| Summary: | Approximately 40% of women will experience at least one urinary tract infection (UTI) in their lifetime and 25% of these women will go on to suffer recurrent UTIs. After the menopause, the risk of developing a recurrent UTI doubles, which is putatively linked to reduced estrogen levels. Topical vaginal estrogen treatment in postmenopausal women has been shown to reduce the incidence of UTIs. However, the mechanism by which vaginal estrogen helps to protect against UTIs is not well understood. Antimicrobial peptides (AMPs), secreted in response to infection and functioning via bacterial cell lysis, are an important component of the host innate immune response to infection. The aim of this project was to investigate the effects of estrogen treatment on vaginal epithelial AMP expression and synthesis. Immortalised vaginal epithelial cells (VK2 E6/E7), used to model the vaginal epithelium, were treated with 4nM 17β-estradiol for seven days and then challenged with 50ng/ml flagellin (isolated from Escherichia coli clinical UTI isolate) for 24 hours. Combined estrogen pretreatment plus flagellin resulted in a 2.3- and 2.1-fold increase in expression of the AMPs human β-defensin 2 (hBD2) and hBD3, respectively, above that observed with flagellin challenge alone (p-values < 0.001). Microarray analyses identified upregulation of the AMPs LCN2, RNase 7, S100A7, S100A12, SLPI, by estrogen pretreatment plus flagellin in addition to hBD2 and hBD3. Furthermore, several genes relating to keratinisation (for example keratin and SPRR genes) and inflammation (for example SERPINB4, S100A8 and S100A9) were also upregulated suggesting that estrogen pretreatment stimulated multiple protective responses in VK2 cells. A hBD2 luciferase reporter vector containing a 2032bp hBD2 promoter region was used to measure hBD2 expression in response to estrogen and flagellin treatments. Reporter activity in VK2 cells significantly increased 2.1-fold following seven day estrogen pretreatment (4nM) plus flagellin (50ng/ml) challenge compared to flagellin challenge without estrogen pretreatment (p=0.0367). Six estrogen response elements (EREs) were identified in the hBD2 promoter and were mutated by site-directed mutagenesis. Mutation of each of the EREs abolished hBD2 gene expression potentiation by estrogen pretreatment plus flagellin, suggesting that hBD2 is regulated through ER-α or ER-β binding to EREs in the hBD2 promoter. Pathway analysis of the microarray data identified IL-17A as a potential regulator of AMP expression. Thus, VK2 cells were challenged with exogenous IL-17A in concentrations ranging from 0.1ng/ml to 100ng/ml for 24 hours. The expression of hBD2, LCN2, RNase 7, and S100A7 was significantly increased between 2- and 21- fold in an IL-17A dose dependent manner (p-values < 0.001). In addition, estrogen pretreatment of VK2 cells prior to challenge with IL-17A (100ng/ml) plus flagellin (50ng/ml) significantly increased the expression of hBD2, hBD3 and S100A7 by between 1.4- and 1.6-fold compared to IL-17A plus flagellin without estrogen (p-values < 0.05). These data indicated an important role for estrogen in AMP expression even in the presence of proinflammatory cytokines, such as IL-17A. Altogether, these data indicated that estrogen is an important regulator of innate epithelial defences of the urogenital tract. These results suggest that estrogen protects against UTIs by augmenting the vaginal antimicrobial response to infection and by strengthening the epithelial barrier to infections. Hence, loss of estrogen following the menopause leaves postmenopausal women susceptible to repeated UTIs.
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