The host response to urinary tract infection

Infect Dis Clin N Am 17 (2003) 279–301

The host response to urinary tract infection Bjo¨rn Wullt, MD, PhD*, G. Bergsten, MSc, H. Fischer, PhD, G. Godaly, PhD, D. Karpman, MD, PhD, I. Leijonhufvud, RN, A.-C. Lundstedt, MSc, P. Samuelsson, MSc, M. Samuelsson, MSc, M.-L. Svensson, C. Svanborg, MD, PhD Department of Microbiology, Immunology and Glycobiology, and Department of Urology, Institute of Laboratory Medicine, Lund University, So¨lvegatan 23, Lund 223 62, Sweden

The normal urinary tract is sterile, because of an efficient antibacterial defense. Bacteriuria is extremely common in all age groups, however, and symptomatic urinary tract infections (UTIs) are a major cause of morbidity and mortality. This paradox illustrates the inherent variation in disease susceptibility in the population. During the last decades, innate host defense mechanisms have been shown to control the susceptibility to UTI [1]. This article concerns the molecular mechanisms of host response activation and the defense effector functions that maintain tissue integrity. The authors show in experimental models that specific response pathways determine the severity of acute UTI, and that the inactivation of the corresponding host response genes may result in asymptomatic bacteriuria (ABU). Also shown is that the quality of the innate host defense determines the severity of acute pyelonephritis and the progression to chronic disease, because mutations, which disrupt neutrophil function, cause the entire syndrome of acute pyelonephritis and renal scarring. Finally, it is demonstrated that patients prone to UTI carry distinct mutations in the innate host response genes. The implications for diagnosis and therapy are also discussed. Bacteria frequently enter the urinary tract, and may establish significant bacteriuria if they overcome the host defense. Symptomatic infections vary

* Corresponding author. E-mail address: [email protected] (B. Wullt). 0891-5520/03/$ - see front matter Ó 2003, Elsevier Inc. All rights reserved. doi:10.1016/S0891-5520(03)00028-X

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in severity depending on the site and magnitude of the host response. The symptoms of acute pyelonephritis emanate both from the kidneys and from systemic compartments that become involved. Acute cystitis is often restricted to the lower urinary tract, and local symptoms include dysuria and frequency. Bacteriuria does not necessarily activate a host response or cause disease. Rather to the contrary, ABU is the most frequent end result, occurring in about 1% of girls, 2% of pregnant women, and 20% of elderly individuals [2]. If left untreated, a single bacterial strain may persist for months or years without side effects and without significant host response induction. The asymptomatic carrier state has even been shown to protect against symptomatic infection [3,4]. Early studies demonstrated that differences in disease severity reflect the properties of the infecting bacterial strain. Uropathogenic Escherichia coli were classified with the help of cellular markers (serotyping) like lipopolysaccharide (LPS), capsular, and flagellar antigens [5,6] showing that certain OKH serogroups caused acute pyelonephritis, whereas others were found in ABU. This difference in prevalence was shown to reflect the virulence of the infecting strains, and the expression of virulence factors mediating adherence [7], toxin production, iron sequestering, and so forth [8]. The genes encoding these and other virulence factors were later localized to chromosomal pathogenicity islands and their expression during disease pathogenesis is tightly regulated [9]. Adherence mechanisms have received special attention, because epithelial cell adherence is the first step in the tissue attack process, marking the onset of disease pathogenesis [10,11]. By adherence and activation of the target cell the uropathogen breaks the inertia of the mucosal barrier, initiating the mucosal inflammatory response. Pyelonephritis strains express multiple, tissue-specific adhesins, enabling them successively to establish molecular interactions with different host cell populations. The ABU strains, in contrast, stop expressing adherence factors once they have established bacteriuria [12]. This switch to a nonadhesive phenotype may provide them with an efficient survival mechanism, avoiding the antibacterial defense. The severity of UTI reflects the host response to the infecting strain. The classical virulence factors of uropathogenic E coli act by triggering an inflammatory response in the urinary tract [13]. The extent of mucosal inflammation depends on the response repertoire of the host, which discriminates asymptomatic carriage from symptomatic disease, and resolution of infection from tissue damage. To improve diagnosis and therapy, one must understand the molecular mechanisms of host response induction and the causes of tissue damage. The two-step model The initiation of the host response (step 1) determines if the inertia of the mucosal barrier is maintained, or if the bacteria succeed in triggering the

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Fig. 1. The two-step model. Adhering bacteria activate the uroepithelial cells (step 1), to release chemokines that recruit neutrophils to the site of infection. During their exodus across the mucosa, the neutrophils kill the bacteria (step 2). (From Svanborg C, Bergsten G, Fischer H, Frendeus B, Godaly G, Gustafsson E, et al. The ÔinnateÕ host response protects and damages the infected urinary tract. Ann Med 2001;33:563–70; with permission.)

innate host response, causing mucosal inflammation (Fig. 1). Activation involves distinct signaling pathways in the host cell, and may be avoided if the infecting strain fails to trigger the response or if the host response pathways are disrupted by mutations of TLR4 or other mechanisms. Inflammatory mediators provide a direct link between the microbe and the host in disease pathogenesis [14]. As a result of step 1, mucosal cells produce chemokines that recruit neutrophils and other defense effector cells to the urinary tract. Step 2 involves the migration of cells through the tissues across the mucosal barrier and into the urine, where they cause pyuria. Step 2 is crucial for neutrophil-dependent clearance of bacteria from the tissues and for maintaining tissue integrity, by removing lingering inflammatory cells and bacteria. Disruption of step 2, by mutations or other mechanisms, disturbs these functions, and poses a great threat to the tissues [15]. Step 1: initiation of the host response Uropathogenic E coli establish contact with the mucosa through specific adherence mechanisms and trigger mucosal inflammation (Fig. 2) [10,11,16]. Cells in the urinary tract mucosa are usually the first to respond to microbial challenge, and then orchestrate the subsequent host response by the release of cytokines and other mediators of inflammation and immunity. Epidemiologic studies have shown strong associations between adherence and disease severity [7], and experimental studies have identified adherence as essential for host response induction (step 1) [14]. Because nonadhesive ABU strains do not initiate a response, they avoid breaking the inertia of the mucosal barrier, and persist without completing step 1.

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Fig. 2. Mechanisms of step 1. Bacterial adhesins bind to specific receptors on the surface of the uroepithelial cells, and the adhesin-receptor interaction activates different transmembrane signaling pathways. Uropathogenic Escherichia coli expressing P fimbriae bind to the specific glycosphingolipids receptors on the uroepithelial cells, and recruit the TLR4 receptor for transmembrane signaling. A dysfunctional TLR4 receptor hinders inflammation.

The uropathogenic E coli strains use specific adherence both for tissue targeting and for host response activation. E coli strains causing pyelonephritis express several different types of adhesins (Dr and afa, Stype 1, and P fimbriae), all of which may bind to different host cell receptors during disease pathogenesis [16,17]. P fimbriae–mediated adherence has been used as a model system to understand step 1 better, because P fimbriae show the most clear-cut disease association. P fimbrial expression characterizes the most virulent strains, including 70% to 90% of acute pyelonephritis but less than 20% of ABU strains [18–21]. The expression of type 1 fimbriae is not associated with human disease because virtually all uropathogenic E coli carry the fim gene cluster [20,22]. Yet, a fimH deletion mutant showed dramatically reduced virulence in the murine UTI model, which favors type 1–dependent interactions [23]. Subsequent experimental studies have proposed type 1 fimbriae as an invasion factor in the dome cells of the rat urinary bladder [24]. Host response induction: lessons from P fimbriated Escherichia coli P fimbriae target host cells by binding specific glycosphingolipid (GSL) receptor motifs and recruit TLR4 for transmembrane signaling [25]. As a consequence, the response may be disrupted either by depletion of specific receptors or by mutational inactivation of TLR4 (see Fig. 2). Adherence is mediated by the PapG tip adhesins, which recognize receptor epitopes defined by the Gala1-4Galb disaccharide in the globoseries GSL. The specificity of binding has been extensively documented, and several members of the GSL receptor family provide binding

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sites for P fimbriae with variant isoreceptor recognition sites [16]. The receptor GSLs are abundant in uroepithelium and in kidney tissue, where the individual repertoire of receptor GSLs varies depending on the P blood group and secretor state [26]. This variation provides a basis for the blood group–related difference in susceptibility to infection with P fimbriated E coli (see later). The membrane GSLs are enriched in the caveolae and the membrane anchoring domain is ceramide, in the outer leaflet of the lipid bilayer. The coupling of P fimbriated E coli to their receptors triggers ceramide release and the ceramide signaling pathway is involved in cell activation by P fimbriated bacteria [27,28]. The carbohydrate head group is essential for binding of the fimbriae, and ceramide acts as a signaling intermediate but the exact mechanism needs to be defined. P fimbriated E coli also rely on the TLR4 signaling pathway for cell activation [25]. The involvement of TLR4 in transmembrane signaling was deduced from early studies in LPS nonresponder mice, later identified as TLR4 mutant mice [29–32]. Different TLR4-deficient mouse strains do not respond to infection with recombinant P fimbriated strains. The cellular basis for TLR4 recruitment has been studied in human uroepithelial cells. After exposure to P fimbriated strains TLR4 mRNA levels increased, and by confocal microscopy TLR4 was tentatively identified in caveolae, adjacent to the GSL receptors [25]. It still remains unclear if P fimbriae trigger the response by delivering LPS to the tissues, and if LPS participates in epithelial cell activation. Such an effect is expected, because LPS is the classical activator of TLR4-dependent responses. There are studies in support of a role of LPS in epithelial cell activation, and others suggesting that the fimbriae activate the cells directly, through different pathways. LPS responses involve the CD14 receptor, the MD2 complex, and TLR4 on the responding cell, and soluble cofactors like the LPS binding protein. Uroepithelial cells are CD14 negative, however, and respond poorly to free LPS [27,33]. The surface-bound CD14 may be substituted by soluble CD14, allowing LPS to trigger the TLR4 pathway. To address this question, the authors inactivated the endotoxic lipid A portion of LPS by mutating the msbB gene that encodes an acyltransferase coupling myristic acid to the lipid IVA precursor [34], and expressed P fimbriae in these two backgrounds. Interestingly, no evidence was found for LPS involvement with this approach [33]. Uroepithelial cells were rendered LPS responsive in the presence of soluble CD14; however the P fimbriae appear to use an LPS-like mechanism to activate cells, which lack CD14 and are refractory to LPS itself. Evidence for P fimbriae-dependent activation of the urinary tract host response has been obtained in the human urinary tract inoculation model [35]. Patients were subjected to intravesical inoculation with a nonfimbriated E coli strain or transformants of this strain expressing P fimbriae. The inflammatory response was analyzed as a function of P fimbrial expression.

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The P fimbriated transformants invariably caused higher interleukin (IL)-8, IL-6, and neutrophil responses in the urinary tract than the ABU strain. The human studies allow one to conclude that P fimbriae confer on a nonfimbriated, avirulent strain the ability to induce a host response in the human urinary tract [35].

Other mechanisms triggering step 1 The P fimbriae exemplify how the pathogenic strains are able to activate the host response. Other adherence factors, like type 1 fimbriae, have been proposed to have a similar effect. Type 1 fimbriated E coli trigger a cytokine response in uroepithelial cells, and enhance the mucosal inflammatory response in experimental UTI models. Mutational inactivation of the FimH adhesin was shown to attenuate the virulence of a fully virulent strain in the mouse urinary tract [23]. Studies on the transmembrane signaling mechanisms of type 1 fimbriated E coli have given results that resemble the P fimbriae. The adhesins bind to mannosylated receptors, and trigger a receptor-specific signal through as yet undefined mechanisms [36]. In addition, the fimbriae deliver an LPSdependent signal that includes the TLR4 pathway [37,38]. There is little evidence to support a direct role of type 1 fimbriae for the induction of a urinary tract host response. Human inoculation studies similar to those performed with P fimbriae are required to understand if type 1 fimbriae are capable of host response induction, or if type 1 fimbriae actually perform other functions in disease pathogenesis. Such functions include colonization of the gut, invasion of dome cells in the murine bladder epithelium, and activation of inflammatory cells, once the response has been initiated by other mechanisms.

Summary of step 1 Mucosal surfaces in the urinary tract respond to bacterial challenge by activation of distinct signaling pathways. The end result is the production of inflammatory mediators that orchestrate the subsequent events in disease pathogenesis. Different virulence factors may contribute to the repertoire of inflammatory mediators and give the spectrum of host responses that is unique for each pathogen and each host. As a metaphor, one may use the hand of the pianist whose fingers strike different cords and produce such diverse music. P fimbriae use cell surface GSLs as the primary receptors for adherence to epithelial cells and they activate these cells by recruiting TLR4 as co-receptors for signal transduction. Because the first step in cell activation relies on these two receptors, genetic variation in GSL or TLR4 receptor repertoire should influence the in vivo response to experimental UTI, and the tendency of patients to develop symptomatic disease or asymptomatic carriage (see later).

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Step 2: neutrophil recruitment and bacterial clearance The activated uroepithelial cells synthesize inflammatory mediators that may be secreted into the urine, and can be measured as a sign of infection. More importantly, basolateral secretion allows the epithelial cells to propagate the signals to adjacent cells in the mucosa or to recruit inflammatory cells from the circulation. Infection causes an increase in chemokine secretion, especially of chemokines like IL-8, which are chemotactic for neutrophils, and of other chemokines that interact with monocytes or lymphocytes [39]. A chemotactic gradient is created, and in response to the gradient, cells leave the bloodstream and migrate toward the mucosal barrier, where they exert their effects. Neutrophils are crucial for bacterial clearance from the urinary tract in vivo [30,31,40]. They migrate through the tissues and cross the epithelial barrier into the lumen. The last step of exit across the mucosa depends on IL-8 and on the receptors for IL-8. IL-8 seems to be the main driving force for neutrophils to cross the human urinary tact epithelium, and macrophage inflammatory protein (MIP)-2 plays a similar role in the murine urinary tract [41–43]. It should be noticed, however, that several different neutrophil chemoattractants are secreted by epithelial cells, and that additional studies are needed to understand their function in the response to UTI. These molecular and cellular interactions explain the emergence of leucocytes in urine, known as pyuria, which is a classical sign of UTI (see Fig. 2). Interleukin-8 and other neutrophil-activating chemokines (CXC) exert their effects by binding to G protein coupled cell surface receptors. The CXC chemokine family, which includes IL-8, binds the CXCR chemokine receptors, which have been studied on neutrophils and endothelial cells [44–46]. The authors have shown that infection stimulates CXCR1 and CXCR2 expression on epithelial cells, and that CXCR1 accounts for the increased neutrophil migration across infected cell layers in vitro [47]. The exit phase of the neutrophils is essential for bacterial clearance and tissue integrity. During this phase, the neutrophils phagocytose the bacteria at the mucosa, through mechanisms that are not fully understood. Disruption of the neutrophil response by short-term treatment with antineutrophil antibodies was shown to impair bacterial clearance. Finally, genetic inactivation of the IL-8 receptor causes a dramatic phenotype in mIL-8Rh KO mice (see later). Summary of step 2 The activated epithelial cells recruit neutrophils that are essential for bacterial clearance. The IL-8 receptor is required for neutrophils to leave the tissues across the epithelial barrier. Blocking of neutrophil exit jeopardizes the tissue integrity by forcing the neutrophils to disintegrate in the tissues, thereby causing extensive tissue damage. These studies predict that the

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inactivation of epithelial chemokine receptor expression is hazardous for tissue repair after infection, and possibly precipitates chronic disease.

Genetic determinants of host susceptibility: evidence from the murine UTI model Disruptions of TLR4 signaling causes ABU Early studies showed that C3H-HeJ mice failed to respond to infection and were unable to clear bacteria from the urinary tract (Fig. 3) [30,31]. Instead, they developed an asymptomatic carrier state. The authors concluded that the failure to activate the host response was protective, in that the animals could avoid developing asymptomatic disease. Furthermore, the lack of antibacterial defense permitted bacterial persistence, but apparently without causing side effects or tissue damage. The C3H-HeJ mice have since been shown to carry a mutation in the signaling domain of TLR4, and additional studies in C57 BL mice lacking TLR4 have confirmed that this pathway controls host response induction in the urinary tract [32]. Thus, TLR4-dependant signaling mechanisms determine if a host response is activated and if the mice develop symptoms or remain asymptomatic. The IL-8 receptor knock-out causes acute pyelonephritis and renal scarring The IL-8 chemokines and chemokine receptors were shown to be crucial for bacterial clearance and tissue integrity in the murine UTI model, using IL-8 receptor knock-out (mIL-8Rh KO) mice (see Fig. 3). The mIL-8Rh KO were constructed by insertion of a neomycin cassette in the murine IL-8 receptor gene [48]. Their neutrophils fail to respond to the IL-8 homologues in general, but retain responses to other chemotactic signals. Because all of the murine IL-8 homologues converge on one murine IL-8 receptor, inactivation of this gene should cause a peripheral neutrophil migration deficiency. The mIL-Rh KO mice were found to develop severe disease (see Fig. 4). The KO mice showed abrogated neutrophil exit from the kidneys, with massive subepithelial accumulation of neutrophil cells (Fig. 4). In addition, there were increasing bacterial tissue counts and the animals developed bacteremia. Neutrophil accumulation resulted in abscess formation throughout the kidney parenchyma, and with time the kidneys shrunk in size. By histology, extensive tissue damage with fibrosis and other signs of renal scarring was detected [49,50]. In control mice, neutrophils appeared in the kidneys within a few hours after infection, and were seen crossing the epithelial barrier into the lumen. In the process, infection was cleared with no evidence of tissue damage.

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The authors conclude that a single gene deletion in mice causes both the syndrome of acute pyelonephritis with bacteremia and the chronic syndrome with renal scar development. Mutational inactivation of the CXCR1 gene was sufficient to impair bacterial clearance, and to cause both acute pyelonephritis and chronic tissue damage in KO mice.

Genetic determinants of host susceptibility: evidence from patients with different forms of UTI The results from the murine model suggest at least three ways in which host receptor expression might influence the inflammatory response. Variations in step 1 may be caused by TLR4 or GSL receptor expression. The GSL receptors should influence if fimbriae find their primary receptor, and TLR4 expression if cell signaling occurs, and both these mechanisms influence the functionality of the epithelial cell response. Variations in step 2 may be caused by chemokine receptor expression, and may play a crucial role for the development of kidney scarring after pyelonephritis. Fig. 5 shows a flow-chart suggesting how steps 1 and 2 could explain individual susceptibility in different kinds of UTI. Genetics of IL-8 receptor expression in pyelonephritis-prone children It has often been discussed if the susceptibility to UTI is inherited, and HLA typing has been performed on limited populations, but until the CXCR1 deficiency was described, no genetic basis for disease had been found. The authors now propose that the CXCR1 gene is polymorphic and that mutations or insertions may underlie differences in the susceptibility to acute pyelonephritis and renal scarring. The progression from acute disease to renal scarring in the mIL-8Rh KO mice prompted the authors to test if similar mechanisms might underlie the susceptibility to acute pyelonephritis in children. In a prospective clinical study, CXCR1 expression was compared between children with at least one episode of acute pyelonephritis and age-matched controls with no history of UTI. CXCR1 expression was dramatically lower in the patients than in the controls [50,51]. Subsequently, the low expression of CXCR1 has been confirmed in other pyelonephritis-prone patients. The authors conclude that pyelonephritis-prone children have low chemokine receptor expression. Single nucleotide polymorphisms (SNPs) in the Human CXCR1 gene Genomic DNA was extracted from peripheral blood neutrophils from patients and healthy controls. CXCR1 sequence analysis revealed a base pair insertion between positions –725 and –726 when compared with the published sequence. The insertion was present in the promotor region. There

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was no disease association, however, because the insertion was present in all patient and control DNAs. Sequencing of the entire CXCR1 gene revealed single nucleotide polymorphisms in about 40% of the patients, but not in the controls. The SNPs were observed in the intron, the coding region, and in the 39UTR region. No mutations were found in the control group. Genetics of glycolipid receptor variation as deduced from the P blood group The authors’ studies predict that low receptor expression hinders both attachment and host response activation. The expression of receptors for P fimbriae reflects the P blood group, because the receptor structures also act as P blood group determinants. Patients prone to UTI show a higher density of epithelial cell receptors and individuals of blood group P1 run an increased risk of developing recurrent pyelonephritis [26,52]. The receptor repertoire may also influence which fimbrial type can cause infection. Individuals of blood group A1P1 express the globo-A receptor and become infected with bacteria recognizing this receptor [53]. In theory, individuals lacking receptors would be resistant to UTI, but there are too few receptornegative individuals to investigate this hypothesis. Genetics of TLR4 expression and induction of the host response The TLR4 signaling deficiency of C3H-HeJ mice disrupts the inflammatory response, even though there are primary receptors for fimbriae on the mucosa, demonstrating that the TLR4 co-receptor can override the primary receptor as a regulator of cell activation. The unresponsiveness had two effects. The mice failed to develop symptoms of infection, and were unable to clear the infection. As a consequence, they developed ABU, with high bacterial counts in the urine for several months. This carrier state resembled ABU patients who maintain high bacterial counts for months and years, if left untreated, and suggested that mutations b Fig. 3. Experimental urinary tract infection (UTI) in TLR4-negative and CXCR1 KO mice. TLR4 determines the host response induction (step 1). (A) Experimental UTI does not trigger a host response in the TLR4 mutant mice. In the TLR4+ control mice the neutrophil influx in urine reaches a peak after 6 hours, and then declines. (B) Deficient bacterial clearance causes persistent infection in the TLR4 mutant mice, compared with the TLR4+ mice. (Data from Svanborg-Ede´n C, Hagberg L, Briles D, McGhee J, Michalek S. Susceptibility to Escherichia coli urinary tract infection and LPS responsiveness. In: Skamene E, editor. Genetic control of host resistance to infection and malignancy. New York: Alan R Liss; 1985. p. 385–91.) The IL-8 receptor influences bacterial clearance and tissue integrity (step 2). (C) Low urine neutrophil counts in the mIL-8R KO mice, compared with the controls. (D) Increasing bacterial numbers in kidney tissue mIL-8R KO mice. The control mice clear the infection. (From Frendeus B, Godaly G, Hang L, Karpman D, Lundstedt AC, Svanborg C. Interleukin 8 receptor deficiency confers susceptibility to acute experimental pyelonephritis and may have a human counter part. J Exp Med 2000;192:881–90; with permission.)

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Fig. 4. Evidence that a single gene defect can cause acute pyelonephritis and renal scarring. IL8Rh KO mice develop acute pyelonephritis 7 days after infection, with a neutrophil infiltrate and bacteremia. Because the neutrophils normally depend on the IL-8 receptor to cross the epithelial barrier, and this receptor is missing, they are trapped in the tissues, and eventually cause tissue damage. (From Hang L, Frendeus B, Godaly G, Svanborg C. Interleukin-8 receptor knockout mice have subepithelial neutrophil entrapment and renal scarring following acute pyelonephritis. J Infect Dis 2000;182:1738–48; with permission.)

in TLR4 might underlie the unresponsiveness in ABU patients. Prospective clinical studies of the TLR4 genotype in patients with a history of primary ABU have supported this hypothesis.

Genetic tools in the identification of risk patients in the clinic Urinary tract infections are among the most common bacterial infections in all age groups. Although there are considerable intra-individual differences in the susceptibility to UTI, few molecular explanations have been

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offered. The authors propose that the new results on host response induction and the step 1–step 2 model may be used in the future to identify patients prone to pyelonephritis or ABU (see Fig. 5). Low CXCR1 expression may be detected phenotypically by flow cytometry of peripheral blood neutrophils, and patients with a low phenotype may then be subjected to DNA sequencing to identify the polymorphism. The TLR4 genotype may be deduced in a similar manner, using a first phenotypic screening step and a second genotyping approach. Ideally, the diagnosis should be coupled to therapies that compensate the patients for their deficiency.

Fig. 5. Flow chart illustrating how the two-step model could explain individual susceptibility to UTI. ABU ¼ asymptomatic bacteriuria.

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New approaches to strengthening host resistance Treatment and prophylaxis in patients with UTI have improved over the last decades, but serious therapeutic dilemmas remain. The traditional antibiotics often provide an efficient cure of acute disease but resistance has started to appear and new therapeutic alternatives are needed. Furthermore, diagnostic tools for the identification of patients at risk of developing chronic disease or scarring are lacking. Some novel treatment approaches that use the new information about the host response are summarized next. Inhibitors of GSL receptor expression (step 1) In theory, individuals lacking GSL receptors should be resistant to infection with P fimbriated bacteria, but there are too few receptor-negative individuals (blood group p) to investigate this hypothesis. As an alternative approach, pharmacologic inhibition of epithelial receptor expression was tested in vivo in the murine UTI model. The glucose analogue N-butyldeoxynojirimycin blocks the ceramide-specific glycosyltransferase involved in receptor biosynthesis [54,55], and causes a depletion of the globoseries of GSLs on epithelial cells and in kidney tissue. The studies showed protection against colonization and inflammation, confirming the importance of the primary receptor for the host response in vivo. The glucose analogues have been shown to be well tolerated in humans, and might be explored as a novel approach to the prevention of recurrent disease in UTI-prone patients. Blocking of TLR4-dependent signaling (step 1) The results from TLR4 mutant mice are quite important, because they emphasize the protective potential of mucosal unresponsiveness. By maintaining mucosal inertia and avoiding host response induction, the host benefits from the protective aspects of bacteriuria, without risking symptomatic disease. The carrier state is also likely to benefit the bacteria, which thrive in a monoculture at a site where there is much less competition for nutrients and less hostility from other members of the indigenous flora than in the gut or the respiratory tract. Can this beneficial carrier state be induced in hosts who have normal TLR4 responses? The molecular studies on TLR4 variants in ABU will provide insights into possible approaches to achieve this goal in patients prone to recurrent symptomatic UTI. Enhancement of neutrophil function in CXCR1-deficient patients (step 2) The marked effect of the single gene deletion in mIL-8Rh KO mice clearly suggested that reconstitution of this function should bring back the normal

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resistance to infection. Furthermore, the low CXCR1 expression in pyelonephritis-prone patients coupled to polymorphisms in CXCR1 suggest that this approach is just as relevant in humans. We propose that enhancement of CXCR1 expression should be made a target of novel therapeutics against recurrent UTI. CXCR1 itself or regulatory genes involved in tissue-specific expression may be an interesting and important target for therapies that enhance the beneficial aspects of the host response. Induction of ABU in patients with recurrent symptomatic UTI Long-term follow-up of children with ABU has shown that the nontreatment approach is safe, and results in fewer symptomatic UTI recurrences, than if antibiotic treatment is given [3,4]. Based on these findings, the authors have developed a strategy deliberately to establish ABU in patients prone to recurrent UTI, and who do not spontaneously develop ABU (Fig. 6) [56,57]. Patients with recurrent symptomatic UTI, refractory to conventional therapy, were invited to receive this treatment. Most of them had residual urine because of neurogenic bladder disorders. The patients were subjected to deliberate intravesical inoculation with a nonvirulent strain of E coli. The ABU strain E coli 83972 is exquisitely adapted for survival in the urinary tract and has been shown to establish long-term bacteriuria following intravesical inoculation in human patients [58]. The strain carries several adhesin gene clusters [59], but like most ABU strains it does not express the fimbriae, and evokes virtually no host response despite high numbers in human urine. ABU strains often carry genes encoding adherence factors including P fimbriae, and are thought to express virulence-colonization factors during the early establishment in the urinary tract, but then stop expressing the phenotype, and persist in a way that does not break the inertia of the mucosal barrier. In the absence of fimbrial expression E coli 83972 establishes bacteriuria only in patients with incomplete bladder voiding, where residual urine allows the bacteria to remain and multiply. A more complete virulence phenotype, including P fimbriae, is required to establish significant bacteriuria in patients with complete voiding. After transformation with gene clusters encoding P fimbriae, the initial establishment of bacteriuria in hosts without residual urine was enhanced but had only a marginal effect in the group with neurogenic bladder disorders. The P fimbriae clearly influenced the host response in all inoculated patients. The P fimbriated strain caused higher cytokine and neutrophil responses than the ABU strain (see Fig. 6) [35,60]. The successfully colonized patients report a fundamental improvement of their daily life, and they experience no or very few symptomatic superinfections while carrying the ABU strain [58,61]. The therapeutic effects of deliberate inoculation with E coli 83972, however, need to be assessed in a double-blind, placebo-controlled trial protocol.

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UTI vaccines and specific immunity (steps 1 and 2) The protection against microbial infections is usually achieved by the cooperation between the specific and the innate defenses. The urinary tract is a part of the mucosal immune system, but this normally sterile site does not maintain a population of immune cells at all times. Rather, the urinary tract response has to be activated by each infection, because there is no memory and the response to each episode is short-lived. Specific immunity requires antigenic exposure, homing of T and B lymphocyte to the urinary tract mucosa, local maturation of plasma cells or effector lymphocytes, and effects on bacteria through antiadherence or bactericidal effector functions. Early experimental studies on the resistance to UTI focused on the role of specific immune mechanisms. In acute pyelonephritis, both serum and urine antibodies titers are elevated. The serum antibody response is dominated by the IgM and IgG isotypes [62], whereas the urinary antibody response primarily involves secretory IgA [63], which plays a mayor role in the local specific defense of the human urinary tract [64]. Induced antibodies are directed to integral parts of LPS (eg, the O and K antigens [65,66]) or adhesive organelles like P and type 1 fimbriae [67–69]. Specific immunity does not seem to be important for the early clearance of bacteria from the urinary tract. Immunodeficient mice lacking B or T

b Fig. 6. Bacterial interference in the human urinary tract. (A) The human colonization protocol. Before inoculation the patients were treated with antibiotics to sterilize the urinary tract. After an antibiotic-free interval, the patients were catheterized and E coli 83972 (30 mL, 105 colonyforming units per milliliter) was deposited into the bladder once daily for 3 days [35,57,58]. (B) Transformation of the nonfimbriated E coli 83972 with the pap sequences, and with a gfp reporter plasmid. E coli 83972 was transformed with the plasmid pRHu1280 carrying a single copy of the papIA2 gene encoding the P fimbriae [60]. To monitor the papIA2 transcription activity, a reporter plasmid pGFP1 was introduced in E coli 83972 pap+. The plasmid GFP1 carried the gene sequences for green fluorescent protein (GFP). The expression of fimbriae and green fluorescent protein by E coli 83972 pap+gfp+ was shown by FACS. (C) E coli pap+gfp+ adheres to human uroepithelial cells in vivo. After inoculation in the human urinary tract, adherence of E coli 83972 pap+gfp+ to exfoliated uroepithelial cells was demonstrated by fluorescence microscopy (upper panel) and by light microscopy (lower panel). (D) P fimbriae enhance the early establishment of E coli 83972. Seventeen patients were subjected to 31 colonization attempts with E coli 83972 and 15 colonization attempts with pap+ transformants. Each colonization attempt consisted of three daily inoculations ("). The pap+ transformant established bacteriuria ([105 colony-forming units per milliliter) more rapidly (P = .021), and achieved higher bacterial numbers (P\.0001) during the first 3 days of colonization, than E coli 83972. (From Wullt B, Bergsten G, Connell H, Rollano P, Gebretsadik N, Hull R, et al. P fimbriae enhance the early establishment of Escherichia coli in the human urinary tract. Mol Microbiol 2000;38:456–64; with permission.) (E) P fimbriae trigger the local host responses to E coli in the human urinary tract. The neutrophil, IL-6, and IL-8 concentrations during the first 6 days of successful colonizations with the nonfimbriated E coli 83972 and the P fimbriated transformant E coli 83972 pap+ are shown. (From Wullt B, Bergsten G, Connell H, Rollano P, Gebratsedik N, Hang L, et al. P-fimbriae trigger mucosal responses to Escherichia coli in the human urinary tract. Cell Microbiol 2001;3:255–64; with permission.)

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lymphocytes do not show an enhanced UTI susceptibility [70,71]. Studies in the experimental UTI model confirmed the inefficiency of the specific immune response in UTI. Nude, xid, and SCID mice with defective Tlymphocyte, immunoglobulin, or B- and T-lymphocyte function are resistant to experimental UTI as are TCR a/b, TCR c/d, and RAG KO mice [70–72].

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Earlier experiments in mice have shown no correlation between circulating and local antibodies in mice and the outcome of infection [73,74]. In contrast, bacteria often persist despite the presence of high titers of specific antibodies in serum [75]. In addition, the protective function of SIgA against UTI strains might be neutralized by the production of bacterial IgA proteases capable of cleaving IgA [76]. Specific immunity may be important for the later phases of chronic UTI, when the acute innate host defense has failed. In the IL-8 KO mice, the first wave of neutrophil cells is succeeded by a lymphocytic infiltrate, with maturation of plasma cells, as shown by histology. In the bladder, the emergence of follicular cystitis is well known, and the follicles have been shown to consist of lymphocytic aggregates resembling the Peyer’s patches of the gut. The follicles disappear if bacteriuria is cleared. The authors propose from these observations that the role of specific immunity is in the control of chronic infection of kidneys and bladder, rather than in the defense against acute bacterial infection. Despite the unclear role of the specific immune response, vaccination is often advocated as a way of preventing acute or recurrent UTI. In humans, systemic, peroral, or local (vaginal) application of antigen has been tested. Antigen mixtures from uropathogenic E coli have been tested in parallel with highly defined antigens like LPS, fimbriae, or capsular polysaccharides, and several studies have indicated that hyperimmunization can protect against challenge with the strain bearing the vaccine epitope [77]. Intravesical P fimbriae adhesin exposition was shown to protect against recurrent UTI in a monkey model [78]. Intravesical inoculation with the FimH adhesin, isolated from the type 1 fimbriae, protected against subsequent experimental UTI in four monkeys. This was interpreted to suggest that the FimH protein could be used as a common antigen in the development of a UTI vaccine [69,79–81]. The authors conclude that there is little evidence for vaccine-mediated protection against recurrent UTI. This is explained both by the antigenic variation of uropathogenic strains, and by the short-lived immune response in the urinary tract. Summary The authors use the UTI model to identify basic mechanisms of disease pathogenesis, host response induction, and defense. Their studies hold the promise to provide a molecular and genetic explanation for susceptibility to UTI, and to offer more precise tools for diagnosis and therapy of these infections. There are few infections where the host response is understood in such detail and where pathologic host responses can be linked to distinct disease states. The susceptibility to UTI varies greatly in the population. The studies suggest that distinct molecular defects can cause the clinical entity of acute pyelonephritis with renal scarring, and suggest that the susceptibility to UTI

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in certain patient groups may have a genetic basis. In addition, the distinct signal transduction pathways explain the development of symptoms, and propose that defects in those signaling mechanisms may occur in patients with ABU. In the future, it may be useful to include these host response parameters in the diagnostic arsenal, to help in early detection of patients susceptible to recurrent UTI and renal scarring. These patients may then be offered therapies that strengthen their defense, and be offered close surveillance for recurrences and other complications. References [1] Svanborg C, Frendeus B, Godaly G, Hang L, Hedlund M, Wachtler C. Toll-like receptor signaling and chemokine receptor expression influence the severity of urinary tract infection. J Infect Dis 2001;183(suppl 1):S61–5. [2] Kunin CM. Urinary tract infections: detection, prevention and management. 5th edition. Baltimore: Williams and Wilkins; 1997. [3] Hansson S, Jodal U, Lincoln K, Svanborg-Eden C. Untreated asymptomatic bacteriuria in girls: II–Effect of phenoxymethylpenicillin and erythromycin given for intercurrent infections. BMJ 1989;298:856–9. [4] Lindberg U. Asymptomatic bacteriuria in school girls. V. The clinical course and response to treatment. Acta Paediatr Scand 1975;64:718–24. [5] Lindberg U, Hansson LA˚, Jodal U, Lidin-Janson G, Lincoln K, Olling S. Asymptomatic bacteriuria in schoolgirls. II. Differences in Escherichia coli causing asymptomatic and symptomatic bacteriuria. Acta Paediatr Scand 1975;64:432–6. [6] Mabeck C, Orskov F, Orskov I. Escherichia coli serotypes and renal involvement in urinary-tract infection. Lancet 1971b;1:1312–4. [7] Svanborg-Ede´n C, Hanson LA, Jodal U, Lindberg U, Sohl-A˚kerlund A. Variable adherence to normal urinary tract epithelial cells of Escherichia coli strains associated with various forms of urinary tract infections. Lancet 1976;2:490–2. [8] Johnson J. Virulence factors in Escherichia coli urinary tract infection. Clin Microbial Rev 1991;4:80–128. [9] Hacker J, Blum-Oehler G, Muhldorfer I, Tschape H. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 1997;23:1089–97. [10] Hedges S, de Man P, Linder H, van Kooten C, Svanborg-Ede´n C. Interleukin-6 is secreted by epithelial cells in response to Gram-negative bacterial challenge. In: Macdonald T, editor. Advances in mucosal immunology. International Conference of Mucosal Immunity. London: Kluwer; 1990. p. 144–8. [11] Hedges S, Svensson M, Svanborg C. Interleukin-6 response of epithelial cell lines to bacterial stimulation in vitro. Infect Immun 1992;60:1295–301. [12] Plos K, Connell H, Jodal U, Marklund B, Ma˚rild S, Wettergren B, et al. Intestinal carriage of P fimbriated Escherichia coli and the susceptibility to urinary tract infection in young children. J Infect Dis 1995;171:625–31. [13] de Man P, Jodal U, Lincoln K, Svanborg-Ede´n C. Bacterial attachment and inflammation in the urinary tract. J Infect Dis 1988;158:29–35. [14] Svanborg C, Godaly G, Hedlund M. Cytokine responses during mucosal infections: role in disease pathogenesis and host defense. Curr Opin Microbiol 1999;2:99–105. [15] Svanborg C, Bergsten G, Fischer H, Frendeus B, Godaly G, Gustafsson E, et al. The Ôinnate’ host response protects and damages the infected urinary tract. Ann Med 2001;33:563–70. [16] Leffler H, Svanborg-Ede´n C. Chemical identification of a glycosphingolipid receptor for Escherichia coli attaching to human urinary tract epithelial cells and agglutinating human erythrocytes. FEMS Microbiol Lett 1980;8:127–34.

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