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The Journal of Immunology, 2006, 177: 7225-7232.
Copyright © 2006 by The American Association of Immunologists, Inc.

Urothelial CD44 Facilitates Escherichia coli Infection of the Murine Urinary Tract1

Kasper M. A. Rouschop2,*, Marc Sylva*, Gwendoline J. D. Teske*, Inge Hoedemaeker*, Steven T. Pals*, Jan J. Weening*, Tom van der Poll{dagger} and Sandrine Florquin2,*

* Department of Pathology and {dagger} Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Escherichia coli is the most common pathogen found in urinary tract infections (UTIs), mainly affecting children and women. We report that CD44, a hyaluronic acid (HA) binding protein that mediates cell-cell and cell-matrix interactions, facilitates the interaction of E. coli with urothelial cells and thus the infection of the host. We found that CD44 is constitutively expressed on urothelial cells and that HA accumulates in E. coli-induced UTI. In CD44-deficient mice, the bacterial outgrowth was dramatically less compared with wild-type mice despite similar granulocyte influx in the bladder and in the kidney as well as comparable cytokines/chemokines levels in both genotypes. E. coli was able to bind HA, which adhered to CD44-positive tubular epithelial cells. Most importantly, the interaction of CD44 on tubular epithelial cells with HA facilitated the migration of E. coli through the epithelial monolayer. The results provide evidence that CD44 on urothelial cells facilitates E. coli UTI. Disruption of the interaction between CD44 and HA in the bladder may provide a new approach to prevent and to treat UTI.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Urinary tract infections (UTIs)3 are among the most common bacterial infections, affecting ~50% of women at one point of their lifetime. UTIs also frequently occur during childhood, and inflammation of the renal pelvis (pyelonephritis) is a significant factor in the development of end-stage renal failure in children and young adults (1, 2). The Gram-negative bacterium Escherichia coli is the most common pathogen found in UTI. The outcome of UTI is determined by the pathogen, which may or may not express specific virulence factors, including hemolysin, adhesive fimbriae, iron acquisition systems, and toxins (3), and by the host itself. Far from being just a physical barrier between urine and tissue, urothelial cells play an active role in coordinating the innate immune response to infection (4). Urothelial cells are able to produce proteins (such as mucin and Tamm-Horsfall protein) that interfere with bacterial adhesiveness, as well as cytokines/chemokines that initiate migration of neutrophils to the luminal side. Urothelial cells also express TLR2, TLR4, and TLR11 (4, 5) to detect the presence of microorganisms.

The adhesion molecule CD44 is constitutively expressed on urothelium (6) but not on tubular epithelial cells (TECs). However, upon injury CD44 is rapidly up-regulated at the cell surface of TECs (7, 8, 9, 10, 11, 12, 13). The strategic position of CD44 on the urinary mucosal surface raises the possibility that this molecule may play a role during UTI.

CD44 is a family of type I transmembrane glycoproteins with a wide tissue distribution implicated in many physiological and pathological processes, including cell-cell and cell-matrix interactions, leukocyte extravasation, wound-healing/scarring, cell migration, lymphocyte activation, and binding/presentation of growth factors (14, 15, 16, 17). All CD44 isoforms contain a binding site for hyaluronic acid (HA), which is a ubiquitously expressed polysaccharide and a major ligand of CD44 (15, 18). Although all CD44 variants contain a HA-binding domain, HA binding is regulated by a complex of modifications involving alternative splicing (19, 20, 21, 22, 23). Posttranslational modifications such as addition of heparan sulfate (23), keratin sulfate (24), and chondroitin sulfate (25) may decrease affinity for HA due to their negative charge. Moreover, CD44 requires a conformational change to bind HA (26, 27, 28, 29, 30). In B cells the transition is considered to be glycosylation dependent (31, 32), but for other cell types, the transition remains unclear (33). Additionally, phosphorylation of CD44 (34) and intracellular interactions with the cytoskeleton may be required for HA binding (28, 35).

Although CD44 has been implicated in a variety of inflammatory processes, only a few studies have investigated the potential role of CD44 during infections with pathogenic microorganisms. Depending on the microorganisms used and the organ of investigation, the function of CD44 seems to be variable. Using an inactivating Ab directed against CD44, Blass et al. (36) suggested that this adhesion molecule is not needed for resistance against infection with Toxoplasma gondii. Others showed that CD44 facilitates the intracellular proliferation of Listeria monocytogenes (37). In models of pneumonia, CD44 deficiency resulted in enhanced inflammation in E. coli-induced but not Streptococcus pneumoniae-induced lung inflammation despite equal clearance of these organisms (38). Interestingly, independent studies provided evidence that CD44 serves as a binding site for microorganisms. Cywes et al. (39, 40) identified CD44 as a receptor for group A Streptococcus in a model of pharyngitis. Leemans et al. (41) identified CD44 as a new macrophage-binding site for Mycobacterium tuberculosis that mediates mycobacterial phagocytosis, macrophage recruitment, and protective immunity against pulmonary tuberculosis. The aim of the present study was to investigate the role of CD44 in E. coli-induced UTI.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and experimental protocol

CD44 knockout (CD44–/–) mice on C57BL/6 background (42) and C57BL/6 wild-type mice (CD44+/+) were bred in our animal facility. Bacterial inoculation was performed under general anesthesia (0.07 ml/10 gram mouse of fentanyl, fluanisone, and midazolam mixture containing: 0.08 mg/ml fentanyl citrate, 2.5 mg/ml fluanisone (Janssen Pharmaceutica), 1.25 mg/ml midazolam (Roche)) in 8- to 10-wk-old female mice. For instillation of the bacteria we used a radiopaque catheter with a diameter of 0.55 mm (Abbott Laboratories). In these experiments, E. coli 1677 was used, which was isolated from a patient with UTI and was previously proven to be uropathogenic in mice during experimental pyelonephritis (43, 44, 45). At 6, 24, and 48 h after infection, mice (n = 8 per group) were anesthetized by fentanyl, fluanisone, and midazolam mixture and were killed by cardiac exsanguination. The left kidney was homogenized at 4°C in 4 volumes of sterile saline. Viable counts of the inoculum and bacterial outgrowth from renal tissue and blood was determined using blood-agar plates. Mice killed at 6 and 24 h after inoculation received 8.0 x 108 CFU per mouse. In a different experiment, mice sacrificed at 48 h after inoculation received 9.6 x 108 CFU per mouse. Mice receiving Ab treatment received 4 x 108 CFU per mouse, which was simultaneously administered with the Ab. Mice were treated with either 50 µg of anti-CD44 (clone IM7.8.1, rat IgG2b; American Type Culture Collection (ATCC)) that interacts with the HA-binding site of CD44 (46) and may induce shedding of CD44 (47), or control IgG (rat IgG2b; ATCC). These mice were killed 24 h after inoculation. All experimental procedures were approved by the Animal Care and Use Committee of the University of Amsterdam.

Immunohistochemistry

Renal and bladder tissues were fixed in 10% formalin for 12 h and embedded in paraffin. Immunohistochemical staining was performed using specific Abs directed against: CD44 (clone IM7.8.1; ATCC), neutrophils (Ly-6G, (GR-1); BD Pharmingen), and HA (biotinylated HA binding protein; Calbiochem)), as previously described (12, 13). As a negative control we used species and isotype-matched Abs (BD Pharmingen).

Neutrophils in bladder tissue were counted in a randomly chosen nonoverlapping total of 10 fields (magnification, x100). Data are expressed per square millimeter.

Cytokine, chemokine, and myeloperoxidase (MPO) ELISAs

MIP-2, keratinocyte-derived chemokine (KC/Gro-{alpha}), IL-1β (all R&D Systems), and mouse MPO (HyCult Biotechnology) were measured in renal homogenates as previously described (13) by specific ELISAs according to the manufacturer’s instructions. Protein concentrations of the renal homogenates were determined using a Bradford protein assay (Bio-Rad).

E. coli phagocytosis and oxidative burst by neutrophils

E. coli 1677 was labeled using CellTracker Orange (Molecular Probes) in medium. Excess label was washed away and quenched by addition of serum as suggested by the manufacturer. Fluorescent E. coli 1677 was used for phagocytosis experiments. Expression of enhanced GFP (eGFP) in E. coli strains Top-10 and DH10B (both Invitrogen Life Technologies) was induced by transfection with pBBR1MCS-5-eGFP (clone pMP2463) (48), which was provided by Dr. H. Spaink, Leiden University (Leiden, the Netherlands). Bacteria were grown under high selective pressure (40 µg/ml gentamicin; Sigma-Aldrich). Enhanced GFP expression was confirmed by flow cytometry and fluorescent microscopy (data not shown). Phagocytosis capacity of CD44+/+ and CD44–/– neutrophils was determined by flow cytometry. In short, heparin blood was withdrawn from CD44+/+ and CD44–/– mice by cardiac exsanguination. Fluorescent bacteria (50 x 106 CFU/ml) and blood were incubated for 0, 5, 10, 15, and 20 min at 37°C. RBC were lysed using a hypotonic lysis buffer, and neutrophils were labeled using anti-Ly6-G allophycocyanin (BD Pharmingen).

Oxidative burst of neutrophils was assessed by dihydrorhodamine 123 (Sigma-Aldrich) measurement according to Kampen et al. (49). Circulating cells (50 µl of whole blood) were loaded with 1.5 mg/ml dihydrorhodamine 123 for 30 min. Neutrophils were stimulated by addition of 50 x 106 CFU E. coli 1677/ml for 30 min, followed by RBC lysis. Conversion of dihydrorhodamine was determined by flow cytometry.

Flow cytometric analysis of HA binding to E. coli 1677

E. coli 1677 was grown in an overnight culture in tryptic soy broth. Approximately 50 x 106 CFU/ml were incubated with various concentrations of HA-FITC (0, 10, 100 and 1000 µg/ml; Sigma-Aldrich) in PBS for 15 min and analyzed by flow cytometry.

Isolation of CD44+/+ and CD44–/– TECs

Immortomice (CBA/ca x C57BL/10 hybrid; Charles River Breeding Laboratories) were crossed with CD44+/+ and CD44–/– for over four generations. CD44+/+ and CD44–/– mice homozygous for the temperature-sensitive SV40 large T cell Ag (H-2kb-tsA58) were anesthetized. Removal of the glomeruli was performed as described by Takemoto et al. (50). In short, kidneys were perfused with 10 x 107 magnetic beads (diameter 4.5 µm; Sigma-Aldrich) in PBS. Kidneys were cut into small pieces (~1 mm3) and digested in 1 mg/ml collagenase A (Roche Diagnostics) in HBSS for 15 min at 37°C. The digested kidneys were pressed through a 100-µm cell strainer. Glomeruli were removed using a magnetic particle concentrator. In addition, the remaining cell suspension was filtered through a 40-µm cell strainer to remove any residual glomeruli.

TECs were isolated by outgrowth in selective medium consisting of a 1:1 ratio of DMEM (Invitrogen Life Technologies) and Ham-F12 (Invitrogen Life Technologies) supplemented with 5% heat-inactivated FCS (Invitrogen Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/L L-glutamine, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 20 ng/ml triiodothyronine, 5 ng/ml hydrocortisone and 5 ng/ml PGE1 (all Sigma-Aldrich). In permissive conditions cells were grown at 33°C with addition of 25 ng/ml IFN-{gamma} (R&D Systems) until confluence. Confluent cells were then placed for 8 days at 37°C without addition of IFN-{gamma}.

Adhesion and migration assays

For determination of E. coli adhesion to CD44, CD44+/+, and CD44–/–, TECs were grown until confluence in a six-well plate (Costar) and were allowed to differentiate for 8 days. Hyaluronidase treatment was performed as previously described (12). In short, HA was removed from the cellular surface of TECs by incubation in a phosphate buffered solution (pH 7.0), containing 300 µg/ml hyaluronidase type IV-s (Sigma-Aldrich) for 10 min. Control cells were incubated in the identical buffer without hyaluronidase. Cells were then washed and incubated with 2.5 x 106 CFU E. coli 1677 in serum-free medium with or without addition of HA (derived from rooster comb; Sigma-Aldrich) and if indicated 50 µg of control IgG (rat IgG2b; ATCC) or anti-CD44 (clone IM7.8.1, rat IgG2b; ATCC) for 30 min. Cells were extensively washed and scraped, and cell suspensions were plated on blood-agar for the determination of viable E. coli 1677 count.

Expression of HA on TEC monolayers was assessed using biotinylated HA binding protein (Calbiochem) after extensively washing the cell layers and fixation in 2% paraformaldehyde (Sigma-Aldrich). Visualization of the bound HA binding protein was done using streptavidin-FITC (DakoCytomation).

CD44-dependent migration of E. coli 1677 was determined using Transwell porous filters (Costar) with a 0.4-µm pore diameter. Cells were grown until confluence and differentiated for 8 days. The apical side (treated with hyaluronidase if indicated) was extensively washed and incubated with 2.5 x 106 CFU E. coli 1677 in serum-free medium, with or without addition of HA. The basal side contained medium with 10% FCS. After 1 h, medium of the basal side was plated on blood-agar to determine viable E. coli counts.

Statistical analysis

All data were analyzed by unpaired Student’s t test comparison. Multiple comparisons were performed using a repeated-measures ANOVA corrected by a Bonferroni posthoc test. A value for p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD44 is constitutively expressed on urothelium

In contrast to TECs, urothelial cells constitutively expressed CD44 on their cytoplasmic membranes (Fig. 1A). Upon UTI, urothelial expression remained stable (Fig. 1B). HA, one of the major ligands of CD44, was not detectable on bladder urothelium in normal conditions (Fig. 1C) but increased strikingly upon UTI (Fig. 1D). This accumulation of HA on urothelium was dependent on CD44 expression on urothelial cells because bladders of CD44-deficient mice did not display any HA accumulation during UTI (Fig. 1E). The constitutive expression of CD44 and accumulation of HA on urothelium upon infection may imply that these molecules participate in infection of the urinary tract by E. coli.


Figure 1
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  		FIGURE 1. CD44 is constitutively expressed on urothelium, and HA is expressed on CD44+/+ but not on CD44–/– urothelial cells after experimental pyelonephritis. Representative immunostainings for CD44 on bladders of CD44+/+ mice before (A) and at 24 h (B) after intravesical inoculation of E. coli 1677. HA is expressed in the lamina propria but not expressed by urothelial cells in control bladder (C). At 24 h after intravesical inoculation, HA is up-regulated on urothelial cells of CD44+/+ bladders (D), but not on CD44–/– urothelial cells (E). Magnification is x40. Images are representative for n = 8 mice per group. *, urinary space.

 
CD44 deficiency leads to decreased bacterial counts during UTI

We examined whether the absence of CD44 would alter susceptibility to infection by E. coli. As shown in Fig. 2, the number of CFUs of E. coli recovered from the kidney at 24 and 48 h (Fig. 2, top) after infection was dramatically lower in CD44–/– than in CD44+/+ mice. Bacterial counts in the blood were significantly decreased in CD44–/– compared with CD44+/+ 6 h after infection (Fig. 2, bottom).


Figure 2
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  		FIGURE 2. CD44 deficiency decreases bacterial outgrowth from the kidney and blood. Bacterial outgrowth was assessed in renal tissue (top) and blood (bottom) after intravesical inoculation of E. coli 1677 in CD44+/+ ({square}) and CD44–/– ({blacksquare}) mice. Because mice killed at 48 h after inoculation received more CFUs compared with mice killed at 6 and 24 h after inoculation, graphs show a separation. *, p < 0.05 for CD44+/+ compared with CD44–/– mice. Data are presented as the mean ± SEM (n = 8 mice).

 
Neutrophil accumulation is comparable in CD44–/– and CD44+/+ mice

To overcome sampling errors due to very heterogeneous distribution of neutrophils around the pyelum, neutrophil migration was assessed by measuring their activity (MPO assay) in renal homogenates (Fig. 3A). In the bladder, neutrophil infiltration was assessed by counting Ly6G-positive cells on histological slides (Fig. 3B). No difference in neutrophil accumulation was detected between CD44–/– and CD44+/+ mice despite better bacterial clearance in CD44–/– mice.


Figure 3
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  		FIGURE 3. Comparable neutrophil migration in kidneys and bladder of CD44+/+ and CD44–/– mice after experimental pyelonephritis. A, MPO assay on renal tissue revealed comparable neutrophil migration in CD44+/+ ({square}) and CD44–/– ({blacksquare}) mice. The maximum of neutrophil infiltration was reached at 6 h after inoculation. B, Neutrophil migration into the infected bladder was maximal 24 h postinfection. No differences were detected in neutrophil migration in CD44+/+ and CD44–/– bladders. Data are presented as the mean ± SEM (n = 8 mice per group).

 
Proinflammatory cytokine and chemokine levels are similar in CD44–/– and CD44+/+ mice

MIP-2 (Fig. 4A), keratinocyte-derived chemokine (Fig. 4B), and IL-1β (Fig. 4C) levels in kidney homogenates were similar in both genotypes, although the trend was observed that cytokines were more expressed in CD44+/+ compared with CD44–/– at 24 and 48 h after inoculation. This observation might be due to the higher bacterial load of the kidneys at these time points.


Figure 4
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  		FIGURE 4. Proinflammatory cytokine and chemokine levels are comparable in CD44+/+ and CD44–/– kidneys. MIP-2 (A), keratinocyte-derived chemokine (KC) (B), and IL-1β (C) levels were determined by ELISA in renal homogenates of CD44+/+ ({square}) and CD44–/– ({blacksquare}) mice and corrected for quantity of protein. Data represent the mean ± SEM (n = 8 mice per group).

 
Similar phagocytosis capacity of CD44–/– and CD44+/+ neutrophils

The phagocytosis capacity of neutrophils was assessed by FACS analysis using CellTracker Orange-labeled E. coli 1677. As shown in Fig. 5A, the percentage of CD44–/– and CD44+/+ neutrophils that phagocytosed E. coli was similar. Moreover the number of phagocytosed E. coli per neutrophil (as reflected by mean fluorescence intensity) was comparable (Fig. 5B). E. coli phagocytosis of two different serotypes of eGFP-expressing E. coli strains, DH10B and Top10, was also assessed and revealed a comparable phagocytosis by CD44+/+ and CD44–/– neutrophils (data not shown). Additionally, the oxidative burst after E. coli 1677 stimulation of CD44–/– and CD44+/+ neutrophils was also identical (Fig. 5C). These data suggest an identical E. coli clearance capacity of CD44+/+ and CD44–/– neutrophils.


Figure 5
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  		FIGURE 5. Comparable E. coli clearance capacity of CD44+/+ and CD44–/– neutrophils. E. coli phagocytosis by neutrophils was determined by coincubation of CellTracker Orange-labeled E. coli 1677 with CD44+/+ ({square}) and CD44–/– ({blacksquare}) whole blood. A, Response of neutrophils is presented as the percentage of neutrophils that phagocytosed fluorescent-labeled bacteria. B, The mean fluorescence intensity (MFI) reflects the quantity of phagocytosed CellTracker Orange-E. coli per neutrophil. C, Oxidative burst of neutrophils after E. coli 1677 stimulation was comparable in CD44+/+ ({square}) and CD44–/– ({blacksquare}) neutrophils. Data are presented as the mean ± SEM (n = 3 mice).

 
E. coli attachment to and transmigration through TEC monolayers is facilitated by HA-CD44 interaction

E. coli did not express HA (data not shown), the major ligand of CD44. However, E. coli was able to bind HA in a dose-dependent manner as shown by flow cytometry. (Fig. 6). Other E. coli strains (DH10B and Top10) were also capable of binding HA-FITC, although in a lesser extent, suggesting a common feature of HA binding by E. coli (data not shown). In addition, preincubation of E. coli 1677 with unlabeled HA prevented binding of HA-FITC (data not shown).


Figure 6
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  		FIGURE 6. E. coli binds HA. Under normal conditions, HA is not expressed by E. coli. Coincubation of E. coli 1677 with 10, 100, or 1 mg/ml FITC-labeled HA revealed a dose-dependent binding of E. coli 1677 with HA. Bacterial counts are shown. Data are representative for n = 3.

 
In an adhesion assay using CD44–/– and CD44+/+ TECs, we found that E. coli adhesiveness to TECs decreased significantly after competitive inhibition of E. coli interactions with cellular HA by addition of soluble HA to the coculture (Fig. 7A). Moreover, removal of cellular HA by hyaluronidase treatment decreased E. coli adhesion, which could be restored by adding HA (Fig. 7B). Restoring E. coli adhesion by addition of HA was enhanced in the presence of CD44.


Figure 7
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  		FIGURE 7. Cellular HA facilitates adhesion of E. coli 1677 to TECs. Adhesion of E. coli 1677 to TECs was determined by coculturing E. coli 1677 with CD44+/+ ({square}) and CD44–/– ({blacksquare}) TECs. E. coli adhered to a comparable extent to CD44+/+ and CD44–/– TECs. A, Addition of HA to the coculture decreased E. coli adhesion. B, Removal of endogenous HA by hyaluronidase (HA-ase) treatment, before coculturing, decreased E. coli adhesion, which could be restored by addition of HA to the coculture. E. coli adhesion in the presence of CD44 could be restored at lower HA concentrations. Data are presented as the mean percentage ± SEM (n = 3). #, p < 0.05 CD44+/+ and CD44–/– compared with TEC; *, p < 0.05 CD44+/+ compared with CD44–/–; +, p < 0.05 CD44+/+ compared with hyaluronidase-treated; {wedge}, p < 0.05 CD44+/+ compared with TEC. C, Expression of HA on CD44+/+ and CD44–/– TEC monolayers was confirmed using HA binding protein. Pretreatment with hyaluronidase decreased HA binding protein to the TEC monolayers. Micrographs are representative for n = 3.

 
Finally, using a Transwell system, we demonstrated that E. coli migration through TEC monolayer required the interaction between CD44 on TECs and HA. In agreement with the adhesion experiments, addition of HA decreased bacterial transmigration (Fig. 8A). In contrast, absence of CD44 reduced transmigration by 75%. Hyaluronidase treatment reduced transmigration through a CD44+/+ monolayer to comparable numbers as observed in CD44–/– monolayers (Fig. 8B). Addition of HA was able to restore transmigration of E. coli through the CD44+/+ but not through the CD44–/– monolayer.


Figure 8
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  		FIGURE 8. E. coli 1677 require CD44-HA interaction for transepithelial migration. A, Transepithelial migration of E. coli was decreased in the absence of CD44 (CD44–/– ({blacksquare}) compared with CD44+/+ ({square})). Transmigration across CD44+/+ TECs was decreased in a dose-dependent manner after addition of HA. B, Removal of endogenous HA, by hyaluronidase (HA-ase) treatment, from the cellular surface of TECs by hyaluronidase treatment decreased E. coli transmigration, which was rapidly restored after HA addition to the coculture. Data are presented as mean percentage ± SEM (n = 3). *, p < 0.05 CD44+/+ compared with CD44–/– TECs; +, p < 0.05 CD44+/+ compared with hyaluronidase-treated TECs.

 
Inhibition of the HA-CD44 interaction by Ab treatment decreased bacterial load of the kidney

To address specificity and importance of the CD44-HA interaction on binding of E. coli to epithelial cells, we added anti-CD44 (interacts with the HA binding site of CD44 (46) and may induce shedding of CD44 (47)) to the coculture of murine proximal TECs and E. coli (Fig. 9A). Addition of anti-CD44 decreased the number of bacteria that were bound to the murine proximal TEC layer compared with cells treated with control IgG (p < 0.09). The strongest differences in binding of bacteria were observed when the present HA was first removed from the cellular surface of murine proximal TECs with hyaluronidase. Coincubation of a hyaluronidase-treated murine proximal TEC layer with E. coli in the presence of anti-CD44 and HA reduced bacterial binding by 40% compared with cells treated with a control Ab. In addition, in vivo administration of anti-CD44 decreased the bacterial outgrowth of the kidneys compared with control IgG-treated mice (Fig. 9B) (p < 0.07).


Figure 9
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  		FIGURE 9. Blockade of the CD44-HA interaction reduces renal bacterial outgrowth. A, Adhesion of E. coli 1677 to TECs was determined by coculturing E. coli 1677 with CD44+/+ TECs in the presence of control IgG or anti-CD44. E. coli adhered less (p < 0.09) to TECs in the presence of anti-CD44. Removal of endogenous HA by hyaluronidase (HA-ase) treatment, before coculturing, decreased E. coli adhesion (#, p < 0.05 compared with untreated cells), which could be restored by addition of HA to the coculture. E. coli adhesion could be restored by addition of HA, but not in the presence of anti-CD44. Data are presented as the mean percentage ± SEM (n = 3). *, p < 0.05 TECs hyaluronidase-treated and coincubated with HA and anti-CD44 vs TECs hyaluronidase-treated and coincubated with HA and control IgG. B, Bacterial outgrowth was assessed in renal tissue with intravesical inoculation of E. coli 1677 in CD44+/+ mice with addition of control IgG ({square}) or anti-CD44 (Figure 9). Mice were sacrificed 24 h after inoculation. Data are presented as the mean ± SEM, n = 8 for anti-CD44 treatment and n = 7 for control IgG treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we describe that CD44 facilitates infection of the urinary tract by E. coli and strong evidence suggested that E. coli via HA binding uses CD44 to invade the host. Our results provide new insights in the way microorganisms may use CD44 to invade the host.

In agreement with previous reports (6), CD44 was found to be constitutively expressed on urothelium. Its expression was not affected by UTI. HA accumulates in different inflammatory states including pneumonia and tubulointerstitial nephritis (12, 15, 51). Upon UTI, HA accumulated only on urothelial cells in the presence of CD44, suggesting HA binding by CD44 on the surface of urothelial cells preventing washout of HA.

Neutrophil recruitment and extravasation into the site of infection is the corner-stone of the innate immune response to remove extracellular bacteria. In a number of previous studies, interaction of CD44 with HA has been associated with adhesion and emigration of neutrophils (12, 52, 53). In contrast to the bladder tissue, neutrophil distribution along the renal pyelum is less homogeneously distributed. Therefore neutrophil migration into the renal tissue was assessed by activity measurements (MPO determination) rather than absolute neutrophil counts. Surprisingly, in our model of E. coli induced UTI, we did not find any difference in neutrophil activity between CD44–/– and CD44+/+ mice. Taking into account the higher bacterial count in CD44+/+ vs CD44–/– mice, we can even argue that CD44 deficiency leads to a relatively increased influx and activity of neutrophils to the site of inflammation. This reasoning is in line with the report from Wang et al. (38) in which CD44 deficiency led to enhanced neutrophil migration in E. coli-induced pneumonia in mice. The same authors showed that CD44-deficient granulocytes migrated through Matrigel-containing HA faster and in greater numbers than wild-type neutrophils. Another in vitro study investigating the role of CD44 in neutrophil transmigration across epithelial monolayers showed that incubation of neutrophils with either an activating CD44 Ab or HA impaired transmigration (54). Furthermore, in a model of Ag-induced arthritis expression of L-selectin, but not CD44, was required for early neutrophil extravasation (55). Altogether it seems that the role of CD44 in neutrophil adhesion, extravasation, and migration is at variance depending on the site of inflammation and the type of inflammatory diseases.

Release of proinflammatory cytokines and chemokines by urothelial cells plays a beneficial role in the rapid induction of an appropriate inflammatory response to remove invading microorganisms during UTI (3, 45, 56). One of the mechanisms implicated in the activation of urothelial cells and their secretion of cytokines/chemokines is the interaction between CD44 and HA that transforms CD44-positive TECs into an inflammatory phenotype characterized by the secretion of MCP-1, RANTES, and TNF-{alpha} (9). The chemokines we examined (MIP-2, keratinocyte-derived chemokine) are the most potent attractors for neutrophils in mice. Because neutrophils are predominantly involved in removal of E. coli we chose to examine these cytokines. IL-1β in contrast has been shown to be differently expressed in CD44-deficient mice in a model of cerebral ischemia-reperfusion (57) and has been shown to up-regulate VCAM-1 and ICAM-1. In accordance with a similar influx of neutrophils, no difference in cytokine/chemokine levels was measured in kidney homogenates of both genotypes.

CD44 could also theoretically affect the oxidative burst or phagocytosis capacity of granulocytes. Indeed, CD44 has been, for example, implicated in the capacity of macrophages to phagocytose apoptotic neutrophils in vitro (58). However, no difference could be detected that may explain the better clearance of E. coli in CD44–/– mice.

As described at the start, CD44 has been identified as a receptor for group A Streptococcus (39, 40) and M. tuberculosis (41). For M. tuberculosis, a DNA-binding protein-1 participates in mycobacterium-lung epithelial cell interaction through HA (59). In contrast to group A Streptococcus (46), E. coli does not present HA on its surface but is able to bind HA similar to group C streptococci (60). Because HA is excreted in the urine and accumulates in the bladder upon UTI, E. coli must be coated with HA in vivo. Adhesion and migration assays provided strong arguments that HA-coated E. coli use CD44 on TECs to adhere and that the presence of CD44 on TECs facilitates the migration of E. coli through an epithelial monolayer. We consider it likely that this mechanism takes place in vivo and contributes to the relative protection of CD44-deficient mice against UTI. Additionally anti-CD44 treatment of the urothelial cells decreased the bacterial adherence, compared with the control IgG-treated cells. In vivo administration of anti-CD44 simultaneously with the inoculation of E. coli reduced bacterial outgrowth from the kidneys, compared with control IgG-treated mice (p < 0.07). This study has provided new insights in the interaction between host and pathogen, which may have impact on clinical practice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants 907-00-004 and 015-001-061 from the Netherlands Organisation for Scientific Research and Grant PC 125 from the Dutch Kidney Foundation. Back

2 Address correspondence and reprint requests to Dr. Kasper Rouschop, Department of Pathology, Academic Medical Center, room M2-108, P.O. Box 22660, 1100 DD, Amsterdam, The Netherlands. E-mail address: Kasper.Rouschop{at}maastro.unimaas.nl or reprint requests to Dr. Sandrine Florquin, Department of Pathology, Academic Medical Center, Room M2-108, P.O. Box 22660, 1100 DD, Amsterdam, The Netherlands. E-mail address: S.Florquin{at}amc.uva.nl Back

3 Abbreviations used in this paper: UTI, urinary tract infection; HA, hyaluronic acid; TEC, tubular epithelial cell; MPO, myeloperoxidase; eGFP, enhanced GFP. Back

Received for publication September 12, 2005. Accepted for publication August 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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