Abstract
The female climacteric or menopausal process characterised by reduced estrogen, associates with an increased risk of recurrent urinary tract infections (rUTIs) linked to uropathogenic Escherichia coli (UPEC). Clinically, topical vaginal estrogen treatment has a prophylactic effect against such infections. The aim of this study was to investigate, in vitro, the effects of a topical estrogen treatment on vaginal epithelial responses following challenge with E.coli flagellin mimicking an UPEC challenge. Immortalised vaginal epithelial cells (VK2 E6/E7), modelling the vaginal epithelium were treated with either 4 nM 17β-estradiol (E) for seven days, 50 ng/ml E.coli flagellin (F) for 12 h, or 4 nM 17β-estradiol plus 50 ng/ml flagellin (E + F(12 h)). RNA was analysed by microarray gene profiling using the Illumina HumanHT-12 v 4 Expression Beadchip. Following E + F treatments expression of genes encoding host defence molecules including DEFβ4A, DEFB103A, LCN2 as well as those associated with keratinisation eg CNFN and SPRR family genes were significantly enhanced (P < 0.05) compared to either E or F treatments alone. Mutation of estrogen responsive elements (EREs) identified in the DEFβ4 gene promoter abolished the augmented gene expression suggesting estrogen functioned directly through a regulatory mechanism involving ESR1/2. Ingenuity pathway analyses also suggested the pro-inflammatory cytokine IL-17A to regulate the vaginal host defences during infection. Pre-treating VK2 E6/E7 cells with estrogen (4 nM) and challenging with 1L-17A & F (12 h) significantly enhanced DEFβ4, DEF103A and S100A7 expression (P < 0.05). Origins of vaginal IL-17 in vivo remain unclear, but patient biopsies support γδ T cells located within the vaginal epithelium. These data suggest that the vaginal antimicrobial response induced by flagellin activation of Toll-like Receptor 5 cell signalling is augmented following topical estrogen application.
Highlights
Decreased circulating concentrations of the steroid hormone estrogen characterise the female climacteric, which in some women coincides with the onset of urogenital pathologies, including vaginal atrophy and recurrent urinary tract infections (rUTIs) Topical vaginal estrogen treatments, but not systemic estrogen therapy have been shown to alleviate rUTIs in post-menopausal women[1,2]
Microarray data relating to the E, F (12 h) and E + F (12 h) challenges were mined and gene probes shown to be differentially expressed in response to either estrogen pretreatment (E), or Flagellin (F12) and/or E + F12 treatments identified (Fig. 1A & B)
Five genes (Fig. 1B), LCN2, S100A8, S100A9, SPRR2A & SPRR2F encoding proteins involved in host defence and keratinisation were differentially expressed in response to each of the three treatments (E12, F12, and E + F12)
Summary
Decreased circulating concentrations of the steroid hormone estrogen characterise the female climacteric, which in some women coincides with the onset of urogenital pathologies, including vaginal atrophy and rUTIs Topical vaginal estrogen treatments, but not systemic estrogen therapy have been shown to alleviate rUTIs in post-menopausal women[1,2]. Uncomplicated rUTIs experienced by post-menopausal women are generally characterised as ascending infections that, in more than 75% of cases, link to Esherichia coli[3] Such bacteria referred to as uropathogenic E.coli (UPEC) originate in the gut, colonise the vaginal epithelium and migrate via the urethra to the bladder where they cause symptomatic infections[4,5]. Protection of the female urogenital tract from such uropathogens, pre-menopausally, is mediated by an array of constitutive and induced host innate defence factors as well as the vaginal microbiota This microbiota is dominated by Lactobacilli[7,8], which metabolise glycogen derived simple sugars to lactic acid, resulting in an www.nature.com/scientificreports acid micro-environment hostile to UPEC survival[9]. The effects of estrogen on the vaginal host innate defences during periods associated with potential UPEC infections are less well described
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