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Estrogenic action on innate defense mechanisms in the urinary tract
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ARTICLE IN PRESS Maturitas xxx (2013) xxx–xxx
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Review
Estrogenic action on innate defense mechanisms in the urinary tract Petra Lüthje a , Angelica Lindén Hirschberg b , Annelie Brauner a,∗ a Department of Microbiology, Tumor and Cell Biology, Division of Clinical Microbiology, Karolinska Institutet and Karolinska University Hospital, 17176 Stockholm, Sweden b Department of Women’s and Children’s Health, Division of Obstetrics and Gynecology, Karolinska University Hospital and Karolinska Institutet, 17177 Stockholm, Sweden
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Article history: Received 18 October 2013 Accepted 27 October 2013 Available online xxx Keywords: Recurrent urinary tract infection Estrogen supplementation Innate immunity Antimicrobial peptides Epithelial differentiation Uropathogenic Escherichia coli
a b s t r a c t Clinical data suggest an impact of estrogen on the pathogenesis of urinary tract infections (UTI). In particular, women after menopause often suffer from recurrent UTI, characterized by at least three acute UTI episodes within a year. Aside from bacterial factors promoting persistence within the urinary bladder, the low estrogen levels induce structural and chemical changes in the urogenital tract which facilitate UTI. Increased residual urine volume and changes in the vaginal microflora are well documented risk factors. Local supplementation with estrogen can at least partly reverse these changes. Treatment allows the re-establishment of a lactobacilli-dominated vaginal microflora and improves epithelial differentiation and integrity in the urogenital tract. This estrogenic effect on the epithelium is marked by an increased production of antimicrobial peptides and a tighter intercellular connection, preventing bacteria from reaching cells where they can hide and later emerge and cause a new infection. Estrogen in the dosages and applications used to date is considered safe for the endometrium in the majority of women. Based on the actions and safety of estrogen, local supplementation thus offers a treatment option for postmenopausal women suffering from recurrent UTI. © 2013 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial persistence in the urinary tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk factors for UTI after menopause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The urinary bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The vaginal microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Effect of estrogen supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The vaginal mucosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Expression of antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The urothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Epithelial differentiation and integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Expression of antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Considerations with local estrogen supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors and their role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Provenance and peer review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗ Corresponding author at: Division of Clinical Microbiology, Karolinska University Hospital, SE-171 76 Stockholm, Sweden. Tel.: +46 8 5177 0000; fax: +46 8 308099. E-mail address: [email protected] (A. Brauner). 0378-5122/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.maturitas.2013.10.018
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1. Introduction Infections of the urinary tract belong to the most common infectious diseases worldwide and are a major economic burden for the public health care system [1]. Remarkably, urinary tract infections (UTI)1 affect mainly women. About 50% of women experience at least one UTI during their lifetime, and more than 25% of these initial infections recur. Besides the close proximity of the female urethra to the vagina and anus, an impact of estrogen on UTI pathogenesis is generally accepted, although the underlying mechanisms are still incompletely understood. While estrogen appears to be a risk factor for infection in young women [2], the same hormone also supports defense mechanisms to eradicate bacteria from the urinary tract [3]. This is mirrored by the increased risk to develop recurrent infections after menopause when estrogen levels are low. Estrogen, in addition to its well-known effect on growth and differentiation of the female reproductive system, acts also on non-reproductive tissues. The activity is exhibited via two types of estrogen receptors (ER), ER␣ and ER, with differential tissue expression patterns and functions. In the urinary bladder, ER is the major ER type [4] and is involved in epithelial differentiation and maintenance [5,6], whereas ER␣ is dominating in the vagina. This review intends to summarize well acknowledged risk factors for UTI related to the hypoestrogenic condition after menopause; and to give an overview about clinically or experimentally confirmed mechanisms, by which exogenous estrogen may improve the situation for postmenopausal women suffering from recurrent infections. 2. Bacterial persistence in the urinary tract Uncomplicated UTI are primarily caused by uropathogenic Escherichia coli (UPEC) [1]. This group of extraintestinal E. coli is defined based solely on its association to an infection of the urinary tract. UPEC present in fact a highly heterogeneous group of strains adapted to survive in the urinary tract [7]. With increasing resistance to conventional antimicrobial agents, alternative treatment strategies are highly needed. To boost the endogenous immune defense appears an attractive option [3,8], with a low risk for bacteria to develop resistances. Although antimicrobial therapy selects for resistant bacteria, the ability to persist is not necessarily associated with antimicrobial resistance. UPEC has for a long time been considered an extracellular pathogen, but now it is well recognized that these bacteria invade and persist inside epithelial cells, a niche protected from antibiotics as well as host defense mechanisms. Thus, bacterial factors enabling E. coli to invade the host cell and to persist intracellularly are highly relevant for persistent infections to occur. A number of studies have demonstrated the association of certain virulence factors for E. coli to persist in the host. Virulence factors mediating adhesion, biofilm formation or involved in iron acquisition appear of particular importance [7,9–11]. 3. Risk factors for UTI after menopause After menopause, declining levels of estrogen induce a condition referred to as vulvovaginal atrophy in 25–50% of women [12]. Symptoms include vaginal dryness, itching and a rise in vaginal pH as well as urinary frequency and incontinence. These changes impair also the defense against invading pathogens and thus may
1
CAMP, cathelicidin antimicrobial peptide; ER, estrogen receptor; hBD, human -defensin; NGF, nerve growth factor; RNase, ribonuclease; UPEC, uropathogenic Escherichia coli; UTI, urinary tract infection; VEGF, vascular endothelial growth factor.
contribute to the increased risk for UTI after menopause. The prevalence rate of UTI is 8–10% in postmenopausal women, and 5% of these women will have a recurrent infection within one year [13]. 3.1. The urinary bladder Incomplete emptying of the urinary bladder, expressed by the volume of residual urine after voiding is one factor amongst those suggested to increase the risk of recurrent UTIs after menopause. The association between low estrogen and high residual urine is well established [14,15]. Studies on rabbit bladders demonstrate that estrogen increases the volume of smooth muscle cells and vascularization in the bladder body, while decreasing collagen, resulting in higher contractility of the bladder [16]. Residual urine and a reduction in urine flow in the absence of estrogen [15], impairs the mechanical clearance of bacteria from the bladder and facilitates pathogens to colonize the tissue and to establish an infection. 3.2. The vaginal microflora The microflora in healthy menstruating women is dominated by lactobacilli. They produce lactic acid, hydrogen peroxide and bactericidal proteins, which restrict the growth of pathogenic bacteria such as E. coli [17]. This normal microflora thus confers protection against invading pathogens, while an abnormal colonization is associated with UTI [18]. Estrogen promotes lactobacilli growth by increasing the storage of glycogen in the cells of the vaginal epithelium [19], the substrate for acid production by these bacteria. With declining glycogen availability the number of lactobacilli declines and the vaginal pH rises. In postmenopausal women a multispecies microflora linked to a higher vaginal pH is therefore more likely to occur [20,21], facilitating the growth of potential pathogens. It has also been proposed that estrogen regulates the vaginal pH by acting on the epithelial cells directly [22]. However, most studies support the hypothesis that a low vaginal pH depends on the presence of acid-generating bacteria [23]. 4. Effect of estrogen supplementation 4.1. Clinical studies The results from clinical studies about the effect of estrogen supplementation after menopause on recurrent UTI are divergent [13]. While two placebo-controlled studies demonstrate that locally applied estrogen reduces recurrence [24,25], studies using oral supplementation overall do not show significant improvements compared to placebo [13]. The low efficiency of oral supplementation is supported by studies, which were not restricted to cases of recurrent UTI. However, in one study involving postmenopausal women suffering from urinary symptoms including UTI [26], local estrogen treatment was strongly associated to low UTI frequencies, while this linkage was only weak for systemic treatment. In a group of postmenopausal women with or without UTI, oral hormone replacement therapy was associated with a lower residual urine volume, which in turn was associated to a lower risk of UTI [14]. Functional studies in postmenopausal women are spars and cannot prove significant effects [27], but improvement of bladder functions by estrogen has been demonstrated in animals [16]. 4.2. The vaginal mucosa It is well established that estrogen supplementation provides relief from vaginal symptoms associated with postmenopausal
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Fig. 1. Potential mechanisms of estrogenic actions on the defense mechanisms in the urogenital epithelium. The vaginal mucosa in postmenopausal women is often colonized by a multispecies flora. Estrogen increases the glycogen storage, thereby promoting growth of lactobacilli (blue rods). Bactericidal peptides, hydrogen peroxide (H2 O2 ) and a low pH reduce the growth of potential pathogens such as E. coli (green rods). E. coli reaching the bladder epithelium invades the cells and forms intracellular bacterial communities (IBC) within the superficial umbrella cells. From here, they emerge and re-infect cells from deeper layers, exposed after exfoliation of infected umbrella cells. Within these cells, E. coli forms quiescent intracellular reservoirs (QIR), the potential source of a recurrent infection. Estrogen hampers this infection process by promoting epithelial integrity and the production of antimicrobial peptides. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
hypoestrogenism, such as dryness, itching and dyspareunia [28]. Furthermore, local estrogen provides greater benefit than systemic treatment. Apart from the reduction of clinical symptoms, a decrease in atrophy and increased maturation of the vaginal epithelium can be noted. The improved condition of the vaginal mucosa by estrogen supplementation most likely has a positive impact on the local defense mechanisms and thus against invading uropathogens. Some parameters have been studied intensively.
antimicrobial peptide human -defensin (hBD) 2 by vaginal cells in response to the bacterial endotoxin lipopolysaccharide [34]. A direct stimulation of hBD1-4 as well as ␣-defensin 2 by endogenous estrogen could be detected in mouse vaginal tissue, with expression peaks during the pro- and/or estrus when estrogen levels are high [35]. Whether estrogen modulates the vaginal microflora via expression of antimicrobial peptides and thereby indirectly impacts on UTI susceptibility remains to be explored.
4.2.1. Microflora The correlation between a Lactobacillus-dominated microflora associated with a low vaginal pH and protection against UTI is recognized. Only one study directly demonstrates the linkage between estrogen, vaginal colonization with lactobacilli and reduced incidence of UTI after menopause [24]. However, other studies support this finding by correlating a decreased vaginal pH in postmenopausal women at the end of a 12-week period of oral estrogen supplementation with a lower incidence of UTI [29]; and demonstrate prominent differences between the vaginal microflora of postmenopausal women receiving hormone replacement therapy or not [20,30]. Raz et al. performed another study in which estriol was applied via vaginal pessaries [31]. This low potency estrogen treatment did not confer the drastic improvement reported previously by other vaginal preparations. In addition, it is interesting to note that the vaginal pH and microflora remained unaffected by estriol-containing vaginal pessaries. This finding indirectly illustrates the important contribution of a Lactobacillus-dominated microflora to the defense against recurrent UTI after menopause.
4.3. The urothelium
4.2.2. Expression of antimicrobial peptides While the expression of antimicrobial peptides in the genital tract is demonstrated [32], the relation to estrogen is less well studied. In the non-sterile environment of the vagina, antimicrobial peptides participate in the maintenance of a healthy microflora. The production of antimicrobial peptides is reduced during bacterial vaginosis [33], a condition which may predispose for UTI. In vitro, estrogen has been shown to enhance the production of the
The anatomic localization of the urinary bladder precludes extensive studies on primary tissue, especially under the situation of infection. This might explain why the impact of estrogen is investigated only in a limited number of studies. The luminal surface of the urinary bladder is covered by a layer of glycosaminoglycans, which are thus the first barrier for bacteria to reach the underlying cells. The relevance of this defense strategy is supported by an infection-induced up-regulation of certain genes involved in glycosaminoglycans biosynthesis [36,37]. A significant impact of estrogen on the thickness and the expression of glycosaminoglycans could however not be established so far [6,37]. Fig. 1 illustrates the potential mechanisms of estrogenic actions on the epithelial surfaces of the urogenital tract. 4.3.1. Epithelial differentiation and integrity The differentiation of the urothelium is closely linked to instant defense strategies. Only terminally differentiated cells express receptors mediating bacteria-induced apoptosis and shedding of infected cells [7]. A fully differentiated epithelium on the other hand is more robust and thus provides better protection of the underlying tissue. Similar to the anti-atrophic effect of estrogen on the vaginal mucosa, estrogen promotes proliferation of epithelial cells of the lower urinary tract. Comparing tissue from pre- and postmenopausal women and postmenopausal women receiving hormone replacement therapy, Blakeman et al. detected a strict
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relation between estrogen status and the proliferation of the urethral epithelium [38]. The marker used could not be detected in the bladder of any patient, probably based on the low proliferation index of normal bladder epithelial cells. However, estrogen was shown to promote bladder epithelial cell proliferation via ER signaling in vitro [5]. The addition of estrogen to cultures of primary urothelial cells clearly induced proliferation. One study related this effect to an estrogen-induced up-regulation of the nerve growth factor (NGF) [39], while another study on rat bladders identified the urothelial-derived vascular endothelial growth factor (VEGF) as the proliferative stimuli [40]. Alongside with differentiation, estrogen induces the expression of intercellular junction proteins including E-cadherin in bladder epithelial cells [3]. Interestingly, expression of E-cadherin was much lower in patients with recurrent UTI compared to control patients [41]. These few studies on urothelial cells are supported by similar findings in epithelial cells of the mammary gland [42] or the colon [43].
supplementation is considered safe for the endometrium and concomitant use of progestogens is not required [28,47]. Despite the general safety with local estrogen supplementation, the treatment is contraindicated in some women, such as those with undiagnosed vaginal/uterine bleeding and in women with breast cancer using adjuvant therapy with aromatase inhibitors. The efficacy of aromatase inhibitors is based on blocking of the estrogen synthesis, leading to very low or undetectable estradiol levels. Since vaginal estrogen may result in a small and transient increase in circulating estradiol, this may influence the efficacy of aromatase inhibitors [50]. In other women with estrogen-dependent cancer, vaginal estrogen may be considered after discussion with the patient’s oncologist. The management of women who have been treated for nonhormone-dependent cancer is similar to women with no history of cancer. These women can therefore be treated with local estrogen.
4.3.2. Expression of antimicrobial peptides The involvement of antimicrobial peptides in the innate defense mechanisms of the urinary tract is known [32,44,45]. However, only few studies target the influence of estrogen on their expression in urothelial cells. In urinary cells derived from postmenopausal women before and after local supplementation with estrogen, we found a significant up-regulation of hBD3 mRNA, which was higher in menstruating women compared to levels in postmenopausal women before supplementation [3]. Moreover, in 75% of the women participating in the study, three of the five detectable peptides (hBD1-3, ribonuclease (RNase) 7 and psoriasin) were elevated after estrogen supplementation; and in 50% of the women, expression of even four peptides was enhanced. In addition, estrogen induced expression of all investigated antimicrobial peptides (hBD1-3, cathelicidin antimicrobial peptide (CAMP), RNase 7 and psoriasin) in two urothelial cell lines in vitro. By sequence analyses of the human genome [46], putative estrogen responsive elements have been identified within the promoter region of genes coding for some but not all affected genes. In addition, the overall increase of antimicrobial peptide expression by estrogen indicates a more general mode of action. Since the expression of antimicrobial peptides is linked to the differentiation of cells, i.e. expression increases during differentiation, this appears the logical linkage between estradiol and increased antimicrobial properties in epithelial cells of the urinary bladder.
Local estrogen supplementation is supported in light of the current knowledge of estrogen increasing antimicrobial peptides, epithelial integrity as well as a likely improving of the vaginal flora. Therefore in postmenopausal women with recurrent UTI, local estrogen supplementation should be considered if no contraindication exists.
5. Considerations with local estrogen supplementation In postmenopausal women with recurrent UTI, local estrogen supplementation appears a promising choice. Local application is also in line with the current recommendations of the International Menopause Society Writing Group [47] and the position of The North American Menopause Society [28]. The today recommended local estrogen dosages and administrations avoid a significant increase in serum estradiol [48], and have therefore not the adverse side effects associated to systemic hormone replacement therapy. Moreover, absorption of estradiol was shown to decrease shortly after the start of treatment as the vaginal mucosa recovers from the atrophic state [48,49]. There was no incidence of endometrial hyperplasia or change in endometrial thickness after one year treatment with 10 g of vaginal estradiol [48,49]. Although long-term studies are lacking, no endometrial cancers have been reported by the use of vaginal preparations of low-dose estrogen. Thus, local estrogen
6. Conclusion
Contributors and their role All authors contributed in designing, writing and reviewing the text. Competing interest The authors declare no competing interests. Funding Swedish Research Council (AB, ALH), ALF (AB, ALH), Karolinska Institutet (AB, ALH, PL) Provenance and peer review Commissioned and externally peer reviewed. References [1] Foxman B, Brown P. Epidemiology of urinary tract infections: transmission and risk factors, incidence, and costs. Infect Dis Clin North Am 2003;17:227–41. [2] Hooton TM, Winter C, Tiu F, Stamm WE. Association of acute cystitis with the stage of the menstrual cycle in young women. Clin Infect Dis 1996;23:635–6. [3] Lüthje P, Brauner H, Ramos NL, Övregaard A, Gläser R, Hirschberg AL, et al. Estrogen supports urothelial defense mechanisms. Sci Transl Med 2013;5, 190ra80. [4] Mäkelä S, Strauss L, Kuiper G, Valve E, Salmi S, Santti R, et al. Differential expression of estrogen receptors alpha and beta in adult rat accessory sex glands and lower urinary tract. Mol Cell Endocrinol 2000;170:219–29. [5] Teng J, Wang ZY, Jarrard DF, Bjorling DE. Roles of estrogen receptor alpha and beta in modulating urothelial cell proliferation. Endocr Relat Cancer 2008;15:351–64. [6] Imamov O, Yakimchuk K, Morani A, Schwend T, Wada-Hiraike O, Razumov S, et al. Estrogen receptor beta-deficient female mice develop a bladder phenotype resembling human interstitial cystitis. Proc Natl Acad Sci U S A 2007;104:9806–9. [7] Nielubowicz GR, Mobley HL. Host-pathogen interactions in urinary tract infection. Nat Rev Urol 2010;7:430–41. [8] Hertting O, Holm A, Luthje P, Brauner H, Dyrdak R, Jonasson AF, et al. Vitamin D induction of the human antimicrobial Peptide cathelicidin in the urinary bladder. PLoS One 2010;5:e15580. [9] Norinder BS, Lüthje P, Yadav M, Kadas L, Fang H, Nord CE, et al. Cellulose and PapG are important for Escherichia coli causing recurrent urinary tract infection in women. Infection 2011;39:571–4.
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Review
Estrogenic action on innate defense mechanisms in the urinary tract Petra Lüthje a , Angelica Lindén Hirschberg b , Annelie Brauner a,∗ a Department of Microbiology, Tumor and Cell Biology, Division of Clinical Microbiology, Karolinska Institutet and Karolinska University Hospital, 17176 Stockholm, Sweden b Department of Women’s and Children’s Health, Division of Obstetrics and Gynecology, Karolinska University Hospital and Karolinska Institutet, 17177 Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 18 October 2013 Accepted 27 October 2013 Available online xxx Keywords: Recurrent urinary tract infection Estrogen supplementation Innate immunity Antimicrobial peptides Epithelial differentiation Uropathogenic Escherichia coli
a b s t r a c t Clinical data suggest an impact of estrogen on the pathogenesis of urinary tract infections (UTI). In particular, women after menopause often suffer from recurrent UTI, characterized by at least three acute UTI episodes within a year. Aside from bacterial factors promoting persistence within the urinary bladder, the low estrogen levels induce structural and chemical changes in the urogenital tract which facilitate UTI. Increased residual urine volume and changes in the vaginal microflora are well documented risk factors. Local supplementation with estrogen can at least partly reverse these changes. Treatment allows the re-establishment of a lactobacilli-dominated vaginal microflora and improves epithelial differentiation and integrity in the urogenital tract. This estrogenic effect on the epithelium is marked by an increased production of antimicrobial peptides and a tighter intercellular connection, preventing bacteria from reaching cells where they can hide and later emerge and cause a new infection. Estrogen in the dosages and applications used to date is considered safe for the endometrium in the majority of women. Based on the actions and safety of estrogen, local supplementation thus offers a treatment option for postmenopausal women suffering from recurrent UTI. © 2013 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial persistence in the urinary tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk factors for UTI after menopause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The urinary bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The vaginal microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Effect of estrogen supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The vaginal mucosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Expression of antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The urothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Epithelial differentiation and integrity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2. Expression of antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Considerations with local estrogen supplementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors and their role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Provenance and peer review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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∗ Corresponding author at: Division of Clinical Microbiology, Karolinska University Hospital, SE-171 76 Stockholm, Sweden. Tel.: +46 8 5177 0000; fax: +46 8 308099. E-mail address: [email protected] (A. Brauner). 0378-5122/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.maturitas.2013.10.018
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1. Introduction Infections of the urinary tract belong to the most common infectious diseases worldwide and are a major economic burden for the public health care system [1]. Remarkably, urinary tract infections (UTI)1 affect mainly women. About 50% of women experience at least one UTI during their lifetime, and more than 25% of these initial infections recur. Besides the close proximity of the female urethra to the vagina and anus, an impact of estrogen on UTI pathogenesis is generally accepted, although the underlying mechanisms are still incompletely understood. While estrogen appears to be a risk factor for infection in young women [2], the same hormone also supports defense mechanisms to eradicate bacteria from the urinary tract [3]. This is mirrored by the increased risk to develop recurrent infections after menopause when estrogen levels are low. Estrogen, in addition to its well-known effect on growth and differentiation of the female reproductive system, acts also on non-reproductive tissues. The activity is exhibited via two types of estrogen receptors (ER), ER␣ and ER, with differential tissue expression patterns and functions. In the urinary bladder, ER is the major ER type [4] and is involved in epithelial differentiation and maintenance [5,6], whereas ER␣ is dominating in the vagina. This review intends to summarize well acknowledged risk factors for UTI related to the hypoestrogenic condition after menopause; and to give an overview about clinically or experimentally confirmed mechanisms, by which exogenous estrogen may improve the situation for postmenopausal women suffering from recurrent infections. 2. Bacterial persistence in the urinary tract Uncomplicated UTI are primarily caused by uropathogenic Escherichia coli (UPEC) [1]. This group of extraintestinal E. coli is defined based solely on its association to an infection of the urinary tract. UPEC present in fact a highly heterogeneous group of strains adapted to survive in the urinary tract [7]. With increasing resistance to conventional antimicrobial agents, alternative treatment strategies are highly needed. To boost the endogenous immune defense appears an attractive option [3,8], with a low risk for bacteria to develop resistances. Although antimicrobial therapy selects for resistant bacteria, the ability to persist is not necessarily associated with antimicrobial resistance. UPEC has for a long time been considered an extracellular pathogen, but now it is well recognized that these bacteria invade and persist inside epithelial cells, a niche protected from antibiotics as well as host defense mechanisms. Thus, bacterial factors enabling E. coli to invade the host cell and to persist intracellularly are highly relevant for persistent infections to occur. A number of studies have demonstrated the association of certain virulence factors for E. coli to persist in the host. Virulence factors mediating adhesion, biofilm formation or involved in iron acquisition appear of particular importance [7,9–11]. 3. Risk factors for UTI after menopause After menopause, declining levels of estrogen induce a condition referred to as vulvovaginal atrophy in 25–50% of women [12]. Symptoms include vaginal dryness, itching and a rise in vaginal pH as well as urinary frequency and incontinence. These changes impair also the defense against invading pathogens and thus may
1
CAMP, cathelicidin antimicrobial peptide; ER, estrogen receptor; hBD, human -defensin; NGF, nerve growth factor; RNase, ribonuclease; UPEC, uropathogenic Escherichia coli; UTI, urinary tract infection; VEGF, vascular endothelial growth factor.
contribute to the increased risk for UTI after menopause. The prevalence rate of UTI is 8–10% in postmenopausal women, and 5% of these women will have a recurrent infection within one year [13]. 3.1. The urinary bladder Incomplete emptying of the urinary bladder, expressed by the volume of residual urine after voiding is one factor amongst those suggested to increase the risk of recurrent UTIs after menopause. The association between low estrogen and high residual urine is well established [14,15]. Studies on rabbit bladders demonstrate that estrogen increases the volume of smooth muscle cells and vascularization in the bladder body, while decreasing collagen, resulting in higher contractility of the bladder [16]. Residual urine and a reduction in urine flow in the absence of estrogen [15], impairs the mechanical clearance of bacteria from the bladder and facilitates pathogens to colonize the tissue and to establish an infection. 3.2. The vaginal microflora The microflora in healthy menstruating women is dominated by lactobacilli. They produce lactic acid, hydrogen peroxide and bactericidal proteins, which restrict the growth of pathogenic bacteria such as E. coli [17]. This normal microflora thus confers protection against invading pathogens, while an abnormal colonization is associated with UTI [18]. Estrogen promotes lactobacilli growth by increasing the storage of glycogen in the cells of the vaginal epithelium [19], the substrate for acid production by these bacteria. With declining glycogen availability the number of lactobacilli declines and the vaginal pH rises. In postmenopausal women a multispecies microflora linked to a higher vaginal pH is therefore more likely to occur [20,21], facilitating the growth of potential pathogens. It has also been proposed that estrogen regulates the vaginal pH by acting on the epithelial cells directly [22]. However, most studies support the hypothesis that a low vaginal pH depends on the presence of acid-generating bacteria [23]. 4. Effect of estrogen supplementation 4.1. Clinical studies The results from clinical studies about the effect of estrogen supplementation after menopause on recurrent UTI are divergent [13]. While two placebo-controlled studies demonstrate that locally applied estrogen reduces recurrence [24,25], studies using oral supplementation overall do not show significant improvements compared to placebo [13]. The low efficiency of oral supplementation is supported by studies, which were not restricted to cases of recurrent UTI. However, in one study involving postmenopausal women suffering from urinary symptoms including UTI [26], local estrogen treatment was strongly associated to low UTI frequencies, while this linkage was only weak for systemic treatment. In a group of postmenopausal women with or without UTI, oral hormone replacement therapy was associated with a lower residual urine volume, which in turn was associated to a lower risk of UTI [14]. Functional studies in postmenopausal women are spars and cannot prove significant effects [27], but improvement of bladder functions by estrogen has been demonstrated in animals [16]. 4.2. The vaginal mucosa It is well established that estrogen supplementation provides relief from vaginal symptoms associated with postmenopausal
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Fig. 1. Potential mechanisms of estrogenic actions on the defense mechanisms in the urogenital epithelium. The vaginal mucosa in postmenopausal women is often colonized by a multispecies flora. Estrogen increases the glycogen storage, thereby promoting growth of lactobacilli (blue rods). Bactericidal peptides, hydrogen peroxide (H2 O2 ) and a low pH reduce the growth of potential pathogens such as E. coli (green rods). E. coli reaching the bladder epithelium invades the cells and forms intracellular bacterial communities (IBC) within the superficial umbrella cells. From here, they emerge and re-infect cells from deeper layers, exposed after exfoliation of infected umbrella cells. Within these cells, E. coli forms quiescent intracellular reservoirs (QIR), the potential source of a recurrent infection. Estrogen hampers this infection process by promoting epithelial integrity and the production of antimicrobial peptides. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
hypoestrogenism, such as dryness, itching and dyspareunia [28]. Furthermore, local estrogen provides greater benefit than systemic treatment. Apart from the reduction of clinical symptoms, a decrease in atrophy and increased maturation of the vaginal epithelium can be noted. The improved condition of the vaginal mucosa by estrogen supplementation most likely has a positive impact on the local defense mechanisms and thus against invading uropathogens. Some parameters have been studied intensively.
antimicrobial peptide human -defensin (hBD) 2 by vaginal cells in response to the bacterial endotoxin lipopolysaccharide [34]. A direct stimulation of hBD1-4 as well as ␣-defensin 2 by endogenous estrogen could be detected in mouse vaginal tissue, with expression peaks during the pro- and/or estrus when estrogen levels are high [35]. Whether estrogen modulates the vaginal microflora via expression of antimicrobial peptides and thereby indirectly impacts on UTI susceptibility remains to be explored.
4.2.1. Microflora The correlation between a Lactobacillus-dominated microflora associated with a low vaginal pH and protection against UTI is recognized. Only one study directly demonstrates the linkage between estrogen, vaginal colonization with lactobacilli and reduced incidence of UTI after menopause [24]. However, other studies support this finding by correlating a decreased vaginal pH in postmenopausal women at the end of a 12-week period of oral estrogen supplementation with a lower incidence of UTI [29]; and demonstrate prominent differences between the vaginal microflora of postmenopausal women receiving hormone replacement therapy or not [20,30]. Raz et al. performed another study in which estriol was applied via vaginal pessaries [31]. This low potency estrogen treatment did not confer the drastic improvement reported previously by other vaginal preparations. In addition, it is interesting to note that the vaginal pH and microflora remained unaffected by estriol-containing vaginal pessaries. This finding indirectly illustrates the important contribution of a Lactobacillus-dominated microflora to the defense against recurrent UTI after menopause.
4.3. The urothelium
4.2.2. Expression of antimicrobial peptides While the expression of antimicrobial peptides in the genital tract is demonstrated [32], the relation to estrogen is less well studied. In the non-sterile environment of the vagina, antimicrobial peptides participate in the maintenance of a healthy microflora. The production of antimicrobial peptides is reduced during bacterial vaginosis [33], a condition which may predispose for UTI. In vitro, estrogen has been shown to enhance the production of the
The anatomic localization of the urinary bladder precludes extensive studies on primary tissue, especially under the situation of infection. This might explain why the impact of estrogen is investigated only in a limited number of studies. The luminal surface of the urinary bladder is covered by a layer of glycosaminoglycans, which are thus the first barrier for bacteria to reach the underlying cells. The relevance of this defense strategy is supported by an infection-induced up-regulation of certain genes involved in glycosaminoglycans biosynthesis [36,37]. A significant impact of estrogen on the thickness and the expression of glycosaminoglycans could however not be established so far [6,37]. Fig. 1 illustrates the potential mechanisms of estrogenic actions on the epithelial surfaces of the urogenital tract. 4.3.1. Epithelial differentiation and integrity The differentiation of the urothelium is closely linked to instant defense strategies. Only terminally differentiated cells express receptors mediating bacteria-induced apoptosis and shedding of infected cells [7]. A fully differentiated epithelium on the other hand is more robust and thus provides better protection of the underlying tissue. Similar to the anti-atrophic effect of estrogen on the vaginal mucosa, estrogen promotes proliferation of epithelial cells of the lower urinary tract. Comparing tissue from pre- and postmenopausal women and postmenopausal women receiving hormone replacement therapy, Blakeman et al. detected a strict
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relation between estrogen status and the proliferation of the urethral epithelium [38]. The marker used could not be detected in the bladder of any patient, probably based on the low proliferation index of normal bladder epithelial cells. However, estrogen was shown to promote bladder epithelial cell proliferation via ER signaling in vitro [5]. The addition of estrogen to cultures of primary urothelial cells clearly induced proliferation. One study related this effect to an estrogen-induced up-regulation of the nerve growth factor (NGF) [39], while another study on rat bladders identified the urothelial-derived vascular endothelial growth factor (VEGF) as the proliferative stimuli [40]. Alongside with differentiation, estrogen induces the expression of intercellular junction proteins including E-cadherin in bladder epithelial cells [3]. Interestingly, expression of E-cadherin was much lower in patients with recurrent UTI compared to control patients [41]. These few studies on urothelial cells are supported by similar findings in epithelial cells of the mammary gland [42] or the colon [43].
supplementation is considered safe for the endometrium and concomitant use of progestogens is not required [28,47]. Despite the general safety with local estrogen supplementation, the treatment is contraindicated in some women, such as those with undiagnosed vaginal/uterine bleeding and in women with breast cancer using adjuvant therapy with aromatase inhibitors. The efficacy of aromatase inhibitors is based on blocking of the estrogen synthesis, leading to very low or undetectable estradiol levels. Since vaginal estrogen may result in a small and transient increase in circulating estradiol, this may influence the efficacy of aromatase inhibitors [50]. In other women with estrogen-dependent cancer, vaginal estrogen may be considered after discussion with the patient’s oncologist. The management of women who have been treated for nonhormone-dependent cancer is similar to women with no history of cancer. These women can therefore be treated with local estrogen.
4.3.2. Expression of antimicrobial peptides The involvement of antimicrobial peptides in the innate defense mechanisms of the urinary tract is known [32,44,45]. However, only few studies target the influence of estrogen on their expression in urothelial cells. In urinary cells derived from postmenopausal women before and after local supplementation with estrogen, we found a significant up-regulation of hBD3 mRNA, which was higher in menstruating women compared to levels in postmenopausal women before supplementation [3]. Moreover, in 75% of the women participating in the study, three of the five detectable peptides (hBD1-3, ribonuclease (RNase) 7 and psoriasin) were elevated after estrogen supplementation; and in 50% of the women, expression of even four peptides was enhanced. In addition, estrogen induced expression of all investigated antimicrobial peptides (hBD1-3, cathelicidin antimicrobial peptide (CAMP), RNase 7 and psoriasin) in two urothelial cell lines in vitro. By sequence analyses of the human genome [46], putative estrogen responsive elements have been identified within the promoter region of genes coding for some but not all affected genes. In addition, the overall increase of antimicrobial peptide expression by estrogen indicates a more general mode of action. Since the expression of antimicrobial peptides is linked to the differentiation of cells, i.e. expression increases during differentiation, this appears the logical linkage between estradiol and increased antimicrobial properties in epithelial cells of the urinary bladder.
Local estrogen supplementation is supported in light of the current knowledge of estrogen increasing antimicrobial peptides, epithelial integrity as well as a likely improving of the vaginal flora. Therefore in postmenopausal women with recurrent UTI, local estrogen supplementation should be considered if no contraindication exists.
5. Considerations with local estrogen supplementation In postmenopausal women with recurrent UTI, local estrogen supplementation appears a promising choice. Local application is also in line with the current recommendations of the International Menopause Society Writing Group [47] and the position of The North American Menopause Society [28]. The today recommended local estrogen dosages and administrations avoid a significant increase in serum estradiol [48], and have therefore not the adverse side effects associated to systemic hormone replacement therapy. Moreover, absorption of estradiol was shown to decrease shortly after the start of treatment as the vaginal mucosa recovers from the atrophic state [48,49]. There was no incidence of endometrial hyperplasia or change in endometrial thickness after one year treatment with 10 g of vaginal estradiol [48,49]. Although long-term studies are lacking, no endometrial cancers have been reported by the use of vaginal preparations of low-dose estrogen. Thus, local estrogen
6. Conclusion
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Please cite this article in press as: Lüthje P, et al. Estrogenic action on innate defense mechanisms in the urinary tract. Maturitas (2013), http://dx.doi.org/10.1016/j.maturitas.2013.10.018