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B Through a Lipopolysaccharide-Dependent Innate Intracellular Response and Leads to IL-8 Expression in Epithelial Cells1
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Unité de Pathogénie Microbienne Moléculaire, Institut National de la Santé et de la Recherche Médicale, Unité 389, and
Unité de Biologie Moléculaire de l’Expression Génique, Unité de Recherche Associée 1773 Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is an
important transcriptional regulator of genes involved in inflammation,
we investigated the role of this transcription factor during S.
flexneri infection of epithelial cells. Infection of HeLa cells
with invasive S. flexneri induced NF-B DNA-binding
activity; noninvasive S. flexneri strains did not lead
to this activation. The pathway leading to NF-B activation by
invasive S. flexneri involved the kinases,
NF-B-inducing kinase, IB kinase-1, and IB kinase-2. NF-B
activation was linked to inflammation, because invasive S.
flexneri activated an IL-8 promoter-driven reporter gene, and
the B site within this promoter was indispensable for its induction.
Microinjection of bacterial culture supernatants into HeLa cells
suggested that LPS is responsible for NF-B activation by S.
flexneri infection. In conclusion, the eukaryotic transcription
factor NF-B was activated during S. flexneri
infection of epithelial cells, which suggests a role for this
transcriptional regulator in modulating the immune response during
infection in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although epithelial cells lining the colon are bathed in bacteria and bacterial products, they remain by and large refractory to the presence of these normally inflammatory agents. Epithelial cells are generally unresponsive to LPS (9), and therefore colonic epithelial cells do not detect the potential threat from the normal bacterial flora. Detection of the resident bacterial flora by colonic epithelial cells would have serious consequences, as the colon would be in a state of chronic inflammation. These cells, however, are not completely refractory to bacterial stimulation. Recent studies have demonstrated that epithelial cells produce proinflammatory cytokines, but this occurs only during infection by certain pathogens (10). In particular, IL-8 appears to be a major secreted product of infected epithelial cells (11). This proinflammatory chemokine is a potent chemoattractant for polymorphonuclear cells and can direct recruitment of these cells into the infected site and their infiltration of the epithelial layer (7, 12). The key role played by IL-8 during S. flexneri infection was recently demonstrated in the rabbit model of shigellosis. Using immunohistochemistry on sections of S. flexneri-infected ligated ileal loops, intense IL-8 staining was observed throughout the epithelial layer, whereas rabbits infected with a noninvasive strain showed few IL-8-positive epithelial cells. Pretreatment of rabbits with Abs to IL-8 before bacterial challenge dramatically reduced both fluid accumulation within the infected ileal loop and the level of inflammation in the infected tissue (13).
An important transcriptional regulator of IL-8 gene expression is
NF-B (14). NF-B is an ubiquitous transcription
factor involved in the inducible expression of a number of genes whose
products, including many cytokines/chemokines, cell adhesion molecules,
and acute phase response proteins, are involved in the inflammatory
response (for a review, see Ref. 15). In most cell types
NF-B is held latent in the cytoplasm through its binding to
inhibitory proteins, called
IBs,4 that mask the
nuclear localization signal on NF-B and thus prevent its nuclear
translocation. Most of the signaling pathways stimulated by known
activators of NF-B converge on the activation of a kinase complex
called the IKK signalsome. Once activated, this complex, which consists
of the two kinases, IKK1 and IKK2 (16, 17, 18), and the
regulatory subunit NEMO (IKK
) (19, 20, 21),
phosphorylates residues Ser32 and
Ser36 on the inhibitory IB
protein
(22). This phosphorylation event leads to
polyubiquitination of the IB and subsequent degradation of the
protein by the 26S proteasome (23). NF-B is then free
to translocate to the nucleus, where it binds to specific B elements
in the promoter region of responsive genes (15).
A number of studies have shown activation of NF-B in epithelial
cells by bacterial pathogens, including members of
Salmonella (24), Neisseria
(25), enteropathogenic Escherichia coli (EPEC)
(26), and S. flexneri (27).
Recently, the signaling system leading to NF-B activation by
different enteroinvasive bacteria was investigated (28);
however, neither the pathway nor the bacterial products responsible for
NF-B activation have been clearly elucidated. Because of its
important role in inflammation, we were interested in examining the
signaling pathway leading to the induction of NF-B in epithelial
cells infected by S. flexneri and identifying the bacterial
products that might activate this transcription factor. In addition, we
have examined the role played by NF-B in the induction of the
proinflammatory chemokine, IL-8.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M90T is a wild-type invasive strain of S. flexneri serotype 5a. BS176 is a noninvasive variant of M90T cured of the 220kB virulence plasmid. SC301 and SC300 are derivatives of M90T and BS176, respectively, harboring the plasmid pIL22 that encodes the afimbrial adhesin from uropathogenic Escherichia coli (29). These strains have been described previously (30). The ipa mutants as well as the ipgD and icsA mutants have been described previously (31, 32, 33). The E. coli strain ß2098, which is deficient in the production of N-formylated proteins, was provided by Dr. Didier Mazel (Institut Pasteur, Paris, France), and has been described previously (34). The Bacillus subtilis strain was provided by Dr. Agnes Fouet (Institut Pasteur).
Bacteria-free culture supernatants were prepared by centrifuging overnight cultures at 3000 x g for 15 min and passing the recovered supernatants through a 0.2-µm pore size filter. Phenol-water extracts of LPS from S. flexneri 1a and E. coli O111:B4 were purchased from Sigma (St. Louis, MO). LPS from S. flexneri 5a was purified by the method of Westphal (35). Rhodobacter sphaeroides LPS was purchased from List Biochemicals (Campbell, CA). LPSs were diluted in microinjection buffer (25 mM Tris (pH 7.4), 100 mM KCl, 5 mM MgCl2, and 1 mM EGTA) and sonicated for 30 s before use.
HeLa cells were grown routinely in MEM (Life Technologies, Paisley, U.K.) with 10% FCS (Life Technologies) and supplemented with penicillin and streptomycin (Life Technologies). The intestinal cell line Caco-2 was grown in DMEM (Life Technologies) with 10% FCS and antibiotics. The human embryonic kidney cell line 293 was cultured in MEM with 10% FCS and antibiotics.
Antisera to NEMO and IB were provided by Dr. Robert Weil
(Institut Pasteur), and antisera to p50 and c-Rel were supplied by Dr.
Nancy Rice (Frederick, MD) and have been described previously
(19, 36). Antisera to p65, IBß, and IB
were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-dextran
(10,000 m.w.) was obtained from Molecular Probes (Eugene, OR).
Recombinant human IL-1 and TNF- were purchased from R&D Systems
Europe (Oxon, U.K.). Genistein, GF109203X, H7, wortmannin, aLLnL, and
MG132 were obtained from Biomol (Plymouth Meeting, PA), and all other
reagents were obtained from Sigma unless otherwise specified.
Infection of HeLa cells
Overnight cultures of S. flexneri strains were diluted 1/100 in trypticase soy broth (Diagnostics Pasteur, Marnes la Coquette, France) and grown to midexponential phase at 37°C (37). HeLa cells that had been serum-starved overnight were washed three times, and the medium was replaced with antibiotic-free, serum-free MEM. Bacteria were then added to the cells and incubated for 10 min at room temperature to allow adherence of bacteria to cells under noninvasive conditions. Plates of infected cells were moved to 37°C and incubated for an additional 15 min. Subsequently, cells were washed five times, and the medium was replaced with MEM containing 50 µg/ml gentamicin to kill extracellular bacteria. Incubations were then conducted for the indicated periods of time. The number of intracellular bacteria was determined by lysing HeLa cells with 0.5% sodium deoxycholate in physiological saline and subsequent plating of serially diluted samples on TCA plates.
Plasmids and transient transfections
Plasmids expressing GST-IB1–72(1–72) wild-type and
GST-IB1–72(1–72)S32A/S36A mutant polypeptides have been described
previously (21). Purification of the GST-linked proteins
was conducted using standard protocols (38).
Plasmids expressing dominant negative forms of IKK1 and IKK2 were
provided by Dr. Michael Karin (University of California a San Diego, La
Jolla, CA) and described previously (16). The NIK
KK429–430AA expression plasmid (39) was provided by Dr.
David Wallach (The Weizmann Institute, Rehovot, Israel). The NF-B
reporter Ig-luciferase has been described previously
(40). The wild-type IL-8 promoter-luciferase construct and
IL-8 promoter constructs with site-directed mutations in AP-1, NF-B,
or NF-IL-6 transcription factor binding sites were provided by Drs.
Andrew Keates and Ciaran Kelly (Beth Israel Deaconess Medical Center,
Boston, MA).
For transfections, HeLa cells were plated in six-well plates at a
density of 1 x 105 cells/ml and transfected
the following day using FuGene reagent (Roche, Indianapolis, IN) as
recommended by the manufacturer, with 0.5 µg of Ig-luciferase
reporter and either the vector or the effector plasmid. Cotransfection
with 0.5 µg of a ß-galactosidase reporter plasmid was used to
normalize transfection efficiencies. Cells were infected with bacteria
as described above or treated with TNF- (100 ng/ml) or IL-1 (10
ng/ml) 36 h posttransfection. Following 5 h with the
indicated treatments, cells were lysed in luciferase lysis buffer (25
mM Tris (pH 7.4), 8 mM MgCl2, 1 mM DTT, 1% (v/v)
Triton X-100, and 15% (v/v) glycerol), and luciferase activity was
determined. Experiments were performed in duplicate or triplicate and
were repeated at least three times. Values represent the mean ±
SD. Student’s t test was used to determine statistical
significance between the different groups.
Preparation of nuclear and cytoplasmic extracts
Following the indicated treatments, nuclear and cytoplasmic extracts were prepared as previously described (41). Cells were washed twice in ice-cold PBS and scraped from the dishes, and cell pellets were resuspended in hypotonic buffer (20 mM HEPES (pH 7.8), 10 mM KCl, 1 mM DTT, 0.5 mM EDTA, 0.5 mM EGTA, and 1 µg/ml each of leupeptin, aprotinin, and pepstatin). Following 10-min incubation on ice, Nonidet P-40 was added to a final concentration of 1%, and samples were centrifuged for 20 s in a microfuge at maximum speed. The supernatant, which represented the cytoplasmic fraction, was recovered and stored at -80°C until further use. The nuclear pellet was washed briefly with hypotonic buffer and resuspended in extraction buffer (10 mM HEPES (pH 7.8), 400 mM NaCl, 0.1 mM EDTA, 1 mM DTT, supplementary protease inhibitors, and 25% glycerol). Following a 30-min incubation at 4°C with occasional vortex mixing, samples were centrifuged at maximum speed for 5 min. The supernatant, containing the nuclear fraction, was recovered and immediately frozen at -80°C until use.
EMSA
Five micrograms of nuclear extracts were combined with binding
buffer (10 mM HEPES (pH 7.8), 100 mM NaCl, 1 mM EDTA, and 10%
glycerol), 1 µg poly(dI-dC), and 0.5 ng of
32P-labeled DNA probe corresponding to the B
site of the H-2KB promoter (42). Where indicated, EMSAs
were also performed using a 32P-labeled DNA probe
corresponding to the region between bp -84 and -68 of the IL-8
promoter: 5'-TCGTGGAATTTCCTCTG (14). Following 30-min
incubation at room temperature, samples were run on a 5%
polyacrylamide gel in 1x TBE. The gel was dried, exposed to a
PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA), and analyzed
using ImageQuant software (Molecular Dynamics).
Western blot analysis
For detection of IB proteins, cytoplasmic fractions equalized
based on protein content were run on 12% SDS-PAGE gels and transferred
to nitrocellulose, and specific Ab binding was revealed using an
enhanced chemiluminescence detection system (Amersham, Aylesbury,
U.K.).
Immunoprecipitation and kinase assays
For IKK kinase assays, the method described previously was
followed (19). Briefly, cytoplasmic extracts were
subjected to immunoprecipitation with anti-NEMO in TNT buffer (50
mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 100 µM
Na3VO4, and supplementary
protease inhibitors). Samples were mixed with protein G-Sepharose beads
for 1 h at 4°C on a wheel and then washed three times with TNT
and three times with kinase buffer (20 mM HEPES (pH 7.5), 50 mM NaCl,
10 mM MgCl2, 100 µM
Na3VO4, 20 mM
ß-glycerophosphate, 2 mM DTT, and 20 µM ATP). Kinase reactions were
conducted at 30°C for 30 min, using 5 µCi
[-32P]ATP and GST-IB1–72(1–72) wild-type
or GST-IB1–72(1–72)S32A/S36A mutant polypeptides as substrates.
Reaction products were run on 12% SDS-PAGE gels and revealed by
phosphorimaging.
IL-8 protein levels
To measure IL-8 secreted from Shigella-infected HeLa
cells, cells were seeded into 12-well dishes and infected as described
above or treated with 100 ng/ml TNF- for various times. IL-8 protein
levels were determined by ELISA using Abs and human recombinant IL-8
from R&D Systems Europe.
Immunodepletion of bacterial supernatants
Fifteen microliters of bacteria-free supernatants from overnight cultures of wild-type S. flexneri serotype 5a were mixed with 5 µg of either dimeric IgA specific for S. flexneri 5a LPS or IgA specific for Salmonella typhimurium LPS for 1 h at 4°C. These Abs have been described previously (43). Protein G-Sepharose beads were then added, and the samples were left rotating overnight at 4°C. Samples were spun at 14,000 rpm in an Eppendorf microfuge, and 5 µg of additional Abs were then added to the supernatants. This process was repeated for a total of three cycles of immunodepletion; each of the subsequent incubations with the Abs was conducted for 1 h. A significant decrease (>4-fold) in the amount of LPS in the supernatants immunodepleted with the S. flexneri LPS 5a-specific Abs, but not the S. typhimurium LPS-specific Abs, was confirmed by SDS-PAGE and silver staining (44).
Microinjection of HeLa and Caco-2 cells
HeLa or Caco-2 cells were plated onto coverslips, and
microinjection was performed the following day in serum-free
conditions. Samples containing FITC-dextran were loaded into glass
capillary micropipettes. Approximately 100 cells were microinjected
within a 5-min period using a Narishige 200 microinjection system
(Narishige Group, London, U.K.) and an IMT-2 inverted microscope
(Olympus Optical, Tokyo, Japan). Following incubation for 30 min at
37°C in 5% CO2, cells were fixed with 3.7%
paraformaldehyde in PBS for 10 min, permeabilized with 0.5% Triton-X
in PBS containing 2% BSA, and subsequently stained with Abs to the p65
subunit of NF-B. Following labeling with Cy3-linked anti-rabbit
Abs (Jackson ImmunoResearch Laboratory, West Grove, PA), coverslips
were mounted and viewed with a conventional immunofluorescence
microscope (BX50, Olympus Optical).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B response in HeLa cells
The NF-B response in S. flexneri-infected HeLa cells
was investigated by EMSA. Fig. 1
A shows that wild-type
S. flexneri, M90T, expressing the afrimbrial adhesin
activated NF-B DNA binding activity in HeLa cells 45 min
postinfection. This activation was specific to invasive S.
flexneri and not to mere adherence to HeLa cells, because the
noninvasive plasmid-cured strain also expressing the adhesin did not
stimulate NF-B DNA binding activity. In addition, these studies were
performed in the absence of serum to avoid LPS activation through
soluble CD14 and the LPS binding protein (9). Moreover,
E. coli LPS or S. flexneri 5a LPS added
extracellularly at concentrations up to 10 µg/ml did not activate
NF-B (data not shown). NF-B DNA binding induced by S.
flexneri was specific, because the addition of excess unlabeled
probe competed the binding activity. In contrast, the addition of
excess unlabeled DNA probe corresponding to the STAT3 binding site did
not affect the NF-B complex induced by the bacteria (data not
shown).
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B DNA binding activity. As few as 104
CFU/106 HeLa cells of intracellular S.
flexneri (a multiplicity of infection of 0.1) were capable of
stimulating NF-B DNA binding activity; however, at 45 min
postinfection, this activation was relatively weak (data not shown). An
initial inoculum corresponding to an MOI of 50 resulted in
107 CFU/106 HeLa cells of
intracellular S. flexneri and a strong activation of NF-B
following 45 min of infection that was comparable to the stimulation
induced by TNF-. Therefore, this inoculum was used in all other
subsequent experiments.
To ensure that the S. flexneri-induced NF-B response was
not limited to HeLa cells, the intestinal cell line Caco-2 and the
human embryonic kidney cell line 293 were infected, and NF-B DNA
binding activity was examined by EMSA. S. flexneri
stimulated NF-B DNA binding activity in CaCo-2 cells with a similar
time course as that observed in HeLa cells (data not shown). 293 cells,
recently shown to be unresponsive to high concentrations of LPS in the
absence of serum (45), showed similar NF-B induction in
response to S. flexneri infection compared with HeLa
epithelial cells (data not shown).
Supershift experiments were performed with Abs to the various NF-B
proteins to determine which components of the S.
flexneri-induced NF-B complex were binding to the consensus
B DNA element from the promoter region of the H-2KB gene. Two
distinct DNA binding complexes were evident in extracts prepared from
S. flexneri-infected HeLa cells (Fig. 1
B). These
two complexes were resolved when the gel was run for extended time
periods (i.e., 2.5 h vs 1.5 h). Addition of Abs to the p50
component of NF-B shifted both complexes, whereas Abs to the p65
(RelA) protein shifted the top complex only. Abs to c-Rel did not lead
to any appreciable change in the S. flexneri-induced NF-B
complexes (data not shown). This indicated that S. flexneri
infection resulted in the formation of NF-B complexes that were
composed mainly of p50/p65 heterodimers (the complex commonly referred
to as NF-B) and homodimers of the non-trans-activating
complex p50/p50.
To fully characterize the NF-B response in HeLa epithelial cells
infected with wild-type S. flexneri, time-course experiments
examining NF-B DNA binding by EMSA were performed and compared with
the time course of NF-B activation with TNF-. As shown in Fig. 2A, NF-B DNA binding
activity was induced after 45 min of S. flexneri infection
that was sustained 4 h postinfection and was evident 6 h
postinfection, which was the latest time point examined (data not
shown). In contrast, NF-B DNA binding activity was induced 15 min
after addition of TNF- and was down-regulated by 2 h
posttreatment (Fig. 2A). To rule out the possibility that
S. flexneri-infected cells were producing a signal that was,
in turn, responsible for NF-B activation, time-course studies
similar to those described above were conducted in the presence of 20
µg/ml cycloheximide to block host cell protein synthesis.
Cycloheximide had no effect on the activation of NF-B by S.
flexneri, indicating that infection directly activates NF-B
(data not shown).
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B
proteins in the cytoplasm of infected HeLa cells and cells treated with
TNF- (Fig. 2B). S. flexneri infection induced
the degradation of IB, IBß, and IB 45 min
postinfection, and this degradation was sustained 4 h
postinfection, thus paralleling what was observed for NF-B DNA
binding activity induced by S. flexneri infection seen in
Fig. 2A. TNF- induced degradation of IB, IBß,
and IB 15 min posttreatment. Although levels of IBß and
IB remained low 4 h posttreatment, levels of IB
increased after 2-h treatment with TNF-.
Characterization of the signaling pathway leading to NF-B
activation in S. flexneri-infected HeLa
cells
To characterize the signaling pathway induced by invasive S.
flexneri leading to NF-B activation, a number of inhibitors
were used to investigate whether they could block NF-B DNA binding
activity in response to S. flexneri infection and TNF-
treatment. Neither inhibitors of protein tyrosine kinases, including
genistein and herbimycin, nor inhibitors of protein kinases, including
H7 and GF109203X, had any effect on NF-B DNA binding activity
induced by S. flexneri infection or TNF- treatment. In
addition, wortmannin, an inhibitor of PI3 kinase recently shown to
inhibit pervanadate-induced (46) and IL-1-induced
(47) NF-B activity, did not affect either S.
flexneri- or TNF--induced NF-B DNA binding activity. NF-B
activation induced by both S. flexneri infection and TNF-
treatment, however, was inhibited by the anti-oxidants,
N-acetyl-cysteine and pyrrolidine dithiocarbamate, as well
as the proteasome inhibitors, aLLnL and MG132. These data suggested
that, like TNF-, NF-B activation by S. flexneri
infection involved the classical pathway, resulting in the
antioxidant-sensitive phosphorylation of IBs through the IKK
signalsome and subsequent ubiquitination and degradation of this
protein by the 26S proteasome.
To more thoroughly examine this S. flexneri-activated
signaling pathway, an approach was taken in which cells were
transiently transfected with vector constructs expressing dominant
negative versions of signaling molecules that have been identified as
key regulators of NF-B activation in response to cytokines. Since
IKK1 and IKK2 have been shown to be important kinases within the IKK
signalsome, we examined the roles of these two proteins in S.
flexneri-induced NF-B activity on an NF-B-responsive
luciferase gene reporter. The IKK1(KM) and IKK2(KA) mutant proteins are
catalytically inactive, since they have methionine or alanine,
respectively, substituted for the lysine codon at position 44, which
presumably results in defective ATP binding (16). Fig. 3A shows the effects of
overexpression of IKK1(KM) and IKK2(KA) in HeLa cells infected with
S. flexneri or treated with TNF-. Expression of IKK1(KM)
or IKK2(KA) in infected cells resulted in a 76 ± 7 or 69 ±
12% decrease, respectively in luciferase activity compared with that
in cells transfected with the vector alone (Fig. 3A). As
previously reported (16), TNF--treated cells expressing
IKK1(KM) or IKK2(KA) showed a 63 ± 8 or 60 ± 20% decrease,
respectively, in relative luciferase activity compared with cells
transfected with the vector alone (Fig. 3A). Transfection of
both IKK1(KM) and IKK2(KA) led to similar decreases in luciferase
activity in S. flexneri-infected cells (77 ± 9%) and
TNF--treated cells (70 ± 14%), suggesting that either of
these dominant negative proteins expressed alone resulted in maximum
inhibition of NF-B activity.
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B in S.
flexneri- and TNF--treated cells. NIK activates the IKK
signalsome and has been shown to be involved in cytokine-induced
NF-B induction (39). Fig. 3B shows that
overexpression of dominant negative NIK significantly decreased both
S. flexneri- and TNF--induced activation of NF-B.
Dominant negative NIK inhibited S. flexneri-induced
activation of the luciferase reporter by 70 ± 16% compared with
infected cells transfected with the vector alone. Dominant negative NIK
also efficiently blocked NF-B activation by TNF- as previously
reported (41) (84 ± 9% decrease in reporter
activation compared with cells transfected with vector alone). Activation of the IKK complex by S. flexneri infection of HeLa cells
To obtain direct evidence that S. flexneri infection
activates the IKK complex in HeLa cells, immune complex kinase assays
were performed. Abs to NEMO, a component of the IKK complex, were used
to immunoprecipitate the complex from untreated, S.
flexneri-infected, or TNF--treated cells, and kinase activity
was then determined by analyzing the incorporation of
32P into IB wild-type and IB mutant
polypeptides. Fig. 4A shows
that immunoprecipitated IKK from cells infected with S.
flexneri for 30 min specifically phosphorylated the wild-type, but
not the mutant, IB polypeptide. TNF- treatment for 30 min
showed a similar level of IKK activation that was specific for the
wild-type IB (Fig. 4A).
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treatment. An increase in IKK activity induced by infection with
S. flexneri was observed 15 min postinfection; maximal
activity was seen at 45 min postinfection (Fig. 4B). At 90
min postinfection, levels of IKK activity were similar to levels in
untreated cells. IKK activation by TNF- treatment demonstrated a
different pattern of kinetics; maximum levels of IKK activity were
observed 15 min posttreatment, and IKK activity was similar to that in
untreated cells by 45 min posttreatment (Fig. 4B).
NF-B induced by S. flexneri infection binds the
B site in the IL-8 promoter
To determine whether there was a link between NF-B activation
and IL-8 production, we first investigated whether the NF-B
complexes induced by S. flexneri infection of HeLa cells
bound the IL-8 promoter. To this end, EMSAs were performed using the
-84 to -68 bp region of the IL-8 promoter that encompass the B
site (14). Fig. 5A shows a representative EMSA
as well as a supershift assay used to reveal the different NF-B
complexes that bound to the B site of the IL-8 promoter. Three
different NF-B complexes from nuclear extracts of S.
flexneri-infected HeLa cells were observed to bind to this DNA
probe. The lower complex was nonspecific, since it was also present in
uninfected cells. Abs to the p50 subunit of NF-B shifted the middle
complex, whereas Abs to the p65 subunit shifted both the top and middle
complexes. Abs to c-Rel did not appreciably alter the NF-B complexes
induced following S. flexneri infection. Therefore, it
appears that p65 homodimers and p50/p65 heterodimers are the main
NF-B complexes binding the B site of the IL-8 promoter following
S. flexneri infection.
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B complex binding to the IL-8 promoter
was surprising, since previous studies have shown that, in addition to
p65 and p50, c-Rel is a component of the IL-8 promoter-specific NF-B
complex (48). However, similar to the findings of Brasier
et al. (49), Western blot analysis revealed increased
levels of c-Rel in nuclear extracts from S.
flexneri-infected cells, while in uninfected cells, c-Rel remained
in the cytoplasmic fraction (data not shown). Thus, it is possible that
c-Rel was also part of the complex, but was not detected in supershift
assays, perhaps due to epitope masking.
The B site in the IL-8 promoter is indispensable for IL-8
promoter-driven luciferase activity induced by S.
flexneri
We next examined whether S. flexneri infection of HeLa
cells could activate an IL-8 reporter gene and what role NF-B played
in this activation. The 5'-flanking region of the IL-8 gene contains
transcription factor binding sites for AP-1, NF-IL-6, and NF-B
located 
120 bp upstream from the TATA box. When the wild-type
construct was transfected into HeLa cells, which were subsequently
infected with invasive S. flexneri for 5 h, an
3-fold activation of luciferase activity was observed compared with
that in uninfected cells (Fig. 5B). TNF- treatment of
these cells for 5 h resulted in a 6-fold activation of luciferase
activity. Infection with the noninvasive plasmid-cured S.
flexneri strain did not lead to an increase in luciferase activity
(Fig. 5B). To determine which of the transcription factor
binding sites was essential for responsiveness to S.
flexneri infection, HeLa cells were transfected with plasmid
constructs that contained point mutations in the AP-1, NF-IL-6, or
NF-B binding sites. Point mutations in the AP-1 site did not affect
the ability of S. flexneri infection or TNF- treatment to
activate the IL-8 reporter gene. Mutation of the NF-IL-6 site did not
affect S. flexneri-induced activation of the reporter, but
decreased TNF--induced activity by 50%, similar to previous
studies (14). Mutation of the NF-B site, however,
blocked both S. flexneri- and TNF--induced activation of
the IL-8 reporter, demonstrating that this site is indispensable for
activation by both these inducers of IL-8 gene expression (Fig. 5B).
IL-8 is secreted by HeLa cells infected with S. flexneri
To examine whether HeLa cells secreted IL-8 in response to
S. flexneri infection, IL-8 ELISAs were performed on
conditioned medium from HeLa cells infected with either wild-type
S. flexneri or the plasmid-cured strain or treated with 100
ng/ml of TNF- for various time periods (Table I). Small amounts of IL-8 were detected
as early as 2 h postinfection with wild-type S.
flexneri, and IL-8 levels increased to 325 ± 51 pg/ml
following 6 h of infection. TNF- treatment also led to
increases in IL-8 protein levels (228 ± 27 pg/ml following 6
h of treatment). In contrast, infection with the noninvasive
plasmid-cured strain of S. flexneri led to relatively small
amounts of IL-8 secreted from the infected cells (34 ± 19 pg/ml
after 6 h of infection). Transfection of cells with a
nondegradable form of IKB (IB-A32/36) before infection led to
a 31% decrease in IL-8 protein levels secreted by S.
flexneri-infected cells compared with infected cells transfected
with vector alone. These data complement the above findings and stress
the importance of NF-B as a regulator of IL-8 gene expression during
S. flexneri infection.
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B
activation
Possible bacterial factors capable of inducing NF-B activity
were analyzed by using mutant S. flexneri strains deficient
in certain virulence factors, including IpaA, IpaB, IpaC, IpaD, IpgD,
IcsA, and a S. flexneri strain cured of the virulence
plasmid. Fig. 6A shows that
mutant strains of S. flexneri that are noninvasive are
unable to stimulate NF-B DNA binding activity. IpaA strains, which
exhibit an entry efficiency 10% that of wild-type S.
flexneri (50), still activated NF-B. Likewise,
invasive strains carrying a mutation in ipgD or
icsA activated NF-B DNA binding activity to similar
extents as the wild-type organism (data not shown). The role of
bacterial entry in the activation of NF-B was examined more
thoroughly using cytochalasin B. This inhibitor of S.
flexneri entry caps actin filaments and prevents the dynamic
cytoskeletal rearrangements that are necessary for S.
flexneri invasion of epithelial cells (30).
Cytochalasin B (1 µg/ml) added to the epithelial monolayer at the
same time as S. flexneri infection prevented NF-B
activation (Fig. 6B). Gentamicin protection assays revealed
200 CFU/monolayer treated with cytochalasin B, whereas without this
inhibitor, 5 x 106 CFU/monolayer was
present. This same concentration of cytochalasin B had only a small
effect on NF-B activation by TNF-. Taken together, these studies
show a role for bacterial entry in NF-B activation of epithelial
cells by S. flexneri.
|
B in HeLa cells, microinjection of bacterial supernatants
containing these secreted virulence proteins was performed.
Bacteria-free supernatants from overnight cultures of wild-type M90T or
the plasmid-cured strain BS176 were microinjected into individual HeLa
cells and compared with cells microinjected with broth medium alone.
Following subsequent staining with Abs to the p65 subunit of NF-B,
immunofluorescence revealed activated NF-B in the nucleus of cells
microinjected with supernatants from M90T (Fig. 7A) and BS176 (data not
shown). Cells microinjected with medium alone showed only cytoplasmic
staining of NF-B (Fig. 7B). NF-B-inducing activity in
the supernatants of wild-type and plasmid-cured S. flexneri
strains appeared to be common to Gram-negative organisms, because the
microinjection of supernatants from the E. coli strain
DH5 into HeLa cells resulted in localization of NF-B in the
nucleus of microinjected cells (data not shown). Supernatants from the
Gram-positive organism B. subtilis were also tested in this
system, but nuclear staining of NF-B was only observed when these
supernatants were concentrated 100-fold (data not shown). Addition
of supernatants from S. flexneri, E. coli, or
B. subtilis to the cell medium (1:2 ratio) did not activate
NF-B. To investigate whether the chemotactic bacterial peptide,
fMLP, was responsible for this activation, supernatants from an
E. coli strain deficient in the production of
N-formylated proteins (34) were microinjected
into HeLa cells. Nuclear staining of NF-B was detected in cells
microinjected with these supernatants, ruling out this possibility
(data not shown).
|
B-inducing factor in supernatants
from M90T was conducted. Diluting the supernatants 1/4 in PBS
significantly reduced NF-B-inducing activity, and the activity was
undetectable at a dilution of 1/16. The activity of supernatants from
E. coli DH5 was higher than that in wild-type S.
flexneri supernatants, in that loss of NF-B-inducing activity
was observed only after diluting the supernatants 1/256 with PBS. All
subsequent characterizations of the NF-B-inducing activity of
wild-type S. flexneri supernatants were performed on samples
diluted 1/4. The NF-B-inducing activity of M90T supernatants was not
affected by 1) boiling for 60 min, 2) treatment with proteinase K (150
µg/ml for 1 h at 60°C, overnight at 37°C, and subsequently
boiled), or 3) DNase (1 µg/ml for 15 min at 37°C and subsequently
boiled; data not shown). In addition, the NF-B-inducing activity of
the bacterial supernatants was stable for extended periods at 4°C.
These observations suggested that LPS might be responsible for
activating NF-B. Immunodepletion of LPS from the bacteria-free
supernatants with dimeric IgA Abs specific for S. flexneri
5a LPS resulted in a partial loss of NF-B-inducing activity with
<50% of microinjected cells showing localization of NF-B in the
nucleus (Fig. 7C). M90T supernatants immunodepleted of LPS
with IgA Abs specific for Salmonella LPS showed localization
of NF-B in the nucleus of microinjected cells (data not shown).
Microinjection of purified LPS from different bacterial species was
then conducted. Purified S. flexneri LPS from serotypes 5a
(Fig. 7D) and 1a, R. sphaeroides LPS (data not
shown), and E. coli LPS from strain O111:B4 (Fig. 7E) all activated NF-B following microinjection. The
NF-B-inducing activity of S. flexneri LPS from both 5a
and 1a as well as LPS derived from R. sphaeroides was weak,
in that 50% of the microinjected cells showed nuclear localization
of NF-B. This was in contrast to cells microinjected with LPS from
E. coli strain O111:B4, which consistently showed NF-B
activation (100% of cells). The insolubility of purified lipid A
prevented us from microinjecting this product. However, microinjection
of detoxified (i.e., delipidated) LPS derived from E. coli
O111:B4 did not lead to activated NF-B in the nucleus, suggesting
that the lipid portion of LPS was likely to be responsible for this
activation (data not shown).
To ensure that this response was not limited to HeLa epithelial cells,
Caco-2 cells were grown on coverslips and microinjected with
bacteria-free supernatants or purified LPS. Similar to what was
observed in HeLa cells, the microinjection of bacteria-free
supernatants from overnight cultures of M90T (data not shown) or the
microinjection of purified LPS from E. coli O111:B4 (Fig. 7F) led to the translocation of NF-B into the nucleus.
Cells microinjected with either medium or buffer alone did not show
activated NF-B in the nucleus (data not shown).
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Previous studies have shown NF-B
activation in epithelial cells infected with S. flexneri
(27) as well as other Gram-negative pathogens, including
Salmonella (24), Neisseria
(25), and EPEC (26). However, neither the
pathway leading to NF-B activation by these organisms nor the
bacterial products involved have been clearly defined. In this study we
characterized the NF-B response in epithelial cells infected with
S. flexneri and compared its activation with that induced by
TNF-. TNF- treatment induced rapid and transient NF-B DNA
binding activity. Down-regulation of NF-B following TNF-
treatment probably occurs because of three events. First, we found that
the kinase activity of the IKK complex was rapidly down-regulated by 30
min post-TNF- treatment, thus halting the activation cascade.
Negative regulation of IKK activity was recently shown to be due to the
phosphorylation of C-terminal serines on the IKK2 subunit
(51). Second, because NF-B induces the expression of
IB (52), newly synthesized IB translocates to
the nucleus, where it sequesters NF-B and shuts down the activation
of NF-B-regulated gene expression (53). Indeed, we
showed that IB reappears in the cytoplasm of TNF--treated
cells by 2 h posttreatment and is thus available to down-regulate
activation of NF-B. Finally, down-regulation of NF-B activation
following TNF- treatment is also attributed to endocytosis of
ligand-bound receptors, thus preventing reactivation (54, 55).
In S. flexneri-infected cells, however, this negative
regulatory loop was apparently missing. We found that S.
flexneri infection resulted in sustained activity of the IKK
complex, similar to what has been previously observed in human
monocytic cells treated with LPS (56). Cytoplasmic levels
of IB remained undetectable for the duration of the time course
studied, and accordingly, NF-B DNA binding activity was also
sustained. The level at which S. flexneri infection disrupts
the negative regulation of this cascade is not yet known. One
possibility is that the NF-B-inducing factor from S.
flexneri is constantly presented to the cell, and this continually
drives the activation of the cascade. Alternatively, S.
flexneri infection could lead to inefficient phosphorylation of
the negative regulatory domain of IKK2, resulting in its sustained
activation. This inability of infected cells to down-regulate NF-B
activation may have important implications during infection in vivo;
sustained NF-B activity could cause inflammation to go unchecked,
resulting in the severe damage to the epithelial layer seen during
shigellosis.
NF-B is an important transcriptional regulator of IL-8
(14), a proinflammatory chemokine produced by infected
epithelial cells. The production of this chemokine appears to be part
of an innate program initiated by bacterial infection (10, 11, 28, 57). The importance of IL-8 during S. flexneri
infection was recently demonstrated in the rabbit ileal loop model of
shigellosis (13). Induction of IL-8 by infected epithelial
cells was shown to drive the massive inflammatory response and tissue
destruction following S. flexneri infection. This response,
although destructive to the host, is necessary to clear the organism
from the infected tissue and prevent bacteriemia. In the present study
S. flexneri infection was shown to activate an IL-8
promoter-driven reporter gene, and IL-8 protein levels were also shown
to increase following infection. In addition, S.
flexneri-induced IL-8 promoter activity relied on the NF-B
binding site within the promoter region. These findings stress the
importance of NF-B in regulating IL-8 induction during S.
flexneri infection and implicate this transcription factor as a
key regulator of the inflammatory process during infection in
vivo.
The transcription factor NF-B has been known for more than a decade;
however, the complex signaling pathway leading to its activation is
just beginning to be elucidated. The signaling pathways leading from
TNF- and IL-1 receptor activation to NF-B have been best
characterized. The recently described human Toll-like receptors (TLR)
are members of the IL-1 receptor family of proteins, and signaling from
these receptors appears to be identical with IL-1 signaling
(58). Although the upstream components of activation are
distinct, signaling to NF-B by TNF- and Toll/IL-1 involves
activation of NIK and the IKK complex (19, 39). By using
transient transfection to overexpress dominant negative versions of
these proteins in HeLa cells we were able to examine whether S.
flexneri infection could activate a NF-B-dependent reporter.
Like TNF- and Toll/IL-1 signaling to NF-B, activation of NF-B
by invasive S. flexneri required IKK1, IKK2, and NIK.
However, our current studies suggest that the S.
flexneri-induced signaling pathway upstream of NIK is potentially
novel; TNF-- or IL-1/TLR-specific signaling molecules, such as TRAF2
or MyD88 and IRAK1, do not play a role in NF-B activation by
S. flexneri (D. J. Philpott, X. Li, M. Mavris, G.
Stark, and P. J. Sansonetti, manuscript in preparation).
Innate immune recognition of bacterial products is an ancient system of host defense that shares striking similarities in organisms as diverse as humans, fruit flies, and, to a certain extent, plants (reviewed in Ref. 59). It is not surprising, therefore, that the bacterial products that are recognized are invariant molecules including structural components such as LPS of Gram-negative organisms and peptidoglycan from the cell walls of Gram-positive organisms (60). More recently, bacterial lipoproteins have also been shown to trigger host defense mechanisms (61, 62). In both Drosophila and humans, a family of proteins termed Toll or TLRs is central to innate immunity. In mammals, innate recognition of microbial pathogens probably proceeds through a system by which CD14, one example of a pattern recognition receptor (PRR), detects LPS and by an unknown mechanism activates TLR4, thereby transducing the signal. A number of human TLRs have been identified in the database, leading to speculation that different TLRs may be involved in detecting different bacterial products to bring about distinct cellular responses.
Our findings show that epithelial cells do not respond to extracellular
LPS, either purified or in the context of noninvasive S.
flexneri strains. The Toll status of three intestinal epithelial
cells lines was recently investigated, and although these cells express
TLR4 and TLR2 in the case of T84 and Caco-2 cells, these cell lines are
unresponsive to LPS at least in the absence of serum (63).
On the other hand, our data suggest that LPS presented inside
epithelial cells, either through the release of LPS by intracellular
S. flexneri or by microinjection of the purified product,
activates NF-B. This response was not only observed in HeLa cells,
but was also reproduced in the intestinal cell line Caco-2. It seems
quite plausible, therefore, that a cytoplasmic PRR exists to detect
intracellular LPS and that activation of this protein initiates a
cascade leading to NF-B activation.
During infection, S. flexneri enters the cell by a process resembling macropinocytosis, and once within the cell, lyses the membrane-bound vacuole. Free LPS is probably shed from the bacterium once inside the host cell (our unpublished observation) as is the case during S. typhimurium infection of HeLa cells (64). Because of its amphiphilic nature, free LPS is probably associated with membrane vesicles (64). It is possible that LPS reacts with the putative PRR within these vesicles or, alternatively, these LPS-containing vesicles could be trafficked to a specific compartment where this recognition takes place. The role of lipid trafficking in LPS activation of monocytes was recently examined. It was demonstrated that R. sphaeroides LPS, which is normally an LPS antagonist, becomes an LPS agonist upon treatment of cells with cationic membrane-active compounds (65). These compounds alter the packing geometry of R. sphaeroides LPS in the membrane and, in turn, its intracellular trafficking, which then correlates with the ability of this LPS to activate human monocytes. It was speculated that sorting of LPS and subsequent involvement in the activation of signaling cascades may be mediated by microbial pattern recognition receptors (65). Interestingly, we also found that R. sphaeroides LPS acts as an LPS agonist if microinjected into HeLa cells. Microinjection of R. sphaeroides LPS may allow it to be sorted in such a way that this LPS is then recognized and available to activate these cells.
Although we cannot rule out the possibility that other factors present
in bacteria-free supernatants or associated with invasive S.
flexneri activate NF-B, several properties of the
NF-B-inducing factor strongly suggest that it is LPS. Firstly,
microinjection of supernatants from both invasive and the plasmid-cured
strains of S. flexneri activated NF-B, ruling out the
possibility that a plasmid-encoded virulence factor was responsible for
this activation. Moreover, microinjection of supernatants from
nonpathogenic E. coli were also able to induce NF-B
activation. Secondly, the NF-B-inducing activity was insensitive to
boiling and proteinase K and DNase treatment, suggesting that the
factor is a lipid. The only treatment that significantly affected the
NF-B-inducing activity of the bacteria-free supernatants was
immunodepletion of LPS. Finally, activation of NF-B is a common
response of epithelial cells to infection with a number of
Gram-negative bacteria (24, 25, 26), including S.
flexneri (27), implying that the responsible factor
is probably a conserved component of this diverse set of pathogens.
Bacterial lipoproteins also share these properties with LPS; however,
its insolubility prevented us from testing this compound in
microinjection experiments. We also found that microinjection of
supernatants from the Gram-positive organism, B. subtilis,
into HeLa cells activated NF-B. However, positive activation was
observed only after concentrating the supernatants 100-fold.
Nevertheless, this finding raises the possibility that epithelial cells
also possess a detection system for Gram-positive associated factors.
Alternatively, the same receptor that detects intracellular LPS could
bind Gram-positive peptidoglycan, as is the case for CD14
(66).
The different bacterial products tested by microinjection varied in
their potency to induce NF-B. Microinjection of bacteria-free
supernatants invariably led to the translocation of NF-B to the
nucleus, whereas purified LPSs from different bacterial species varied
in their potency to stimulate NF-B. In terms of the bacterial
supernatants tested, E. coli DH5 supernatants had the
highest NF-B-inducing activity, which correlated with the fact that
the amount of released LPS in these supernatants was greater than that
in S. flexneri supernatants as determined by silver-stained
gels (data not shown). As for the purified LPSs tested, LPS from
E. coli strain O111:B4 had the highest activity, which
correlates with its high critical aggregation concentration and a
tendency to form nonlamellar structures in solution (67).
This property has been correlated with increased biological potency of
LPS (68). What was surprising, however, was the fact that
the amount of LPS in the purified samples was much greater than that
observed in the bacterial supernatants, yet the supernatants exhibited
higher NF-B-inducing activity. This discrepancy in activity vs the
concentration of LPS is possibly due to differences in the presentation
of LPS in its purified form vs that released from bacteria. We
speculate that LPS in bacteria-free supernatants may be in a form that
is more available to interact with potential receptors because of its
association with bacterial membrane or other bacterial surface
components. The association of LPS with these factors is likely to
inhibit lameller formation, and this may enhance the ability of LPS to
interact with an intracellular receptor. In support of this idea, it
was shown that LPS in bacterial supernatants is more biologically
potent in stimulating monocytes than the same amount of purified LPS
(69). Therefore, the biological context of LPS is
extremely relevant and impacts on the potency of LPS released by
S. flexneri during infection in vivo.
In conclusion, our studies of the mechanism of NF-B activation by
the invasive pathogen, S. flexneri, have suggested the
presence of an intracellular pattern recognition receptor that detects
LPS in the cytoplasm of infected epithelial cells. In colonic
epithelial cells, such an intracellular receptor seems logical. It
would be detrimental to the organism if colonic epithelial cells were
capable of responding to extracellular LPS in the context of the normal
resident flora. However, by triggering a response once a pathogen
invades the cell, an intracellular PRR could initiate an inflammatory
cascade in an attempt to control the threat of infection. These
findings open up the possibility for a novel system of host defense
against invasive enteric pathogens and may also impact on the potential
of modulating the immune response to intestinal infections through this
common mode of bacterial detection.
|
Acknowledgments |
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|
Footnotes |
|---|
2 Current address: Department of Microbiology, Tokyo Medical and Dental University School of Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan.
3 Address correspondence and reprint requests to Dr. Philippe Sansonetti, Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cédex 15, France.
4 Abbreviations used in this paper: IB, inhibitory B; NIK, NF-B-inducing kinase; IKK, IB kinase; IRAK-2, IL-1 receptor-associated kinase-2; TLR, Toll-like receptor; PRR, pattern recognition receptor.
Received for publication October 13, 1999. Accepted for publication April 27, 2000.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B and cis-regulatory enhancer binding protein-like factor binding elements in activation the interleukin-8 gene by pro-inflammatory cytokines. J. Biol. Chem. 265:21128.B and Rel proteins: evolutionary conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B kinase complex (IKK) contains two kinase subunits, IKK and IKKß, necessary for IB phosphorylation and NF-B activation. Cell. 91:243.[Medline]
B kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
B kinases essential for NF-B activation. Science 278:860.B kinase complex essential for NF-B activation. Cell 93:1231.[Medline]
B kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex. Mol. Cell. Biol. 19:1526. is an essential regulatory subunit of the IB kinase complex. Nature 395:297.[Medline]
B- proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485.B requires site-specific ubiquitination. Proc. Natl. Acad. Sci. USA 92:11259.B and activator protein 1 and the induction of inflammatory cytokines. J. Exp. Med. 186:247.B in intestinal epithelial cells by enteropathogenic Escherichia coli. Am. J. Physiol. 273:C1160.
B DNA-binding activity. Infect. Immun. 61:4427.B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J. Immunol. 163:1457.B p50 has IB-like functions. Cell 71:243.[Medline]
B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B through dissociation of cytoplasmic complexes containing p105 or p100 but does not induce degradation of IBa/MAD3. J. Virol. 68:8035.B in response to multiple stimuli. Mol. Cell. Biol. 17:1441.[Abstract]
B-like enhancer binding activity which displaces constitutive factors. EMBO J. 8:3793.[Medline]
B activation. Proc. Natl. Acad. Sci. USA 96:429.B and AP-1 activation. J. Biol. Chem. 272:29167.B. Mol. Cell. Biol. 13:7191.-induced interleukin-8 transcription in type II pulmonary epithelial cells. J. Biol. Chem. 273:3551.B kinase activity through IKKB subunit phosphorylation. Science 284:309.B controls expression of inhibitor IB: evidence for an inducible autoregulatory pathway. Science 259:1912.B negatively regulates DNA binding and transcriptional activities of NF-B. Mol. Cell. Biol. 15:2689.[Abstract]
receptor on human monocytes and internalization of receptor bound ligand. J. Immunol. 139:2989.[Abstract]
receptor in MCF7 cells. FASEB J. 3:2633.[Abstract]
B and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6). J. Exp. Med. 187:2097.This article has been cited by other articles:
|
|
 |
|
  J. Zheng, J. Meng, S. Zhao, R. Singh, and W. Song Campylobacter-Induced Interleukin-8 Secretion in Polarized Human Intestinal Epithelial Cells Requires Campylobacter-Secreted Cytolethal Distending Toxin- and Toll-Like Receptor-Mediated Activation of NF-{kappa}B Infect. Immun., October 1, 2008; 76(10): 4498 - 4508. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. L. Mumy, X. Chen, C. P. Kelly, and B. A. McCormick Saccharomyces boulardii interferes with Shigella pathogenesis by postinvasion signaling events Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G599 - G609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. N. Schroeder and H. Hilbi Molecular Pathogenesis of Shigella spp.: Controlling Host Cell Signaling, Invasion, and Death by Type III Secretion Clin. Microbiol. Rev., January 1, 2008; 21(1): 134 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. V. Zurawski, K. L. Mumy, L. Badea, J. A. Prentice, E. L. Hartland, B. A. McCormick, and A. T. Maurelli The NleE/OspZ Family of Effector Proteins Is Required for Polymorphonuclear Transepithelial Migration, a Characteristic Shared by Enteropathogenic Escherichia coli and Shigella flexneri Infections Infect. Immun., January 1, 2008; 76(1): 369 - 379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chowers, J. Kirschner, N. Keller, I. Barshack, S. Bar-Meir, S. Ashkenazi, R. Schneerson, J. Robbins, and J. H. Passwell O-Specific polysaccharide conjugate vaccine-induced IgG antibodies prevent invasion of Shigella into Caco-2 cells and may be curative PNAS, February 13, 2007; 104(7): 2396 - 2401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Boutet, J. Sulon, R. Closset, J. Detilleux, J.-F. Beckers, F. Bureau, and P. Lekeux Prolactin-Induced Activation of Nuclear Factor {kappa}B in Bovine Mammary Epithelial Cells: Role in Chronic Mastitis J Dairy Sci, January 1, 2007; 90(1): 155 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Fu, K. Bettega, S. Carroll, K. R. Buchholz, and T. E. Machen Role of Ca2+ in responses of airway epithelia to Pseudomonas aeruginosa, flagellin, ATP, and thapsigargin Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L353 - L364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Yamamoto-Furusho, N. Barnich, R. Xavier, T. Hisamatsu, and D. K. Podolsky Centaurin beta1 Down-regulates Nucleotide-binding Oligomerization Domains 1- and 2-dependent NF-{kappa}B Activation J. Biol. Chem., November 24, 2006; 281(47): 36060 - 36070. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Kufer, E. Kremmer, D. J. Banks, and D. J. Philpott Role for erbin in bacterial activation of nod2. Infect. Immun., June 1, 2006; 74(6): 3115 - 3124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Khan, C. Ma, L. A. Knodler, Y. Valdez, C. M. Rosenberger, W. Deng, B. B. Finlay, and B. A. Vallance Toll-Like Receptor 4 Contributes to Colitis Development but Not to Host Defense during Citrobacter rodentium Infection in Mice. Infect. Immun., May 1, 2006; 74(5): 2522 - 2536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-T. Tien, S. E. Girardin, B. Regnault, L. Le Bourhis, M.-A. Dillies, J.-Y. Coppee, R. Bourdet-Sicard, P. J. Sansonetti, and T. Pedron Anti-Inflammatory Effect of Lactobacillus casei on Shigella-Infected Human Intestinal Epithelial Cells J. Immunol., January 15, 2006; 176(2): 1228 - 1237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Lee, S. Y. Kim, B. C. Jeong, Y. R. Kim, S. J. Bae, O. S. Ahn, J.-J. Lee, H.-C. Song, J. M. Kim, H. E. Choy, et al. A Bacterial Flagellin, Vibrio vulnificus FlaB, Has a Strong Mucosal Adjuvant Activity To Induce Protective Immunity Infect. Immun., January 1, 2006; 74(1): 694 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Kim, G. Lenzen, A.-L. Page, P. Legrain, P. J. Sansonetti, and C. Parsot The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes PNAS, September 27, 2005; 102(39): 14046 - 14051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Bambou, A. Giraud, S. Menard, B. Begue, S. Rakotobe, M. Heyman, F. Taddei, N. Cerf-Bensussan, and V. Gaboriau-Routhiau In Vitro and ex Vivo Activation of the TLR5 Signaling Pathway in Intestinal Epithelial Cells by a Commensal Escherichia coli Strain J. Biol. Chem., October 8, 2004; 279(41): 42984 - 42992. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Singer and P. J. Sansonetti IL-8 Is a Key Chemokine Regulating Neutrophil Recruitment in a New Mouse Model of Shigella-Induced Colitis J. Immunol., September 15, 2004; 173(6): 4197 - 4206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Guillot, S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, and M. Si-Tahar Response of Human Pulmonary Epithelial Cells to Lipopolysaccharide Involves Toll-like Receptor 4 (TLR4)-dependent Signaling Pathways: EVIDENCE FOR AN INTRACELLULAR COMPARTMENTALIZATION OF TLR4 J. Biol. Chem., January 23, 2004; 279(4): 2712 - 2718. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cersini, M. C. Martino, I. Martini, G. Rossi, and M. L. Bernardini Analysis of Virulence and Inflammatory Potential of Shigella flexneri Purine Biosynthesis Mutants Infect. Immun., December 1, 2003; 71(12): 7002 - 7013. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Rogers, A. W. Paton, S. R. McColl, and J. C. Paton Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli Infect. Immun., October 1, 2003; 71(10): 5623 - 5632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Pedron, C. Thibault, and P. J. Sansonetti The Invasive Phenotype of Shigella flexneri Directs a Distinct Gene Expression Pattern in the Human Intestinal Epithelial Cell Line Caco-2 J. Biol. Chem., September 5, 2003; 278(36): 33878 - 33886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hisamatsu, M. Suzuki, and D. K. Podolsky Interferon-{gamma} Augments CARD4/NOD1 Gene and Protein Expression through Interferon Regulatory Factor-1 in Intestinal Epithelial Cells J. Biol. Chem., August 29, 2003; 278(35): 32962 - 32968. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D Berrebi, R Maudinas, J-P Hugot, M Chamaillard, F Chareyre, P De Lagausie, C Yang, P Desreumaux, M Giovannini, J-P Cezard, et al. Card15 gene overexpression in mononuclear and epithelial cells of the inflamed Crohn's disease colon Gut, June 1, 2003; 52(6): 840 - 846. [Abstract] [Full Text]
|
|
|
|
|
|
D. Boulanger, F. Bureau, D. Melotte, J. Mainil, and P. Lekeux Increased Nuclear Factor {kappa}B Activity in Milk Cells of Mastitis-Affected Cows J Dairy Sci, April 1, 2003; 86(4): 1259 - 1267. [Abstract] [Full Text] [PDF]
|
|
|
|
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S. E. Girardin, I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas, D. J. Philpott, and P. J. Sansonetti Nod2 Is a General Sensor of Peptidoglycan through Muramyl Dipeptide (MDP) Detection J. Biol. Chem., March 7, 2003; 278(11): 8869 - 8872. [Abstract] [Full Text] [PDF]
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J Berkes, V K Viswanathan, S D Savkovic, and G Hecht Intestinal epithelial responses to enteric pathogens: effects on the tight junction barrier, ion transport, and inflammation Gut, March 1, 2003; 52(3): 439 - 451. [Abstract] [Full Text] [PDF]
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M. O'Riordan, C. H. Yi, R. Gonzales, K.-D. Lee, and D. A. Portnoy Innate recognition of bacteria by a macrophage cytosolic surveillance pathway PNAS, October 15, 2002; 99(21): 13861 - 13866. [Abstract] [Full Text] [PDF]
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 K. H. Mellits, J. Mullen, M. Wand, G. Armbruster, A. Patel, P. L. Connerton, M. Skelly, and I. F. Connerton Activation of the transcription factor NF-{kappa}B by Campylobacter jejuni Microbiology, September 1, 2002; 148(9): 2753 - 2763. [Abstract] [Full Text] [PDF]
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M. Innocenti, A.-C. Thoreson, R. L. Ferrero, E. Stromberg, I. Bolin, L. Eriksson, A.-M. Svennerholm, and M. Quiding-Jarbrink Helicobacter pylori-Induced Activation of Human Endothelial Cells Infect. Immun., August 1, 2002; 70(8): 4581 - 4590. [Abstract] [Full Text] [PDF]
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J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
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C. M. Tato and C. A. Hunter Host-Pathogen Interactions: Subversion and Utilization of the NF-{kappa}B Pathway during Infection Infect. Immun., July 1, 2002; 70(7): 3311 - 3317. [Full Text] [PDF]
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H. d'Hauteville, S. Khan, D. J. Maskell, A. Kussak, A. Weintraub, J. Mathison, R. J. Ulevitch, N. Wuscher, C. Parsot, and P. J. Sansonetti Two msbB Genes Encoding Maximal Acylation of Lipid A Are Required for Invasive Shigella flexneri to Mediate Inflammatory Rupture and Destruction of the Intestinal Epithelium J. Immunol., May 15, 2002; 168(10): 5240 - 5251. [Abstract] [Full Text] [PDF]
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S. Dahan, V. Busuttil, V. Imbert, J.-F. Peyron, P. Rampal, and D. Czerucka Enterohemorrhagic Escherichia coli Infection Induces Interleukin-8 Production via Activation of Mitogen-Activated Protein Kinases and the Transcription Factors NF-{kappa}B and AP-1 in T84 Cells Infect. Immun., May 1, 2002; 70(5): 2304 - 2310. [Abstract] [Full Text] [PDF]
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T. Vasselon and P. A. Detmers Toll Receptors: a Central Element in Innate Immune Responses Infect. Immun., March 1, 2002; 70(3): 1033 - 1041. [Full Text] [PDF]
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H. Kohler, S. P. Rodrigues, and B. A. McCormick Shigella flexneri Interactions with the Basolateral Membrane Domain of Polarized Model Intestinal Epithelium: Role of Lipopolysaccharide in Cell Invasion and in Activation of the Mitogen-Activated Protein Kinase ERK Infect. Immun., March 1, 2002; 70(3): 1150 - 1158. [Abstract] [Full Text] [PDF]
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S. Houalet-Jeanne, P. Pellen-Mussi, S. Tricot-Doleux, J. Apiou, and M. Bonnaure-Mallet Assessment of Internalization and Viability of Porphyromonas gingivalis in KB Epithelial Cells by Confocal Microscopy Infect. Immun., November 1, 2001; 69(11): 7146 - 7151. [Abstract] [Full Text] [PDF]
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P. J. Sansonetti Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions: III. Shigellosis: from symptoms to molecular pathogenesis Am J Physiol Gastrointest Liver Physiol, March 1, 2001; 280(3): G319 - G323. [Abstract] [Full Text] [PDF]
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N. Inohara, Y. Ogura, F. F. Chen, A. Muto, and G. Nunez Human Nod1 Confers Responsiveness to Bacterial Lipopolysaccharides J. Biol. Chem., January 19, 2001; 276(4): 2551 - 2554. [Abstract] [Full Text] [PDF]
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J. da Silva Correia and R. J. Ulevitch MD-2 and TLR4 N-Linked Glycosylations Are Important for a Functional Lipopolysaccharide Receptor J. Biol. Chem., January 11, 2002; 277(3): 1845 - 1854. [Abstract] [Full Text] [PDF]
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) or
TNF-
). HeLa cells were transiently cotransfected with
0.5 µg of NF-
) drive IL-8 promoter-driven luciferase activity that is
dependent upon the 
). HeLa cells were transiently
transfected with 0.5 µg of the IL-8 promoter constructs, and 36
h later were infected with wild-type or plasmid-cured S.
flexneri or treated with TNF-
