of March 24, 2018.This information is current as

-Independent Inflammatory Pathwaysby Activating Distinct TLR4-Dependent and

Escherichia colito Pyelonephritis-Associated Renal Collecting Duct Epithelial Cells React

Dominique Buzoni-Gatel and Alain VandewalleCatherine Werts, Eric Ogier-Denis, Chantal Le Bouguénec,Laurence du Merle, Marcelle Bens, Françoise Cluzeaud, Cécilia Chassin, Jean-Michel Goujon, Sylvie Darche,

http://www.jimmunol.org/content/177/7/4773doi: 10.4049/jimmunol.177.7.4773

2006; 177:4773-4784; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/177/7/4773.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Renal Collecting Duct Epithelial Cells React toPyelonephritis-Associated Escherichia coli by ActivatingDistinct TLR4-Dependent and -Independent InflammatoryPathways1

Cecilia Chassin,* Jean-Michel Goujon,† Sylvie Darche,‡ Laurence du Merle,§ Marcelle Bens,*Francoise Cluzeaud,* Catherine Werts,‡ Eric Ogier-Denis,* Chantal Le Bouguenec,2§

Dominique Buzoni-Gatel,2‡ and Alain Vandewalle2,3*

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggestedthat renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses.In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentiallyadhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lpsd) mice carryingan inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators ascompared with infected wild-type C3H/HeOuJ (Lpsn) mice. However, the level of expression of the leukocyte chemoattractantsMIP-2 and TNF-� still remained greater in UPEC-infected than in naive C3H/HeJ (Lpsd) mice. Using primary cultures ofmicrodissected Lpsn MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECsstimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-�-independent pathway anda TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathwayin Lpsn MCDs leads to the activation of NF-�B, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. Thesefindings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via severaldistinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing thekidneys. The Journal of Immunology, 2006, 177: 4773–4784.

E scherichia coli is the bacterial pathogen most frequentlyresponsible for urinary tract infections (UTIs)4 and pye-lonephritis, which constitute a major cause of morbidity

and mortality in humans (1, 2). Bacterial attachment to mucosalepithelial cells represents the initial step in E. coli pathogenicity.

Various fimbrial and afimbrial adhesins are produced by uropatho-genic E. coli (UPEC) (2). The expression and binding of thesevarious adhesins to epithelial cell receptors determine tissue spec-ificity and allow UPEC isolates to ascend to the lower urinary tractand the kidney in experimental models of UTI (3–8).

Recognition of UPEC isolates by the mucosal cells lining theurinary tract elicits potent inflammatory responses. This processinvolves TLR4, a pattern-recognition receptor that recognizesLPS, the major cell wall constituent of all Gram-negative bacteria(9). LPS recognition by TLR4 results in the recruitment of multiplecytoplasmic signaling molecules, including MyD88 and theTNFR-associated factor (TRAF) 6, which, in turn, recruit and as-semble additional molecules to activate downstream signalingcomponents involving the transcription factors NF-�B, MAPKp38, ERK1/2, and JNK, leading to the production of proinflam-matory cytokines and chemokines (10, 11). C3H/HeJ mice (Lpsd),which have a loss-of-function mutation in the tlr4 gene (12), areunresponsive to LPS and fail to clear Gram-negative bacteria col-onizing the lower urinary tract and kidneys (13).

Type 1 piliated E. coli has been shown to enhance the inflam-matory response of the bladder epithelium by mediating bacterialinvasion via a LPS recognition, TLR4-mediated pathway (14).Fisher et al. (15) also showed that the binding of type 1 or Pfimbriae to their respective uroepithelial cell receptors activates aTLR4-mediated mucosal response, as assessed by analyses of neu-trophil recruitment to the urinary tract of infected mice, throughthe requirement of distinct adaptor proteins. These findings sug-gested that the recognition of fimbrial adhesins by cell surface

*Institut National de la Sante et de la Recherche Medicale U773, Centre de RechercheBiomedicale Bichat-Beaujon (CRB3), Paris France; Universite Paris 7, Denis Diderot,Paris, France; †Service d’Anatomie et Cytologie Pathologiques, Centre HospitalierUniversitaire de Poitiers, Poitiers, France; ‡Unite de Reponses Precoces aux Parasiteset Immunopathologie, Institut National de la Recherche Agronomique, Paris, France;and §Unite de Pathogenie Bacterienne des Muqueuses, Institut Pasteur, Paris, France

Received for publication April 5, 2006. Accepted for publication July 12, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was funded by Institut National de la Sante et de la Recherche Medicale(INSERM) and by Institut Pasteur Grant PTR 165 (to C.L.B. and D.B.-G.). A.V. wasin receipt of an Interface INSERM-AP-HP fellowship.2 C.L.B, D.B.-G, and A.V. made equal contributions to this work.3 Address correspondence and reprint requests to Dr. Alain Vandewalle, Institut Na-tional de la Sante et de la Recherche Medicale U773, Centre de Recherche Biomedi-cale Bichat-Beaujon CRB3, Unite de Formation et de Recherche de Medecine XavierBichat, BP 416, 16 Rue Henri Huchard, F-75870 Paris Cedex 18, France. E-mailaddress: vandewal@bichat.inserm.fr4 Abbreviations used in this paper: UTI, urinary tract infection; AQP-2, aquaporin-2;ASK1, apoptosis signal-regulatory kinase 1; CFTR, cystic fibrosis transmembraneconductance regulator; ClC-5, chloride channel 5; ENaC, epithelial sodium channel;iNOS, inducible NO synthase; MAPKAPK-2, MAPK-activated protein kinase 2;MCD, medullary collecting duct; ROS, reactive oxygen species; TRAF, TNF recep-tor-associated factor; TRIF, TIR domain-containing adaptor inducing IFN-�; UPEC,uropathogenic Escherichia coli.

The Journal of Immunology

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00

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mucosal receptors may directly activate, independently of LPS, theTLR4 signaling pathway.

The mechanisms of interaction between UPECs and bladderepithelial cells have been extensively investigated (14, 16, 17).UPECs can also interact with renal tubule cells. UPECs may formmicrocolonies at the surface of renal proximal tubule cells from thekidneys of young rats and provoke Ca2� oscillations induced bythe secreted toxin �-hemolysin, which, in addition, stimulates theproduction of IL-6 and IL-8 in cultured human renal epithelialA498 cells (18). However, the exact contribution of renal epithelialcells, as primary or additional sources of chemokine and cytokineproduction, to increasing the immune response still remains to beclarified. Recent studies using the hemopoietic chimeric Lpsn andLpsd mice demonstrated that bladder epithelial cells (19), as wellas intrinsic renal epithelial cells (20), contribute together with bonemarrow-derived cells to the initiation of antibacterial immunity.Murine and human renal tubule cells express some of the TLRexpressed in hemopoietic cells and secrete chemokines upon LPSstimulation (21, 22). In addition, TLR2 and TLR4 have beenshown to be up-regulated in the distal nephron of inflamed, post-ischemic, reperfused kidneys (23). These data indirectly suggestthat the most distal renal tubule segment, i.e., the medullary col-lecting duct (MCD), which is the first to come into contact withascending UPEC strains, might be a site of bacterial adherenceand, thus, is involved in triggering early inflammatory response.

In this study we analyzed the role of tubule epithelial cells dur-ing kidney colonization by two pyelonephritic UPEC strains lack-ing �-hemolysin, and we characterized the resulting innate im-mune response. The role of TLR4 in the initiation of immuneresponse was evaluated in whole kidneys and confluent cultures ofMCDs microdissected from adult LPS-sensitive C3H/HeOuJ(Lpsn) and LPS-nonresponsive C3H/HeJ (Lpsd) mice. Experimentswere also conducted with MyD88-deficient (MyD88�/�) mice(24), which exhibit no inflammatory response to the TLR4 ligandLPS (25), and Lps2 mutant (Lps2�/�) mice (26), which are defi-cient for the TIR domain-containing adaptor inducing IFN-�(TRIF), an adaptor protein that regulates an MyD88-independentsignaling pathway upon stimulation by TLR3 and TLR4 ligands(26, 27). We show that UPEC isolates colonized kidneys aftertransurethral inoculation and preferentially and constantly adheredto apical membranes of MCD intercalated cells expressing TLR4.UPECs induced the expression of proinflammatory mediators inMCD cells and stimulated bipolarized secretion of the chemokineMIP-2 (CXCL2/MIP-2) via both TLR4-mediated MyD88-depen-dent and TLR4-independent pathways requiring distinct MAPK-related signaling molecules. These findings indicate that MCDcells are involved in UPEC recognition and actively participate tothe initiation of immune innate response by triggering rapid stim-ulation of proinflammatory mediators in injured kidneys.

Materials and MethodsBacteria

Two uropathogenic strains of E. coli (AL511 and AL10) isolated from theurine of women presenting pyelonephritis were used. They belong to twodifferent O serogroups (O9 for AL511, and O101 for AL10), carry pap andafa-8 adhesin-encoding genes, and lack the hly �-hemolysin-encoding gene(28). In addition, the AL511 isolate carries f17Ac adhesin genes and pro-duces factors contributing to serum resistance. These UPEC isolates wereselected on the basis of their in vitro interactions with and ability to stim-ulate cytokine/chemokine mRNA expression in immortalized mouse col-lecting duct mpkCCDcl4 cells (not shown). The nonpathogenic E. colistrain MG1655 was used as the control (29). The strains were grown instatic Luria-Bertani broth at 37°C for 24 h.

Animals and infections

Eight- to 10-wk-old female C3H/HeOuJ (Lpsn), C3H/HeJ (Lpsd), and wild-type C57BL/6J mice were obtained from The Jackson Laboratory. TLR2(tlr2�/�) and MyD88 (MyD88�/�) knockout mice (24, 30), originally ob-tained from S. Akira (Osaka University, Osaka, Japan), have been furtherbackcrossed eight times into C57BL/6 mice to ensure a similar geneticbackground. C57BL/6 Lps2�/� mice (26) were provided by D. J. Philpott(Institut Pasteur, Paris, France). Mice submitted to sanitary control tests toensure proper pathogen-free status were housed in the same animal facilityat the Institut Pasteur for 1 wk before the experiments began. Lpsn and Lpsd

mice, subjected to water restriction for 12 h, were anesthetized and theninfected with 50 �l of bacterial suspension (108 bacteria) in sterile PBSintroduced via the transurethral route into the bladder. A soft polyethylenecatheter (Insyte Autoguard soft catheter, 0.7-mm external diameter; Vy-gon) was used as described (5, 8). Two days after bacterial inoculation themice were sacrificed, and the kidneys and bladder were removed asepti-cally. The two halves of one kidney were fixed or quick frozen in liquidnitrogen. The contralateral kidney was homogenized, diluted in sterilePBS, and plated on Luria-Bertani agar plates to enumerate the number ofCFUs. For in vivo TNF-� neutralization experiments, Lpsn and Lpsd micewere i.p. injected 1 h before bacterial inoculation with 1 ml of PBS con-taining 1 mg of purified anti-mouse TNF-� from the MP6-XT3 mAb orwith 1 mg of the rat IgG1 isotype control (eBioscience) (31). All experi-ments were performed in accordance with the guidelines of the FrenchAgricultural Office and in compliance with the legislation governing ani-mal studies.

Microdissection and culture of MCDs

Kidneys from naive Lpsn and Lpsd mice or wild-type C57BL/6 (tlr2�/�),tlr2�/�, or MyD88�/� mice were rapidly removed under sterile conditionsand incubated in modified defined medium supplemented with 0.1% col-lagenase (Roche Diagnostics) for 45 min at 37°C. MCDs were then mi-crodissected under sterile conditions as described (32, 33). Pools of iso-lated MCDs (8–12 fragments) were seeded onto collagen-coated 48-welltrays or onto Transwell filters (0.4- or 3-�m pore size, 0.33-cm2 insertgrowth area; Corning Costar). Isolated MCDs were grown to confluencefor 15 days at 37°C in a 5% CO2-95% air atmosphere in the same modifieddefined medium (33). E. coli strains AL10 or AL511 (5 � 105 bacteria/filter) were added to the apical medium bathing confluent cell layers.Transepithelial resistance and potential were measured as described (33).

Histological and immunohistochemical studies

Kidneys were fixed in Dubosc-Brazil solution, rinsed in PBS, embedded inparaffin, and stained with H&E or periodic acid-Schiff. Immunohistochem-ical studies using Abs raised against E. coli (1/200; Interchim), murineTLR4 (1/200; provided by M. W. Hornef, University of Freiburg, Freiburg,Germany) (34), aquaporin-2 (AQP-2), and the chloride channel 5 (ClC-5)(1/200) (35) were performed using avidin-biotin blocking kit and alkalinephosphatase and peroxidase substrate kits (Vector Laboratories) accordingto the manufacturer’s instructions. Alkaline phosphatase and peroxidaseactivities were revealed with diaminobenzidine (brown reaction), nitrobluetetrazolium (purple reaction) (Lab Vision), or Vector Novared (red reac-tion) (Vector Laboratories) substrate solutions. Indirect immunofluores-cence studies were also conducted on cultured MCDs using Abs againstNF-�B (Santa Cruz Biotechnology), E. coli, cytokeratins K8–K18, andClC-5 and species-specific Alexa 488- and Cy3-conjugated IgG as second-ary Abs (Jackson ImmunoResearch Laboratories). Cells were stained withphalloidin to visualize F-actin. Specimens were examined using a confocallaser-scanning microscope (CLSM-510-META; Zeiss) and photographed.

Scanning microscopy

E. coli strains (5 � 105 bacteria/filter) were added to the apical side ofconfluent cultures of MCDs grown on permeable filters for 3 h at 37°C.Cell layers were then rinsed in PBS, fixed in 4% glutaraldehyde in 0.1 Mphosphate buffer for 1 h at 4°C, dehydrated in ascending concentrations ofacetone, critical point dried, coated with gold, and then examined under aJEOL-JSM840A scanning electron microscope.

Real-time and RT-PCR

Total RNA was extracted from whole kidneys or cultured MCDs using theRNeasy mini kit (Qiagen), and reverse transcribed using Moloney murineleukemia virus reverse transcriptase (Invitrogen Life Technologies). cDNAwas subjected to real-time PCR by using a ABI 7000 sequence detector(Applied Biosystems) (36). The mouse primers (GenBank accession nos.in parentheses) and TaqMan probes used were as follows: IL-1�(NM_008361), nt 527–551, nt 678–658, and probe, nt 629–656; IL-6

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(NM_031168), nt 526–546, nt 608–587, and probe, nt 555–583; MIP-2(NM_009140), nt 181–204, nt 262–237, and probe, nt 210–228; MCP-1(NM_011333), nt 107–125, nt 233–213, and probe, nt 160–180; RANTES(NM_013653), nt 224–245, nt 294–268, and probe, nt 247–266; inducibleNO synthase (iNOS) (NM_010927), nt 2176–2196, nt 2270–2250, andprobe, nt 2198–2220; TNF-� (NM_013693), nt 401–425, nt 575–553, andprobe, nt 436–461; and �-actin (NM_007393), nt 694–713, nt 831–811,and probe, nt 764–786. PCR data were reported as the relative increase inmRNA transcripts vs that found in kidneys of naive mice or untreatedcultured MCDs and corrected by the respective levels of �-actin mRNAused as the internal standard (36). For RT-PCR, cDNA and non-reversetranscribed RNA (400 ng) from cultured Lpsn MCDs were amplified for30–35 cycles in 40 �l of total PCR buffer (50 mM KCl and 20 mMTris-HCl (pH 8.4)) containing 100 �M dNTP, 1 or 1.5 mM MgCl2, 1 U ofTaq polymerase, 10 pmol of TLR4 (BC029856, nt 1999–2020 and nt2309–2288), 10 pmol of MD-2 (NM_016923, nt 12–34, and nt 327–306),5 pmol of MyD88, 10 pmol of CD14 (NM_009841, nt 223–243 and nt926–902), 5 pmol of TLR2 (NM_011905, nt 2335–2354 and nt 2816–2835), 30 pmol of CD45 (NM_011210, nt 3127–3146 and nt 3531–3551),30 pmol of the �-subunit of the epithelial sodium channel (ENaC), and 35pmol of the cystic fibrosis transmembrane conductance regulator (CFTR)primers. The primers used for MyD88, �-ENaC, and CFTR were the sameas those previously described (33, 37). The thermal cycling program was94°C for 30 s, 61°C (TLR2), 60°C (TLR4 and CFTR), 55°C (MD-2, CD14,and CD45), 53°C (MyD88), or 54°C (�-ENaC) for 30 s and then 72°C for1 min. Amplification products were run on a 2% agarose gel and thenstained with ethidium bromide and autoradiographed.

Immunoblot analysis

For the detection of TLR4, the kidneys from a naive Lpsn mouse wereremoved and frozen in liquid nitrogen. An aliquot of homogenized proteins(50 �g of total protein) in Laemmli buffer was then processed for Westernblotting using an anti-TLR4 Ab (34). Confluent MCD cells grown in 48-well plates precoated with rat tail collagen were lysed in 50 �l of 62.5 mMTris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM DTT and sonicatedfor 15 s at 4°C. For each condition tested, cell lysates from six separatewells were pooled. Samples (15 �g/lane) were electrophoresed using 10%SDS-PAGE and transferred to a nitrocellulose membrane. Abs againstIkB-�, p38, TRAF2, MAPK-activated protein kinase 2 (MAPKAPK-2),c-Jun (Cell Signaling Technology), TRIF (Imgenex), JNK, TRAF6, apo-ptosis signal regulatory kinase 1 (ASK1), ERK1/2 (Santa Cruz Biotech-nology), and �-actin (Sigma-Aldrich) were used to detect the correspond-ing Ags. Protein phosphorylation was analyzed using Abs againstphosphorylated p38, JNK, ERK1/2, MAPKAPK-2, c-Jun, and ASK1 (CellSignaling Technology). Protein bands were revealed using peroxidase-con-jugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Lab-oratories) and detected using the ECL Plus Western blotting detection sys-tem (Amersham Biosciences).

ELISA

Cells were incubated for 3 h with UPECs alone and with anti-mouseTNF-� mAb or rat IgG1 isotype control (eBioscience) or with or withoutspecific cell-permeable MAPK inhibitors (Calbiochem). MIP-2 and TNF-�secreted in cell supernatants were then determined using ELISA kits (R&DSystems) according to the manufacturer’s instructions. For in vivo TNF-�measurements, kidneys from Lpsn and Lpsd mice infected with AL511isolates were homogenized in 1 ml of PBS and kept at �80°C until use.Tissue samples were then thawed and assayed to measure the levels ofTNF-� production. Results were standardized to the amount of proteindetected for each sample using the Bio-Rad protein assay with BSA asstandard.

Statistical analysis

Results are expressed as means � SEM. The significance of the differenceswas analyzed using Student’s t test ( p � 0.05 was considered to besignificant).

ResultsPyelonephritic E. coli strains interact with renal collecting ductintercalated cells

The UPEC strains AL10 and AL511 and the nonpathogenic strainMG1655 used as control were inoculated into the bladder ofhealthy Lpsn mice via the transurethral route. Bacteria in bladderand kidney tissues were counted 48 h postinfection. The AL10 andAL511 isolates were found to colonize the bladder and kidneys

more effectively ( p � 0.05) than MG1655 (Fig. 1A). All of thekidneys from mice inoculated with the two UPEC isolates werecolonized with bacteria, whereas no (50% of inoculated mice) oronly a few bacteria were detected in the kidneys following thetransurethral inoculation of the nonpathogenic E. coli MG1655.Differences between the nonpathogenic strain and the UPEC iso-lates were also observed when kidney sections were examined byimmunohistochemistry using an anti-E. coli polyclonal Ab. Nopositive E. coli immunostaining was detected in kidneys of naive

FIGURE 1. Renal colonization by UPECs. A, Bacterial counts in blad-ders and kidneys from Lpsn mice 2 days after transurethral inoculationswith the UPEC AL10 or AL511 strains or the commensal E. coli MG1655strain. Values are means � SEM from 8 to 14 infected mice in each group.Experiments were performed on two (for E. coli MG1655) and three dif-ferent sets (for UPEC AL10 or AL511 strains) of mice at 6-mo intervals.�, p � 0.05 vs MG1655 values. B–F, E. coli immunostaining in kidneysections from mice inoculated with E. coli MG1655 (B), AL10 (C, arrow-heads; D, arrow), or AL511 (E, arrowheads; F, double arrow) isolates. Gand H, Illustrations of collecting duct cells double labeled with anti-E. coli(purple; arrows and arrowheads) and anti-ClC-5 (G, red) or anti-AQP-2 (H,red) Abs. Original magnifications: �200 (B, C, E, and I) and �400 (D, F,G, and H).

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mice (not shown), and almost no staining was detected in kidneysfrom mice inoculated with MG1655 bacteria (Fig. 1B). Positive E.coli immunostaining was detected in the kidneys colonized withthe AL10 and AL511 isolates; the two UPEC isolates were ob-served adhering to the luminal surface of some, but not all, cellsfrom the collecting duct sections (Fig. 1, C–F). Positive cytoplas-mic staining was also observed in collecting duct cells from thekidneys of mice infected with AL511 isolates (Fig. 1F). To furthercharacterize the bacterial attachment to collecting duct cells com-posed of principal and intercalated cells, sections were doublestained with Abs raised against E. coli and the water channelAQP-2 or the chloride channel ClC-5 expressed in the principaland intercalated cells, respectively (35, 38). AL10 (Fig. 1G) andAL511 (not shown) isolates were observed adhering to ClC-5-expressing intercalated cells. In contrast, few or no bacteria ad-hered to AQP-2-expressing principal cells (Fig. 1H).

We then tested whether collecting duct cells expressed TLR4 byusing a specific rabbit antiserum raised against the mouse TLR4(34) to recognize a protein band of the predicted size of �96 kDain the mouse kidney (Fig. 2A). Immunohistochemical studies usingthis TLR4 Ab showed that most if not all cells from tubule sec-tions, including collecting ducts, expressed TLR4, mainly in thecytoplasm (Fig. 2B). In addition, some of the collecting duct cellsappeared intensely stained (Fig. 2B). As control, the staining wasnot detected when tissue sections were incubated with the anti-TLR4 Ab plus an excess of the peptide used for immunization(Fig. 2C). Examination of kidney sections from mice infected withthe AL511 isolates also revealed that the collecting duct cells ex-hibiting intense positive E. coli staining (i.e., intercalated cells)corresponded to the more intensely TLR4-stained cells (Fig. 2, Dand E). The preferential binding of UPECs to intercalated cells wasalso observed in ex vivo experiments using isolated MCDs micro-dissected from the kidneys of Lpsn mice. The MCDs formed con-

fluent cell layers expressing K8–K18 cytokeratins and the tightjunction-associated protein ZO-1 (Fig. 3A). Indirect immunofluo-rescence studies revealed that �50% of the MCD cell layers ex-pressed the ClC-5 Cl� channel (Fig. 3A). Cell layers were notcontaminated by hemopoietic cells, as they did not express anymRNA transcripts of the bone marrow-derived cell marker CD45(Fig. 3B). Cultured MCDs expressed mRNAs for TLR4 and itsaccessory protein MD-2 and for the TLR4 adaptor proteinsMyD88 and CD14 (Fig. 3C). They also expressed mRNAs forTLR2 and for the ENaC �-subunit and CFTR Cl� channel, both ofwhich were expressed in principal and intercalated cells (39, 40)(Fig. 3C). MCDs grown to confluence on filters developed hightransepithelial electrical resistance (1941 � 32 � � cm2, n � 28),and a negative potential (�32 � 8 mV, n � 28), both of which arefeatures of collecting duct cells (33). Overall, these results indi-cated that the confluent cultures of microdissected MCDs formedhighly purified tight epithelial cell layers composed of �50%ENaC-expressing principal cells and 50% ClC-5-expressing inter-calated cells. Three hours after the apical addition of E. coli AL511isolates to confluent MCDs grown on filters, bacteria were foundto be preferentially associated with the apical surface of the cells(Fig. 3, D-G) corresponding to ClC-5-positive stained cells (Fig.3F, inset).

The induction of proinflammatory mediators caused by UPECisolates is mediated by TLR4-dependent and -independentpathways

Lpsd and Lpsn mice were then inoculated with the E. coli strainsAL10 and AL511 to determine the consequence of kidney colo-nization on the induction of proinflammatory mediators and towhat extent the resulting inflammatory response was dependentupon TLR4 expression. Lpsd mice were more susceptible to infec-tion by both of these pathogenic strains of E. coli, because the

FIGURE 2. Immunolocalization of TLR4 in epithe-lial renal tubule cells. A, Western blot of kidney celllysate using the anti-TLR4 Ab revealed a major band of96 kDa (arrow), which was not detected in the presenceof an excess of the peptide used for the rabbit immu-nization. B, Representative illustration of cellular TLR4immunostaining in the renal medulla of a naive mouse.Gl, glomerulus; PT, proximal tubule; CD, collectingduct. Note that some collecting duct cells appearedheavily labeled (B, arrowheads). As control, no stainingwas detected when the section was incubated with anexcess of the peptide used for immunization (C). D andE, Illustrations of collecting duct cells (arrowheads)from an AL511-infected kidney double labeled with anti-E. coli (purple) and anti-TLR4 (brown) Abs. Originalmagnifications: �200 (B) and �400 (C, D, and E).

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renal bacterial burden at day 2 after challenge was significantly�63-fold (AL10) to �108-fold (AL511) greater ( p � 0.05) thanin kidneys from Lpsn mice (Fig. 4A). As in Lpsn kidneys, the

AL511 (and AL10; data not shown) isolates were found to beconcentrated at the cell surface of collecting ducts cells (Fig. 4B,upper panels). Infiltrating cells surrounding tubules, includingMCDs, were detected in Lpsn kidneys and also to a lower extent inLpsd kidneys colonized with the AL511 (and AL10; data notshown) isolates (Fig. 4B, middle and lower panels).

The results from real-time PCR revealed that the UPEC strainsinduced differing degrees of significant activation ( p � 0.01) ofmRNA expression of MIP-2, RANTES, and TNF-� in kidneys ofinfected Lpsn mice; AL511 isolates caused an increase in a broaderrange of proinflammatory mediators, including IL-1�, MCP-1, andiNOS, than the AL10 isolates (Fig. 4C). In contrast, mRNA ex-pression of all the proinflammatory mediators was dramaticallyreduced in infected Lpsd mouse kidneys, but this was less pro-nounced after infection with AL511 than with AL10 isolates (Fig.4C). As a result, the expression of MIP-2, IL-1�, and TNF-� re-mained greater in AL511-infected than in AL10-infected Lpsd

mouse kidneys (Fig. 4C), suggesting that some UPEC strains suchas AL511 may induce significant TLR4-independent stimulationof proinflammatory mediators.

FIGURE 3. Binding of the UPEC strain AL511 to MCD cells. A, Con-fluent cultures of Lpsn MCDs formed layers of cuboid cells expressingcytokeratins K8–K18 (C. K8-K18) (red) and the tight junction-associatedprotein ZO-1 (green). Approximately 50% of the cells corresponded tointercalated cells expressing the ClC-5 Cl� channel. B, Confluent epithelialLpsn MCDs cell layers did not express CD45 mRNA. As controls, ampli-fied products of expected size (405 bp) were detected in peritoneal mac-rophages, and no band was detected when cDNA was omitted. C, Identi-fication by RT-PCR of amplified products for TLR4 (311 bp), MD-2 (316bp), MyD88 (660 bp), ENaC �-subunit (564 bp), CFTR (636 bp), or CD14(703 bp) mRNA. No amplified product was detected using non-reverse-transcribed (non RT) RNA for TLR4, CD14, and TLR2 or when cDNAwas omitted (Control). D–F, Illustrations showing AL511 bacterial adhe-sion (arrow) to the apical surface of cultured Lpsn MCDs examined byscanning electron microscopy (D and E) and E. coli immunostaining(green) F, MCD cells were stained with phalloidin (red). Inset, Doubleimmunofluorescence staining showing AL511 isolates (green) preferen-tially adhering to the apical surface of a ClC-5-stained intercalated cell (�).G, Confocal microscopy images (x-z planes) showing bacterial (green)attachment to the apical surface of MCD cells (red). Bars,10 �m.

FIGURE 4. Bacterial counts, interstitial cell infiltrates, and induction ofproinflammatory mediators in kidneys from Lpsn and Lpsd mice challengedwith UPEC isolates. A, Bars are means � SEM from bacterial countsperformed on kidney sections from seven infected Lpsn and nine infectedLpsd mice. CD, collecting duct. B, Illustrations of E. coli immunostaining(upper panels) to collecting duct cells and cellular infiltrates in interstitialmedulla (middle panels) surrounding tubules (lower panels, arrowheads) inUPEC AL511-infected Lpsn and Lpsd mice kidneys. CD: collecting duct.C, Expression of proinflammatory mediators analyzed in kidneys fromLpsn (open bars) and Lpsd (black bars) mice 2 days after inoculation withUPEC strains AL10 and AL511. The relative fold increase of each mRNAlevel compared with that found in naive mice is reported as means � SEMfrom duplicate measurements performed on 7–9 different kidneys in eachgroup tested. �, p � 0.05 vs Lpsn kidney values. Original magnifications inB: �400 (upper and lower panels) and �200 (middle panel).

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MCD cells are involved in the induction of the inflammatoryresponse to UPEC isolates

Because UPECs specifically interact with the apical surface ofMCD cells expressing TLR4, we addressed the question ofwhether these epithelial cells directly contribute to the inflamma-tory response and chemoattraction of immune cells. Since in vivoexperiments did not allow us to determine the exact role of par-enchymatous renal cells in these processes, our experiments wereconducted using primary cultures of MCDs microdissected fromkidneys of naive Lpsn and Lpsd mice. Indirect immunofluorescencestudies using the anti-ClC-5 Ab revealed that Lpsd MCDs exhib-ited the same percentage (�50%) of intercalated cells as in Lpsn

MCDs (data not shown). Apical addition of AL10 isolates to con-fluent cultures of Lpsn MCDs grown on filters caused dramatic andpreferential increases in the expression of MIP-2 and TNF-� com-pared with untreated Lpsn MCDs (Fig. 5A). Apical addition ofAL511 isolates induced an even wider increase in the expressionof proinflammatory mediators, including IL-1�, MIP-2, MCP-1,iNOS, and TNF-� (Fig. 5A). AL511 isolates, and to a lesser extentAL10 isolates, rapidly stimulated the expression of MCP-1 in Lpsn

MCDs; but, in contrast to that observed in the day 2 postinfectedkidneys, UPECs did not significantly stimulate the expression ofRANTES in cultured Lpsn MCDs (Fig. 5A). Whichever strain of E.coli was used, the expression of proinflammatory mediators wasconsiderably lower in Lpsd MCDs than in Lpsn MCDs (Fig. 5A).However, both UPEC isolates still induced significantly greater

expression of MIP-2 (�13- to �15-fold) and TNF-� (�6.4- to�7.2-fold) mRNA in Lpsd MCDs as compared with untreated Lpsd

MCDs. The induction profiles of proinflammatory mediators trig-gered by UPECs closely resemble those observed in whole kidneyscolonized by UPECs.

Interaction between UPEC isolates and the apical membranes ofcollecting duct intercalated cells induces bidirectional secretionof MIP-2

MIP-2 is required for neutrophil passage across the epithelial bar-rier of the infected urinary tract in experimental models of UTI(41, 42). The question arises of whether collecting duct cells mayattract leukocytes by secreting chemokines toward their basal(i.e., interstitium) and/or apical (i.e., tubule lumen) sides. AddingUPECs to the apical side of confluent Lpsn MCDs grown on filtersresulted in apical and basal secretion of MIP-2 (Fig. 5B, left panel).AL10 and AL511 isolates also stimulated apical and basal secre-tion of MIP-2 by confluent cultures of Lpsd MCDs, although to alesser extent than in Lpsn MCDs (Fig. 5B, left panel). In contrast,the inactivation of the tlr2 gene did not affect the secretion ofMIP-2. Apical and basal secretion of MIP-2 stimulated by UPECsremained identical in cultured MCDs dissected from wild-type andtlr2�/� mouse kidneys (Fig. 5B, right panel).

UPEC isolates activate both NF-�B-dependent and -independentsignaling pathways in MCD cells

TLR-mediated recognition of bacterial components by immunecells induces a cascade of signaling events that finally leads to theactivation of the transcriptional factor NF-�B, which is critical inthe regulation of genes involved in inflammation (43). Signals thatinduce NF-�B activity will cause the phosphorylation of the in-hibitor proteins I�Bs by I�B kinases as well as their dissociationand subsequent degradation, which then allows NF-�B to translo-cate into the nucleus (44, 45). In Lpsn MCDs, AL511 bacteriainduced time-dependent degradation of I�B-� and concomitantNF-�B nuclear translocation that could be prevented by preincu-bating the cells with the specific NF-�B inhibitor SN50 (Fig. 6A).In contrast, AL511 (and AL10; data not shown) isolates did notcause nuclear translocation of NF-�B and did not alter the amountof I�B-� in Lpsd MCDs (Fig. 6A). The cellular activation causedby UPECs resulted in a subsequent transcriptional up-regulation ofMIP-2 in Lpsn MCDs (Fig. 6A). The two UPEC strains also stim-ulated the secretion of MIP-2 independently of NF-�B activationin Lpsd MCDs (Fig. 6B). The specific NF-�B inhibitor SN50 re-duced the production of MIP-2 elicited by the two UPEC strains inLpsn MCDs by �50% but had no significant inhibitory action onMIP-2 produced by Lpsd MCDs (Fig. 6B).

Although MyD88 is required for antibacterial effector function,urine neutrophil recruitment still occurred in MyD88�/� mice in-fected with P fimbriated E. coli (15). These results raised thequestion of whether the stimulated secretion of MIP-2 caused byUPECs in Lpsn MCDs occurs through MyD88-dependent and/orMyD88-independent pathways. The adaptor molecule TRIF hasbeen shown to play a key role in TLR4-mediated (and TLR3-mediated) MyD88-independent responses, which lead to the acti-vation of IFN regulatory factor 3 and the induction of IFN-� (26,27, 46–48). To address the role of MyD88 as well TRIF in theTLR4-mediated pathway of MCDs, the production of MIP-2 elic-ited by the two UPEC strains was measured in primary culturedMCDs dissected from the kidneys of MyD88�/� and TRIF-deficient Lps2�/� mice. The production of MIP-2 stimulated byUPECs was significantly reduced ( p � 0.01) in primary culturedMyD88�/� MCDs as compared with that measured in wild-typeC57BL/6 counterparts (Fig. 6C). Similarly to what is observed in

FIGURE 5. Differential effects of UPEC isolates on proinflammatorymediators and polarized secretion of MIP-2 in Lpsn and Lpsd MCD cells.A, The relative mRNA expression levels of proinflammatory mediators wasquantified by real-time PCR in confluent cultures of untreated MCDs andafter the apical addition of isolates of UPEC strains AL10 or AL511 (5 �105 bacteria per filter for 3 h). Values are means � SEM from duplicatemeasurements performed on 9–13 separate cultures of Lpsn and Lpsd

MCDs dissected from four or five different mice in each group tested. �,p � 0.05 vs Lpsn MCD values. B, The production of MIP-2 was measuredin the apical and basal medium bathing confluent cultures of Lpsn and Lpsd

MCDs (A) or wild-type (tlr2�/�) and tlr2�/� mice (B) grown on filtersbefore (None) and after the apical addition of isolates of the UPEC strainsAL10 or AL511 (5 � 105 bacteria per filter for 3 h). Values are means �SEM from 8 to 11 separate cultures of MCDs dissected from the kidneysof two or three mice in each group tested. �, p � 0.05 vs Lpsn MCD values.

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Lpsd MCDs, SN50 had no significant inhibitory action on MIP-2produced by cultured MyD88�/� MCDs (Fig. 6C). In contrast, theproduction of MIP-2 stimulated by UPECs remained identical inwild-type C57BL/6 and Lps2�/� MCDs (Fig. 6C). SN50 reducedto the same extent (�50%) the production of MIP-2 elicited by thetwo UPEC strains in wild-type C57BL/6 and Lps2�/� MCDs (Fig.6C). These results indicate that the TLR4-mediated stimulation ofMIP-2 occurs through a MyD88-dependent pathway but notthrough a TLR4-mediated MyD88-independent pathway. In addi-tion, �50% of secreted MIP-2 occurs through a TLR4-indepen-dent activated pathway.

E. coli AL511 isolate activates MAPK pathways in MCD cells

The detection of NF-�B-independent secretion of MIP-2 by Lpsn

and Lpsd MCDs raised the question of which other signaling path-ways are activated by UPECs. All subsequent experiments wereconducted using the AL511 strain of E. coli. AL511 did not stim-

ulate the expression of TRIF in both Lpsn and Lpsd MCDs (Fig. 7,A and C). In contrast, AL511 caused time-dependent activation ofboth phosphorylated p38 and ERK1/2 MAPKs in Lpsn MCDs butnot in Lpsd MCDs (Fig. 7, A and C). In any case, AL511 did notaffect the total amounts of ERK1/2 and p38. It is important to notethat this pathogenic isolate had different effects on the expressionof TRAF6 and TRAF2, two signaling adapter molecules shared bythe IL-1R/TLR family and the TNFR superfamily that control NF-�B, p38, and JNK signaling cascades (49). AL511 induced a time-dependent increase in TRAF6 when compared with the levels of�-actin, used as internal standard, in Lpsn MCDs only (Fig. 7, Aand C). In contrast, AL511 induced a time-dependent increase inTRAF2 in both Lpsn and Lpsd MCDs (Fig. 7, B and C). The factthat the adapter molecule TRAF2, which is involved in the TNF-induced activation of JNK independently of NF-�B (50–52), isstimulated by AL511 Lpsd MCDs suggests that UPECs may di-rectly activate JNK independently of TLR4 and TRAF6. AL511

FIGURE 7. Differential activation of TRAF2,TRAF6, and MAPKs by the UPEC AL511 strain inLpsn and Lpsd MCD cells. Western blot analyses of thetime-dependent expression of TRIF-, TRAF6-, and�-actin-labeled, phosphorylated (p-) and total p38-la-beled, and ERK1/2-labeled bands (A) and TRAF2,�-actin, and phosphorylated (p-) and total ASK1 andJNK (B) in cultured Lpsn and Lpsd MCDs incubatedwith AL511 (5 � 105 bacteria per well) for 3 h. C, Barsare mean ratio values (arbitrary units) of densitometricanalyses of phosphorylated (p-) over total p38-, ERK1/2-, ASK1-, and JNK-labeled bands and TRAF2 orTRAF6 over �-actin-labeled bands. Values aremeans � SEM from three or four separate cultures ofMCDs dissected from the kidneys of 2–4 mice in eachgroup tested. �, p � 0.05 vs time 0 values.

FIGURE 6. NF-�B-dependent and -independent andMyD88-dependent and -independent regulations ofMIP-2 secreted by MCD cells. Expression of NF-�Band I�B-� degradation (A) and secretion of MIP-2 (B)in confluent cultures of Lpsn and Lpsd MCDs after add-ing AL511 isolates (5 � 105 bacteria per filter for 3 h).A, Cytoplasmic and nuclear immunolocalization ofNF-�B (green) in Lpsn (upper panels) and Lpsd (lowerpanels) MCDs incubated without AL511 (None), withAL511 alone, or with AL511 plus SN50 (18 �M) for6 h. B, Western blot analyses of the time-dependentexpression of I�B-� and �-actin in Lpsn or Lpsd MCDsincubated with AL511 isolates. C, Secretion of MIP-2in confluent cultures of wild-type C57BL/6 MCDs,MyD88�/�, and Lps2�/� MCDs incubated without orwith UPEC AL10 or AL511 and without or with SN50(18 �M). Values are means � SEM from four to sevenseparate cultures of MCDs dissected from the kidneysof two or three mice in each group tested. �, p � 0.05vs E. coli-stimulated values.

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produced a time-dependent increase in the amount of phosphory-lated JNK without altering the amount of total JNK, not only inLpsn MCDs but also in Lpsd MCDs (Fig. 7, B and C). Furtherevidence that this UPEC strain may activate TRAF2 independentlyof TLR4 was provided by the fact that AL511 also stimulated thephosphorylated form of ASK1, which is known to associate rap-idly with TRAF2 in a TNF-dependent manner (53) in both Lpsn

and Lpsd MCDs, (Fig. 7, B and C).We then tested the effects of MAPK inhibitors on the secretion

of MIP-2 stimulated by AL511 in Lpsn and Lpsd MCDs. The se-cretion of MIP-2 stimulated by AL511 was consistently inhibited( p � 0.05) by �50% when Lpsn MCDs were preincubated withthe JNK inhibitor SP600125 and the p38 inhibitor SB203580 aswell as the MEK1/2 kinase inhibitor PD98059 (Fig. 8A). As con-trols, the phosphorylation of c-Jun and MAPKAPK-2, which aredirect substrates of JNK and p38, respectively, and the phosphor-ylation of ERK1/2 were tested to assess the selectivity of theMAPK inhibitors at the concentration used in this study. In allcases, each of the inhibitors tested selectively inhibited their cor-responding MAPK substrate without altering the phosphorylationof the other MAPKs (Fig. 8B). In contrast to what was observed inLpsn MCDs, the secretion of MIP-2 caused by AL511, which wasnot significantly affected by SB203580 and PD98059, was almostcompletely inhibited when Lpsd MCDs were preincubated with the

JNK inhibitor SP600125 (Fig. 8A). The MEK1/2, p38, and JNKinhibitors had very similar inhibitory effects on the secretion ofMIP-2 elicited by AL511 in cultured wild-type C57BL/6(MyD88�/�) MCDs and MyD88�/� MCDs, respectively (notshown). As in Lpsd MCDs, the JNK inhibitor SP600125 inhibitedby 84% the production of MIP-2 elicited by AL511 in MyD88�/�

MCDs (AL511, 1103 � 107; AL511 plus SP600125, 173 � 28pg/ml, n � 6; p � 0.01).

Because the expression of TNF-� was not totally abolished inLpsd MCDs incubated with UPECs (see Fig. 4), the persistent pro-duction of TNF-� should be sufficient to promote MIP-2 secretionvia the TRAF2-ASK1-JNK pathway. The stimulated production ofTNF-�, although lower in AL511-treated Lpsd MCDs than inAL511-treated Lpsn MCDs, was still significantly greater ( p �0.05) than in untreated Lpsd MCDs (Fig. 8C). Preincubating thecells with an anti-mouse TNF-� Ab significantly reduced ( p �0.05) the secretion of MIP-2 by 35 and 63% in Lpsn and Lpsd

MCDs, respectively (Fig. 8C). Pretreatment of MCDs with theanti-mouse TNF-� Ab also almost totally abolished the increase inphosphorylated ASK1 caused by AL511 in both Lpsn and Lpsd

MCDs (Fig. 8D). These findings demonstrate that the TNF-� pro-duced by activated MCD cells may directly stimulate the secretionof MIP-2 by activating a TRAF2-ASK1-JNK signaling pathway

FIGURE 8. Effects of MAPKs inhibitors and the role of TNF-� in the secretion of MIP-2 in Lpsn and Lpsd MCD cells and the occurrence ofinflammatory lesions in kidneys of Lpsn and Lpsd mice challenged with AL511 isolates. A, The secretion of MIP-2 was measured in Lpsn and Lpsd MCDsincubated with AL511 isolates (5 � 105 bacteria per well for 3 h) and with or without SP600125 (40 �M), SB203580 (10 �M), or PD98059 (10 �M).B, As controls, the phosphorylation of MAPKAPK-2 (a substrate of p38), c-Jun (a substrate of JNK), and ERK1/2 was analyzed in Lpsn MCDs incubatedwithout or with AL511 and without or with each of the MAPK inhibitors tested. C, The secretion of TNF-� and MIP-2 was measured in cultured Lpsn andLpsd MCDs incubated with or without AL511 and, in the case of MIP-2, after preincubating cells with a mouse anti-TNF-� Ab or a rat IgG1 isotype control30 min before adding the bacteria (lower panel). D, Western blot analysis of the expression of phosphorylated (p-) and total ASK1 in Lpsn and Lpsd MCDsincubated without or with AL511 alone or after preincubating with the anti-TNF-� Ab for 30 min before adding the bacteria. E–G, TNF-� production (E),bacterial counts (F), and histological alterations (G) in the day 2 AL511 postinfected kidneys of Lpsd mice treated with rat IgG1 isotype control orneutralizing anti-mouse TNF-� Abs. Note the presence of numerous microabscesses in Lpsd kidney following TNF neutralization. Original magnification:�200. Values are means � SEM from 3–5 separate cultures of MCDs dissected from the kidneys of two or three mice and counts from eight different kidneyhomogenates in each group tested. �, p � 0.05 between groups or vs AL511-stimulated values.

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and therefore account for the recruitment of leukocytes in UPEC-infected Lpsd mouse kidneys. The production of TNF-� was dra-matically increased in the kidneys of AL511-infected Lpsn miceand to a lesser extent in those of infected Lpsd mice as comparedwith that measured in the kidneys of naive mice (Fig. 8E). Tobetter assess the participation of TNF-� in renal bacterial clear-ance, bacterial counts and histological examinations were then per-formed on kidneys from Lpsn and Lpsd mice pretreated with theneutralizing anti-TNF-� Ab 1 h before the intravesical inoculationof AL511 isolates. The kidneys from Lpsd mice treated with theanti-TNF-� Ab were much more susceptible to AL511 infectionthan those from untreated Lpsd mice, because the renal bacterialburden at day 2 after challenge was significantly �104-fold greaterthan that for kidneys of untreated Lpsd mice infected with AL511(Fig. 8F). Numerous intrarenal abscesses were more frequentlydetected in the infected kidneys from Lpsd mice treated with theneutralizing anti-TNF-� Ab (seven of eight infected mice) than inthose from untreated Lpsd mice (one of eight infected mice; datanot shown) or Lpsd mice pretreated with the rat IgG1 isotype con-trol (none of eight infected mice) (Fig. 8G). Neutralization ofTNF-� also led to significant greater increase (�127-fold) in thenumber of CFU in AL511-infected Lpsn kidneys than in the kid-neys from untreated or IgG1 isotype control-pretreated Lpsn mice(not shown). However, no significant difference was observed inrenal inflammatory reaction (data not shown). These resultsstrongly suggest that, in addition to the activation of the mainTLR4-dependent pathway, the activation of the TLR4-independentsignaling pathway mediated by TNF-� also participates in the re-nal clearance of UPECs. Fig. 9 summarizes the TLR4-dependentand -independent pathways and corresponding signaling moleculesleading to stimulated secretion of MIP-2 by UPEC AL511 bacteriain mouse renal collecting duct cells.

DiscussionEpithelial cells from the upper urinary tract form a barrier to patho-gen entry and are active players in the innate immune system byproducing cytokines and chemokines in response to bacterial stim-ulation (54). The fact that all bone marrow-derived cells and epi-

thelial cells either express or lack functional TLR4 in Lpsn or Lpsd

mice makes it difficult to define the exact role of renal tubuleepithelial cells in initiating the innate immune response. Usinghemopoietic chimeric Lpsn and Lpsd mice to compare the specificcontributions of TLR4 expressed by hemopoietic cells and paren-chymatous bladder epithelial cells, Schilling et al. (19) have dem-onstrated that bladder epithelial cells expressing TLR4 can ac-tively clear bacteria during an acute infection but require thepresence of TLR4-expressing hemopoietic cells to elicit a full in-nate immune response. Using a similar approach, Patole et al. (20)have also reported that TLR4 is required in both intrinsic renaltubule cells and bone marrow-derived cells to initiate chemokine-driven, renal neutrophil recruitment.

Previous studies have shown that renal epithelial cells are sen-sitive to purified LPS or intact UPECs. For example, the pyelone-phritogenic E. coli ARD6 strain interacts with proximal tubulecells from young rats to induce large Ca2� oscillations (19).Tsuboi et al. (21) also reported that LPS mediates the productionof MCP-1 and RANTES in primary cultures of mouse renal tubuleepithelial cells exhibiting a proximal phenotype. We provide hereevidence that collecting duct epithelial cells may directly partici-pate in the recognition and rapid induction of proinflammatorymediators in response to UPEC strains. We show that two pyelo-nephritis-associated E. coli isolates, AL10 and AL511, both ofwhich colonize kidneys in the mouse model of the upper UTI andlack the virulent toxin �-hemolysin, interact with the apical mem-brane of collecting duct cells and, more specifically, with interca-lated cells, suggesting that this particular cell type may be apreferential site for bacterial attachment. The profile of stimulatedproinflammatory mediator expression caused by UPECs is quitesimilar in the day 2 postinfected kidneys and in cultured Lpsn

MCDs. However, UPECs that stimulate the expression ofRANTES in whole kidneys did not activate its expression incultured MCDs. LPS has been shown to stimulate the productionof both MCP-1 and RANTES in cultured mouse primary renalproximal tubule cells (21). Although the NF-�B activation seemsto be essential for LPS-mediated up-regulation of these two che-mokines, the inhibition of JNK and p38 MAPK impairs theexpression of RANTES but not of MCP-1 (21), suggesting thatRANTES and MCP-1 are differently regulated. Such dissimilarityin the regulation of these two mononuclear-directed chemokinesmay also account for the differences observed in day 2 postinfectedkidneys exhibiting predominant expression of RANTES in inter-stitial cells on one hand and a preferential expression of MCP-1 in the3-h infected MCDs on the other hand. Alternatively, the disparityobserved may also reflect differences in the stimulated rates ofinduction and/or degradation of the two chemoattractant chemokines.

The molecular mechanism(s) by which E. coli strains activaterenal epithelial cells still remain not fully elucidated. In C3H/HeJLpsd mice the renal inflammatory response to P fimbriated E. coliis TLR4 dependent (55, 56), and this activation signaling in epi-thelial A498 cells lacking CD14 was independent of LPS and lipidA myristoylation (56). More recently, Fischer et al. (15), usingmurine models of ascending UTI, have shown that P and type 1fimbriated E. coli may use different adaptor molecules to influenceneutrophil activation and bacterial clearance, but that in both casesMyD88 is required for efficient bacterial clearance. These resultssuggested that P and type 1 fimbriae, through their recognition bytheir respective epithelial cell receptors, may engage specificTLR4-associated proteins for the induction of innate immune re-sponse in the urinary tract (15). In this study, we show that murinewild-type MCDs in primary culture constitutionally express afunctional TLR4 and its accessory molecules, including CD14, andare an important source of chemokines and cytokines following the

FIGURE 9. Schematic representation of the TLR4-dependent and -in-dependent pathways activated by UPEC AL511 isolates in renal collectingduct intercalated cells.

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adhesion of UPECs at their cell surface. The interaction of the twoUPEC isolates with collecting duct intercalated cells induces anearly and strong inflammatory response characterized by the up-regulation of proinflammatory mediators, including proinflamma-tory cytokines (IL-1�, IL-6, and TNF-�), chemokines (MIP-2,RANTES, and MCP-1), and iNOS in the infected kidneys fromLpsn mice. Consistent with what is observed in UPECs-infectedbladder epithelial cells (14, 19), the inflammatory response in kid-neys colonized by the AL10 and AL511 UPEC isolates appears tobe mediated mainly by TLR4. However, the two UPEC strainsinduced markedly different stimulation profiles of proinflammatorymediators in the kidneys of Lpsn and Lpsd mice, suggesting thatdifferent bacterial factors produced by the UPECs may be impli-cated in the activation of distinct TLR4-dependent and -indepen-dent signaling pathways. MIP-2 and its human counterpart IL-8play key roles in the migration of neutrophils to infected mucosalsites to protect them against invading pathogens (41, 42, 57). Thetwo UPECs stimulated MIP-2 mRNA expression and protein se-cretion in both Lpsn kidney and cultured Lpsn MCDs through amain TLR4-dependent pathway. The results obtained with culturedMCDs dissected from the kidneys of MyD88�/� and Lps2�/�

mice also strongly suggest that UPECs activate TLR4 pathwaythrough a predominant, MyD88-dependent, mediated pathway.The persistent activation of proinflammatory mediators (mainlyMIP-2 and TNF-�) in Lpsd mouse kidneys, together with the per-sistent stimulation of basal and apical secretions of MIP-2 in Lpsd

MCDs caused by both UPEC strains, further suggested that UPECsmay also activate MCD cells via nonmediated TLR4 cellular sig-naling pathways.

In mammalian cells, the recognition of LPS by TLR4 results inthe recruitment of multiple signaling molecules including TRAF6,which activates downstream components involving NF-�B, p38,and JNK (58). UPEC AL511 isolates activate the TLR4-mediatedERK1/2 signaling pathway and increase the abundance of TRAF6and down-stream activation of NF-�B, p38, and JNK in Lpsn

MCDs only (Fig. 9). Conversely, UPEC AL511 isolates had nostimulatory effect on these signaling molecules in TLR4-defectiveLpsd MCDs. As a result of the TLR4-mediated, TRAF6-activatedpathway, the stimulated secretion of MIP-2 in Lpsn MCDs is par-tially or almost entirely abolished by the inhibitors of NF-�B, p38,or MEK1/2. We also demonstrate that the NF-�B-independentstimulation of MIP-2 caused by AL511 depends mainly, if notexclusively, on the activation of JNK in Lpsd MCDs (Fig. 9). JNKis activated by TNF-� and IL-1 and by exposing cells to a varietyof environmental stress conditions (58). TRAF2 transduces the sig-nals required for TNF-mediated activation of NF-�B (59), p38,and JNK (50, 60). However, distinct activated cellular mechanismsmay lead to diversification of TRAF2 signaling. Analysis of em-bryonic fibroblasts from TRAF2-deficient mice that reveals a se-vere reduction in TNF-mediated JNK activation and only a mildeffect on NF-�B activation suggests that a TRAF2-independentpathway of NF-�B activation must exist (51). Habelhah et al. (61)have shown that TNF-induced ubiquitination of TRAF2, resultingfrom its translocation into insoluble lipid rafts, is required for theactivation of JNK, but not for that of p38 and I�B kinases. UPECstrain AL511 also stimulates TRAF2 and activates JNK, but notNF-�B or p38, in TLR4-defective Lpsd MCDs. The fact that theselective inhibition of JNK by SP600125 almost completely pre-vented the stimulation of the secretion of MIP-2 caused by UPECsin Lpsd MCDs provides further evidence that the TRAF2-JNKpathway corresponds to the TLR4-independent pathway activatedby UPECs in Lpsd MCDs.

The interaction between UPECs and both Lpsn and Lpsd MCDscaused increased expression of ASK1. This evolutionarily con-

served MAPK kinase kinase is activated in response to variouscytotoxic stresses such as TNF-� and reactive oxygen species(ROS) (62). ASK1 is selectively required for the TLR4-mediated,LPS-induced activation of both JNK and p38 (63). The stimulationof TRAF6 and ASK1 caused by strain AL511 therefore must ac-count for the activation of p38 in Lpsn MCDs. In contrast, theUPEC isolate does not affect TRAF6 but does stimulate TRAF2and activates ASK1 in Lpsd MCDs. These findings strongly sug-gest that the formation of a TRAF2-ASK1 complex leads to thesubsequent activation of JNK and, consequently, to stimulated pro-duction of MIP-2 in Lpsd MCDs. Recently, the antioxidant pyrro-lidine dithiocarbamate has been shown to induce NF-�B-indepen-dent MIP-2 promoter activation mediated via the JNK pathwayand the subsequent activation of the AP-1 transcription factor inmouse macrophage RAW 264.7 cells (64). These findings suggestthat the TLR4-independent secretion of MIP-2 stimulated byUPECs results from the activation of AP-1 mediated via theTRAF2-ASK1-JNK pathway. We also provide some evidencesuggesting that the UPEC isolates stimulate the production ofTNF-�, particularly in Lpsn MCDs, but also, although to a muchlesser extent, in Lpsd MCDs. Interestingly, the neutralization ofrenal TNF-� production significantly altered the production ofMIP-2 and blunted the induction of phosphorylated ASK1 causedby the AL511 isolate in both Lpsn and Lpsd MCDs. These datatherefore provide convincing evidence that the stimulation ofTNF-� production by epithelial collecting duct cells activated byUPECs may directly activate the TLR4-independent production ofMIP-2. Matsuzawa et al. (65) have demonstrated that ASK1specifically mediates LPS-induced TLR4 signaling via a ROS-dependent activation of the TRAF6-ASK1-p38 pathway. Thismeans that we cannot rule out the possibility that ROS producedlocally by polymorphonuclear neutrophils or collecting duct cellsmay activate the TLR4-dependent TRAF6-ASK1-p38 pathway inLpsn MCDs and, together with TNF-�, activate the TLR4-independent, ASK1-JNK-activated pathway that we have identi-fied in Lpsd MCDs. Alternatively, UPECs might activate TLR4-dependent and/or TLR4-independent pathway through theirrecognition by other pattern recognition receptors. TLR11, whichis expressed in murine tubule epithelial cells but not in humans,has also been shown to play a key role in the renal bacterialclearance from mouse kidneys through the activation of identicalTLR4-dependent signaling pathways (66). Nevertheless, our re-sults strongly suggest that the TLR4-independent pathway acti-vated by UPECs also participates in the protection of the kidneysagainst ascending pathogens; after blocking TNF-�, TLR4-deficient Lpsd mice as well as Lpsn mice are much more suscep-tible to renal bacterial invasion than their respective untreated Lpsn

and Lpsd mice counterparts and develop more rapid and extendedrenal inflammatory lesions. In human pathology, uncomplicatedUTIs and acute pyelonephritis caused by UPECs are the mostcommon forms of bacterial infection in renal transplant recipients.UTIs are thought to be directly attributable to exposure to patho-gens during the early postoperative period and to immunosuppres-sive therapy (67). A recent major retrospective study has revealedthat UTIs occurring after renal transplantation are often associatedwith an increased risk of septic shock and subsequent death (68).Therefore, it cannot be excluded that the frequent occurrence ofascending pyelonephritis caused by UPECs in renal transplantedpatients may be favored, at least in part, by alterations in theactivation of cellular signaling pathways due to prolonged immu-nosuppressive therapy. Further clinical studies will be needed totest this hypothesis.

To summarize, the present study contributes to elucidation ofthe role played by collecting duct intercalated cells in recognizing

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pyelonephritic E. coli isolates and provides evidence that collect-ing duct cells actively participate to the renal clearance of bacteriaby mediating a reliable immune response via TLR4-mediated NF-�B-, ERK1/2-, p38-, and JNK-activated signaling pathways or viaan alternative TLR4-independent, TNF-mediated, TRAF2-ASK1-JNK-activated pathway.

AcknowledgmentsWe thank B. Fernandez, N. Quellard, and P. Levillain for their expertassistance with scanning microscopy, C. Pouzet for expert technical assis-tance with confocal microscopy, and R. Ruimy for making real-time PCRfacilities available. We thank T. J. Jentsch for the anti-ClC-5 Ab, M. W.Hornef for the anti-TLR4 Ab, and D. Paulin for the anti-cytokeratins K8–K18 Ab. We also thank S. Akira for the permission to use tlr2�/� andMyD88�/� mice and M. Chignard for housing facilities. We are grateful toG. Richet for unfailing support and to M. W. Hornef and C. Hechard-Lafleuriel for their critical reading of the manuscript.

DisclosuresThe authors have no financial conflict of interest.

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