We report that VLF from women with BV is deficient in AMP and antimicrobial activity compared to VLF from healthy women or women with VVC. The deficiency is not merely due to dilution but reflects altered composition with reduced concentrations of small cationic peptides that migrate rapidly in both acid-urea-PAGE (Fig. ) and SDS-PAGE (Fig. ). Quantitative analysis corroborates these results (Fig. ), where nearly all of the AMP were two- to fourfold lower in BV fluid compared to H and VVC fluids. The vaginal AMP originates from different sites and cell types. The α-defensins 1 to 3 (HNP) originate from neutrophils, the β-defensin HBD-2 is induced by inflammation in epithelial cells, and SLPI is predominantly secreted into cervical mucus. Our observation of decreases in all three components suggests that BV is associated with a local impairment of multiple innate immune pathways. The antimicrobial activity of BV VLF is also much reduced (Fig. ). Based on our previous studies (38
), the antimicrobial activity of vaginal fluid depends predominantly on its lactic acid content with a smaller contribution from AMP, and both of these factors are reduced in BV. Reduced cationic polypeptide concentrations, including SLPI, would also be expected to decrease the resistance to HIV transmission (19
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 (more ...)
We considered the possibility that decreased levels of AMP in women with BV reflected a genetic deficiency. However, the AMP levels observed in women with BV rose to the range observed in healthy women after successful medical treatment, suggesting that the women were genetically competent to produce normal amounts of AMP. VVC-VLF, in contrast, showed elevated protein and AMP compared to BV VLF, likely due to the proinflammatory effect of yeast infections. Although detailed comparison of H and VVC polypeptide concentrations was not one of the goals of the present study, it is notable that lysozyme and HBD-2 concentrations were significantly higher in VVC than in H samples (Fig. , P = 0.04 and 0.01, respectively). Because of within-group variations, comparisons of the H, BV, and VVC groups to each other are less sensitive to disease-related changes in AMP concentrations than paired pre- and posttreatment comparisons, where each patient serves as her own control. These analyses in VVC revealed significant posttreatment decreases of AMP (Fig. and ), as well as total protein levels, and the normalization of electrophoretic protein patterns after treatment. The increase in AMP during VVC is in sharp contrast to the decrease in AMP during BV and undoubtedly reflects the differential effects of the respective microbial agents on innate immunity.
BV is characterized by a microbial shift from predominantly lactobacilli to a mixed anaerobic milieu. These anaerobes are smaller in size and much more numerous than lactobacilli (34
), leading to an increased bacterial surface area. Most AMP require a threshold concentration for effective disruption of microbial membranes and the resulting antimicrobial activity (31
). We speculated that the cationic AMP could be removed from the fluid phase by binding to the increased surface area of the anionic bacterial membranes. To test this hypothesis, we extracted protein from VLF cell pellets, which contained bacteria and epithelial cells, but were unable to recover any low-molecular-weight cationic proteins (Fig. ), indicating that the missing AMP were not trapped in the cellular fraction of BV VLF.
We next examined whether the change in the composition of VLF reflects differences in the modulatory effects of vaginal microbial flora on cytokine and AMP production. Other investigators have reported that BV generates a very low tissue inflammatory response so that 50% of women with BV are asymptomatic (14
). Our analysis of local cytokine and chemokines responses found elevated IL-1β but not a concomitant elevation in IL-8, which is the primary chemotactic factor for neutrophils (Fig. ). These findings are in agreement with previous reports (5
). The low IL-8 concentration in the vaginal fluid of BV patients may explain the relatively low neutrophil influx in BV (5
), as well as the low levels of the neutrophil-specific HNP defensins. Although the detailed mechanisms have not been identified, it has been suggested that factors secreted by the microbial flora in BV inhibit the IL-8 response despite the elevation of IL-1 concentrations (5
The reasons for the deficiency of HBD-2 and other epithelially secreted polypeptides in BV appear to be more complex. In previous studies of epithelial production of HBD-2 in organotypic epidermal cultures, IL-1 was found to be a potent inducer of HBD-2 synthesis, as well as the predominant HBD-2-inducing component of the secretions of lipopolysaccharide-stimulated monocytes (15
). We therefore examined the possibility that the HBD-2 deficiency in BV samples was due to either a low concentration of IL-1 or a high concentration of its principal antagonist, IL-1ra. Surprisingly, IL-1ra was not induced in BV compared to H or VVC VLF, so that the ratio of IL-1ra to IL-1in BV VLF was lower than in the H or VVC samples (Fig. ). We therefore used an in vitro model of VE to examine whether the bacterial flora can modify HBD-2 production independently of IL-1. In these experiments, L. jensenii
or G. vaginalis
was placed on the model VE surface. Although the complexity of microbial flora in healthy women and women with BV is well established (13
), L. jensenii
and G. vaginalis
were chosen as model organisms because they are common and abundant in healthy women and women with BV, respectively: ca. 30 to 40% of healthy women are colonized by L. jensenii
), and G. vaginalis
is found in up to 94% of all cases of BV (18
). The lactobacilli induced HBD-2, IL-1, and IL-8 (Fig. ), but comparable densities of Gardnerella
did not induce HBD-2 or IL-8 and only minimally induced IL-1. In addition, lactobacilli were more effective inducers of HBD-2 and IL-8 than was the relatively high concentration (20 ng/ml) of IL-1β. In another experiment, when excess of IL-1ra (200 ng/ml) was added to the culture medium before the addition of bacteria to the surface of the tissue, IL-1ra did not influence the bacterium-induced HBD-2 or IL-8 concentrations (data not shown). Taken together, these data suggest that the composition of microbial flora affects the concentrations of AMP through mechanisms distinct from the IL-1-dependent pathway.
Microbes can directly induce AMP and cytokines by interactions of their characteristic macromolecules (“patterns”) with Toll-like receptors and other “pattern” recognition systems (36
). G. vaginalis
is a gram-positive bacterium, as indicated by ultrastructural studies and the lack of lipopolysaccharide (29
), but its peptidoglycan content is unusually low. These characteristics may contribute to its ability to avoid triggering the inflammatory response that is induced by other microbes that cause vaginal infections, such as Candida albicans
. It has also been suggested that G. vaginalis
may actively suppress inflammation, but the specific mechanisms involved are still speculative.
In vivo, BV flora appears to be less stimulatory than normal flora for the production of innate immune mediators, and this difference is replicated in vitro when organotypic vaginal epithelium is exposed to the BV agent G. vaginalis compared to the normally resident L. jensenii. By decreasing the stimulus to innate immunity, BV may produce a state of local immunosuppression that could increase susceptibility to HIV and other sexually transmitted diseases.