Are beta defensin 1 and beta defensin 2 key innate immune effector peptides against urinary tract infection in women?

Abstract

Introduction & Hypothesis Recurrent urinary tract infection (rUTI) is a debilitating problem affecting 5% of women. Current treatment using intermittent or long-term antibiotics gives limited symptomatic benefit and encourages bacterial resistance. The aetiology of rUTI is unclear but may involve altered innate defence mechanisms in susceptible individuals. Colonisation of vaginal mucosa by uropathogenic Escherichia coli (UPEC) is the initiating event for UTI, with subsequent migration up the urethra and attachment to bladder epithelium. Protective innate immunological mechanisms include epithelial synthesis of cationic antimicrobial peptides (AMPs) such as the beta defensins. These may be expressed constitutively or induced via Toll-like-receptor (TLR) activation by pathogen associated molecular patterns (PAMPs). This study investigates the hypothesis that women suffering from rUTI have altered tolerance to infecting bacteria related to differences in expression of endogenous AMPs and that identification of such deficiency gives a potentially useful opportunity for novel preventive therapy. Methods and Patients A synergistic methodological approach of in vitro modelling with validation in clinical samples from the relevant patient group and controls was used. In vitro, cell culture was carried out using RT4 immortalised urothelial-cells, VK2 E6/E7 immortalised vaginal cells and finite primary culture of normal human urothelium. Cells were challenged with E. coli and PAMPs. Assays for Beta defensin AMP gene expression, secretion and antimicrobial activity were carried out. Clinically, 98 women (60 rUTI, 38 controls) were recruited with ethical approval. All subjects provided symptom and health state questionnaires; blood for single nucleotide polymorphism (SNP) analysis; vaginal and bladder biopsies for AMP gene expression analysis; plus vaginal washings and overnight urine samples for AMP peptide secretion assays. Results In vitro, cell culture experiments demonstrated that beta defensin 1 (BD1) was constitutively expressed and secreted in urothelial and vaginal cells. Beta defensin 2 (BD2) expression and secretion was induced by E. coli flagellin and is a potent antimicrobial against UPEC. In vaginal cells, BD2 expression and secretion was enhanced by estrogen. Clinically, women with rUTI were identified as having significantly lower basal levels of vaginal BD2 expression and secretion than controls but no difference in BD1 expression. Postmenopausal women had significantly lower BD2 levels than pre-menopausal women. 392Stop During active UTI, women with history of rUTI and the TLR5 SNP showed significantly lower BD2 expression and secretion in both the bladder and vagina than women with wild type TLR5 gene and rUTI. Discussion This study identifies flagellin induced BD2 expression as a novel and important urogenital innate immune response against invading E. coli, which is reduced in a significant

proportion of women with rUTI particularly those with the TLR5 392Stop SNP. Observations in vitro on the BD2 inducing effect of estrogen, and clinically in pre- and post-menopausal women, raise the possibility that BD2 expression can be modulated by exogenous factors.