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Enteroinvasive BacteriaResponse Induced by Infection withIntestinal Epithelial Cell Innate Immune

B Is a Central Regulator of theκNF-

Truong, Lars Eckmann and Martin F. KagnoffDirk Elewaut, Joseph A. DiDonato, Jung Mogg Kim, Francis

http://www.jimmunol.org/content/163/3/14571999; 163:1457-1466; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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NF-kB Is a Central Regulator of the Intestinal Epithelial CellInnate Immune Response Induced by Infection withEnteroinvasive Bacteria1

Dirk Elewaut, 2,3 Joseph A. DiDonato,3,4 Jung Mogg Kim,5 Francis Truong, Lars Eckmann, andMartin F. Kagnoff 6

Human intestinal epithelial cells up-regulate the expression of an inflammatory gene program in response to infection with aspectrum of different strains of enteroinvasive bacteria. The conserved nature of this program suggested that diverse signals,which are activated by enteroinvasive bacteria, can be integrated into a common signaling pathway that activates a set ofproinflammatory genes in infected host cells. Human intestinal epithelial cell lines, HT-29, Caco-2, and T84, were infected withinvasive bacteria that use different strategies to induce their uptake and have different intracellular localizations (i.e., Salmonelladublin, enteroinvasiveEscherichia coli,or Yersinia enterocolitica). Infection with each of these bacteria resulted in the activationof TNF receptor associated factors, two recently described serine kinases, IkB kinase (IKK) a and IKK b, and increased NF-kBDNA binding activity. This was paralleled by partial degradation of IkBa and IkBe in bacteria-infected Caco-2 cells. Mutantproteins that act as superrepressors of IKKb and IkBa inhibited the up-regulated transcription and expression of downstreamtargets genes of NF-kB that are key components of the epithelial inflammatory gene program (i.e., IL-8, growth-related onco-gene-a, monocyte chemoattractant protein-1, TNF-a, cyclooxygenase-2, nitric oxide synthase-2, ICAM-1) activated by thoseenteroinvasive bacteria. These studies position NF-kB as a central regulator of the epithelial cell innate immune response toinfection with enteroinvasive bacteria. The Journal of Immunology,1999, 163: 1457–1466.

Epithelial cells that line the intestinal mucosa are an initialsite of interaction between enteroinvasive microbialpathogens and the host. After infection with several

strains of enteroinvasive bacteria (e.g.,Salmonella,enteroinvasiveEscherichia coli, Yersinia, Shigella, Listeria), intestinal epithelialcells rapidly (within 2–3 h) up-regulate the expression of a pro-gram of host genes, the products of which activate mucosal in-flammatory and immune responses and alter epithelial cell func-tions (1–8). This epithelial cell inflammatory program includes theup-regulated expression and production of proinflammatory andchemoattractant cytokines (e.g., TNF-a, IL-8, growth-related on-cogene-a (GROa),7 and monocyte chemoattractant protein-1

(MCP-1)), an inducible isoform of cyclooxygenase (COX),COX-2, and prostaglandins E2 and F2a, an inducible form of nitricoxide synthase (NOS), NOS2, and nitric oxide, and increased sur-face expression of the adhesion molecule ICAM-1 on the apicalcell membrane (1–5, 7).

Enteroinvasive bacterial pathogens use a variety of strategies toinduce their uptake into nonphagocytic intestinal epithelial cells(9). For example, the uptake ofSalmonellainvolves the formationof large membrane folds or ruffles and macropinocytosis (10). Incontrast, internalization ofYersiniainvolves the binding of a bac-terial outer membrane protein, invasin, tob1 integrins on the ep-ithelial cell surface and a “zippering” mechanism, which is char-acterized by the close juxtaposition of the mammalian cellmembrane around bacteria that are being internalized (10, 11). Ineach case, interaction of bacteria with the host epithelial cell re-sults in morphological changes in the epithelial cell membrane andunderlying cytoskeleton. After entry into epithelial cells, differentstrains of bacteria reside in different intracellular localizations(e.g., after entry,Salmonellaand Yersinia reside in membrane-bound vesicles, whereasShigella and Listeria rapidly lyse suchvesicles and move freely within the cytoplasm) (10). Despite di-verse mechanisms of bacterial invasion and the different intracel-lular localization of bacteria within the cytoplasm, the array ofgenes and mediators that are consistently up-regulated as compo-nents of the epithelial cell innate immune response is relativelylimited (1, 7). This suggested that a common signal transductionpathway might underlie the up-regulated expression of the proin-flammatory genes and mediators that are activated in intestinalepithelial cells in response to infection with enteroinvasivebacteria.

Laboratory of Mucosal Immunology, Department of Medicine, University of Cali-fornia at San Diego, La Jolla, CA 92093

Received for publication March 22, 1999. Accepted for publication May 17, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grant DK35108. D.E. wassupported by the Belgian American Educational Foundation, D. Collen ResearchFoundation, and the Francqui Foundation. L.E. was supported by a Career Develop-ment Award from the Crohn’s and Colitis Foundation of America.2 Current address: La Jolla Institute for Allergy and Immunology, La Jolla, CA.3 D.E. and J.A.D. contributed equally to this work.4 Current address: Department of Cancer Biology, Cleveland Clinic Foundation,Cleveland, OH.5 Current address: Department of Microbiology, Hanyang University, Seoul, Korea.6 Address correspondence and reprint requests to Dr. Martin F. Kagnoff, Laboratoryof Mucosal Immunology, University of California, San Diego, 9500 Gilman Drive, LaJolla, CA 92093-0623. E-mail address: mkagnoff@ucsd.edu7 Abbreviations used in this paper: GROa, growth-related oncogene-a; MCP-1,monocyte chemoattractant protein-1; COX, cyclooxygenase; NOS2, nitric oxide syn-thase-2; IKK, IkB kinase; MAP3, mitogen-activated protein-3; NIK, NF-kB-inducingkinase; MEKK-1, MEK kinase 1; TRAF, TNF receptor-associated factor; UCSD,University of California, San Diego; MOI, multiplicity of infection; CAT, chloram-

phenicol acetyltransferase; HA, hemagglutinin; EMSA, electrophoretic mobility shiftassay; AP-1, activator protein 1; LTbR, lymphotoxinb receptor .

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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Many of the genes that are activated in intestinal epithelial cellsafter bacterial invasion are target genes of the transcription factorNF-kB. NF-kB is a dimeric transcription factor composed of ho-modimers and heterodimers of Rel proteins, of which there are fivefamily members in mammalian cells (i.e., RelA (p65), c-Rel, RelB,NF-kB1 (p50), and NF-kB2 (p52)) (12, 13). NF-kB dimers areheld in the cytoplasm in an inactive state by inhibitory proteins, theIkBs. There are seven IkBs (i.e., IkBa, IkBb, IkBe, IkBg, Bcl-3,p100, and p105) (14) that preferentially associate with various Relfamily protein dimers (e.g., IkBa and IkBb predominantly asso-ciate with p65/p50 and p50/c-Rel heterodimers, IkBe preferen-tially associates with p65 and c-Rel homodimers, and Bcl-3 asso-ciates with nuclear p50 and p52 homodimers). Heterodimers ofp65 and p50 are the predominant NF-kB subunits that translocateto the nucleus after cytokine stimulation of intestinal epithelialcells (15).

Stimulation of cells with IL-1 or TNF-a activates a signalingcascade that culminates in the phosphorylation of IkBs (16, 17).Two recently described IkB kinases, IkB kinase (IKK) a andIKK b, directly phosphorylate IkBs on serine residues and are com-ponents of a high molecular weight cytoplasmic IKK complex (16,18, 19). Activation of IKK appears to be mediated through themitogen-activated protein-3 (MAP3) kinases, NF-kB-inducing ki-nase (NIK), and MEK kinase 1 (MEKK-1) (20–24). Phosphory-lation of IkBs on conserved serine residues targets the IkBs forsubsequent ubiquitination and degradation (25, 26). This freesdimers of NF-kB (e.g., p65/p50) to translocate to the nucleuswhere theytrans-activate NF-kB target genes. NF-kB can alsotrans-activate transcription of its own inhibitor, IkBa (27). Thisnegative feedback loop results in the restoration of IkBa proteinlevels, which complex cytoplasmic NF-kB and thereby down-reg-ulate NF-kB activation (28, 29). Activation of NF-kB in responseto extracellular signaling through TNF receptor family membersinvolves TNF receptor-associated factors (TRAFs) that serve asadaptor molecules in these signal transduction pathways (e.g.,TRAF2 and TRAF5 are involved in signaling through TNF andlymphotoxinb receptors (LTbR), respectively) (30, 31).

The present studies asked whether intestinal epithelial cells usea final common signal transduction pathway to activate the host’searly inflammatory response to a diverse array of enteroinvasivebacteria. NF-kB is shown herein to be a central regulator of theactivation of the intestinal epithelial cell inflammatory responseafter infection with a spectrum of enteroinvasive bacteria. More-over, activation of NF-kB after epithelial cell infection with en-teroinvasive bacteria is shown to involve the activation of TRAF2,independent of extracellular signaling through the TNF receptor,and to be preceded by the activation of IKKa and IKKb and thedegradation of IkBs. Consistent with this, expression of superre-pressor alleles of IkBa or IKKb inhibited the up-regulated expres-sion of key components of the epithelial cell-inflammatory geneprogram (e.g., IL-8, GROa, TNF-a, COX-2, NOS2, ICAM-1) inintestinal epithelial cells infected with enteroinvasive bacteria.

Materials and MethodsCell lines

HT-29 human colon epithelial cells (American Type Culture Collection(ATCC) HTB 38) and Caco-2 human ileocecal epithelial cells (ATCCHTB 37) were grown in DME (Life Technologies, Gaithersburg, MD)supplemented with 10% FCS, 2 mM glutamine, and 25 mM HEPES. T84cells were grown in 50% DMEM and 50% Ham’s F-12 medium supple-mented with 5% newborn calf serum and 2 mM glutamine, as describedbefore (1, 2).

Bacteria, cytokines, and other reagents

S. dublinlane strain (1) andYersinia enterocolitica08 (1) were providedby J. Fierer (University of California, San Diego, CA (UCSD)). Enteroin-vasiveEscherichia coli(serotype O29:NM) were obtained from the ATCC(No. 43892), and nonpathogenicE. coli DH5a were purchased from LifeTechnologies. Recombinant human TNF-a and IL-1a were from R&DSystems (Minneapolis, MN). IFN-g was from BioSource International,Camarillo, CA.

Bacterial infection

Human colon epithelial cell lines were infected with bacteria at multiplic-ities of infection (MOIs) ranging from 50 to 100 as described before (1, 5,32). Briefly, cells were grown to confluence in six-well plates containing;106 cells/well. Cells were incubated with bacteria for up to 1 h, afterwhich whole cell or nuclear extracts were prepared (16, 17, 33). For longerincubations, extracellular bacteria were removed by washing after 1 h, andthe cells were incubated for an additional period in the presence of 50mg/ml gentamicin to kill the remaining extracellular, but not intracellular,bacteria. Those cells were harvested for isolation of total RNA for RT-PCRor for analysis of reporter gene expression (see below).

Plasmids, transfections, chloramphenicol acetyltransferase(CAT) assays, and luciferase assays

A mammalian expression vector encoding a hemagglutinin (HA) epitope-tagged mutant IkBa having substitutions of serine residues at positions 32and 36 with alanine residues was used to block NF-kB activation (17). Themutant protein cannot be phosphorylated by IkB kinases at those positionsand acts as an IkB superrepressor. An expression vector that encodesFLAG-tagged IKKb in which lysine present at position 44 is replaced byalanine, which inactivates kinase activity in the mutant molecule and actsas a superrepressor of IKK, was a gift of F. Mercurio (Signal Pharmaceu-ticals, San Diego, CA) (19). Expression vectors for the superrepressor ver-sions of TRAF2 and TRAF5 (amino-terminal deletions) (34, 35) were giftsfrom D. Goeddel, (Tularik Corp., South San Francisco, CA) and H. Nakano(Juntendo Univ, Tokyo, Japan), respectively. An expression vector for aNIK catalytic mutant that has a double replacement of alanine residues tolysine residues at positions 429 and 430 and lacks kinase activity (21) wasa gift of Z. G. Liu and M. Karin, UCSD. An expression vector for A20 (36)was a gift of G. Natoli. IL-8-luciferase, 2XNF-kB-luciferase, and RSV-b-galactosidase transcriptional reporters were constructed as described before(33, 37). A full length ICAM-1 promoter construct containing 1.4 kb ofICAM-1 59-flanking DNA linked to the luciferase gene was provided by K.Roebuck (38). CAT transcriptional reporters containing various 59-flankingdeletions of the IL-8 promoter (39) were a gift of K. Matsushima. Cells insix-well dishes were transfected with 1.5mg of plasmid DNA, using Li-pofectamine Plus (Life Technologies), according to the manufacturer’s in-structions. Luciferase activity was determined and normalized relative tob-galactosidase expression as described before (37, 40). CAT-assays wereperformed with an enzyme immunoassay (CAT ELISA, Boehringer Mann-heim, Indianapolis, IN).

Recombinant adenovirus and adenovirus infection

Recombinant adenovirus containing an IkBa-AA superrepressor (Ad5IkB-A32/36) or theE. coli b-galactosidase gene (Ad5LacZ) was constructed asdescribed before (41). Ad5IkB-A32/36 expresses a HA-epitope tagged mu-tant form of IkBa in which serine residues 32 and 36 are replaced byalanine residues as described above. The mutant IkBa cannot be phos-phorylated at positions 32 and 36 and acts as a superrepressor. The HAepitope tag enables identification of the exogenous superrepressor withanti-hemagglutinin Abs. Viral titers were determined by plaque assay.Recombinant virus was stored in PBS containing 10% (v/v) glycerol at280°C.

HT-29 cells grown to confluence in six-well tissue culture plates(Costar, Cambridge, MA) were infected with Ad5IkB-A32/36 or Ad5LacZin serum-free media (Opti-MEM, Life Technologies) at MOI 100 for 16 h.At this MOI, Ad5IkB-A32/36 or Ad5LacZ infected.80% HT-29 cells,and infected cells expressed IkBa-A32/36 andb-galactosidase, respec-tively, at high levels as assessed by staining forb-galactosidase and im-munostaining for HA-tagged IkB-A32/36 (data not shown). After infec-tion, adenovirus was removed by washing, fresh medium containing serumwas added, and cells were incubated for an additional 12 h before bacterialinfection or stimulation with TNF-a.

Cell extracts

Cells were harvested, and whole cell and nuclear extracts were prepared asdescribed before (16, 17, 33). Briefly, cell pellets were resuspended and

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lysed at 4°C for 25 min in whole cell extract lysis buffer (20 mM HEPES,0.4 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, and 1 mMDTT containing phosphatase inhibitors (40 mMb-glycerophosphate, 20mM NaF, 1 mM Na3VO4, 20 mM p-nitrophenyl phosphate (Calbiochem,San Diego, CA)); and protease inhibitors (aprotinin 10mg/ml, leupeptin 10mg/ml, bestatin 10mg/ml, and pepstatin 10mg/ml) (Calbiochem) and 1mM phenylmethylsulfonyl chloride (Sigma, St. Louis, MO). Lysates werecentrifuged at 13,0003 g for 15 min in the cold, and the resulting super-natants were transferred to fresh tubes. Protein concentrations in the su-pernatants were determined by the Bradford assay (Bio-Rad,Hercules, CA).

Immunoblotting for IkBs

Cell lysates containing 20mg of protein were electrophoresed on SDS-10%polyacrylamide gels with a 4% polyacrylamide stacking gel. After transferto Immobilon-P membranes (Millipore, Marlborough, MA), membraneswere blocked with a solution of 5% (w/v) dry milk in PBS-T (PBS con-taining 0.05% Tween 20) for 1 h at room temperature. Membranes wereprobed with murine mAbs to IkBa, IkBb (Santa Cruz Biotechnology,Santa Cruz, CA), and IkBe (gift of N. Rice) and visualized using an ECLdetection kit (Amersham, Arlington Heights, IL) and exposure to x-ray film(XAR5, Eastman Kodak, Rochester, NY).

Electrophoretic mobility shift assays

For electrophoretic mobility shift assays (EMSA), either 20mg of wholecell extract or 6mg of nuclear extract were incubated for 30 min at 4°Cwith 5 mg of polyoligonucleotides (dI-dC) and 23 104 cpm (;0.2 ng) ofa labeled oligonucleotide probe corresponding to a consensus NF-kB bind-ing site (33). After incubation, bound and free DNAs were resolved in 5%native polyacrylamide gels as described before (33).

IKK assay

Kinase activity was assayed in 20 mM HEPES, 20 mMb-glycerophos-phate, 10 mM MgCl2, 10 mM p-nitrophenyl phosphate, 100mM Na3VO4,2 mM DDT, 20mM ATP, 10 mg/ml aprotinin, 50–200 mM NaCl, pH 7.5,and 1–10mCi [g-32P]ATP at 30°C for 30 min. IkB-substrate proteins wereexpressed and purified fromE. coli as described before (17). IKKa- andIKK b-containing complexes were immunoprecipitated with specific mAbsto IKKa (PharMingen, San Diego, CA.) or monospecific rabbit polyclonalAb to carboxy-terminal IKKb. Immune complexes were isolated andwashed in kinase buffer containing 1.5 M urea (16, 18). Kinase activity wasdetermined using GST-IkBa (1-54) wild type as substrate as describedbefore (17). Kinase specificity was established with mutant GST-IkBa(1-54-AA) in which serines 32 and 36 were substituted with alanines (16).Fold induction of IKKa and IKKb kinase activities was determined afterphosphorimaging of the dried SDS-PAGE-fractionated GST-IkBprotein-containing gels.

Oligonucleotide primers for PCR amplification

Oligonucleotide primers used for PCR amplification and the size of thePCR products obtained from target cellular RNA are shown in Table I.Exon-spanning primers were designed to amplify cDNA, but not genomicDNA. Primers were designed and synthesized as described previously(2–4) or were obtained commercially.

Reverse transcription PCR

Total cellular RNA was extracted from cells with Trizol reagent (LifeTechnologies) and reverse transcribed as described previously (2). PCR

amplification consisted of 35 cycles of 1 min denaturation at 95°C, 2.5 minannealing, and extension at 60°C (IL-8, COX-2, GROa, ICAM-1, NOS2),65°C (MCP-1, TGF-b1), or 72°C (b-actin, TNF-a, TGF-a). A hot start inwhich samples were preheated to 95°C before addition ofTaqpolymerase(Stratagene, San Diego, CA) was used to increase specificity of the am-plification. Each experiment included negative controls in which RNA wasomitted from the reverse transcription mixture, and cDNA was omittedfrom the PCR reaction.

Cytokine ELISAs

Cytokines in culture supernatants were assayed by ELISA as describedbefore (2, 3). The IL-8 and the GROa ELISAs were sensitive to 20 and 30pg/ml, respectively.

Flow cytometry

Monolayers of colon epithelial cells were detached by incubation with0.25% EDTA in calcium- and magnesium-free PBS (pH 7.2) as describedbefore (5). Cell viability was.95% as assessed by trypan blue dye ex-clusion. For flow cytometry,;5 3 105 cells were incubated with optimalconcentrations of anti-CD54 (ICAM-1) (murine IgG1, AMAC, Westbrook,ME) at 4°C for 60 min after which cells were washed, incubated with anoptimal concentration of a PE-labeled goat anti-mouse IgG (H1 L)(Southern Biotechnology Associates, Birmingham, AL) at 4°C for 60 min,and analyzed using a flow cytometer (FACScan, Becton Dickinson,Sunnyvale, CA).

ResultsEnteroinvasive bacteria activate NF-kB in HT-29, Caco-2, andT84 human colon epithelial cells

The transcription factor NF-kB has a role in the transcriptionalactivation of genes the mRNA expression of which is known to beincreased by infection of human colon epithelial cells with variousenteroinvasive bacteria (e.g., TNF-a, the C-X-C chemokines IL-8and GROa, the C-C chemokine MCP-1, as well as COX-2 andNOS2) (1–5, 7, 12, 42, 43). To determine whether enteroinvasivebacteria activate NF-kB in human colon epithelial cells, DNAbinding studies were performed using cell extracts obtained at var-ious times after infection of HT-29, Caco-2, and T84 cells withS.dublin, Y. enterocolitica, or enteroinvasiveE. coli. Infection ofthose cells with each of these enteroinvasive bacteria increasedNF-kB DNA binding, as shown by EMSAs (Fig. 1). Maximumbinding activity occurred within 30–45 min after bacterial infec-tion. For comparison, maximum binding after stimulation ofHT-29 and T84 cells with TNF-a, a potent activator of NF-kB,was more rapid (i.e., within 10 min). NF-kB binding to the NF-kBoligonucleotide probe was mediated predominantly by het-erodimers of p65, as demonstrated in supershift assays in whichirrelevant rabbit Ab, anti-p65, or anti-p50 Abs were added to cellextracts (data not shown). Nonpathogenic noninvasiveE. coliDH5a resulted in little if any increase in NF-kB DNA bindingactivity in the same epithelial cell lines (Fig. 1). These studiesshowed that activation of NF-kB parallels activation of the epi-thelial cell inflammatory program. This suggested that NF-kB may

Table I. Oligonucleotide primers and PCR product sizes

mRNA Species 59-Primer 39-PrimerSize of PCRProduct (bp)

IL-8 59-ATGACTTCCAAGCTGGCCGTGGCT-39 59-TCTCAGCCCTCTTCAAAAACTTCTC-39 289GROa 59ACTCAAGAATGGGCGGAAAG-39 59-TGGCATGTTGCAGGCTCCT-39 468MCP-1 59-TCTGTGCCTGCTGCTCATAGC-39 59-GGGTAGAACTGTGGTTCAAGAGG-39 510COX-2 59TTCAAATGAGATTGTGGGAAAATTGCT-39 59-AGATCATCTCTGCCTGAGTATCTT-39 305TGF-a 59-ATGGTCCCCTCGGCTGGACAGCTCGCC-39 59-GATGGCCTGCTTCTTCTGGCTGGCAGC-39 300TGF-b1 59-GCCCTGGACACCAACTATTGCT-39 59-AGGCTCCAAATGTAGGGGCAGG-39 161TNF-a 59-CGGGACGTGGAGCTGGCCGAGGAG-39 59-CACCAGCTGGTTATCTCTCAGCTC-39 355iNOS 59-CGGTGCTGTATTTCCTTACGAGGCGAAGAAGG-3959GGTGCTGCTTGTTAGGAGGTCAAGTAAAGGGC-39259ICAM-1 59-GATGCTGACCCTGGAGAGCA-39 59-AGCACTTGCGGTCCACGATG-39 409b-Actin 59-TGACGGGGTCACCCACACTGTGCCCATCTA-3959-CTAGAAGCATTGCGGTGGACGATGGAGGG-39 661

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be an essential transcription factor for integrating the host responseof intestinal epithelial cells to infection with a variety of entero-invasive bacteria that are known to use a spectrum of differentreceptors and signaling pathways to induce their uptake into thesenonphagocytic cells.

Degradation of IkBa in human colon epithelial cells afterinfection with enteroinvasive bacteria

One of the major pathways for NF-kB activation involves thephosphorylation of IkBa on serine residues 32 and 36, which isfollowed by IkBa degradation and the subsequent migration ofNF-kB dimers from the cytoplasm to the nucleus. To determinewhether this is also the major pathway for NF-kB activation afterbacterial infection of human intestinal epithelial cells, we assayedthe kinetics of IkBa degradation, as well as the degradation ofIkBe and IkBb, by immunoblot analysis, in HT-29, Caco-2, andT84 cells after infection withS. dublin, Y. enterocolitica, and en-teroinvasiveE. coli, or the nonpathogenicE. coli DH5a. Infectionof Caco-2 cells withS. dublinor Y. enterocoliticaresulted in therapid but transient degradation of IkBa, whereas the kinetics ofIkBa degradation was slower after enteroinvasiveE. coli infection(Fig. 1). Further, relative to IkBa, IkBe was more slowly degradedin those cells. Consistent with the known lack of response ofCaco-2 cells to TNF-a stimulation (44), little degradation of IkBaand IkBe was seen in TNF-a-stimulated Caco-2 cells. IkBa andIkBe degradation was less marked and incomplete in bacteria-in-fected HT-29 and T84 cells (Fig. 1). The latter findings are con-sistent with the incomplete degradation of IkBa in IL-1-stimulatedHT-29 and T84 cells (15). In contrast, however, TNF-a stimula-tion of HT-29 and T84 cells resulted in rapid and nearly completedegradation of IkBa (Fig. 1). In HT-29 or T84 cells, IkBb was notdegraded in response to infection with the enteroinvasive bacteria,although it was partially degraded in Caco-2 cells (data notshown). Taken together, the data show that NF-kB is activated ineach cell line in response to infection with several different en-teroinvasive bacteria and in HT-29 and T84 cells stimulated withTNF-a, despite differences among the cell lines in the extent of

degradation of IkBs in response to different stimuli. Infection ofthe cell lines with nonpathogenicE. coli DH5a resulted in little ifany degradation of IkBa, IkBe, or IkBb (Fig. 1 and data notshown).

Activation of IKK precedes NF-kB activation

IKK contains two subunits, IKKa and IKKb, that can directlyphosphorylate IkBs (45). Therefore, we assessed whether IKKaand IKKb were activated in intestinal epithelial cells in response toinfection with enteroinvasive bacteria. Kinase activity was deter-mined with a GST-IkBa fusion protein as a substrate that can bephosphorylated by IKK on serine residues 32 and 36. A GST-IkBafusion protein with alanine substitutions at positions 32 and 36,which cannot be phosphorylated by IKK, was used in parallel todemonstrate IKK specificity. As shown in Fig. 2, IKKa and IKKbwere activated within 15 min after infection of HT-29, Caco-2, andT84 cells withS. dublin,Y. enterocoliticaor enteroinvasiveE. coli.Moreover, the time course of IKKa and IKKb activation in re-sponse to infection with these bacteria preceded the time course ofIkBa degradation, as shown in Fig. 1. IKKa and IKKb were ac-tivated weakly, if at all, above baseline after infection with non-pathogenicE. coli DH5a.

Enteroinvasive bacteria activate IL-8 and ICAM reporter genes

We previously showed that infection of HT-29, Caco-2, and T84cells with S. dublin,Y. enterocolitica, or enteroinvasiveE. coli,up-regulated IL-8 and ICAM-1 mRNA levels, as well as IL-8 se-cretion and membrane ICAM-1 expression (1, 2, 5). To determinewhether increased IL-8 and ICAM-1 expression in response tobacterial infection was paralleled by activation of those genes,HT-29 cells were transiently transfected with IL-8-luciferase (37),ICAM-1-luciferase (38) or NF-kB-luciferase transcriptional re-porter genes (33), after which cells were infected withS. dublin,Y.enterocolitica,or enteroinvasiveE. coli. As shown in Table II,infection with those enteroinvasive bacteria markedly increasedluciferase activity in cells transfected with the IL-8, ICAM-1, and23 NF-kB promoter plasmids, but not in cells transfected with a

FIGURE 1. Enteroinvasive bacteria activatesNF-kB in human colon epithelial cells. Human co-lon epithelial cell lines HT-29 (top), Caco-2 (mid-dle), or T84 (bottom) were infected with the en-teroinvasive bacterial strainsS. dublin, Y.enterocoliticaor E. coli O29:NM, the noninvasivebacterial strainE. coli DH5a, or stimulated withTNF-a as a control. NF-kB DNA binding activitywas assessed by EMSA at the indicated times up to60 min after infection. Background levels ofNF-kB binding at time 0, immediately before in-fection, are shown immediately adjacent to the col-umn for TNF-a stimulation. Immunoblots (IB)showing concurrent IkBa and IkBe levels underthe same set of conditions are provided beneatheach EMSA time point. The single major band forIkBa represents the nonphosphorylated molecule,whereas the second closely running band noted at45 and 60 min afterS. dublininfection of Caco-2cells represents phosphorylated IkBa. IkBe existsas two closely related phosphoisoforms as mostclearly shown for Caco-2 cells. Caco-2 cells showlittle, if any, response to TNF-a stimulation (44)and, consistent with this, TNF-a stimulation was aweak activator of IkBa degradation and NF-kBDNA binding in Caco-2 cells. The results are rep-resentative of three or more repeated experiments.

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control b-actin-luciferase reporter gene construct. NonpathogenicE. coli DH5a did not significantly activate expression of the testedreporter constructs.

Activation of IL-8 and ICAM-1 reporter genes in response toenteroinvasive bacterial infection is inhibited by IkBa and IKKbsuperrepressors

We asked whether activation of IKKs and degradation of IkBawere components of the signaling pathway that culminates in in-creased IL-8 and ICAM-1 expression following infection of hu-man colon epithelial cells with enteroinvasive bacteria. For thesestudies, IL-8-, ICAM-1-, and NF-kB-luciferase reporters weretransiently transfected into HT-29 cells alone, or together witheither an IKKb-AA expression plasmid that encodes a catalyticallyinactive IKKb that acts as a superrepressor or an IkBa-A32/36expression plasmid that encodes a mutant IkBa in which serineresidues at positions 32 and 36 are replaced by alanine residues toprevent its stimulus-induced phosphorylation and subsequent deg-radation. Activation of the IL-8, ICAM-1, and NF-kB transcrip-tional reporters was inhibited in cells cotransfected with the IKKb

and IkBa superrepressor plasmids (Table II), but not in cells co-transfected with control plasmid (data not shown).

The IL-8 promoter contains a binding site for NF-kB which islocated between nucleotides280 to269. However, 59 of the NF-kB-binding site, the IL-8 promoter also contains binding sites forother transcription factors (39, 46) that might play a role in tran-scriptional activation of the IL-8 promoter. We therefore deter-mined whether the nucleotide sequences in the IL-8 promoter thatencompass the NF-kB-binding site were required for the activationof the IL-8 promoter following bacterial infection, or whether 59upstream sequences that encoded an activator protein 1 (AP-1)binding site (2120 to 2126), a glucocorticoid response element(-325 to2330), and an IFN regulatory factor-1 binding site (-420to 2425) were also required. A series of IL8-CAT transcriptionalreporters containing 59 flanking region deletions, as shown in Ta-ble III, were transiently transfected into HT-29 cells, after whichcells were infected withS. dublinor Y. enterocoliticaor stimulatedwith TNF-a. Increased CAT expression in response to bacterialinfection was greatest in cells transfected with the298-CAT de-letion mutant, that contains the NF-kB binding site required for

FIGURE 2. Enteroinvasive bacteria activate IKKaand IKKb kinase activity. HT-29 (top), Caco-2 (mid-dle), and T84 (bottom) cells were infected with theenteroinvasive bacterial strainsS. dublin, Y. enteroco-litica, or E. coli O29:NM, the noninvasive bacterialstrain E. coli DH5a, or stimulated with TNF-a (20ng/ml) as a control. Whole cell lysates were obtainedbefore or up to 60 min after infection or TNF-a stim-ulation as indicated, and IKKa and IKKb were iso-lated from cell lysates by immunoprecipitation as de-scribed inMaterials and Methods. IkB kinase activityof each specific immunocomplex was determined us-ing GST- IkBa (1-54) as a substrate. A GST-IkB-A32/36 fusion protein that contains alanine substitu-tions at residues 32 and 36 and is not phosphorylatedby IKKa and IKKb was used as a specificity control,and data using this substrate are shown in the rightlane for lysates obtained 10 min after TNF-a stimu-lation (10AA). Consistent with the lack of response ofCaco-2 cells to TNF-a stimulation, IKKa and IKKbwere not activated after TNF-a stimulation of thosecells. WT, wild type.

Table II. Activation of reporter genes by enteroinvasive bacteria is inhibited by IkBa and IKKb superrepressorsa

LuciferaseReporterConstruct Superrepressor

Stimulus Added

S. dublin Y. enterocolitica E. coliO29:NM E. coli DH5a TNF-a

IL-8 None 8.36 1.9b 9.76 1.5 5.86 1.2 2.26 0.8 9.56 1.4IkBa-AA 1.7 6 0.4 1.56 0.3 2.36 0.8 1.46 0.7 0.96 0.2IKK b-AA 1.2 6 0.3 1.36 0.9 1.36 0.9 1.16 0.8 0.96 0.3

ICAM-1 None 1.86 0.1 2.16 0.2 ND ND 2.16 0.3IkBa-AA 0.8 6 0.2 0.76 0.1 ND ND 0.66 0.1IKK b-AA 0.7 6 0.1 0.76 0.1 ND ND 0.56 0.1

2XNF-kB None 2.26 0.4 3.86 0.8 2.26 0.2 1.56 0.3 2.66 0.4IkBa-AA 0.7 6 0.2 0.56 0.1 0.46 0.1 0.46 0.1 0.36 0.1IKK b-AA 0.7 6 0.2 1.66 0.3 0.96 0.2 0.96 0.2 1.06 0.3

b-Actin None 0.96 0.1 1.16 0.2 1.16 0.1 1.26 0.1 1.06 0.2

a HT-29 cells were transfected with IL-8, ICAM-1, or NF-kB-luciferase transcriptional reporters together with IkBa-AA or IKK b-AA expression vectors or a vector control(none), as indicated. From 30 to 36 h later, cells were infected with the indicated bacteria or stimulated with TNF-a for 8 h.

b Data are the mean fold induction in luciferase activity relative to uninfected or unstimulated controls. Values are the mean6 SEM of three or more experiments.

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IL-8 gene transcription and a NF-IL6 binding site, but lacks theAP-1 binding site located at nucleotides2120 to2126 and otherupstream elements. No increase in CAT activity was seen when all59 sequence upstream of28 was deleted. Parallel findings wereseen after TNF-a stimulation (Table III).

An IkBa superrepressor blocks expression of IL-8, GROa,MCP-1, ICAM-1, COX-2, NOS2, and TNF-a in response tobacterial infection

We next evaluated whether blocking NF-kB activation decreasedthe expression of endogenous genes that are known to be majorcomponents of the epithelial cell response to infection with inva-sive bacteria. HT-29 cells were infected with a recombinantreplication-deficient adenovirus containing either a IkBa superre-pressor, termed Ad5IkB-A32/36 (Ad5-IkB/AA) or the E. colib-galactosidase gene, termed Ad5LacZ, as a control. Ad5-IkB-AApartially to almost completely inhibited the up-regulated expres-sion of ICAM-1, IL-8, GROa, MCP-1, and COX-2 mRNA (Fig.3), as well as NOS2 and TNF-a mRNA (data not shown), in re-sponse to infection with enteroinvasive bacteria, but did not blockmRNA expression of TGFb1, b-actin (Fig. 3) or TGFa (data notshown).

IL-8 and GROa secretion in response toS. dublininfection orTNF-a stimulation of HT-29 cells infected with Ad5IkB-A32/36or the Ad5LacZ control virus was also assessed. As shown inTable IV, IL-8 and GROa secretion was markedly inhibited by theIkBa-AA superrepressor. The low levels of IL-8 and GROa inAd5IkB infected cultures may reflect production by the 20% re-sidual cells that were not infected by the adenovirus vector or alow level of activation of those mediators via other signal-trans-duction pathways. Further, as shown in Fig. 4, increased cell sur-face ICAM-1 expression in response to infection with enteroinva-

sive bacteria, but not in response to IFN-g stimulation, wasinhibited by the IkBa-A32/36 superrepressor.

Bacteria-induced activation of NF-kB requires intracellularsignaling molecules that are also components of pathwaysactivated by TNF receptor family members

Differences in IkB degradation in bacteria-infected, compared withTNF-a-stimulated HT-29 cells, suggested differences in the path-ways leading to activation of NF-kB in response to these stimuli.Nonetheless, many of the NF-kB target genes that are activated in

Table IV. Ad5IkB-AA superrepressor inhibits IL-8 and GROa secretion byS. dublin-infected or TNF-a-stimulated HT-29 cellsa

Additions toCulture

IL-8 (ng/ml) GROa (ng/ml)

Ad5IkB-AA Ad5LacZ

Ad5IkB-AA Ad12LacZ

None 1.26 0.7 1.26 0.7 0.76 0.1 1.16 0.1S. dublin 8.66 2.5 89.66 1.6 1.36 0.1 11.66 0.5TNF-a 3.06 1.8 143.06 20.3 1.06 0.1 36.66 0.3

a Monolayers of HT-29 cells were infected with Ad5IkB-AA or Ad5LacZ as indicated. Twenty-four hours later, cells wereinfected withS. dublin(5 3 108 bacteria/well) or stimulated with TNF-a (20 ng/ml) as described inMaterials and Methods. Culturesupernatants were collected 10 h later, and cytokine concentrations were determined by ELISA. Results are the mean6 SEM oftriplicate cultures from a single experiment. Similar results were obtained in two additional experiments.

Table III. Activity of IL-8 transcriptional reporter deletion mutants inHT-29 cells infected with enteroinvasive bacteria or stimulated withTNF-aa

IL-8-CATConstruct (bp)

S. dublinInfected

Y. enterocoliticaInfected

TNF-aStimulated

21481 4.96 1.8b 7.16 2.1 9.06 3.02546 3.46 1.0 4.96 1.3 5.56 1.42272 3.46 0.7 3.46 0.8 4.86 1.8298 8.66 1.5 10.76 1.9 14.36 5.528 0.66 0.1 0.96 0.2 0.66 0.1

a HT-29 cells were transiently transfected with the various 59-deletion mutantIL-8-CAT transcriptional reporters, as indicated, and infected withS. dublinor Y.enterocoliticaor stimulated with TNF-a (20 ng/ml) as described inMaterials andMethods.

b CAT activity is given as fold increase over unstimulated cells. Data are means6SEM of three or more experiments.

FIGURE 3. An IkBa superrepressor inhibits the up-regulated expres-sion of major components of the host’s endogenous epithelial cell proin-flammatory gene program. HT-29 cells were infected with Ad5IkB-A32/36which expresses an IkBa superrepressor or, as a control, Ad5LacZ whichexpressesb-galactosidase. Twenty-four hours after viral infection, cellswere left untreated or were infected withS. dublin or stimulated withTNF-a as a positive control to up-regulate NF-kB target genes. Threehours later, the expression of genes known to be components of the proin-flammatory program in bacteria-infected human intestinal epithelial cells,and control genes that are not components of that program, were assessedby RT-PCR. The IkBa superrepressor inhibited, partially to almost com-pletely, the up-regulated expression of IL-8, GROa, MCP-1, ICAM-1, andCOX-2 (Fig. 3) as well as NOS2 and TNF-a (not shown) which are com-ponents of the epithelial cell proinflammatory program. In contrast, thesuperrepressor did not inhibit expression of the control genes TGF-b1 orb-actin or TGF-a (not shown) inS. dublin-infected or TNF-a-stimulatedcells. The results are representative of two or more repeated experiments.

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response to bacterial infection of human intestinal epithelial cells,including IL-8, are also activated by signaling through TNF, IL-1or LTbR. We assessed, therefore, whether molecules that play amore proximal role in the signal transduction pathway leading toIKK activation following agonist stimulation via TNF receptors,such as TRAF2 or NIK, a mitogen-activated MAP-3 kinase whichmediates the phosphorylation and activation of IKK (21, 22), arealso required for activation of the NF-kB target gene IL-8 in bac-

teria infected cells. For these studies, an IL-8-luciferase transcrip-tional reporter was cotransfected into HT-29 cells with a TRAF2superrepressor plasmid (30), or with a plasmid that expresses amutant of NIK that is catalytically inactive (21). In addition, somecultures were cotransfected with the IL-8-luciferase reporter to-gether with a plasmid expressing A20, a protein that blocksTRAF2-mediated NF-kB activation (36) or with a plasmid ex-pressing a superrepressor of TRAF5, a protein that is important forthe activation of NF-kB following signal transduction through theLTbR (31). Cultures were subsequently infected with enteroinva-siveS. dublin,Y. enterocolitica, or enteroinvasiveE. coli or stim-ulated with TNF-a as a control. As shown in Table V, increasedluciferase activity in response to stimulation of cells with TNF-awas markedly inhibited by blocking TRAF2, and to a lesser extentby blocking NIK or TRAF5. Blocking of NIK also partially in-hibited bacteria induced activation of the IL-8 reporter. Inhibitionof TRAF5 was as effective as inhibition of TRAF2 in blockingS.dublin or enteroinvasiveE. coli, but notY. enterocoliticainducedactivation of the IL-8 reporter. Consistent with the results ofTRAF2 inhibition described above, expression of A20, which in-hibits TRAF2 mediated NF-kB activation, also inhibited bacteria,and TNF-a induced activation of the IL-8 reporter.

DiscussionSalmonella, enteroinvasiveE. coli, andYersiniause different strat-egies, and activate different signaling pathways, to induce theiruptake into nonphagocytic epithelial cells (10, 32). Nonetheless, inthe early period following infection with those pathogens, intesti-nal epithelial cells up-regulate the expression of a conserved set ofproinflammatory genes and the production of mediators that cansignal the onset of mucosal inflammation and alter epithelial cellfunctions (1–5, 7). The studies herein define NF-kB as a centralregulator for the activation of essential components of the intesti-nal epithelial cell innate immune response following infection witha spectrum of enteroinvasive bacterial pathogens.

IKK (16, 18, 19) was shown to be a key intermediate in theepithelial cell signal transduction pathway leading to NF-kB acti-vation following infection with enteroinvasive bacteria. AlthoughIKK a and IKKb were activated with a similar time course, anIKK b superrepressor alone was sufficient to inhibit the activationof IL-8 and ICAM-1 transcriptional reporters and was moreefficient than an IKKa superrepressor in this regard (data notshown). These findings are consistent with the physiologic roleIKK b is known to have in IkB phosphorylation and NF-kB

FIGURE 4. Flow cytometric analysis of ICAM-1 expression by HT-29cells. HT-29 cells were infected with Ad5IkB-A32/36 (Ad5IkB) whichexpresses an IkBa superrepressor (middle column), with Ad5LacZ whichexpressesb-galactosidase (right column), or were not infected with ade-novirus (control,left column). Twenty-four hours after viral infection, cellswere infected withS. dublin(top row) or Y. enterocolitica(second row) orstimulated with TNF-a (20 ng/ml,third row) or IFN-g (40 ng/ml,bottomrow). After 9 h, cells were detached from the plates, stained, and analyzedby flow cytometry as outlined inMaterials and Methods. Shaded area ineach panel represents ICAM-1 staining of cells that were not bacteria in-fected or stimulated with cytokines. The unshaded area outlined by thedarker line represents ICAM-1 staining in bacteria-infected or cytokine-stimulated cells. As shown, the IkB superrepressor inhibited increasedICAM-1 expression in bacteria-infected or TNF-a-stimulated cells, but notin IFN-g-stimulated cells.

Table V. Activation of IL-8 luciferase reporter gene by enteroinvasive bacteria is inhibited by NIK, TRAF2,and TRAF5 superrepressorsa

Superrepressorsc

Stimulus Added

S. dublin Y. enterocolitica E. coliO29:NM TNF-a

None 7.446 2.32b 9.236 1.1 6.356 1.07 6.696 0.64NIK 2.85 6 0.02 5.996 0.30 2.496 0.93 4.596 0.28TRAF2 2.466 0.29 2.876 0.47 1.636 0.59 1.726 0.27TRAF5 2.696 0.28 4.396 0.25 1.196 0.03 3.696 0.65

a HT-29 cells were transfected with an IL-8-luciferase transcriptional reporter together with an expression vector for a catalyticmutant of NIK, or expression vectors for superrepressors of TRAF2 or TRAF5 or a vector control (none), as indicated. From 30 to36 h later, cells were infected with the indicated bacteria or stimulated with TNF-a for 8 h.

b Data are the mean fold induction in luciferase activity relative to uninfected or unstimulated cells. Values are the mean6 SEMof data from three experiments.

c In a separate experiment, expression of A20 was shown to inhibit both bacteria and TNF-a-induced activation of the IL-8reporter. Mean fold induction in luciferase activity forS. dublin,Y. enterocolitica,E. coli O29:NM, and TNF-a-stimulated cells was1.516 0.08, 1.976 0.12, 1.426 0.01, and 1.086 0.01, respectively.

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activation(18, 19, 45, 46). IKKb in the large m.w. IKK com-plex may function either as an IKKb homo- or IKKabheterooligomer (18, 19, 45, 46).

The promoter regions of the epithelial cell proinflammatorygenes studied herein contain binding sites for several transcriptionfactors in addition to NF-kB. For example, the IL-8 promoter hasbinding sites for AP-1 and NF-IL6 that are functionally importantfor IL-8 gene activation in several cell types (39, 47). However, theAP-1 binding site was not required forS. dublinor Y. enteroco-litica induced IL-8 promoter activity in the three intestinal epithe-lial cell lines studied herein, because a transcriptional IL-8 reporterthat lacks the AP-1 binding site was readily activated in those cellsafter infection with those bacteria. In contrast, there appears to bea requirement for both NF-kB and AP-1 for transcriptional acti-vation of the IL-8 promoter inS. typhimurium-infected Henle-407cells (48). Although NF-kB has an essential role in integrating thehost intestinal epithelial cell response to infection with enteroin-vasive bacteria, additional transcription factors are known to pos-itively modify the transcription of many of the genes that comprisethe epithelial cell innate immune response.

NF-kB was activated in three human colon epithelial cell linesinfected with three different stains of enteroinvasive bacteria, butnot after infection with noninvasiveE. coli. Consistent with this,NF-kB was not activated in T84 cells cultured with a differentnonpathogenic strain ofE. coli although activation was noted incells infected with an enteropathogenicE. coli (49). Activation ofNF-kB in the cytoplasm involves the inducible phosphorylation ofIkBs, which then undergo ubiquitin-mediated proteolysis, therebyreleasing NF-kB dimers to translocate to the nucleus (33, 42, 50).We noted marked differences in the extent of IkBa and IkBe deg-radation among the cell lines, with degradation being more com-plete in enteroinvasive bacteria-infected Caco-2 than in HT-29 orT84 cells. This finding parallels that reported for NF-kB activationand IkBa degradation after IL-1b stimulation of the same celllines (15), in which case incomplete degradation of IkBa was alsosufficient for significant activation of NF-kB.

In contrast to bacterial infection (data herein) or IL-1b stimu-lation (15), degradation of IkBa was almost complete in TNF-a-stimulated HT-29 and T84 cells. The difference between bacterialinfection and TNF stimulation may reflect the fact that only afraction of cells are infected by enteroinvasive bacteria (e.g., 10–50% of HT-29 and T84 cells are infected byS. dublinat the MOI’sused herein). In this case, even complete degradation of IkBa inbacteria-infected cells would be obscured by the lack of degrada-tion in uninfected cells. This would not apply to a soluble cytokinelike TNF-a, which presumably can stimulate a larger fraction ofthe cells. Alternatively, differences in IkBa degradation in re-sponse to bacterial infection, compared with TNF-a stimulation,may reflect an interplay between the different signaling pathwaysactivated by these stimuli, and the relative importance of thosepathways, as also suggested by our findings using superrepressorsof TRAF2 and TRAF5.

Infection of Caco2 cells with bacteria but not stimulation withTNF-a activated NF-kB, indicating that activation of NF-kB likelyis independent of signaling through the extracellular domain of theTNF receptor. Whereas our findings with bacterial infection ofhuman intestinal cells are consistent with those showing activationof NF-kB in a human cervical epithelial cell line, HeLa, infectedwith Shigella flexneri(51), our findings withY. enterocoliticain-fection of human intestinal epithelial cells differ markedly fromthose in mouse macrophages, whereY. enterocoliticapreventedNF-kB activation (52)

The various IkBs can differentially associate and regulate theactivation of NF-kB (53) and the transcriptional activation of var-

ious NF-kB target genes by virtue of binding to different popula-tions of NF-kB dimers in the cytoplasm. IkBe is widely expressedin different human tissues, is known to interact preferentially withp65 and c-Rel members and appears to affect a subset of NF-kBgenes apparently regulated by p65 homodimers (54, 55). ICAM-1and GM-CSF promoters contain NF-kB-binding sites that bindonly p65/c-Rel heterodimers in vitro (56), and the NF-kB site ofthe IL-8 promoter optimally binds and is activated by p65 ho-modimers rather than p50 homodimers or p50/p65 heterodimers(57). Our finding of partial IkBe degradation after bacterial infec-tion of intestinal epithelial cells suggests a role for IkBe in theactivation of several NF-kB target genes that are up-regulated inresponse to bacterial infection of intestinal epithelial cells. A sim-ilar situation may exist for the antimicrobial response inDrosoph-ila, where different NF-kB family members are differentially ac-tivated and imported into the nucleus where they activate selectivepeptide (cecropin) genes (58).

Human intestinal epithelial cells rapidly activate NF-kB and aproinflammatory gene program within a few hours of infectionwith enteroinvasive bacteria. However, in the later period afterinfection (e.g., 12–18 h) human colon epithelial cell lines undergoapoptosis (59). The delayed onset of apoptosis may provide in-vading bacteria sufficient time to adapt to the intracellular epithe-lial cell environment and multiply, before invading deeper mucosallayers. Moreover, the delayed onset of apoptosis might be partlyexplained by the activation of NF-kB after bacterial entry, becauseNF-kB target genes suppress signals for cell death as shown formouse cell lines stimulated with TNF-a (60, 61) and for endothe-lial cells infected byRickettsia rickettsii(62).

NF-kB activation in bacteria-infected cells appears to be inde-pendent of signaling through the extracellular domain of the TNFreceptor. Nonetheless, our data indicate that molecules that areintegral components of the signal transduction pathway leading toNF-kB activation after signaling through members of the TNFreceptor family were involved in signal transduction leading to theactivation of the NF-kB target gene, IL-8, in bacteria-infectedcells. Activation of IKKa and IKKb requires their phosphoryla-tion, which has been reported to be mediated by the MAP3 ki-nases, NIK and MEKK-1 (20–24). As shown herein, NIK appearsto play a role in NF-kB activation after bacterial infection, giventhat overexpression of a catalytically inactive NIK protein partiallyinhibited transcriptional activation of an IL-8 reporter in responseto bacterial infection. Further upstream in the signal transductionpathway activated by TNF-a, NIK interacts with TRAF2, an adap-tor protein known to associate with the TNF receptor family ofproteins (23, 30, 63). Recently, TRAF2 was reported to be in-volved in TNF-a and IL-1b signaling cascades leading to NF-kBactivation and IL-8 expression in HT-29 cells (64). Interference ofTRAF2 signaling by transfection of a superrepressor of TRAF2, orwith A20, which interacts with TRAF2 and blocks TRAF2-medi-ated NF-kB activation (36), markedly inhibited IL-8 reporter geneactivity in response to bacterial infection. Blocking TRAF5, anadaptor molecule important in signaling through other TNF recep-tor family members (e.g., LTbR), also partially blocked IL-8 re-porter gene activity. Taken together, our findings suggest that bac-terial infection may activate a number of different intracellularsignaling pathways also used by TNF and perhaps IL-1 receptorfamily members, that culminate in the activation of NF-kB and itstarget genes.

Intestinal epithelial cells act as sensors of microbial infectionand produce proinflammatory signals that can activate the host’smucosal inflammatory response. The data herein demonstrate thatNF-kB is an essential transcription factor for integrating the hostepithelial cell proinflammatory response to infection with a

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spectrum of enteroinvasive bacteria. Studies in a murine model ofintestinal inflammation (65) and in human intestinal xenograftsinfected withE. histolytica(66) showed that blocking NF-kB ac-tivation in the intestinal mucosa can markedly decrease intestinalinflammation. Our studies in epithelial cells indicate that signaltransduction through NF-kB is a key element in the activation ofthe epithelial cell innate immune response after bacterial infectionand suggest novel therapeutic targets for the prevention and treat-ment of intestinal inflammation.

AcknowledgmentsWe thank J. Smith for expert technical help and R. Lara for assistance inpreparation of the manuscript. We also thank Drs. M. Karin, F. Mercurio,D. Goeddel, H. Nakano, M. Levrero, G. Natoli, Z.G. Liu, andN. Rice for gifts of plasmids and Ab, and D. Brenner for sharing therecombinant adenoviruses.

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