Toll-like receptors recognize uropathogenic Escherichia coli and trigger inflammation in the urinary tract
Hans-Joachim Anders and
Prashant S. Patole
Nephrological Center, Medical Policlinic, Ludwig-Maximilians-University, Munich, Germany
Correspondence and offprint requests to: Dr H.-J. Anders, Medizinische Poliklinik der LMU, Pettenkoferstr. 8a, D-80336 Munich, Germany. Email: hjanders{at}med.uni-muenchen.de
Keywords: chemokines; immunity; infection; leukocytes
 |
Introduction
|
---|
Urinary tract infection (UTI) is one of the most common types of infection. In the USA, >US$1.6 billion per year are spent in the management of UTI [1]. Amongst the pathogens that can cause UTI, uropathogenic Escherichia coli (UPEC) has been found to be the causative organism in
80% of cases. A number of pathogen-related virulence factors have been identified that relate mostly to pathogen adhesion to the epithelial lining of the urinary tract [24]. In addition, patient-related factors determine the prevalence and severity of UTI. Diabetes, immunosuppression, reflux and obstruction due to structural abnormalities of the urinary tract or pregnancy are known to predispose to UTI. These conditions affect either physiological urine flow or cellular immunity against uropathogenic agents. However, the patient-related risk for UTI may also be determined by pathogen recognition and pathogen-induced signalling for host defence. Host defence in UTI is based on innate immunity. Thus, which receptors recognize uropathogenic pathogens, and what are the subsequent signalling cascades that concert antibacterial immunity? The recent discovery of the Toll-like receptor (TLR) family has identified a group of such receptors that appear to have important functions not only for pathogen recognition but also for innate immune activation [5]. In this article, we present the current view on TLRs in UTI. Based on these data, we propose a working hypothesis and raise new questions that deserve further study in this field.
 |
Toll-like receptors are innate pathogen recognition receptors
|
---|
Innate immune recognition is based on so-called pattern recognition receptors (PRRs). Important families of PRRs include scavenger receptors, complement receptors and mannose receptors. PRRs recognize pathogen-associated molecular patterns (PAMPs). Several PRRs can recognize the same pathogen through different PAMPs. Contrarily, various classes of pathogens can be recognized by the same PRR. Recently, a new family of PRRs was discovered, the TLRs [6]. The TLR family consists of 12 members, but ligands and functions for TLR10 and TLR12 have not yet been identified (Figure 1). A subgroup of the TLRs, namely TLR3, TLR7, TLR8 and TLR9, recognize various types of nucleic acids. Nucleic acid-specific TLRs are mostly localized in intracellular endosomes, allowing the recognition of complexed nucleic acids that are delivered to that compartment [7]. TLR1 and TLR6 are localized on the cell surface and activate different signalling pathways upon heterodimerization with TLR2 [8]. TLR1/TLR2 and TLR6/TLR2 heterodimers recognize various forms of lipopeptides that occur on both Gram-positive and Gram-negative bacteria [9]. TLR5 recognizes flagellin, and its localization at basolateral membranes of enterocytes allows selective recognition of enteroinvasive forms of flagellated pathogens in the intestinal tract [10]. Finally, TLR4 is a critical component of the lipopolysaccharide (LPS) receptor complex, which forms a detection system for Gram-negative bacteria [11]. TLR11 was only recently described and responds to an as yet unknown UPEC-associated molecule [12].

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1. Toll-like receptors: ligands and adaptor molecules. TLRs recognize specific pathogen-associated molecular patterns by homophilic or heterophilic interaction. A subset of nucleic acid-specific TLRs is localized in intracellular endosomes. TLRs signal through a group of cytosolic adaptor molecules that redistribute to the intracellular Toll/interleukin-1 receptor (TIR) domain upon activation of the receptor complex.
|
|
 |
TLR4 and TLR11 signal for renal neutrophil recruitment in mice with UTI
|
---|
It has long been known that so-called LPS-hyporesponsive C3H/HeJ mice lack renal neutrophil recruitment and leukocyturia upon challenge with UPEC [13]. After the discovery of the TLR family, it was noted that the functional defect in LPS-hyporesponsive C3H/HeJ mice results from a non-functional product of a Tlr4 gene mutation [11,14]. Upon ascending UPEC infection, TLR4 mutant mice fail to induce renal expression of chemokines that signal for neutrophil recruitment in UTI, i.e. CXCL2 [15,16]. CXCL2, the murine homologue of interleukin-8 (IL-8), is expressed by immune cells as well as renal tubular epithelial cells [15]. We used intravital microscopy in order to test the hypothesis that TLR4LPS interaction is required for local neutrophil recruitment after LPS exposure. Upon challenge with UPEC LPS, TLR4 mutant mice were found to lack endovascular adhesion and transendothelial migration of leukocytes [15]. After local exposure to CXCL2, TLR4-deficient mice showed normal leukocyte recruitment, indicating that LPS recognition through TLR4 is a proximal event in that process.
TLR11 may have a similar role because TLR11-deficient mice, similarly to TLR4 mutants, lack appropriate cellular immunity upon UPEC infection [12]. TLR11 is expressed on cells of the monocytemacrophage lineage but also at high levels in the healthy mouse kidney [12]. This raises the question of whether intrinsic renal cells, i.e. renal tubular epithelia, signal UPEC infection through TLRs.
 |
Do intrinsic renal cells signal through TLRs during UPEC infection?
|
---|
Epithelial cells represent a structural barrier and functional interface for pathogenhost interaction during infection [17]. Thus, it was hypothesized that TLRs expressed by the epithelial lining of the lower and upper urinary tract could recognize UPEC during UTI. Tsuboi et al. demonstrated that tubular epithelial cells isolated from TLR4 mutant mice lack CC-chemokine expression upon exposure to E.coli LPS [18]. We generated chimeric mice by bone marrow cross-transplantation from TLR4 mutant and wild-type mice in order to determine the contribution of TLR4 on intrinsic renal cells for neutrophil recruitment in infective pyelonephritis [15]. After UPEC infection, mice that lack a functional TLR4 in intrinsic renal cells were unable to signal for neutrophil recruitment. In these mice, renal abcess formation was absent, despite high UPEC counts in the kidney. These findings are consistent with a similar study that identified a role for TLR4 in bladder epithelia during UTI of the lower urinary tract [19]. Both studies also revealed that TLR4 activation on infiltrating haematopoietic cells is also required for mounting leukocyte recruitment in UTI [15,19]. These data convincingly suggest that TLR4 on intrinsic renal cells as well as on immune cells directs immunity against UPEC. In the urinary tract, TLR4 signals for local neutrophil recruitment as a major mechanism of infection control (Figure 2). Apart from TLR4, TLR11 appears to be expressed by renal tubular epithelial cells in mice. Because TLR11-deficient mice also show defective immunity in UPEC pyelonephritis, a similar role for UPEC recognition has been suggested [12].

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 2. Proposed working model for UPEC recognition during UTI. UPEC bind to uroepithelia in the lower urinary tract and to renal tubular epithelial cells in the kidney by adhesion through P fimbriae and other factors. Cross-linking of glycosphingolipid receptors with TLR4 overcomes the lack of CD14 in these epithelia. Ligation of the LPS receptor complex (and possibly other TLRs) induces local expression of CXCL2/IL8 as well as activation and upregulation of adhesion molecules on adjacent endothelial cells of peritubular capillaries. Local expression of chemokines and adhesion molecules is followed by local neutrophil recruitment, renal abcess formation and leukocyturia.
|
|
 |
Are data from mouse models relevant for human UTI?
|
---|
The relevance of data derived from murine studies for human UTI remains a matter of debate. Zhang et al. reported stop codons in the genomic sequence of TLR11, suggesting that TLR11 might not be expressed in humans [12]. TLR4 expression in the human kidney has also been questioned, because studies with human kidney carcinoma cells yielded inconsistent results in terms of TLR4 expression in vitro [20,21]. However, human biopsies stained for TLR4 revealed expression in epithelial cells lining the entire human urinary tract, including the renal tubular epithelium [22]. Besides TLR4, the LPS receptor complex includes other proteins such as CD14 and MD-2 [23,24]. TLR4, MD-2 and CD14 are all present on mouse tubular epithelial cells [18,25], but their expression in the human urinary tract is less clear. Immunostaining for CD14 was found to be negative on biopsies of the human urinary tract [22]. However, UPEC LPS can activate human uroepithelial cells in the absence of CD14 [21,26]. In UPEC, LPS becomes complexed with P fimbriae that interact with glycosphingolipid receptors on uroepithelia. It has been proposed that TLR4 may act as a co-receptor for that interaction and signal despite the absence of CD14 (Figure 2 [16]).
 |
Future directions
|
---|
The discovery of TLRs holds great promise for a better understanding of pathogen-induced immunity and autoimmunity in the field of nephrology [27]. In contrast to other organs that are exposed to huge loads of commensals and pathogens, the urinary tract is supposed to be sterile. Therefore, it is suspected that TLRs in the urinary tract represent pathogen detection receptors. However, the recently discovered role of TLR4 and TLR11 in UTI raises new questions. The natural ligand for TLR11, a presumed component of UPEC, is unknown. Do humans express a functional TLR11? Does TLR11 ligation compete with or contribute to the effects of TLR4 signalling in UTI? Do genetic polymorphisms that modulate the functions of TLRs affect the susceptibility to UTI or asymptomatic bacteriuria? Do other TLRs mediate UPEC recognition or do other TLRs recognize other uropathogenic microbes, i.e. Gram-positive bacteria? What factors modulate TLR4 signalling towards selective neutrophil recruitment, as TLR4 ligation leads to production of monocyte and T-cell-attracting chemokines? What is the role of modulatory molecules of TLR signalling? For example, SIGGIR, the Tir8 gene product and a negative regulator of TLR signalling, is expressed at high levels in the kidney but at low levels in the bladder ([28], own unpublished observation). Is UPEC adhesion through fimbriae required before TLR activation can occur? How do glycosphingolipid receptors and TLRs interact during UPEC infection? Are their additional modifiers of TLR-mediated innate immunity in UTI? Tamm-Horsfall protein has been shown to activate dendritic cells through TLR4 [29]. Its role in UTI-driven renal inflammation remains to be studied. UTI is a major medical problem. A better understanding of pathogen recognition and immune activation in the urinary tract may enable us to identify new targets for infection control.
 |
Acknowledgments
|
---|
The work was supported by grants from the Deutsche Forschungsgemeinschaft (AN 372/4-1) and the Fritz-Thyssen Foundation to H.J.A.
Conflict of interest statement. None declared.
 |
References
|
---|
- Foxman B, Barlow R, D'Arcy H, Gillespie B, Sobel JD. Urinary tract infection: self-reported incidence and associated costs. Ann Epidemiol 2000; 10: 509515[CrossRef][ISI][Medline]
- Hedlund M, Duan RD, Nilsson A, Svensson M, Karpman D, Svanborg C. Fimbriae, transmembrane signaling, and cell activation. J Infect Dis 2001; 183 [Suppl 1]: S47S50[CrossRef][Medline]
- Johnson JR. Microbial virulence determinants and the pathogenesis of urinary tract infection. Infect Dis Clin North Am 2003; 17: 261278[CrossRef][ISI][Medline]
- Mulvey MA. Adhesion and entry of uropathogenic Escherichia coli. Cell Microbiol 2002; 4: 257271
- Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20: 197216[CrossRef][ISI][Medline]
- Underhill DM, Ozinsky A. Phagocytosis of microbes: complexity in action. Annu Rev Immunol 2002; 20: 825852[CrossRef][ISI][Medline]
- Marshak-Rothstein A, Busconi L, Rifkin IR, Viglianti GA. The stimulation of Toll-like receptors by nuclear antigens: a link between apoptosis and autoimmunity. Rheum Dis Clin North Am 2004; 30: 559574[CrossRef][ISI][Medline]
- Ozinsky A, Underhill DM, Fontenot JD et al. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between Toll-like receptors. Proc Natl Acad Sci USA 2000; 97: 1376613771[Abstract/Free Full Text]
- Kirschning CJ, Schumann RR. TLR2: cellular sensor for microbial and endogenous molecular patterns. Curr Top Microbiol Immunol 2002; 270: 121144[ISI][Medline]
- Hayashi F, Smith KD, Ozinsky A et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001; 410: 10991103[CrossRef][ISI][Medline]
- Poltorak A, He X, Smirnova I et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998; 282: 20852088[Abstract/Free Full Text]
- Zhang D, Zhang G, Hayden MS et al. A Toll-like receptor that prevents infection by uropathogenic bacteria. Science 2004; 303: 15221526[Abstract/Free Full Text]
- Shahin RD, Engberg I, Hagberg L, Svanborg Eden C. Neutrophil recruitment and bacterial clearance correlated with LPS responsiveness in local gram-negative infection. J Immunol 1987; 138: 34753480[Abstract/Free Full Text]
- Hoshino K, Takeuchi O, Kawai T et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999; 162: 37493752[Abstract/Free Full Text]
- Patole PS, Schubert S, Meßmer K et al. Toll-like receptor-4: intrinsic renal cells and bone marrow-derived cells signal for neutrophil recruitment during infective pyelonephritis. Submitted
- 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]: S61S65[CrossRef][Medline]
- Chowdhury P, Sacks SH, Sheerin NS. Functions of the renal tract epithelium in coordinating the innate immune response to infection. Kidney Int 2004; 66: 13341344[CrossRef][ISI][Medline]
- Tsuboi N, Yoshikai Y, Matsuo S et al. Roles of toll-like receptors in C-C chemokine production by renal tubular epithelial cells. J Immunol 2002; 169: 20262033[Abstract/Free Full Text]
- Schilling JD, Martin SM, Hung CS, Lorenz RG, Hultgren SJ. Toll-like receptor 4 on stromal and hematopoietic cells mediates innate resistance to uropathogenic Escherichia coli. Proc Natl Acad Sci USA 2003; 100: 42034208[CrossRef]
- Bäckhed F, Soderhall M, Ekman P, Normark S, Richter-Dahlfors A. Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ- and cell-specific expression of Toll-like receptors within the human urinary tract. Cell Microbiol 2001; 3: 153158[CrossRef][ISI][Medline]
- Hedlund M, Frendeus B, Wachtler C, Hang L, Fischer H, Svanborg C. Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells. Mol Microbiol 2001; 39: 542552[CrossRef][ISI][Medline]
- Samuelsson P, Hang L, Wullt B, Irjala H, Svanborg C. Toll-like receptor 4 expression and cytokine responses in the human urinary tract mucosa. Infect Immun 2004; 72: 31793186[Abstract/Free Full Text]
- Akashi S, Ogata H, Kirikae F et al. Regulatory roles for CD14 and phosphatidylinositol in the signaling via Toll-like receptor 4-MD-2. Biochem Biophys Res Commun 2000; 268: 172177[CrossRef][ISI][Medline]
- Shimazu R, Akashi S, Ogata H et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 1999; 189: 17771782[Abstract/Free Full Text]
- Morrissey J, Guo G, McCracken R, Tolley T, Klahr S. Induction of CD14 in tubular epithelial cells during kidney disease. J Am Soc Nephrol 2000; 11: 16811690[Abstract/Free Full Text]
- Frendeus B, Wachtler C, Hedlund M et al. Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation. Mol Microbiol 2001; 40: 3751[CrossRef][ISI][Medline]
- Anders HJ, Banas B, Schlondorff D. Signaling danger: Toll-like receptors and their potential roles in kidney disease. J Am Soc Nephrol 2004; 15: 854867[Abstract/Free Full Text]
- Wald D, Qin J, Zhao Z et al. SIGIRR, a negative regulator of Toll-like receptorinterleukin 1 receptor signaling. Nat Immunol 2003; 4: 920927[CrossRef][ISI][Medline]
- Saemann MD, Weichhart T, Zeyda M et al. Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4-dependent mechanism. J Clin Invest 2005; 115: 468475[Abstract/Free Full Text]