLF in BV. Paired samples are shown before and after (r) treatment for BV. The migration of HNP (defensin)-1, lysozyme, and HBD-2 standards is as indicated.

FIG. 5.

Bactericidal activity of VF fluid. Median and interquartile ranges of log changes in E. coli 8739 CFU are shown. Exponentially growing E. coli 8739 (ATCC) was added to VLF and incubated for 2 h at 37°C in an environmental shaker. Aliquots at time zero and 2 h were diluted, dotted onto Trypticase soy agar plates, and incubated overnight at room temperature. The number of colonies was multiplied by the dilution factor to determine the CFU. The log change CFU was calculated from the input CFU compared to the CFU at 2 h. A value of zero indicates no change, values above zero indicate bacterial growth, and values below zero indicate bactericidal activity.

FIG. 6.

Cytokine concentrations in VLF determined by enzyme-linked immunosorbent assay. The box-whisker plot shows median values and the 25 to 75% interquartile range as lines within the boxes, the 10 to 90% range as whiskers, and values outside this range as dots. Median BV values were compared to H and VVC by using the rank-sum test. Before- and after-treatment values (BV versus BV-R and VVC versus VVC-R, respectively) were compared by the paired Student t test with P values of ≤0.05 in boldface.

FIG. 7.

Induction of HBD-2, IL-1, and IL-8 by L. jensenii but not G. vaginalis. Bacteria were grown to saturation and then brought to an OD₆₂₀ of 1.0, 0.1, 0.01, or 0.001 with their respective culture medium (MRS for L. jensenii or BHI plus 4% lysed horse blood for G. vaginalis). Aliquots were boiled for 5 min to heat inactivate the bacteria. (A) A total of 20 μl of heat-inactivated or culture medium (M) bacteria was placed onto the top surface of the VE tissue (MatTek), or IL-1β (20 ng/ml) was added to the culture medium. Control (C) tissue was untreated. After 24 h of incubation, tissues were analyzed by quantitative reverse transcription-PCR. Concentrations of cytokine mRNAs are shown relative to the G3PDH mRNA as differences in threshold cycles (ΔC_T). Closed and open symbols indicate L. jensenii and G. vaginalis, respectively. Differences significant at a P of <0.05 between samples treated with the two different bacteria are marked by asterisks. (B) Combinations of L. jensenii and G. vaginalis (20 μl each at an OD₆₂₀ of 1.0, 0.1, 0.01, 0.001, or 0 with their respective culture medium) were placed on the top surface of the VE tissue, and the mRNA was analyzed 24 h later. Concentrations of cytokine mRNAs are shown relative to G3PDH mRNA as differences in threshold cycles (ΔC_T). The OD₆₂₀ values of the added G. vaginalis were as follows: 0, •; 0.001, ○; 0.01, ▾; 0.1, ▿; and 1.0, ▪. Representative error bars are shown. L. jensenii (LbJ) stimulated mRNA expression of HBD-2, IL-1, and IL-8 in a dose-dependent manner. G. vaginalis had no detectable effect on stimulation by L. jensenii.