Tumor Suppressor Foxo3a Is Involved in the Regulation of Lipopolysaccharide-Induced Interleukin-8 in Intestinal HT-29 Cells

Abstract

Enteric bacteria and their products play an important role in intestinal inflammation; however, the complete mechanisms are not elucidated yet. Tumor suppressor Foxo3a regulates gene expression in the nucleus, and its translocation to the cytosol leads to inactivation. Proximally, Foxo3a is regulated by different pathways including the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study was to determine the effect of bacterial infection on Foxo3a in intestinal epithelial cells and to examine the contribution of Foxo3a in intestinal inflammation. Bacterial lipopolysaccharide (LPS) and infection with mouse pathogen Citrobacter rodentium induce translocation of the nuclear Foxo3a into the cytosol, where it degrades in human HT-29 and mouse CMT-93 cells. In colonic epithelia of healthy mice, Foxo3a is localized in the epithelia at the bottom of the crypts in both the nucleus and the cytosol, while in C. rodentium-infected colon Foxo3a is expressed along the crypts and located mainly in the cytosol, suggesting its inactivation. LPS utilized the PI3K pathway to inhibit Foxo3a. Additionally, inhibition of PI3K attenuated LPS-induced proinflammatory interleukin-8 (IL-8). LPS-induced IL-8 is increased in HT-29 cells with silenced Foxo3a. Moreover, in HT-29 cells with silenced Foxo3a, the amount of IkappaBalpha, an NF-kappaB inhibitor, is decreased. In conclusion, LPS and bacterial infection inactivate Foxo3a in intestinal epithelia via the PI3K pathway and inactivated Foxo3a leads to the upregulation of IL-8 by suppressing inhibitory IkappaBalpha.

Full text

INFECTION AND IMMUNITY, Oct. 2008, p. 4677–4685 Vol. 76, No. 10
0019-9567/08/$08.00⫹0 doi:10.1128/IAI.00227-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Tumor Suppressor Foxo3a Is Involved in the Regulation of
Lipopolysaccharide-Induced Interleukin-8 in
Intestinal HT-29 Cells
䌤
Lobke Snoeks,
1
Christopher R. Weber,
2
Jerrold R. Turner,
2
Mitra Bhattacharyya,
1
Kaarin Wasland,
1
and Suzana D. Savkovic
1
*
Department of Medicine, Division of Gastroenterology, Evanston-Northwestern Research Institute, Evanston, Illinois 60201,
1
and
Department of Pathology, The University of Chicago, Chicago, Illinois 60637
2
Received 18 February 2008/Returned for modification 25 March 2008/Accepted 21 July 2008
Enteric bacteria and their products play an important role in intestinal inflammation; however, the complete
mechanisms are not elucidated yet. Tumor suppressor Foxo3a regulates gene expression in the nucleus, and its
translocation to the cytosol leads to inactivation. Proximally, Foxo3a is regulated by different pathways including the
phosphoinositide 3-kinase (PI3K) pathway. The aim of this study was to determine the effect of bacterial infection
on Foxo3a in intestinal epithelial cells and to examine the contribution of Foxo3a in intestinal inflammation.
Bacterial lipopolysaccharide (LPS) and infection with mouse pathogen Citrobacter rodentium induce translocation
of the nuclear Foxo3a into the cytosol, where it degrades in human HT-29 and mouse CMT-93 cells. In colonic
epithelia of healthy mice, Foxo3a is localized in the epithelia at the bottom of the crypts in both the nucleus and the
cytosol, while in C. rodentium-infected colon Foxo3a is expressed along the crypts and located mainly in the cytosol,
suggesting its inactivation. LPS utilized the PI3K pathway to inhibit Foxo3a. Additionally, inhibition of PI3K
attenuated LPS-induced proinflammatory interleukin-8 (IL-8). LPS-induced IL-8 is increased in HT-29 cells with
silenced Foxo3a. Moreover, in HT-29 cells with silenced Foxo3a, the amount of I␬B␣,anNF-␬B inhibitor, is
decreased. In conclusion, LPS and bacterial infection inactivate Foxo3a in intestinal epithelia via the PI3K pathway
and inactivated Foxo3a leads to the upregulation of IL-8 by suppressing inhibitory I␬B␣.
Inflammatory bowel disease, including Crohn’s disease and
ulcerative colitis, is characterized by chronic mucosal injury
and infiltration of inflammatory cells. In the pathogenesis of
intestinal inflammation, luminal bacteria play an important
role (14, 49). Most commonly, bacteria trigger an epithelial
response through toll-like receptors (TLR). TLR generate sig-
nals that activate a specific pattern of genes that elicit an
inflammatory response (1, 2). Downstream of TLR, the inflam-
matory response is controlled by NF-␬B, a transcriptional fac-
tor involved in the regulation of cytokines, chemokines, and
adhesion molecules. In unstimulated cells, NF-␬B is localized
in the cytoplasm and associated with inhibitory proteins known
as I␬Bs (48). Proinflammatory stimuli induce the phosphory-
lation of I␬B proteins, which are mediated by the proximal I␬B
kinase (IKK) complex. The phosphorylated I␬B degrades, and
NF-␬B is released and subsequently translocates to the nucleus
(48). Other molecules that are involved in the regulation of the
NF-␬B pathway also play an important role in controlling in-
flammation.
Tumor suppressor Foxo3a belongs to the Foxo family of Fork-
head transcriptional factors together with Foxo1, Foxo4, and
Foxo6 (10). In the absence of stimuli, Foxo3a is retained in the
nucleus, bound to the DNA or to other transcriptional factors (7).
Foxo3a regulates the expression of specific target genes that mod-
ulate the metabolic state, control cell cycle progression, regulate
the mitotic program, and induce cellular apoptosis (3, 10). Foxo
proteins are regulated by several pathways including phosphoino-
sitide 3-kinase (PI3K), casein kinase 1, DYRK1A, and IKK (4, 7,
20). These pathways phosphorylate Foxo3a, which leads to its
translocation to the cytoplasm and attachment to 14-3-3 proteins
(7, 29, 54). Cytoplasmic Foxo3a may also be regulated by the
proteasome system (4, 20).
Foxo3a-deficient mice develop a spontaneous, multisystemic
inflammatory syndrome, accompanied with increased cytokine
production, increased NF-␬B activation, and hyperactivation of T
cells (35); therefore, we assessed the role of Foxo3a in intestinal
inflammation. We have shown in this paper that bacterial lipo-
polysaccharide (LPS) and infection with Citrobacter rodentium
inactivate Foxo3a in intestinal epithelia, in vitro and in vivo. LPS-
dependent Foxo3a inactivation in intestinal HT-29 cells is con-
trolled by the PI3K pathway. We further demonstrated that
blocking PI3K leads to attenuation of LPS-induced interleukin-8
(IL-8) in intestinal HT-29 cells. Additionally, our data revealed
that LPS-induced IL-8 is increased in HT-29 cells with silenced
Foxo3a. Also, in HT-29 cells with silenced Foxo3a, the amount of
I␬B␣, an inhibitor of NF-␬B, is decreased. Altogether, our results
suggest that bacterial infection inactivates the tumor suppressor
Foxo3a, which additionally increases IL-8 by downregulating in-
hibitory I␬B␣(see Fig. 7 for model).
MATERIALS AND METHODS
Cell culture. Human intestinal epithelial HT-29 cells and mouse intestinal
epithelial CMT-93 cells (American Type Culture Collection, Manassas, VA)
from passages 14 to 25 were used in these studies. HT-29 cells were propagated
in McCoy’s 5A medium (Sigma-Aldrich, Saint Louis, MO), and CMT-93 cells
were propagated in Dulbecco-Vogt modified Eagle medium (Gibco-Invitrogen,
* Corresponding author. Mailing address: Evanston-Northwestern
Research Institute, Department of Medicine, Division of Gastroenter-
ology, 1001 University Place, Room 314, Evanston, IL 60201. Phone:
(224) 364-7404. Fax: (847) 733-5041. E-mail: SSavkovic@enh.org.
䌤
Published ahead of print on 4 August 2008.
4677

Carlsbad, CA) with 10% fetal bovine serum (Gibco). For protein analysis, cells
were plated in six-well plates and used when 60 to 70% confluent, while for
cytokine analysis, cells were plated in 12-well plates and used as 50% confluent
monolayers. Monolayers were serum deprived overnight before use in experi-
ments.
LPS treatment and bacterial infection. Monolayers of human HT-29 cells
were treated with LPS purified from Escherichia coli serotype O111:B4 (Sigma)
at a concentration of 100 ␮g/ml.
Monolayers of mouse CMT-93 cells were infected with C. rodentium DBS100
(American Type Culture Collection, Manassas, VA). C. rodentium cultures were
grown overnight with shaking in Luria-Bertani broth (LB) at 37°C, diluted (1:33)
in serum-free and antibiotic-free tissue culture medium containing 0.5% man-
nose, and grown at 37°C with aeration to mid-log growth phase (5 ⫻10
8
cells/
ml). Bacteria were spun down and resuspended in fresh serum-free medium;
monolayers of CMT-93 cells were then infected with ⬃100 bacteria/cell (37°C in
5% CO
2
) for designated time periods. For cytokine analysis, C. rodentium culture
grown in serum-free, antibiotic-free tissue culture medium was spun down and
the supernatant was sterilized by filtration through 0.22-␮m filters.
Treatment with PI3K inhibitors. For inhibitor studies, HT-29 cells were pre-
treated for 1 h with 200 nM wortmannin or 30 ␮M LY294002 (Calbiochem, San
Diego, CA) and then treated with LPS in the presence of an inhibitor for various
time periods. These concentrations of inhibitors were based on the most effective
inhibition of PI3K in other cell lines (19, 37, 38, 56).
Immunofluorescent staining. Monolayers of cells grown on coverslips were
LPS treated, washed with phosphate-buffered saline (PBS), fixed with 3.7%
paraformaldehyde, and permeabilized with 0.2% Triton X-100 in PBS. Following
permeabilization, monolayers were blocked in 2.5% bovine serum albumin and
incubated with anti-Foxo3a antibody (1:200; Upstate Biotechnology, Temecula,
CA). After being washed with PBS, monolayers were incubated with secondary
anti-rabbit immunoglobulin G antibody conjugated with Alexa 488 (Molecular
Probes-Invitrogen). Monolayers were mounted with Prolong Gold antifade re-
agent (Molecular Probes) and assessed using a Nikon Opti-Photo microscope.
Images were captured using a Spot RT-slider camera (Diagnostic Instruments,
Sterling Heights, MI), and images were managed with Image Pro software
(Media Cybernetics, San Diego, CA).
Protein extraction and immunoblot assays. Total proteins were extracted with
lysis buffer (Cell Signaling, Beverly, MA), in the presence of protease inhibitor
cocktail (Sigma).
Proteins (40 ␮g) were separated by 10% sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to
nitrocellulose membranes (Bio-Rad, Hercules, CA). Blots were incubated in
blocking solution and sequentially with a primary antibody against total Foxo3a
or phosphorylated Foxo3a (both from Upstate Biotechnology), actin (Santa Cruz
Biotechnology, Santa Cruz, CA), or I␬B␣(Cell Signaling) according to the
manufacturer’s protocols for antibodies. The blots were washed and incubated
with appropriate dilutions of secondary antibodies conjugated with horseradish
peroxidase for 1 h, and detection was achieved with ECL Plus Western blotting
detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).
siRNA of Foxo3a. Two different sequences of Foxo3a small interfering RNA
(siRNA; 30 nM) or equal amounts of negative-control oligonucleotides were
incubated in 50 ␮l of Opti-MEM containing Lipofectamine RNAiMax (Invitro-
gen) for 20 min at room temperature. The complexes were then added to
⬃90,000 HT-29 cells plated in 12-well plates and kept at 37°C for 24, 48, or 72 h.
The initial experiment confirmed that a period of 48 h leads to optimal silencing
of Foxo3a; thus, this time point was further used for experiments.
Overexpression of Foxo3a. To create CMT-93 cells that overexpress Foxo3a,
we employed pMX-Foxo3a-IRES-GFP and pMX-IRES-GFP retroviral vectors
kindly provided by Stanford Peng (Roche Palo Alto LLC, Palo Alto, CA).
Purified plasmids (10 ␮g) were used to transfect Phoenix packaging cell lines
(Clontech, Mountain View, CA) by Lipofectamine RNAiMax (Invitrogen).
These packaging cells are able to assemble the amphotropic viral particles with
a genome containing pMX constructs. Retroviral particles were harvested from
the medium of the packaging cells after 48 h and used to infect mouse intestinal
CMT-93 cells. Infected CMT-93 cells were allowed to express Foxo3a for a
period of 48 h.
Cytokine quantification. The amounts of IL-8 and macrophage inflammatory
protein 2 (MIP-2) were determined using enzyme-linked immunosorbent assay
(R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol.
Statistical analysis. All data are represented as the means ⫾standard errors.
Data comparisons were made with Student’s ttest. Differences were considered
significant when the Pvalues were ⱕ0.05.
Mouse infection. Mice of strain C57BL/J6, aged 4 to 6 weeks, were obtained
from the Jackson Laboratory (Bar Harbor, ME) and were maintained in the
Biological Resources Laboratory at the University of Illinois at Chicago. Main-
tenance and experiments were performed according to standard animal care
protocols. Approximately 2 ⫻10
8
C. rodentium bacteria in 200 ␮l of PBS were
introduced to animals by gavage using a 4-cm-long curved needle with a steel ball
at the tip. Control animals received 200 ␮l of PBS. Mice were returned to
microisolator cages with free access to food and water. After 14 days of infection,
animals were euthanized and the intestinal tissue was used for further analysis.
Histological analysis. For histological analysis, colon tissues of experimental
mice were washed with PBS, fixed in formalin, processed by a Tissue-Tek VIP 5
processor (Sakura Finetek, Torrance, CA), and embedded in paraffin. Tissue
sections 5 ␮m thick were stained with hematoxylin and eosin (H&E) solutions.
Images were acquired by use of a DMLB microscope equipped with Fluotar
objectives (Leica Microsystems Inc., Bannockburn, IL) and a Micropublisher 5.0
camera (Q Imaging, Burnaby, British Columbia, Canada). Images were collected
by use of QCapture software 2.6.
Immunohistostaining. Antigen retrieval was performed in a pressure cooker
using citrate buffer (Millipore, Bedford, United Kingdom). Immunohistochem-
istry was done on a Dako Autostainer universal staining system using a two-step
indirect immunoperoxidase technique. Foxo3a primary antibody was applied
(1:150, as determined by antibody titration) for1hatroom temperature. Next,
labeled polymer rabbit horseradish peroxidase was added for 1 h, followed by
incubation with diaminobenzidine-positive chromogen, and then counterstained
with hematoxylin. All tissues were then dehydrated in graded alcohol and xylene
and coverslipped using Permount (Biomeda, Foster City, CA).
RESULTS
LPS induces Foxo3a translocation and degradation in hu-
man intestinal epithelial cells. Nuclear Foxo3a, as a transcrip-
tion factor, is actively involved in the regulation of gene ex-
pression (3, 10, 12). Upon stimulation with growth factors,
nuclear Foxo3a translocates to the cytosol and becomes inac-
tive (7, 29, 54). The role of Foxo3a in inflammation in colonic
epithelia is not yet defined. To address whether bacteria and
their products affect Foxo3a, human colonic epithelial HT-29
cells were treated with LPS and Foxo3a localization was ex-
amined by immunofluorescent staining. In untreated HT-29
cells, Foxo3a is localized in the nucleus as is expected (Fig.
1A). During LPS treatment, Foxo3a translocates from the nu-
cleus to the cytosol (at 30 min), and the immunofluorescent
signal appears dim during the course of the LPS treatment,
suggesting a decreased amount of Foxo3a. Therefore, we ex-
amined the effect of LPS on the amount of Foxo3a in HT-29
cells. The total amount of Foxo3a remained the same in the
first 30 min of LPS treatment (Fig. 1B). However, after 45 min
of LPS treatment, the amount of Foxo3a decreased, implying
degradation of Foxo3a in HT-29 cells during LPS treatment.
These data show that bacterial LPS induces Foxo3a transloca-
tion and degradation in colonic HT-29 cells, which suggests its
inactivation.
Infection with C. rodentium induces translocation and deg-
radation of Foxo3a in mouse intestinal epithelial cells. To
further examine the effect of bacterial infection on Foxo3a, we
utilized in vitro and in vivo infection with the mouse enteric
pathogen Citrobacter rodentium, known to induce colonic in-
flammation (8). In mouse colonic CMT-93 cells, infected with
C. rodentium, Foxo3a starts to degrade after2hofinfection,
which further progressed during the course of infection (Fig.
2A). This delayed Foxo3a degradation upon C. rodentium in-
fection, compared to LPS treatment, might be the result of
LPS treatment versus infection or a difference in response
between HT-29 and CMT-93 cells. To address this, we treated
mouse CMT-93 cells with LPS. Figure 2B shows that LPS
induces Foxo3a degradation in mouse CMT-93 cells with ki-
4678 SNOEKS ET AL. INFECT.IMMUN.

netics similar to the pattern seen in human HT-29 cells. Hence,
we speculate that the delayed response to C. rodentium infec-
tion is due to the fact that bacterial infection takes a longer
time to elicit a response from the host cells.
We examined whether Foxo3a distribution is affected in
colonic epithelia of mice after 14 days of C. rodentium infec-
tion. H&E staining revealed severely active inflammation in
the colonic mucosa of C. rodentium-infected mice (Fig. 2C),
compared to control lamina propria; inflammatory cells, in-
cluding granulocytes and lymphoplasmatic cells, were also in-
creased. In control, immunohistostaining revealed that nuclear
Foxo3a is present in crypts and surface epithelial cells. How-
ever, there was more cytoplasmic staining in crypt epithelial
cells than in apical epithelial cells (Fig. 2C). In contrast, in C.
rodentium-infected colon Foxo3a is expressed along the entire
crypt-villous axis and is primarily cytosolic with no nuclear
staining. We noticed that the infiltrated immune cells in the
lamina propria of the C. rodentium-infected colon also stained
positive for Foxo3a (Fig. 2C, 63⫻). These data demonstrate
that C. rodentium infection alters Foxo3a localization in mouse
colonic epithelia in vitro and in vivo, suggesting its inactivation.
LPS-induced Foxo3a phosphorylation is PI3K dependent.
Prior to Foxo3a translocation from the nucleus to the cytosol,
Foxo3a must be phosphorylated (7, 20). Several upstream sig-
naling pathways, including the PI3K pathway, regulate Foxo3a
phosphorylation (7). Consequently, we assessed the role of
PI3K in the phosphorylation of Foxo3a in intestinal epithelial
cells treated with LPS. We employed an antibody that recog-
nizes Foxo3a phosphorylation on the Thr32 site, known to be
phosphorylated by the PI3K pathway (7). Figure 3 shows the
basal level of Foxo3a phosphorylation in untreated HT-29
cells. We speculated that because HT-29 cells are colon cancer
cells and PI3K is activated in colon cancer (42), it is more likely
to have a basal level of PI3K activity in these cells, which
contributes to the basal level of Foxo3a phosphorylation. Fur-
thermore, LPS increases Foxo3a phosphorylation on the Thr32
site threefold in the first 5 min of treatment. C. rodentium
infection also increased Thr32 phosphorylation of Foxo3a in
FIG. 1. LPS induces Foxo3a translocation and degradation in human HT-29 intestinal epithelial cells. (A) HT-29 cells treated with LPS were
fixed and immunofluorescently stained for Foxo3a (magnification, ⫻60). This experiment was repeated three independent times in triplicate.
(B) Total proteins from HT-29 cells (control cells and cells treated with LPS) were separated by SDS-PAGE and immunoblotted for Foxo3a and
actin. Each experiment was performed three times, and three samples were used per experimental group. Densitometric analysis shows significant
(*) degradation of Foxo3a during the course of LPS treatment compared to untreated cells (P⬍0.05).
VOL. 76, 2008 Foxo3a REGULATES INTESTINAL INFLAMMATION 4679

FIG. 2. Infection with C. rodentium alters Foxo3a status in colonic epithelia. (A and B) Mouse colonic CMT-93 cells were infected with C.
rodentium (A) or treated with LPS (B), and total proteins were separated by SDS-PAGE. Proteins were immunoblotted with Foxo3a and actin
antibodies. These experiments were repeated three times (three samples were used per experimental group), and densitometric analysis reveals
significant (*) Foxo3a degradation during treatment compared with control (P⬍0.05). (C) Colonic tissue from C57BL/J6 mice, both control mice
and those infected with C. rodentium, was initially H&E stained (upper panel). Tissue sections were further immunohistostained for Foxo3a (lower
panels). Magnifications, ⫻10, ⫻20, and ⫻63.
4680

mouse CMT-93 cells (data not shown). We utilized a PI3K
pharmacological inhibitor, wortmannin or LY294002, to assess
the contribution of the PI3K pathway in LPS-induced Foxo3a
phosphorylation. Inhibition of the PI3K pathway with both
inhibitors blocked LPS-induced Foxo3a phosphorylation and
decreased the basal level of Foxo3a phosphorylation in HT-29
cells (Fig. 3 shows inhibition with LY294002). These data show
that LPS-induced Foxo3a phosphorylation in HT-29 cells is
controlled proximally by the PI3K pathway.
Effect of PI3K on IL-8 expression. IL-8 is a proinflammatory
chemokine that is a strong chemoattractant for neutrophils and
T lymphocytes (24, 31, 51). It has been reported that IL-8
expression is in part controlled by the PI3K pathway (18, 43,
57). To study the contribution of the PI3K pathway in LPS-
induced IL-8 regulation in HT-29 cells, we utilized the more
specific PI3K inhibitor LY294002, since wortmannin increases
NF-␬B interactions with DNA, a critical regulator of cytokine
expression (52). LY294002, at a concentration of 30 ␮M, sig-
nificantly attenuated LPS-induced Akt phosphorylation, a tar-
get of PI3K (data not shown); we therefore applied this con-
centration for further studies. We showed that IL-8 is
dramatically increased (12-fold, P⬍0.001) after HT-29 mono-
layers were treated for 6 h with LPS (Fig. 4), which corre-
sponds with data shown by others (33). Inhibition of the PI3K
pathway with LY294002 significantly decreased (30%, P⬍
0.05) LPS-induced IL-8 expression (Fig. 4). These data showed
that active PI3K is involved in the regulation of IL-8 induced
by LPS.
Foxo3a is involved in the regulation of LPS-induced IL-8
expression. To examine the contribution of Foxo3a in the regu-
lation of LPS-induced IL-8 expression in intestinal HT-29 cells,
we performed siRNA experiments. The efficiency of silencing
Foxo3a was greater than 90% (data not shown). Monolayers,
both control layers and those with silent Foxo3a, were stimulated
with LPS for 6 h, and the medium from the different experimental
groups was collected and assayed for IL-8. All monolayers that
were exposed to Lipofectamine produced lower levels of IL-8 in
response to LPS than did the control monolayers exposed to LPS
(2.5-fold). We hypothesize that Lipofectamine rearranges recep-
tors on the surface of the cells and that the cells then become less
responsive to LPS. Silenced Foxo3a insignificantly increased the
basal level of IL-8 in HT-29 monolayers. However, LPS-induced
IL-8 levels were increased on average by 63% (P⬍0.05) in
monolayers with silenced Foxo3a compared with the negative
control (Fig. 5A). These data support a negative regulation of
IL-8 by Foxo3a in HT-29 cells.
To further confirm the role of Foxo3a in the regulation of
expression of proinflammatory cytokines, mouse CMT-93 cells
were transfected with an adenovirus vector that overexpressed
Foxo3a. In these monolayers, Foxo3a expression was increased
by 40% (data not shown). CMT-93 monolayers, both control
layers and those with overexpressed Foxo3a, were treated with
sterile supernatant (SN) from C. rodentium culture; medium
was collected and assayed for MIP-2 (mouse analog of human
IL-8). Treatment with sterile SN from C. rodentium culture
induced a 6.5-fold (P⬍0.001) increase in MIP-2 compared
with untreated CMT-93 monolayers (data not shown). Over-
expressed Foxo3a did not alter the basal level of MIP-2 in
untreated CMT-93 monolayers. However, overexpression of
Foxo3a attenuated C. rodentium-induced MIP-2 expression by
54% (P⬍0.05) (Fig. 5B). These data suggest that overex-
pressed Foxo3a negatively regulates MIP-2 expression induced
by C. rodentium in mouse CMT-93 cells. Altogether, these
experiments show that active Foxo3a repressed cytokine ex-
pression induced by LPS or bacterial infection in colonic epi-
thelial cells.
Foxo3a is involved in regulation of I␬B␣.Jobin et al. dem-
onstrated that in intestinal HT-29 cells IL-8 expression and
NF-␬B activity are controlled by inhibitory I␬B␣molecules
FIG. 2—Continued.
VOL. 76, 2008 Foxo3a REGULATES INTESTINAL INFLAMMATION 4681

(23). Since the IL-8 promoter did not show any putative
Foxo3a binding sites (9, 50), we assessed the effect of Foxo3a
deficiency on I␬B␣. Figure 6 shows that silencing Foxo3a leads
toa⬃40% (P⬍0.05) decrease of I␬B␣in HT-29 cells. These
data correspond with the data of Lin et al. for immune cells
from Foxo3a-deficient mice with decreased amounts of other
I␬B molecules (35). It is unclear at this point whether Foxo3a
regulates I␬Bs directly or indirectly, but its effect on I␬B␣is
obvious.
DISCUSSION
Enteric bacteria and their products play an important role in
intestinal inflammation (13, 17, 55). Here we demonstrated for
the first time that bacterial LPS and infection with C. roden-
tium inactivate tumor suppressor Foxo3a in intestinal epithelia.
LPS-dependent Foxo3a regulation in intestinal epithelial cells
is controlled by the PI3K pathway. Additionally, in HT-29 cells
with silent Foxo3a, LPS-induced IL-8 is increased, while the
amount of I␬B␣is decreased. We hypothesized that Foxo3a
regulates IL-8 through the inhibitory I␬B␣molecule. Alto-
gether this suggests that bacterial infections and their products
regulate Foxo3a tumor suppressor via the PI3K pathway and
that Foxo3a contributes to the regulation of IL-8 expression.
Regulation may occur by I␬B␣either directly or indirectly
(Fig. 7).
The importance of intestinal bacteria in triggering and con-
trolling colitis is well established; however, the underlying
mechanisms are still unclear. Enteric bacteria affect host intes-
FIG. 3. LPS-induced Foxo3a phosphorylation is PI3K dependent. HT-29 cells, with or without pretreatment with a PI3K inhibitor (LY294002), were
incubated with LPS. Total proteins were separated by SDS-PAGE and immunoprobed with an antibody against phosphorylated Foxo3a and actin. This
immunoblot is representative of three independent experiments (three samples were used per experimental group). Densitometric analysis shows a
significant increase (*) in phosphorylated Foxo3a after LPS treatment, which is completely abrogated in the presence of LY294002 (**,P⬍0.05).
FIG. 4. Effect of PI3K inhibitors on LPS-induced IL-8 expression.
HT-29 monolayers were pretreated with 30 ␮M LY294002 (LY) for 1 h
and then incubated with LPS for 6 h. Medium was collected, and IL-8
was quantified by enzyme-linked immunosorbent assay. This experi-
ment was repeated three independent times in triplicate, and the graph
represents one experiment. LY294002 significantly attenuated (*) the
LPS-induced IL-8 response (P⬍0.05).
4682 SNOEKS ET AL. INFECT.IMMUN.

tinal epithelia by using toxins, effector molecules, and/or direct
invasion of the host cell (16, 34). On the other hand, host cells
are equipped with tools that are able to recognize and respond
to bacteria as well. One of the mechanisms that the host uti-
lizes to recognize bacterial product is through TLR. The dif-
ferent members of the TLR family are located on the cell
membrane, and when interacting with their ligands, they gen-
erate signals by which they activate a specific pattern of gene
expression (1, 2). In the pathogenesis of C. rodentium, a model
of bacterium-induced colitis (8, 40), TLR signaling plays an
important role. TLR4-deficient mice infected with C. roden-
tium showed delayed mortality compared with infected wild-
type mice (27). MyD88-deficient mice (MyD88 is downstream
of TLR) showed, upon C. rodentium infection, severe necro-
tizing colitis, high bacterial counts in the colon, and delayed
recruitment of neutrophils (32). Our data suggest that Foxo3a
is a downstream target of TLR4 in intestinal HT-29 cells and
that LPS binding to TLR4 leads to Foxo3a inactivation.
One of the downstream pathways utilized by TLR to control
expression of various genes is the PI3K pathway (5, 45). PI3K
activation mediates several critical cellular responses, includ-
ing cell differentiation, proliferation, and survival (11, 25). The
upregulation of PI3K has been suggested to cause a hyperpro-
liferative epithelium, and its upregulation has been observed in
a majority of colon cancers (26, 42). Also, PI3K participates in
the regulation of cytokines, such as IL-12 in mouse dendritic
cells and IL-8 in response to flagellin or LPS in intestinal
epithelial cells (43, 57). Rhee et al. showed that blocking PI3K
activation substantially inhibits IL-8 induced by flagellin/TLR5
in NCM460 cells (43), while Yu et al. showed opposite data in
T
84
cells (57). Also, Huang et al. suggested that inhibition of
PI3K in T
84
cells results in increased Salmonella enterica-in-
duced IL-8 (21). Our data demonstrated that inhibition of
FIG. 5. Effect of Foxo3a on cytokine expression. (A) HT-29 cells,
with knocked-down Foxo3a, were treated with LPS for 6 h. Medium
was collected for IL-8 quantification. The graph represents the average
IL-8 ratio of three independent experiments performed in triplicate.
The asterisk represents a significant difference between LPS-treated
monolayers with Foxo3a and those without Foxo3a (P⬍0.05).
(B) CMT-93 cells transfected with a retrovirus vector that overex-
pressed Foxo3a were treated with sterile SN from C. rodentium culture,
and medium was collected after 4 h and assayed for MIP-2. The
asterisk represents a significant difference between MIP-2 produced by
monolayers with normal Foxo3a and that produced by monolayers with
overexpressed Foxo3a (n⫽4, P⬍0.05).
FIG. 6. Foxo3a regulates I␬B␣in HT-29 cells. Total proteins from
HT-29 cells with silent Foxo3a were immunoblotted for I␬B␣, Foxo3a,
and actin. A representative immunoblot of four independent experi-
ments shows a decreased amount of I␬B␣in Foxo3a-silenced cells
compared with negative controls. Densitometric analysis shows signif-
icant (*) differences in the amounts of I␬B␣(P⬍0.05).
FIG. 7. Schematic representation of Foxo3a regulation by LPS and
its contribution in inflammation. The data presented here suggest that
LPS activation of PI3K leads to an inactivation of Foxo3a in intestinal
epithelial cells. Inactivated Foxo3a downregulates inhibitory I␬B␣,
which leads to increased NF-␬B activity and increased IL-8 expression.
This provides a new insight into how bacteria regulate tumor suppres-
sor Foxo3a and inflammation in intestinal epithelial cells.
VOL. 76, 2008 Foxo3a REGULATES INTESTINAL INFLAMMATION 4683

PI3K attenuates LPS-induced IL-8 expression in HT-29 cells.
Although the mechanisms of these two different responses are
not clear, we speculate that pathways proximal to NF-␬B might
respond differently to various stimuli.
Downstream, PI3K phosphorylates Foxo3a proteins at sev-
eral sites, which leads to the translocation of Foxo3a to the
cytosol and abrogation of its transcriptional activity (6, 9, 28).
Cytoplasmic localization of Foxo3a is associated with certain
tumor cell lines and also contributes to the pathogenesis and
development of tumors (36, 39, 41). Foxo3a is phosphorylated
in colon carcinoma cell lines (26), and we have also detected a
basal level of Foxo3a phosphorylation in HT-29 cancer cells.
Kristof et al. have demonstrated that LPS controls Foxo3a in
human lung epithelial cells (30), suggesting that in other epi-
thelial cells Foxo3a also may be regulated by TLR4. Moreover,
we have demonstrated a gradient distribution of Foxo3a in
proliferating intestinal epithelia in mouse colon. A similar dis-
tribution is seen with PCNA, one of the markers of prolifera-
tion of colonic epithelia (53). Interestingly, in C. rodentium-
infected colon, the distribution of Foxo3a is along the entire
crypt and it is mainly cytosolic, suggesting an inactivated state.
These data imply that Foxo3a might be involved in the prolif-
eration of normal colonic epithelia while bacterium-induced
inflammation affects the function of Foxo3a. Moreover, we
have demonstrated that Foxo3a contributes in PI3K-depen-
dent IL-8 regulation. Effective silencing of Foxo3a yields an
increase in IL-8. Altogether, this suggests that Foxo3a plays a
role in intestinal inflammation induced by bacteria or their
products.
Several investigators have shown a regulatory network com-
prised of Foxo3a and NF-␬B in immune cells (15, 35, 47). Lin
et al. provided evidence that Foxo3a modulates NF-␬B activity
in lymphoid cells. They have shown that T cells from Foxo3a-
deficient mice displayed markedly increased NF-␬B activity
accompanied with decreased I␬B␤and I␬Bεexpression (35).
In intestinal HT-29 cells treated with cytokines or LPS, I␬B␣
degradation is incomplete and relatively few molecules of
NF-␬B are needed in the nuclei of HT-29 cells to induce high
levels of IL-8 expression (22, 44). Nevertheless, Jobin et al.
demonstrated that inhibition of I␬B␣degradation completely
blocked IL-8-induced expression (23), suggesting that I␬B␣is
an important player in the regulation of NF-␬B-dependent
genes. Our data showed that in HT-29 cells with silent Foxo3a,
the amount of inhibitory I␬B␣is decreased. We speculate that
unstimulated cells with knocked-down Foxo3a equilibrate to a
smaller amount of I␬B␣; thus, IL-8 is not significantly in-
creased. However, we hypothesize that during LPS stimulation
de novo synthesis of I␬B␣is important to keep IL-8 expression
down. Lack of Foxo3a in the nucleus in LPS-stimulated cells
directly or indirectly inhibits I␬B␣; thus, IL-8 expression is
higher. However, the immediate targets of Foxo3a for repress-
ing I␬B␣activity remain unspecified. It is known that Foxo
members and other Foxs can form complexes together with
additional signaling molecules to exert transcriptional effects
(46). Thus, it is possible that Foxo3a could cooperatively bind
and recruit different coactivators or corepressors to control
I␬B␣.
In summary, bacterial LPS and infection with C. rodentium
regulate tumor suppressor Foxo3a in intestinal epithelia and
Foxo3a participates in the regulation of inflammation. It will
be necessary to further define the role of Foxo3a in the patho-
genesis of colon diseases. Understanding how Foxo3a partici-
pates in the pathogenesis of colon diseases may help unlock
novel therapeutic possibilities.
ACKNOWLEDGMENTS
We thank Hemant Roy, Michael Goldberg, and Ramesh Wali for
constructive assistance in preparation of the manuscript. The vector
that overexpresses Foxo3a was a generous gift from Stanford Peng
(Roche Palo Alto LLC, Palo Alto, CA).
This work was in part supported by a Senior Investigator Award
from the Crohn’s and Colitis Foundation of America (CCFA#1953)
and an ENH Pilot Study grant. Lobke Snoeks received a Fulbright
scholarship for her visiting fellowship, and Christopher R. Weber re-
ceived an NIH F32DK082134 fellowship award.
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Editor: B. A. McCormick
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