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B Is a Central Regulator of the Intestinal Epithelial Cell Innate Immune Response Induced by Infection with Enteroinvasive Bacteria1
Laboratory of Mucosal Immunology, Department of Medicine, University of California at San Diego, La Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B kinase (IKK) 
and IKK
, and
increased NF-B DNA binding activity. This was paralleled by partial
degradation of IB and IB
in bacteria-infected Caco-2 cells.
Mutant proteins that act as superrepressors of IKK and IB
inhibited the up-regulated transcription and expression of downstream
targets genes of NF-B that are key components of the epithelial
inflammatory gene program (i.e., IL-8, growth-related oncogene-,
monocyte chemoattractant protein-1, TNF-, cyclooxygenase-2, nitric
oxide synthase-2, ICAM-1) activated by those enteroinvasive bacteria.
These studies position NF-B as a central regulator of the epithelial
cell innate immune response to infection with enteroinvasive
bacteria. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-8, growth-related oncogene-
(GRO),7 and
monocyte chemoattractant protein-1 (MCP-1)), an inducible isoform of
cyclooxygenase (COX), COX-2, and prostaglandins
E2 and F2, an inducible
form of nitric oxide synthase (NOS), NOS2, and nitric oxide, and
increased surface expression of the adhesion molecule ICAM-1 on the
apical cell membrane (1, 2, 3, 4, 5, 7).
Enteroinvasive bacterial pathogens use a variety of strategies to
induce their uptake into nonphagocytic intestinal epithelial cells
(9). For example, the uptake of Salmonella
involves the formation of large membrane folds or ruffles and
macropinocytosis (10). In contrast, internalization of
Yersinia involves the binding of a bacterial outer membrane
protein, invasin, to 1 integrins on the
epithelial cell surface and a "zippering" mechanism, which is
characterized by the close juxtaposition of the mammalian cell membrane
around bacteria that are being internalized (10, 11). In
each case, interaction of bacteria with the host epithelial cell
results in morphological changes in the epithelial cell membrane and
underlying cytoskeleton. After entry into epithelial cells, different
strains of bacteria reside in different intracellular localizations
(e.g., after entry, Salmonella and Yersinia
reside in membrane-bound vesicles, whereas Shigella and
Listeria rapidly lyse such vesicles and move freely within
the cytoplasm) (10). Despite diverse mechanisms of
bacterial invasion and the different intracellular localization of
bacteria within the cytoplasm, the array of genes and mediators that
are consistently up-regulated as components of the epithelial cell
innate immune response is relatively limited (1, 7). This
suggested that a common signal transduction pathway might underlie the
up-regulated expression of the proinflammatory genes and mediators that
are activated in intestinal epithelial cells in response to infection
with enteroinvasive bacteria.
Many of the genes that are activated in intestinal epithelial cells
after bacterial invasion are target genes of the transcription factor
NF-B. NF-B is a dimeric transcription factor composed of
homodimers and heterodimers of Rel proteins, of which there are five
family members in mammalian cells (i.e., RelA (p65), c-Rel, RelB,
NF-B1 (p50), and NF-B2 (p52)) (12, 13). NF-B
dimers are held in the cytoplasm in an inactive state by inhibitory
proteins, the IBs. There are seven IBs (i.e., IB, IB,
IB, IB
, Bcl-3, p100, and p105) (14) that
preferentially associate with various Rel family protein dimers (e.g.,
IB and IB predominantly associate with p65/p50 and
p50/c-Rel heterodimers, IB preferentially associates with p65 and
c-Rel homodimers, and Bcl-3 associates with nuclear p50 and p52
homodimers). Heterodimers of p65 and p50 are the predominant NF-B
subunits that translocate to the nucleus after cytokine stimulation of
intestinal epithelial cells (15).
Stimulation of cells with IL-1 or TNF- activates a signaling cascade
that culminates in the phosphorylation of IBs (16, 17).
Two recently described IB kinases, IB kinase (IKK) and
IKK, directly phosphorylate IBs on serine residues and are
components of a high molecular weight cytoplasmic IKK complex
(16, 18, 19). Activation of IKK appears to be mediated
through the mitogen-activated protein-3 (MAP3) kinases,
NF-B-inducing kinase (NIK), and MEK kinase 1 (MEKK-1)
(20, 21, 22, 23, 24). Phosphorylation of IBs on conserved serine
residues targets the IBs for subsequent ubiquitination and
degradation (25, 26). This frees dimers of NF-B (e.g.,
p65/p50) to translocate to the nucleus where they
trans-activate NF-B target genes. NF-B can also
trans-activate transcription of its own inhibitor, IB
(27). This negative feedback loop results in the
restoration of IB protein levels, which complex cytoplasmic
NF-B and thereby down-regulate NF-B activation (28, 29). Activation of NF-B in response to extracellular
signaling through TNF receptor family members involves TNF
receptor-associated factors (TRAFs) that serve as adaptor molecules in
these signal transduction pathways (e.g., TRAF2 and TRAF5 are involved
in signaling through TNF and lymphotoxin receptors (LTR),
respectively) (30, 31).
The present studies asked whether intestinal epithelial cells use a
final common signal transduction pathway to activate the host’s early
inflammatory response to a diverse array of enteroinvasive bacteria.
NF-B is shown herein to be a central regulator of the activation of
the intestinal epithelial cell inflammatory response after infection
with a spectrum of enteroinvasive bacteria. Moreover, activation of
NF-B after epithelial cell infection with enteroinvasive 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 IKK and IKK and the degradation of IBs.
Consistent with this, expression of superrepressor alleles of IB
or IKK inhibited the up-regulated expression of key components of
the epithelial cell-inflammatory gene program (e.g., IL-8, GRO,
TNF-, COX-2, NOS2, ICAM-1) in intestinal epithelial cells infected
with enteroinvasive bacteria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HT-29 human colon epithelial cells (American Type Culture Collection (ATCC) HTB 38) and Caco-2 human ileocecal epithelial cells (ATCC HTB 37) were grown in DME (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 2 mM glutamine, and 25 mM HEPES. T84 cells were grown in 50% DMEM and 50% Ham’s F-12 medium supplemented with 5% newborn calf serum and 2 mM glutamine, as described before (1, 2).
Bacteria, cytokines, and other reagents
S. dublin lane strain (1) and
Yersinia enterocolitica 08 (1) were
provided by J. Fierer (University of California, San Diego, CA (UCSD)).
Enteroinvasive Escherichia coli (serotype O29:NM) were
obtained from the ATCC (No. 43892), and nonpathogenic E.
coli DH5 were purchased from Life Technologies. Recombinant
human TNF- and IL-1 were from R&D Systems (Minneapolis, MN).
IFN- was from BioSource International, Camarillo, CA.
Bacterial infection
Human colon epithelial cell lines were infected with bacteria at
multiplicities 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, after which
whole cell or nuclear extracts were prepared (16, 17, 33).
For longer incubations, extracellular bacteria were removed by washing
after 1 h, and the cells were incubated for an additional period
in the presence of 50 µg/ml gentamicin to kill the remaining
extracellular, but not intracellular, bacteria. Those cells were
harvested for isolation of total RNA for RT-PCR or 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 IB having substitutions of serine residues
at positions 32 and 36 with alanine residues was used to block NF-B
activation (17). The mutant protein cannot be
phosphorylated by IB kinases at those positions and acts as an IB
superrepressor. An expression vector that encodes FLAG-tagged IKK in
which lysine present at position 44 is replaced by alanine, which
inactivates kinase activity in the mutant molecule and acts as a
superrepressor of IKK, was a gift of F. Mercurio (Signal
Pharmaceuticals, San Diego, CA) (19). Expression vectors
for the superrepressor versions of TRAF2 and TRAF5 (amino-terminal
deletions) (34, 35) were gifts from D. Goeddel, (Tularik
Corp., South San Francisco, CA) and H. Nakano (Juntendo Univ, Tokyo,
Japan), respectively. An expression vector for a NIK catalytic mutant
that has a double replacement of alanine residues to lysine residues at
positions 429 and 430 and lacks kinase activity (21) was a
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-B-luciferase, and RSV--galactosidase transcriptional
reporters were constructed as described before (33, 37). A
full length ICAM-1 promoter construct containing 1.4 kb of ICAM-1
5'-flanking DNA linked to the luciferase gene was provided by K.
Roebuck (38). CAT transcriptional reporters containing
various 5'-flanking deletions of the IL-8 promoter (39)
were a gift of K. Matsushima. Cells in six-well dishes were transfected
with 1.5 µg of plasmid DNA, using Lipofectamine Plus (Life
Technologies), according to the manufacturer’s instructions.
Luciferase activity was determined and normalized relative to
-galactosidase expression as described before (37, 40).
CAT-assays were performed with an enzyme immunoassay (CAT ELISA,
Boehringer Mannheim, Indianapolis, IN).
Recombinant adenovirus and adenovirus infection
Recombinant adenovirus containing an IB-AA superrepressor
(Ad5IB-A32/36) or the E. coli -galactosidase gene
(Ad5LacZ) was constructed as described before (41).
Ad5IB-A32/36 expresses a HA-epitope tagged mutant form of IB
in which serine residues 32 and 36 are replaced by alanine residues as
described above. The mutant IB cannot be phosphorylated at
positions 32 and 36 and acts as a superrepressor. The HA epitope tag
enables identification of the exogenous superrepressor with
anti-hemagglutinin Abs. Viral titers were determined by plaque
assay. Recombinant virus was stored in PBS containing 10% (v/v)
glycerol at -80°C.
HT-29 cells grown to confluence in six-well tissue culture plates
(Costar, Cambridge, MA) were infected with Ad5IB-A32/36 or Ad5LacZ
in serum-free media (Opti-MEM, Life Technologies) at MOI 100 for
16 h. At this MOI, Ad5IB-A32/36 or Ad5LacZ infected >80%
HT-29 cells, and infected cells expressed IB-A32/36 and
-galactosidase, respectively, at high levels as assessed by staining
for -galactosidase and immunostaining for HA-tagged IB-A32/36
(data not shown). After infection, adenovirus was removed by washing,
fresh medium containing serum was added, and cells were incubated for
an additional 12 h before bacterial infection or stimulation with
TNF-.
Cell extracts
Cells were harvested, and whole cell and nuclear extracts were
prepared as described before (16, 17, 33). Briefly, cell
pellets were resuspended and 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 mM DTT
containing phosphatase inhibitors (40 mM -glycerophosphate, 20 mM
NaF, 1 mM Na3VO4, 20 mM
p-nitrophenyl phosphate (Calbiochem, San Diego, CA)); and
protease inhibitors (aprotinin 10 µg/ml, leupeptin 10 µg/ml,
bestatin 10 µg/ml, and pepstatin 10 µg/ml) (Calbiochem) and 1 mM
phenylmethylsulfonyl chloride (Sigma, St. Louis, MO). Lysates were
centrifuged at 13,000 x g for 15 min in the cold, and
the resulting supernatants were transferred to fresh tubes. Protein
concentrations in the supernatants were determined by the Bradford
assay (Bio-Rad, Hercules, CA).
Immunoblotting for IBs
Cell lysates containing 20 µg of protein were electrophoresed
on SDS-10% polyacrylamide gels with a 4% polyacrylamide stacking gel.
After transfer to Immobilon-P membranes (Millipore, Marlborough, MA),
membranes were blocked with a solution of 5% (w/v) dry milk in PBS-T
(PBS containing 0.05% Tween 20) for 1 h at room temperature.
Membranes were probed with murine mAbs to IB, IB (Santa
Cruz Biotechnology, Santa Cruz, CA), and IB (gift of N. Rice) and
visualized using an ECL detection 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 20 µg
of whole cell extract or 6 µg of nuclear extract were incubated for
30 min at 4°C with 5 µg of polyoligonucleotides (dI-dC) and 2
x 104 cpm (0.2 ng) of a labeled
oligonucleotide probe corresponding to a consensus NF-B binding 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 mM
-glycerophosphate, 10 mM MgCl2, 10 mM
p-nitrophenyl phosphate, 100 µM
Na3VO4, 2 mM DDT, 20 µM
ATP, 10 µg/ml aprotinin, 50–200 mM NaCl, pH 7.5, and 1–10 µCi
[-32P]ATP at 30°C for 30 min.
IB-substrate proteins were expressed and purified from E.
coli as described before (17). IKK- and
IKK-containing complexes were immunoprecipitated with specific mAbs
to IKK (PharMingen, San Diego, CA.) or monospecific rabbit
polyclonal Ab to carboxy-terminal IKK. Immune complexes were
isolated and washed in kinase buffer containing 1.5 M urea (16, 18). Kinase activity was determined using GST-IB (1-54)
wild type as substrate as described before (17). Kinase
specificity was established with mutant GST-IB (1-54-AA) in which
serines 32 and 36 were substituted with alanines (16).
Fold induction of IKK and IKK kinase activities was determined
after phosphorimaging of the dried SDS-PAGE-fractionated GST-IB
protein-containing gels.
Oligonucleotide primers for PCR amplification
Oligonucleotide primers used for PCR amplification and the size
of the PCR products obtained from target cellular RNA are shown in
Table I
. Exon-spanning primers were
designed to amplify cDNA, but not genomic DNA. Primers were designed
and synthesized as described previously (2, 3, 4) or were
obtained commercially.
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Total cellular RNA was extracted from cells with Trizol reagent
(Life Technologies) and reverse transcribed as described previously
(2). PCR amplification consisted of 35 cycles of 1 min
denaturation at 95°C, 2.5 min annealing, and extension at 60°C
(IL-8, COX-2, GRO, ICAM-1, NOS2), 65°C (MCP-1, TGF-1), or
72°C (-actin, TNF-, TGF-). A hot start in which samples were
preheated to 95°C before addition of Taq polymerase
(Stratagene, San Diego, CA) was used to increase specificity of the
amplification. Each experiment included negative controls in which RNA
was omitted from the reverse transcription mixture, and cDNA was
omitted from the PCR reaction.
Cytokine ELISAs
Cytokines in culture supernatants were assayed by ELISA as
described before (2, 3). The IL-8 and the GRO ELISAs
were sensitive to 20 and 30 pg/ml, respectively.
Flow cytometry
Monolayers of colon epithelial cells were detached by incubation
with 0.25% EDTA in calcium- and magnesium-free PBS (pH 7.2) as
described before (5). Cell viability was >95% as
assessed by trypan blue dye exclusion. For flow cytometry, 5 x
105 cells were incubated with optimal
concentrations of anti-CD54 (ICAM-1) (murine IgG1, AMAC, Westbrook,
ME) at 4°C for 60 min after which cells were washed, incubated with
an optimal concentration of a PE-labeled goat anti-mouse IgG (H +
L) (Southern Biotechnology Associates, Birmingham, AL) at 4°C for 60
min, and analyzed using a flow cytometer (FACScan, Becton Dickinson,
Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in HT-29, Caco-2, and T84
human colon epithelial cells
The transcription factor NF-B has a role in the transcriptional
activation of genes the mRNA expression of which is known to be
increased by infection of human colon epithelial cells with various
enteroinvasive bacteria (e.g., TNF-, the C-X-C chemokines IL-8 and
GRO, the C-C chemokine MCP-1, as well as COX-2 and NOS2)
(1, 2, 3, 4, 5, 7, 12, 42, 43). To determine whether enteroinvasive
bacteria activate NF-B in human colon epithelial cells, DNA binding
studies were performed using cell extracts obtained at various times
after infection of HT-29, Caco-2, and T84 cells with S.
dublin, Y. enterocolitica, or enteroinvasive
E. coli. Infection of those cells with each of these
enteroinvasive bacteria increased NF-B DNA binding, as shown by
EMSAs (Fig. 1). Maximum binding activity
occurred within 30–45 min after bacterial infection. For comparison,
maximum binding after stimulation of HT-29 and T84 cells with TNF-,
a potent activator of NF-B, was more rapid (i.e., within 10 min).
NF-B binding to the NF-B oligonucleotide probe was mediated
predominantly by heterodimers of p65, as demonstrated in supershift
assays in which irrelevant rabbit Ab, anti-p65, or anti-p50 Abs
were added to cell extracts (data not shown). Nonpathogenic noninvasive
E. coli DH5 resulted in little if any increase in
NF-B DNA binding activity in the same epithelial cell lines (Fig. 1). These studies showed that activation of NF-B parallels
activation of the epithelial cell inflammatory program. This suggested
that NF-B may be an essential transcription factor for integrating
the host response of intestinal epithelial cells to infection with a
variety of enteroinvasive bacteria that are known to use a spectrum of
different receptors and signaling pathways to induce their uptake into
these nonphagocytic cells.
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B in human colon epithelial cells after
infection with enteroinvasive bacteria
One of the major pathways for NF-B activation involves the
phosphorylation of IB on serine residues 32 and 36, which is
followed by IB degradation and the subsequent migration of
NF-B dimers from the cytoplasm to the nucleus. To determine whether
this is also the major pathway for NF-B activation after bacterial
infection of human intestinal epithelial cells, we assayed the kinetics
of IB degradation, as well as the degradation of IB and
IB, by immunoblot analysis, in HT-29, Caco-2, and T84 cells after
infection with S. dublin, Y. enterocolitica, and
enteroinvasive E. coli, or the nonpathogenic E.
coli DH5. Infection of Caco-2 cells with S. dublin
or Y. enterocolitica resulted in the rapid but transient
degradation of IB, whereas the kinetics of IB degradation
was slower after enteroinvasive E. coli infection (Fig. 1
).
Further, relative to IB, IB was more slowly degraded in
those cells. Consistent with the known lack of response of Caco-2 cells
to TNF- stimulation (44), little degradation of
IB and IB was seen in TNF--stimulated Caco-2 cells.
IB and IB degradation was less marked and incomplete in
bacteria-infected HT-29 and T84 cells (Fig. 1). The latter findings are
consistent with the incomplete degradation of IB in
IL-1-stimulated HT-29 and T84 cells (15). In contrast,
however, TNF- stimulation of HT-29 and T84 cells resulted in rapid
and nearly complete degradation of IB (Fig. 1). In HT-29 or T84
cells, IB was not degraded in response to infection with the
enteroinvasive bacteria, although it was partially degraded in Caco-2
cells (data not shown). Taken together, the data show that NF-B is
activated in each cell line in response to infection with several
different enteroinvasive bacteria and in HT-29 and T84 cells stimulated
with TNF-, despite differences among the cell lines in the extent of
degradation of IBs in response to different stimuli. Infection of
the cell lines with nonpathogenic E. coli DH5 resulted in
little if any degradation of IB, IB, or IB (Fig. 1
and data not shown).
Activation of IKK precedes NF-B activation
IKK contains two subunits, IKK and IKK, that can directly
phosphorylate IBs (45). Therefore, we assessed whether
IKK and IKK were activated in intestinal epithelial cells in
response to infection with enteroinvasive bacteria. Kinase activity was
determined with a GST-IB fusion protein as a substrate that can
be phosphorylated by IKK on serine residues 32 and 36. A GST-IB
fusion protein with alanine substitutions at positions 32 and 36, which
cannot be phosphorylated by IKK, was used in parallel to demonstrate
IKK specificity. As shown in Fig. 2,
IKK and IKK were activated within 15 min after infection of
HT-29, Caco-2, and T84 cells with S. dublin, Y.
enterocolitica or enteroinvasive E. coli. Moreover, the
time course of IKK and IKK activation in response to infection
with these bacteria preceded the time course of IB degradation,
as shown in Fig. 1. IKK and IKK were activated weakly, if at all,
above baseline after infection with nonpathogenic E. coli
DH5.
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We previously showed that infection of HT-29, Caco-2, and T84
cells with S. dublin, Y. enterocolitica, or
enteroinvasive E. coli, up-regulated IL-8 and ICAM-1 mRNA
levels, as well as IL-8 secretion and membrane ICAM-1 expression
(1, 2, 5). To determine whether increased IL-8 and ICAM-1
expression in response to bacterial 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-B-luciferase transcriptional reporter genes
(33), after which cells were infected with
S. dublin, Y. enterocolitica, or enteroinvasive
E. coli. As shown in Table II,
infection with those enteroinvasive bacteria markedly increased
luciferase activity in cells transfected with the IL-8, ICAM-1, and 2x
NF-B promoter plasmids, but not in cells transfected with a control
-actin-luciferase reporter gene construct. Nonpathogenic E.
coli DH5 did not significantly activate expression of the
tested reporter constructs.
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B and IKK
superrepressors
We asked whether activation of IKKs and degradation of IB
were components of the signaling pathway that culminates in increased
IL-8 and ICAM-1 expression following infection of human colon
epithelial cells with enteroinvasive bacteria. For these studies,
IL-8-, ICAM-1-, and NF-B-luciferase reporters were transiently
transfected into HT-29 cells alone, or together with either an
IKK-AA expression plasmid that encodes a catalytically inactive
IKK that acts as a superrepressor or an IB-A32/36 expression
plasmid that encodes a mutant IB in which serine residues at
positions 32 and 36 are replaced by alanine residues to prevent its
stimulus-induced phosphorylation and subsequent degradation. Activation
of the IL-8, ICAM-1, and NF-B transcriptional reporters was
inhibited in cells cotransfected with the IKK and IB
superrepressor plasmids (Table II), but not in cells cotransfected with
control plasmid (data not shown).
The IL-8 promoter contains a binding site for NF-B which is located
between nucleotides -80 to -69. However, 5' of the NF-B-binding
site, the IL-8 promoter also contains binding sites for other
transcription factors (39, 46) that might play a role in
transcriptional activation of the IL-8 promoter. We therefore
determined whether the nucleotide sequences in the IL-8 promoter that
encompass the NF-B-binding site were required for the activation of
the IL-8 promoter following bacterial infection, or whether 5' upstream
sequences that encoded an activator protein 1 (AP-1) binding site
(-120 to -126), a glucocorticoid response element (-325 to -330),
and an IFN regulatory factor-1 binding site (-420 to -425) were also
required. A series of IL8-CAT transcriptional reporters containing 5'
flanking region deletions, as shown in Table III, were transiently transfected into
HT-29 cells, after which cells were infected with S. dublin
or Y. enterocolitica or stimulated with TNF-. Increased
CAT expression in response to bacterial infection was greatest in cells
transfected with the -98-CAT deletion mutant, that contains the
NF-B binding site required for IL-8 gene transcription and a NF-IL6
binding site, but lacks the AP-1 binding site located at nucleotides
-120 to -126 and other upstream elements. No increase in CAT activity
was seen when all 5' sequence upstream of -8 was deleted. Parallel
findings were seen after TNF- stimulation (Table III).
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B superrepressor blocks expression of IL-8, GRO,
MCP-1, ICAM-1, COX-2, NOS2, and TNF- in response to bacterial
infection
We next evaluated whether blocking NF-B activation decreased
the expression of endogenous genes that are known to be major
components of the epithelial cell response to infection with invasive
bacteria. HT-29 cells were infected with a recombinant
replication-deficient adenovirus containing either a IB
superrepressor, termed Ad5IB-A32/36 (Ad5-IB/AA) or the E.
coli -galactosidase gene, termed Ad5LacZ, as a control.
Ad5-IB-AA partially to almost completely inhibited the up-regulated
expression of ICAM-1, IL-8, GRO, MCP-1, and COX-2 mRNA (Fig. 3), as well as NOS2 and TNF- mRNA
(data not shown), in response to infection with enteroinvasive
bacteria, but did not block mRNA expression of TGF1, -actin (Fig. 3) or TGF (data not shown).
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secretion in response to S. dublin infection
or TNF- stimulation of HT-29 cells infected with Ad5IB-A32/36 or
the Ad5LacZ control virus was also assessed. As shown in Table IV, IL-8 and GRO secretion was
markedly inhibited by the IB-AA superrepressor. The low levels of
IL-8 and GRO in Ad5IB infected cultures may reflect production by
the 20% residual cells that were not infected by the adenovirus vector
or a low level of activation of those mediators via other
signal-transduction pathways. Further, as shown in Fig. 4, increased cell surface ICAM-1
expression in response to infection with enteroinvasive bacteria, but
not in response to IFN- stimulation, was inhibited by the
IB-A32/36 superrepressor.
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B requires intracellular
signaling molecules that are also components of pathways activated by
TNF receptor family members
Differences in IB degradation in bacteria-infected, compared
with TNF--stimulated HT-29 cells, suggested differences in the
pathways leading to activation of NF-B in response to these stimuli.
Nonetheless, many of the NF-B target genes that are activated in
response to bacterial infection of human intestinal epithelial cells,
including IL-8, are also activated by signaling through TNF, IL-1 or
LTR. We assessed, therefore, whether molecules that play a more
proximal role in the signal transduction pathway leading to IKK
activation following agonist stimulation via TNF receptors, such as
TRAF2 or NIK, a mitogen-activated MAP-3 kinase which mediates the
phosphorylation and activation of IKK (21, 22), are also
required for activation of the NF-B target gene IL-8 in bacteria
infected cells. For these studies, an IL-8-luciferase transcriptional
reporter was cotransfected into HT-29 cells with a TRAF2 superrepressor
plasmid (30), or with a plasmid that expresses a mutant of
NIK that is catalytically inactive (21). In addition, some
cultures were cotransfected with the IL-8-luciferase reporter together
with a plasmid expressing A20, a protein that blocks TRAF2-mediated
NF-B activation (36) or with a plasmid expressing a
superrepressor of TRAF5, a protein that is important for the activation
of NF-B following signal transduction through the LTR
(31). Cultures were subsequently infected with
enteroinvasive S. dublin, Y. enterocolitica, or
enteroinvasive E. coli or stimulated with TNF- as a
control. As shown in Table V, increased
luciferase activity in response to stimulation of cells with TNF-
was markedly inhibited by blocking TRAF2, and to a lesser extent by
blocking NIK or TRAF5. Blocking of NIK also partially inhibited
bacteria induced activation of the IL-8 reporter. Inhibition of TRAF5
was as effective as inhibition of TRAF2 in blocking S.
dublin or enteroinvasive E. coli, but not Y.
enterocolitica induced activation of the IL-8 reporter. Consistent
with the results of TRAF2 inhibition described above, expression of
A20, which inhibits TRAF2 mediated NF-B activation, also inhibited
bacteria, and TNF- induced activation of the IL-8 reporter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B as a central regulator for
the activation of essential components of the intestinal epithelial
cell innate immune response following infection with a spectrum of
enteroinvasive bacterial pathogens.
IKK (16, 18, 19) was shown to be a key intermediate in the
epithelial cell signal transduction pathway leading to NF-B
activation following infection with enteroinvasive bacteria. Although
IKK and IKK were activated with a similar time course, an IKK
superrepressor alone was sufficient to inhibit the activation of IL-8
and ICAM-1 transcriptional reporters and was more efficient than an
IKK superrepressor in this regard (data not shown). These findings
are consistent with the physiologic role IKK is known to have
in IB phosphorylation and NF-B activation
(18, 19, 45, 46). IKK in the large m.w. IKK
complex may function either as an IKK homo- or IKK
heterooligomer (18, 19, 45, 46).
The promoter regions of the epithelial cell proinflammatory genes
studied herein contain binding sites for several transcription factors
in addition to NF-B. For example, the IL-8 promoter has binding
sites for AP-1 and NF-IL6 that are functionally important for IL-8 gene
activation in several cell types (39, 47). However, the
AP-1 binding site was not required for S. dublin or Y.
enterocolitica induced IL-8 promoter activity in the three
intestinal epithelial cell lines studied herein, because a
transcriptional IL-8 reporter that lacks the AP-1 binding site was
readily activated in those cells after infection with those bacteria.
In contrast, there appears to be a requirement for both NF-B and
AP-1 for transcriptional activation of the IL-8 promoter in S.
typhimurium-infected Henle-407 cells (48). Although
NF-B has an essential role in integrating the host intestinal
epithelial cell response to infection with enteroinvasive bacteria,
additional transcription factors are known to positively modify the
transcription of many of the genes that comprise the epithelial cell
innate immune response.
NF-B was activated in three human colon epithelial cell lines
infected with three different stains of enteroinvasive bacteria, but
not after infection with noninvasive E. coli. Consistent
with this, NF-B was not activated in T84 cells cultured with a
different nonpathogenic strain of E. coli although
activation was noted in cells infected with an enteropathogenic
E. coli (49). Activation of NF-B in the
cytoplasm involves the inducible phosphorylation of IBs, which then
undergo ubiquitin-mediated proteolysis, thereby releasing NF-B
dimers to translocate to the nucleus (33, 42, 50). We
noted marked differences in the extent of IB and IB
degradation among the cell lines, with degradation being more complete
in enteroinvasive bacteria-infected Caco-2 than in HT-29 or T84 cells.
This finding parallels that reported for NF-B activation and
IB degradation after IL-1 stimulation of the same cell lines
(15), in which case incomplete degradation of IB was
also sufficient for significant activation of NF-B.
In contrast to bacterial infection (data herein) or IL-1 stimulation
(15), degradation of IB was almost complete in
TNF--stimulated HT-29 and T84 cells. The difference between
bacterial infection and TNF stimulation may reflect the fact that only
a fraction of cells are infected by enteroinvasive bacteria (e.g.,
10–50% of HT-29 and T84 cells are infected by S. dublin at
the MOI’s used herein). In this case, even complete degradation of
IB in bacteria-infected cells would be obscured by the lack of
degradation in uninfected cells. This would not apply to a soluble
cytokine like TNF-, which presumably can stimulate a larger fraction
of the cells. Alternatively, differences in IB degradation in
response to bacterial infection, compared with TNF- stimulation, may
reflect an interplay between the different signaling pathways activated
by these stimuli, and the relative importance of those pathways, as
also suggested by our findings using superrepressors of TRAF2 and
TRAF5.
Infection of Caco2 cells with bacteria but not stimulation with TNF-
activated NF-B, indicating that activation of NF-B likely is
independent of signaling through the extracellular domain of the TNF
receptor. Whereas our findings with bacterial infection of human
intestinal cells are consistent with those showing activation of
NF-B in a human cervical epithelial cell line, HeLa, infected with
Shigella flexneri (51), our findings with
Y. enterocolitica infection of human intestinal epithelial
cells differ markedly from those in mouse macrophages, where Y.
enterocolitica prevented NF-B activation (52)
The various IBs can differentially associate and regulate the
activation of NF-B (53) and the transcriptional
activation of various NF-B target genes by virtue of binding to
different populations of NF-B dimers in the cytoplasm. IB is
widely expressed in different human tissues, is known to interact
preferentially with p65 and c-Rel members and appears to affect a
subset of NF-B genes apparently regulated by p65 homodimers
(54, 55). ICAM-1 and GM-CSF promoters contain
NF-B-binding sites that bind only p65/c-Rel heterodimers in vitro
(56), and the NF-B site of the IL-8 promoter optimally
binds and is activated by p65 homodimers rather than p50 homodimers or
p50/p65 heterodimers (57). Our finding of partial IB
degradation after bacterial infection of intestinal epithelial cells
suggests a role for IB in the activation of several NF-B
target genes that are up-regulated in response to bacterial infection
of intestinal epithelial cells. A similar situation may exist for the
antimicrobial response in Drosophila, where different
NF-B family members are differentially activated and imported into
the nucleus where they activate selective peptide (cecropin) genes
(58).
Human intestinal epithelial cells rapidly activate NF-B and a
proinflammatory gene program within a few hours of infection with
enteroinvasive bacteria. However, in the later period after infection
(e.g., 12–18 h) human colon epithelial cell lines undergo apoptosis
(59). The delayed onset of apoptosis may provide invading
bacteria sufficient time to adapt to the intracellular epithelial cell
environment and multiply, before invading deeper mucosal layers.
Moreover, the delayed onset of apoptosis might be partly explained by
the activation of NF-B after bacterial entry, because NF-B target
genes suppress signals for cell death as shown for mouse cell lines
stimulated with TNF- (60, 61) and for endothelial cells
infected by Rickettsia rickettsii (62).
NF-B activation in bacteria-infected cells appears to be independent
of signaling through the extracellular domain of the TNF receptor.
Nonetheless, our data indicate that molecules that are integral
components of the signal transduction pathway leading to NF-B
activation after signaling through members of the TNF receptor family
were involved in signal transduction leading to the activation of the
NF-B target gene, IL-8, in bacteria-infected cells. Activation of
IKK and IKK requires their phosphorylation, which has been
reported to be mediated by the MAP3 kinases, NIK and MEKK-1
(20, 21, 22, 23, 24). As shown herein, NIK appears to play a role in
NF-B activation after bacterial infection, given that overexpression
of a catalytically inactive NIK protein partially inhibited
transcriptional activation of an IL-8 reporter in response to bacterial
infection. Further upstream in the signal transduction pathway
activated by TNF-, NIK interacts with TRAF2, an adaptor protein
known to associate with the TNF receptor family of proteins (23, 30, 63). Recently, TRAF2 was reported to be involved in TNF-
and IL-1 signaling cascades leading to NF-B activation and IL-8
expression in HT-29 cells (64). Interference of TRAF2
signaling by transfection of a superrepressor of TRAF2, or with
A20, which interacts with TRAF2 and blocks TRAF2-mediated
NF-B activation (36), markedly inhibited IL-8 reporter
gene activity in response to bacterial infection. Blocking TRAF5, an
adaptor molecule important in signaling through other TNF receptor
family members (e.g., LTR), also partially blocked IL-8 reporter
gene activity. Taken together, our findings suggest that bacterial
infection may activate a number of different intracellular signaling
pathways also used by TNF and perhaps IL-1 receptor family members,
that culminate in the activation of NF-B and its target genes.
Intestinal epithelial cells act as sensors of microbial infection and
produce proinflammatory signals that can activate the host’s mucosal
inflammatory response. The data herein demonstrate that NF-B is an
essential transcription factor for integrating the host epithelial cell
proinflammatory response to infection with a spectrum of
enteroinvasive bacteria. Studies in a murine model of intestinal
inflammation (65) and in human intestinal xenografts
infected with E. histolytica (66) showed that
blocking NF-B activation in the intestinal mucosa can markedly
decrease intestinal inflammation. Our studies in epithelial cells
indicate that signal transduction through NF-B is a key element in
the activation of the epithelial cell innate immune response after
bacterial infection and suggest novel therapeutic targets for the
prevention and treatment of intestinal inflammation.
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Acknowledgments |
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Footnotes |
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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, Laboratory of Mucosal Immunology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623. E-mail address:
7 Abbreviations used in this paper: GRO, growth-related oncogene-; MCP-1, monocyte chemoattractant protein-1; COX, cyclooxygenase; NOS2, nitric oxide synthase-2; IKK, IB kinase; MAP3, mitogen-activated protein-3; NIK, NF-B-inducing kinase; MEKK-1, MEK kinase 1; TRAF, TNF receptor-associated factor; UCSD, University of California, San Diego; MOI, multiplicity of infection; CAT, chloramphenicol acetyltransferase; HA, hemagglutinin; EMSA, electrophoretic mobility shift assay; AP-1, activator protein 1; LTR, lymphotoxin receptor .
Received for publication March 22, 1999. Accepted for publication May 17, 1999.
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References |
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S. Dahan, G. Dalmasso, V. Imbert, J.-F. Peyron, P. Rampal, and D. Czerucka Saccharomyces boulardii Interferes with Enterohemorrhagic Escherichia coli-Induced Signaling Pathways in T84 Cells Infect. Immun., February 1, 2003; 71(2): 766 - 773. [Abstract] [Full Text] [PDF]
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M. E. Hobert, K. A. Sands, R. J. Mrsny, and J. L. Madara Cdc42 and Rac1 Regulate Late Events in Salmonella typhimurium-induced Interleukin-8 Secretion from Polarized Epithelial Cells J. Biol. Chem., December 20, 2002; 277(52): 51025 - 51032. [Abstract] [Full Text] [PDF]
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P. M. Cobelens, A. Kavelaars, A. Vroon, M. Ringeling, R. van der Zee, W. van Eden, and C. J. Heijnen The {beta}2-Adrenergic Agonist Salbutamol Potentiates Oral Induction of Tolerance, Suppressing Adjuvant Arthritis and Antigen-Specific Immunity J. Immunol., November 1, 2002; 169(9): 5028 - 5035. [Abstract] [Full Text] [PDF]
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D. Haller, M. P. Russo, R. B. Sartor, and C. Jobin IKKbeta and Phosphatidylinositol 3-Kinase/Akt Participate in Non-pathogenic Gram-negative Enteric Bacteria-induced RelA Phosphorylation and NF-kappa B Activation in Both Primary and Intestinal Epithelial Cell Lines J. Biol. Chem., October 4, 2002; 277(41): 38168 - 38178. [Abstract] [Full Text] [PDF]
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C. A. Dobbin, N. C. Smith, and A. M. Johnson Heat Shock Protein 70 Is a Potential Virulence Factor in Murine Toxoplasma Infection Via Immunomodulation of Host NF-{kappa}B and Nitric Oxide J. Immunol., July 15, 2002; 169(2): 958 - 965. [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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M. T. Abreu, E. T. Arnold, L. S. Thomas, R. Gonsky, Y. Zhou, B. Hu, and M. Arditi TLR4 and MD-2 Expression Is Regulated by Immune-mediated Signals in Human Intestinal Epithelial Cells J. Biol. Chem., May 31, 2002; 277(23): 20431 - 20437. [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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 S J H van Deventer Small therapeutic molecules for the treatment of inflammatory bowel disease Gut, May 1, 2002; 50(90003): iii47 - 53. [Abstract] [Full Text] [PDF]
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M. C. Cerquetti, N. B. Goren, A. J. Ropolo, D. Grasso, M. N. Giacomodonato, and M. I. Vaccaro Nitric Oxide and Apoptosis Induced in Peyer's Patches by Attenuated Strains of Salmonella enterica Serovar Enteritidis Infect. Immun., February 1, 2002; 70(2): 964 - 969. [Abstract] [Full Text] [PDF]
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 W. Altaf, S. Perveen, K. U. Rehman, S. Teichberg, I. Vancurova, R. G. Harper, and R. A. Wapnir Zinc Supplementation in Oral Rehydration Solutions: Experimental Assessment and Mechanisms of Action J. Am. Coll. Nutr., February 1, 2002; 21(1): 26 - 32. [Abstract] [Full Text] [PDF]
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 E. W. Uhl, S. Giguere, T. J. Jack, and T. Hodge Increased Pulmonary Activation of Nuclear Factor-{kappa}B (NF-{kappa}B) in Foals Inoculated with Rhodococcus equi is Associated with Increased Expression of Inflammatory Cytokines Vet. Pathol., January 1, 2002; 39(1): 132 - 136. [Abstract] [Full Text] [PDF]
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F. Sierro, B. Dubois, A. Coste, D. Kaiserlian, J.-P. Kraehenbuhl, and J.-C. Sirard Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells PNAS, November 20, 2001; 98(24): 13722 - 13727. [Abstract] [Full Text] [PDF]
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S. D. Savkovic, A. Ramaswamy, A. Koutsouris, and G. Hecht EPEC-activated ERK1/2 participate in inflammatory response but not tight junction barrier disruption Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G890 - G898. [Abstract] [Full Text] [PDF]
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M. T. Abreu, P. Vora, E. Faure, L. S. Thomas, E. T. Arnold, and M. Arditi Decreased Expression of Toll-Like Receptor-4 and MD-2 Correlates with Intestinal Epithelial Cell Protection Against Dysregulated Proinflammatory Gene Expression in Response to Bacterial Lipopolysaccharide J. Immunol., August 1, 2001; 167(3): 1609 - 1616. [Abstract] [Full Text] [PDF]
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 E. C. CLAUD and W. A. WALKER Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis FASEB J, June 1, 2001; 15(8): 1398 - 1403. [Abstract] [Full Text] [PDF]
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M. C. Berin, M. B. Dwinell, L. Eckmann, and M. F. Kagnoff Production of MDC/CCL22 by human intestinal epithelial cells Am J Physiol Gastrointest Liver Physiol, June 1, 2001; 280(6): G1217 - G1226. [Abstract] [Full Text] [PDF]
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 D. DRUDY, D.P. O'DONOGHUE, A. BAIRD, L. FENELON, and C. O'FARRELLY Flow cytometric analysis of Clostridium difficile adherence to human intestinal epithelial cells J. Med. Microbiol., June 1, 2001; 50(6): 526 - 534. [Abstract] [Full Text] [PDF]
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A. Deb, S. J. Haque, T. Mogensen, R. H. Silverman, and B. R. G. Williams RNA-Dependent Protein Kinase PKR Is Required for Activation of NF-{{kappa}}B by IFN-{{gamma}} in a STAT1-Independent Pathway J. Immunol., May 15, 2001; 166(10): 6170 - 6180. [Abstract] [Full Text] [PDF]
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W. F. Stenson, Z. Zhang, T. Riehl, and S. L. Stanley Jr. Amebic Infection in the Human Colon Induces Cyclooxygenase-2 Infect. Immun., May 1, 2001; 69(5): 3382 - 3388. [Abstract] [Full Text] [PDF]
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 M. Cecilia Berin, L. Eckmann, D. H. Broide, and M. F. Kagnoff Regulated Production of the T Helper 2-Type T-Cell Chemoattractant TARC by Human Bronchial Epithelial Cells In Vitro and in Human Lung Xenografts Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 382 - 389. [Abstract] [Full Text]
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A. Izadpanah, M. B. Dwinell, L. Eckmann, N. M. Varki, and M. F. Kagnoff Regulated MIP-3{alpha}/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity Am J Physiol Gastrointest Liver Physiol, April 1, 2001; 280(4): G710 - G719. [Abstract] [Full Text] [PDF]
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G. T.-J. Huang, D. Kim, J. K.-H. Lee, H. K. Kuramitsu, and S. K. Haake Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms Infect. Immun., March 1, 2001; 69(3): 1364 - 1372. [Abstract] [Full Text] [PDF]
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 A. S. Neish, A. T. Gewirtz, H. Zeng, A. N. Young, M. E. Hobert, V. Karmali, A. S. Rao, and J. L. Madara Prokaryotic Regulation of Epithelial Responses by Inhibition of Ikappa B-alpha Ubiquitination Science, September 1, 2000; 289(5484): 1560 - 1563. [Abstract] [Full Text]
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R. SCHULTE, G. A. GRASSL, S. PREGER, S. FESSELE, C. A. JACOBI, M. SCHALLER, P. J. NELSON, and I. B. AUTENRIETH Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers FASEB J, August 1, 2000; 14(11): 1471 - 1484. [Abstract] [Full Text]
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D. J. Philpott, S. Yamaoka, A. Israel, and P. J. Sansonetti Invasive Shigella flexneri Activates NF-{kappa}B Through a Lipopolysaccharide-Dependent Innate Intracellular Response and Leads to IL-8 Expression in Epithelial Cells J. Immunol., July 15, 2000; 165(2): 903 - 914. [Abstract] [Full Text] [PDF]
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L. Eckmann, J. R. Smith, M. P. Housley, M. B. Dwinell, and M. F. Kagnoff Analysis by High Density cDNA Arrays of Altered Gene Expression in Human Intestinal Epithelial Cells in Response to Infection with the Invasive Enteric Bacteria Salmonella J. Biol. Chem., May 5, 2000; 275(19): 14084 - 14094. [Abstract] [Full Text] [PDF]
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U. Bocker, A. Schottelius, J. M. Watson, L. Holt, L. L. Licato, D. A. Brenner, R. B. Sartor, and C. Jobin Cellular Differentiation Causes a Selective Down-regulation of Interleukin (IL)-1beta -mediated NF-kappa B Activation and IL-8 Gene Expression in Intestinal Epithelial Cells J. Biol. Chem., April 14, 2000; 275(16): 12207 - 12213. [Abstract] [Full Text] [PDF]
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D. F. McCole, L. Eckmann, F. Laurent, and M. F. Kagnoff Intestinal Epithelial Cell Apoptosis following Cryptosporidium parvum Infection Infect. Immun., March 1, 2000; 68(3): 1710 - 1713. [Abstract] [Full Text] [PDF]
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C. Jobin and R. B. Sartor The Ikappa B/NF-kappa B system: a key determinant of mucosal inflammation and protection Am J Physiol Cell Physiol, March 1, 2000; 278(3): C451 - C462. [Abstract] [Full Text] [PDF]
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 M. Zamanian-Daryoush, T. H. Mogensen, J. A. DiDonato, and B. R. G. Williams NF-kappa B Activation by Double-Stranded-RNA-Activated Protein Kinase (PKR) Is Mediated through NF-kappa B-Inducing Kinase and Ikappa B Kinase Mol. Cell. Biol., February 15, 2000; 20(4): 1278 - 1290. [Abstract] [Full Text]
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 B. S. Hall, W. Tam, R. Sen, and M. E. A. Pereira Cell-specific Activation of Nuclear Factor-kappa B by the Parasite Trypanosoma cruzi Promotes Resistance to Intracellular Infection Mol. Biol. Cell, January 1, 2000; 11(1): 153 - 160. [Abstract] [Full Text]
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D. A. O'Neil, E. M. Porter, D. Elewaut, G. M. Anderson, L. Eckmann, T. Ganz, and M. F. Kagnoff Expression and Regulation of the Human {beta}-Defensins hBD-1 and hBD-2 in Intestinal Epithelium J. Immunol., December 15, 1999; 163(12): 6718 - 6724. [Abstract] [Full Text] [PDF]
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O. Steele-Mortimer, L. A. Knodler, S. L. Marcus, M. P. Scheid, B. Goh, C. G. Pfeifer, V. Duronio, and B. B. Finlay Activation of Akt/Protein Kinase B in Epithelial Cells by the Salmonella typhimurium Effector SigD J. Biol. Chem., November 22, 2000; 275(48): 37718 - 37724. [Abstract] [Full Text] [PDF]
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