Introduction

The vaginal microbiome in most reproductive age women is dominated by species of Lactobacilli and vaginal fluid is acidic due to production of lactic acid by these bacteria [1••]. This differs from what is present in all other mammals, including non-human primates, where Lactobacilli are scarce and vaginal pH approaches neutrality. These changes are likely a consequence of the unique behavioral, dietary, and environmental exposures of humans [2, 3]. Vaginal epithelial cells in women have had to adapt to these species differences to optimize a lower genital tract environment most compatible with health and reproductive efficacy. This communication will review properties of human vaginal epithelial cells that monitor and respond to external variations in the local environment.

Epithelial Cell Turnover and Survival in an Acidic Environment

Several layers of rapidly regenerating stratified squamous epithelial cells line the human vagina. The layer closest to the vaginal lumen exfoliates approximately every 4 h [4]. This serves a protective function as any potential microbial pathogen that have gained entry and adhered to the vaginal epithelium will be rapidly disengaged from the vagina and eliminated. In addition, the degradation of sloughed epithelial cells results in the release of glycogen into the vaginal lumen. The breakdown of glycogen by alpha amylase, an enzyme present in human vaginal secretions [5], results in production of small carbohydrates that are utilized by Lactobacilli to preferentially facilitate their proliferation in the vagina [6].

Resident Lactobacilli are the principal source of lactic acid, the major acid in the vagina [7, 8], responsible for maintaining an acidified vaginal lumen. The acidic pH lyses many non-endogenous bacterial species and prevents their maintenance at this site [9, 10]. To remain viable in this acidic environment, the vaginal epithelial cells must be able to regulate the influx and egress of hydrogen ions. This is accomplished by the activity of a protein, monocarboxylate transporter 1 (MCT-1), on the epithelial cell surface. MCT-1 activity requires the presence of an essential co-factor, extracellular matrix metalloproteinase inducer (EMMPRIN) [11]. EMMPRIN is also produced by vaginal epithelial cells and its concentration in vaginal fluid is directly proportional to the lactic acid level [8]. This suggests that the vaginal epithelial cells are sensitive to the level of acidity and respond by adjusting their production of EMMPRIN.

Epithelial Cell Recognition of Bacteria

The dominant Lactobacilli species in most reproductive age women are Lactobacillus crispatus and L. iners. The dominant non-Lactobacillus species is usually Gardnerella vaginalis [12]. Recent studies have demonstrated that the expression of genes in vaginal epithelial cells differs according to the dominant bacterium that is present. One mechanism regulating gene transcription is the epigenetic alteration of the acetylation state of histones that are associated with a particular gene. Acetylated histones do not bind as tightly to chromosomes as do histones whose acetyl groups have been removed. A specific enzyme, histone deacetylase (HDAC), removes acetyl groups from histones and, thereby, inhibits gene activation [13]. In a study of 150 pregnant women, the HDAC concentration in vaginal epithelial cells was lowest when L. crispatus was the dominant vaginal bacterium and significantly elevated when non-Lactobacilli dominated the microbiome [14]. The HDAC level was positively associated with the concentration of matrix metalloproteinase-8 (MMP-8) and the stress-inducible 70 kDa heat shock protein (hsp70) in vaginal secretions. Many studies have concluded that L. crispatus numerical dominance is associated with vaginal health and maintenance of conditions conducive to successful pregnancy progression and outcome [15, 16]. Thus, it appears that the differential regulation of HDAC production by vaginal epithelial cells in response to the bacterium that is dominant in the vagina influences the production and release of specific bioactive compounds from these cells. Extracellular hsp70 is a potent inducer of pro-inflammatory immunity [17], while MMP-8 releases hyaluronan from the extracellular matrix leading to activation of anti-microbial immunity [18]. A second study has provided additional evidence of epigenetic gene regulation in vaginal epithelial cells by Lactobacilli. Expression of the gene coding for human beta defensin-1, DEFB1, in an immortalized vaginal epithelial cells line was shown to vary between different strains of Lactobacilli through species-specific alterations in DNA methylation and histone modification [19].

Production of other compounds by vaginal epithelial cells has also been shown to be sensitive to the bacterial milieu. Neutrophil gelatinase-associated lipocalin (NGAL) production is induced in these cells when Lactobacilli predominate [20]. This protein inhibits bacterial uptake of iron and, therefore, inhibits proliferation of those microorganisms that require iron for growth [21]. G. vaginalis is an iron-dependent bacterium [22], while Lactobacilli can proliferate in the absence of this element [23]. Other iron-binding compounds released from vaginal epithelial cells include lactoferrin [24] and calprotectin [25]. Thus, the capability of vaginal epithelial cells to influence the vaginal level of iron, and possibly other elements, will impact the likelihood of specific bacterial dominance at this site. A direct comparison of levels of compounds in vaginal fluid from pregnant women when either L. crispatus or L. iners was numerically dominant revealed that levels of NGAL and calprotectin, as well as other compounds involved in anti-microbial defense—stress-induced hsp70, MMP-8, and hyaluronan—were preferentially induced by L. iners [26]. This reinforces studies demonstrating that L. iners dominance in the vaginal microbiota is associated with induction of a stress response in the vaginal epithelium [27].

Other anti-microbial compounds differentially released by vaginal epithelial cells in response to the presence of specific microorganisms include secretory leukocyte protease inhibitor [28], mannose-binding lectin [29], beta defensins [30], and cathelicidins [31]. The surface of vagina epithelial cells contains a number of Toll-like receptors (TLR) that recognize invariant molecular patterns on microorganisms [32]. TLR-agonist binding results in the production of a large number of pro-inflammatory cytokines—interleukin (IL)-1, IL-6, IL-8, IL-12, and tumor necrosis factor-alpha [33, 34]. The release of these cytokines from in vitro-cultivated vaginal epithelial cells could not be demonstrated when either L. crispatus or L. jensenii was present. Furthermore, these two Lactobacillus strains inhibited epithelial cell cytokine expression when TLR agonists were added to the cultures [35]. This further highlights the sensitivity of vaginal epithelial cells to the local external environment and its influence on composition of the vaginal milieu.

Epithelial Cell Responses to Stress

Studies in a mouse model have demonstrated that maternal stress changes the composition of the vaginal microbiota, resulting in a decreased level of Lactobacilli [36]. Evidence for a possible involvement of vaginal epithelial cells in the stress-related alteration of vaginal microbial abundance comes from a recent study demonstrating that two human vaginal epithelial cell lines secreted both norepinephrine and dopamine. In addition, these cells recognized and bound exogenous norepinephrine [37]. Interestingly, norepinephrine by itself did not induce production of pro-inflammatory compounds from vaginal epithelial cells, but its presence resulted in a significant upregulation of the release of these mediators in the simultaneous presence of immune system activators. These observations led us to postulate a stress-vaginal dysbiosis relationship based on vaginal epithelial cell responses [1••]. Production of norepinephrine in woman experiencing prolonged stress results in its appearance in the vagina due to transduction from the circulation. Norepinephrine binding to vaginal epithelial cells coupled with its local production by these cells leads to the decrease or loss of Lactobacilli dominance in the vagina. The subsequent increased production of non-Lactobacilli and their induction of inflammation are enhanced by the presence of norepinephrine. Thus, disparities between individual women in the magnitude of their response to various stressors may determine their propensity to develop a symptomatic vaginal disorder. A strain of Lactobacillus, L. salivarius, has very recently been shown to possess receptors for the uptake of neuroactive biogenic amines [38••]. If Lactobacilli in the vagina possess a similar mechanism, still to be determined, then vaginal epithelial cell-Lactobacilli cross-communication may have even a more enhanced influence on the stress-associated vaginal environment.

The stress-induced reduction in the vaginal Lactobacillus level is also a consequence of the effect of cortisol on vaginal epithelial cells [39]. Elevations in cortisol as a result of prolonged stress inhibit glycogen deposition in the epithelial cells. This reduces the glycogen concentration in the vaginal lumen resulting in local conditions that are less than ideal for the preferential proliferation of Lactobacilli.

Vaginal Epithelial Cell Autophagy

Autophagy is a mechanism present in all multicellular organisms to maintain intracellular homeostasis [40]. Misfolded or aggregated proteins, aged mitochondria as well as fungi, bacteria and viruses that have entered the cytoplasm are engulfed in autophagosomes. The subsequent fusion with a lysosome results in degradation of the engulfed macromolecules or microorganisms by lysosomal enzymes and the resulting amino acids, nucleotides, carbohydrates, and lipids are returned to the cytoplasm for reutilization. The induction of a stress response results in upregulation of hsp70, an inhibitor of autophagy [41]. Unlike autophagy, which removes non-functional proteins, hsp70 binds to intracellular proteins to maintain their functional activity and prevent their misfolding [42]. This latter process appears to take precedence over autophagy under non-physiological conditions [43, 44]. A recent study evaluated the level of autophagy in vaginal epithelial cells from pregnant women in relation to bacterial dominance in the vaginal microbiome [45•]. It was determined that L. crispatus dominance was associated with the highest level of epithelial cell autophagy and the lowest intracellular hsp70 concentration. These levels were significantly different from what was observed when L. iners was dominant. The highest hsp70 level and lowest autophagy corresponded to Streptococcus and Bifidobacterium dominance. It thus appears that vaginal epithelial cells modulate their level of autophagy in response to the vaginal bacterial composition and the predominance of L. crispatus at this site results in the maintenance of an optimal intracellular environment. This may account, at least in part, for the association between L. crispatus vaginal dominance and normal pregnancy progression [15, 16]. A low level of autophagy has been associated with elevations in reactive oxygen species, possibly from defective or aged mitochondria, and induction of preterm birth [46]. In addition, vaginal epithelial cell homeostasis would maximize the ability of these cells to recognize and respond to potential pathogens by the induction of innate immunity [47].

Candida albicans that has penetrated into vaginal epithelial cells is sequestered into autophagosomes and destroyed by autophagy [48••]. Inhibition of autophagy in these cells permitted C. albicans to proliferate and kill the infected cell [49]. Conversely, the detection of Streptococci in vaginal secretions from pregnant women is followed by the inhibition of autophagy in vaginal epithelial cells and the upregulation of the autophagy inhibitor, hsp70 [45•, 50]. Thus, the differential capacity of microorganisms that are present in the vagina to either promote or inhibit autophagy modulates this anti-microbial defense mechanism in vaginal epithelial cells.

Conclusion

There is a symbiotic relationship between human vaginal epithelial cells and the resident vaginal microbiota in the exchange of macromolecules and creation of an environment that protects against the invasion and establishment of potential pathogens. Changes in the bacterial environment and the presence of factors that alter the vaginal microbiome induce differential gene activity and the upregulation by vaginal epithelial cells of compounds with anti-microbial and immune-inducing activity. Properties of vaginal epithelial cells that are responsive to variations in the local environment are summarized in Table 1. This adaptation to a milieu—namely vaginal acidity and Lactobacilli dominance—specific to humans maximizes conditions conducive to pregnancy progression.

Table 1 Properties of vaginal epithelial cells responsive to the external milieu
Full size table