Download Th2 Cytokines Down-Regulate TLR Expression and Function

Th2 Cytokines Down-Regulate TLR
Expression and Function in Human Intestinal
Epithelial Cells
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of May 9, 2017.
Tobias Mueller, Tomohiro Terada, Ian M. Rosenberg, Oren
Shibolet and Daniel K. Podolsky
J Immunol 2006; 176:5805-5814; ;
doi: 10.4049/jimmunol.176.10.5805
http://www.jimmunol.org/content/176/10/5805
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References
The Journal of Immunology
Th2 Cytokines Down-Regulate TLR Expression and Function
in Human Intestinal Epithelial Cells1
Tobias Mueller, Tomohiro Terada, Ian M. Rosenberg, Oren Shibolet, and Daniel K. Podolsky2
T
oll-like receptors are pattern recognition receptors which
bind microbial signature molecules and trigger innate and
adaptive immune responses upon stimulation with their
specific ligands (1). Primary human intestinal epithelial cells
(IECs)3 and model IEC lines express membrane-associated and
cytoplasmic TLRs and their associated molecules (2–5), which establish an effective double line of innate immune defense against
noninvasive and invasive pathogens challenging the IEC monolayer (6). TLR signals from IECs may exert direct antibacterial
effects by triggering the secretion of antimicrobial peptides (7) and
provide a critical interrelation between the innate and the adaptive
immunity of the intestinal mucosa by attracting adjacent lamina
propria immune cells for subsequent adaptive immune responses
(8). Recent reports also suggested important nonimmune functions
of TLRs in IECs. Commensal-derived TLR signaling has been
shown to maintain intestinal tissue homeostasis, promoting IEC
proliferation and differentiation, and may also be important for
repair mechanisms following IEC injury (9). Furthermore, TLR2
signaling may enhance the physical barrier function of IECs (10).
TLR signaling must be tightly controlled to prevent unwanted or
prolonged stimulation which might be harmful for the host (11).
Gastrointestinal Unit, Department of Medicine, Center for the Study of Inflammatory
Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114
Received for publication October 14, 2005. Accepted for publication February
28, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Grants DK069344, DK043351, and DK07191 from the
National Institutes of Health (to D.K.P.).
2
Address correspondence and reprint requests to Dr. Daniel K. Podolsky, Gastrointestinal Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114.
E-mail address: [email protected]
3
Abbreviations used in this paper: IEC, intestinal epithelial cell; DAPI, 4⬘,6⬘-diamidino-2-phenylindole; IP, immunoprecipitation; MFI, mean fluorescence intensity;
CT, cycle threshold.
Copyright © 2006 by The American Association of Immunologists, Inc.
This appears to be particularly important for TLR signaling derived from IECs, which are constantly exposed to a vast array of
TLR stimuli from the intestinal microenvironment. TLR ligands in
the intestinal microflora comprise conserved structural components from both potential pathogens and from harmless commensal
microbiota, and the low constitutive expression of key TLRs in
IECs likely limits excessive TLR signaling (2). Moreover, IECs
become hyporesponsive upon prolonged stimulation by ubiquitous
TLR2 and TLR4 ligands, and inhibitory molecules such as Tollinteracting protein have been shown to further restrict TLR signaling in IECs (5).
Dysregulated TLR signaling in IECs may also be an important
pathogenic factor in the development of chronic intestinal inflammation (12). Breaks within the intestinal epithelial barrier likely
facilitate leakage of intraluminal Ags into the lamina propria. Subsequent nonphysiological encounters of TLR-expressing lamina
propria immune cells with TLR ligands derived from the apparently normal intestinal microflora may fuel inappropriate immune
responses and initiate or maintain chronic intestinal inflammation
(13). Following activation by TLR ligands, immature CD4⫹ Th
lymphocytes may differentiate into two functionally distinct Th
cell subsets and secrete characteristic cytokines (14). Th1 cells
predominantly secrete IFN-␥ and TNF-␣, while Th2 cells may be
characterized by IL-4, IL-5, and IL-13 secretion. Imbalance of the
Th cytokine profile toward a Th1 or Th2 cytokine predominance
has been shown to contribute to chronic inflammatory bowel disease in humans and animal models (15) and likely also influences
TLR expression in the intestinal mucosa. Recent studies associated
the mucosal Th1 cytokine profile in patients with Crohn’s disease
with enhanced intestinal TLR expression and signaling (16, 17).
Recent in vitro findings demonstrating that the key Th1 cytokine
IFN-␥ promoted TLR4/MD-2-mediated signaling in model IECs
(4, 18, 19), leading to increased IL-8 secretion upon stimulation
with the specific TLR4 ligand LPS, underscored the important role
of proinflammatory Th1 cytokines in the development and maintenance of chronic intestinal inflammation.
0022-1767/06/$02.00
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TLRs serve important immune and nonimmune functions in human intestinal epithelial cells (IECs). Proinflammatory Th1
cytokines have been shown to promote TLR expression and function in IECs, but the effect of key Th2 cytokines (IL-4, IL-5, IL-13)
on TLR signaling in IECs has not been elucidated so far. We stimulated human model IECs with Th2 cytokines and examined TLR
mRNA and protein expression by Northern blotting, RT-PCR, real-time RT-PCR, Western blotting, and flow cytometry. TLR
function was determined by I-␬B␣ phosphorylation assays, ELISA for IL-8 secretion after stimulation with TLR ligands and flow
cytometry for LPS uptake. IL-4 and IL-13 significantly decreased TLR3 and TLR4 mRNA and protein expression including the
requisite TLR4 coreceptor MD-2. TLR4/MD-2-mediated LPS uptake and TLR ligand-induced I-␬B␣ phosphorylation and IL-8
secretion were significantly diminished in Th2 cytokine-primed IECs. The down-regulatory effect of Th2 cytokines on TLR
expression and function in IECs also counteracted enhanced TLR signaling induced by stimulation with the hallmark Th1 cytokine
IFN-␥. In summary, Th2 cytokines appear to dampen TLR expression and function in resting and Th1 cytokine-primed human
IECs. Diminished TLR function in IECs under the influence of Th2 cytokines may protect the host from excessive TLR signaling,
but likely also impairs the host intestinal innate immune defense and increases IEC susceptibility to chronic inflammation in
response to the intestinal microenvironment. Taken together, our data underscore the important role of Th2 cytokines in balancing
TLR signaling in human IECs. The Journal of Immunology, 2006, 176: 5805–5814.
5806
Th2 CYTOKINES DECREASE TCR SIGNALING IN HUMAN IECs
Primary human IECs and model IEC lines also express abundant
mRNA for the IL-4R (20), which initiates signaling pathways of
two key Th2 cytokines, IL-4 and IL-13. The presence of Th2 cytokine receptors on IECs suggests a regulatory role of Th2 cytokines in the innate immune responsiveness of IECs, but little is
known about the influence of Th2 cytokines on TLR expression
and function in human IECs. We speculated that Th2 cytokines
may modulate TLR signaling in IECs in a manner distinct from the
proinflammatory effects of Th1 cytokines. Therefore, we assessed
the effect of the key Th2 cytokines IL-4, IL-5, and IL-13 on TLR
expression and function in model IEC lines.
(Finnzymes; New England Biolabs). Briefly, 100 ng of reversed transcribed cDNA was used for each PCR with 250 nM forward and reverse
primers. The thermal cycling conditions comprised 4 min at 95°C, followed by 45 cycles at 94°C for 30 s, 58°C for 5 s (GAPDH), and 72°C for
45 s. Annealing temperatures were optimized for each TLR primer pair
used and ranged between 59°C and 62°C. The threshold cycle (CT) values
were obtained for the reactions reflecting the quantity of the template in the
sample. TLR or MD-2 ⌬CT (⌬CT) was calculated by subtracting the calibrator gene GAPDH CT value from the TLR or MD-2 CT value and thus
represented the relative quantity of the target mRNA normalized to the
level of the internal standard GAPDH mRNA level. The TLR or MD-2
mRNA level in untreated IECs was defined as 1 arbitrary unit.
Materials and Methods
IECs were grown in 10-cm plates and stimulated with Th2 and/or Th1
cytokines for 18 h. Medium was removed, and 500 ␮l of lysis buffer (1%
Triton X-100, 0.1 M NaCl, 10 mM HEPES (pH 5.6), 2 mM EDTA, 4 mM
Na3VO4, and 40 mM NaF) supplemented with protease inhibitor mixture
(Complete Mini; Roche Diagnostics) was added. Supernatants of cell lysates were obtained by centrifugation at 12,000 ⫻ g for 10 min. Protein
concentration was determined using the DC protein assay kit (Bio-Rad).
For TLR IP, 4 mg of total protein were precleared for 2 h with 50 ␮l of
Hiptrap protein A/G-Sepharose beads (Calbiochem) at 4°C. Supernatants
were incubated overnight with 2 ␮g of primary Ab at 4°C and immunoprecipitated proteins were separated on 4 –12% Tris-glycine gels (Invitrogen Life Technologies). TLR protein was blotted onto polyvinylidene difluoride membranes followed by blocking with 5% dry milk in TBS
containing 0.1% Tween 20 (TBST). Membranes were incubated overnight
with primary Abs (diluted 1/250 in blocking buffer, 4°C), washed for 60
min and stained with specific secondary Abs (1/5000 in blocking buffer) for
60 min at room temperature. After extensive washing of the membranes,
hybridized bands were detected using an ECL kit (PerkinElmer Life
Sciences).
Abs and reagents
Cell culture and cytokine stimulation assay
The human model IEC lines HT29, Colo205, SW480, T84 and the human
monocytic cell line U937 were obtained from the American Type Culture
Collection. U937 and Colo205 cells were grown in RPMI 1640 (Cellgro
Mediatech). HT29 cells were grown in DMEM (Cellgro Mediatech). T84
and SW480 cells were grown in a 1:1 mixture of DMEM and Ham’s F-12
medium (Cellgro Mediatech). Media were supplemented with 50 U of penicillin/ml and 50 ␮g of streptomycin/ml (Invitrogen Life Technologies) and
10% heat-inactivated FCS (Atlanta Biologicals). T84 cells were grown to
confluence on Transwells (0.4 ␮M; BD Biosciences) and achieved a polarized and differentiated state within 14 –21 days. Transepithelial resistance was measured to determine T84 cell differentiation and ranged from
700 ⍀/cm2 to ⬎1000 ⍀/cm2 for confluent T84 cell monolayers (Millicell
Electrical Resistance System; Millipore). Cytokines were added to the basolateral compartment for the indicated time periods. All other cell lines
were grown to 75–90% confluence in 6-well culture plates (BD Biosciences) and incubated with cytokines for 18 h before the determination of
TLR expression and function.
RNA extraction and Northern blotting
Total RNA was isolated from IECs using TRIzol reagent (Invitrogen Life
Technologies) following the manufacturer’s instruction. For Northern blot
analysis, 20 ␮g of total RNA was subjected to electrophoresis using 1%
agarose-formaldehyde gels, followed by transfer to Nytran SuPerCharge
membranes (Schleicher & Schuell). Human cDNA probes were generated
from PCR products. Probes were labeled with [␣-32P]dCTP using ReadyTo-Go DNA labeling beads (Amersham Biosciences), followed by spin
column removal of unincorporated nucleotides. Membranes were pretreated, hybridized in QuikHyb hybridization solution (Stratagene), washed
with 2⫻ SSC (0.3 M NaCl plus 0.03 M sodium citrate) containing 0.1%
SDS at room temperature for 5 min and then washed thrice with 0.5⫻ SSC
containing 0.1% SDS at 42°C for 10 min. Membranes were exposed to film
for 8 – 48 h at ⫺80°C with intensifying screens.
RT-PCR and real-time RT-PCR
For reverse transcription, 2 ␮g of total RNA were transcribed using SuperScript III reverse transcriptase (Invitrogen Life Technologies). Aliquots
of 2 ␮l of cDNA were subjected to 45 cycles of PCR amplification with
Taq polymerase (Invitrogen Life Technologies). As an internal control,
PCR for GAPDH was performed at 95°C for 120 s, followed by 35 cycles
at 95°C for 60 s, 58°C for 45 s, and 72°C for 60 s. Primers for GAPDH
(440-bp fragment) were forward 5⬘-tcatctctgccccctctgct-3⬘, reverse 5⬘cgacgcctgcttcaccacct-3⬘. Negative controls included the amplification of
samples without prior RT reaction. The primer sequences used for the
different TLR family members and TLR4 coreceptor MD-2 have been published elsewhere (5).
Real-time RT-PCR was performed in the DNA Engine Opticon 2 System (MJ Research) using the DyNAmo SYBR Green qPCR kit
Flow cytometry and immunocytochemistry
IECs grown in 6-well plates were detached using enzyme-free cell dissociation buffer (Invitrogen Life Technologies), washed twice with FACS
buffer (PBS containing 1% FCS and 0.1% NaN3), and blocked for 20 min
with FACS buffer containing 10% horse serum. For direct immunofluorescence staining, single-cell suspensions of IECs and monocytes (5 ⫻ 105
cells/100 ␮l) were incubated at 4°C in FACS buffer containing PE-labeled
anti-human Abs (diluted 1/100) for 30 min. Control experiments were performed with PE-conjugated isotype-matched controls. Flow cytometry
analysis was performed using a FACSCalibur cytometer (BD Biosciences).
For some experiments, cells were fixed and permeabilized using the BD
Cytofix/Cytoperm kit according to the manufacturer’s protocol (BD
Biosciences).
Alexa Fluor 488-labeled LPS was preincubated in cell culture medium
containing 10% FCS for 1 h at 37°C and added to IECs primed with
medium in the absence or presence of Th cytokines in 6-well plates before
the experiments. After the indicated incubation time, IECs were detached
using enzyme-free cell dissociation buffer (Invitrogen Life Technologies).
IECs (5 ⫻ 105 cells per sample) were washed four times with FACS buffer
and stained by propidium iodide (1 ␮g/ml) to eliminate dead cells.
For immunocytochemistry, IECs were grown on sterile permanox coverslips and stained for endogenous TLR expression using monoclonal
mouse anti-human TLR4 Ab (HTA-125; dilution 1/100). Cells were
washed twice with ice-cold PBS and fixed for 20 min with ice-cold methanol at ⫺20°C. After three washes with ice-cold PBS, IECs were saturated
for 30 min with PBS containing 5% donkey serum. IECs were then incubated for 2 h with primary Ab. Immunostaining was performed with FITCconjugated anti-rabbit IgG (Vector Laboratories) secondary Abs and 4⬘,6⬘diamidino-2-phenylindole (DAPI) for nuclear staining.
ELISA
For ELISA of secreted IL-8, 1 ⫻ 105 IECs were plated per well in 12-well
plates, grown to 75–90% confluence and primed with cytokines for 18 h
before stimulation with LPS (100 ng/ml), Pam3CSK4 (50 ␮g/ml),
poly(I:C) (50 ␮g/ml) or flagellin (500 ng/ml). Supernatants were harvested
after the indicated time periods and IL-8 ELISA (BD Pharmingen) was
performed according to the manufacturer’s instructions.
Statistical analysis
Statistical significance between groups was assessed by Student’s t test.
Data are expressed as means ⫾ SDs. Triplicate determinations were performed in the IL-8 ELISA experiments, and all experiments were repeated
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LPS (Escherichia coli serotype O55:B5) was purchased from SigmaAldrich, and Alexa Fluor 488-labeled LPS (E. coli serotype O55:B5) from
Molecular Probes. The TLR ligands Pam3CSK4, poly(I:C), and flagellin
(Salmonella typhimurium) were obtained from InvivoGen. The human cytokines IL-4, IL-5, IL-13, and IFN-␥ were obtained from R&D Systems.
Polyclonal Abs against I-␬B␣ and phosphorylated I-␬B␣ were obtained
from Cell Signaling. Monoclonal mouse anti-human TLR4 Ab (HTA-125),
PE-labeled anti-human Abs against TLR3 (TLR3.7), TLR4 (HTA-125),
CD14, and PE-labeled isotype controls (mouse IgG1,␬ and mouse
IgG2a,␬) were obtained from eBioscience.
TLR protein immunoprecipitation (IP) and immunoblotting
The Journal of Immunology
at least three times. A p value ⱕ0.05 was considered to be statistically
significant.
Results
The signature Th cytokines IL-4 (Th2) and IFN-␥ (Th1) exhibit
opposite effects on mRNA expression of key TLRs in IECs
FIGURE 1. IL-4 and IFN-␥ exert
opposite effects on TLR expression in
T84 cells. A, Northern blot showing
mRNA expression for TLR3 and
TLR4 in unstimulated T84 cells (Medium) or T84 cells primed with 50
ng/ml IL-4 or 50 ng/ml IFN-␥ (overnight). B, Representative Northern
blot showing concentration-dependent down-regulation of TLR4
mRNA expression in T84 cells after
overnight stimulation with increasing
concentrations of IL-4. C, RT-PCR
showing time-dependent down-regulation of TLR4 mRNA in T84 cells
after stimulation with 50 ng/ml IL-4.
Comparable data were generated in
HT29 cells (data not shown). Representative blots from five Northern
blots and four RT-PCRs are shown.
IL-4 and IL-13 decrease TLR3 and TLR4 mRNA expression in
IECs without significant effect on TLR2 and TLR5 mRNA
expression
Real-time RT-PCR was used to quantify the down-regulatory effect of IL-4 and IL-13 on TLR mRNA expression in differentiated
T84 cells. Steady-state TLR mRNA expression (determined from
the TLR:GAPDH mRNA ratio in unstimulated T84 cells) was set
as 1 arbitrary unit and compared with the TLR:GAPDH mRNA
ratio after cytokine stimulation (Fig. 2). IL-4-primed T84 cells
exhibited significantly diminished mRNA expression of TLR3
(60% decrease; p ⫽ 0.03) and TLR4 (54% decrease; p ⫽ 0.02).
Priming with IL-13 similarly decreased mRNA encoding for TLR3
and TLR4 (55% decrease for TLR3, p ⫽ 0.04, respectively, 50%
decrease for TLR4, p ⫽ 0.03). TLR2 mRNA expression also diminished under the influence of Th2 cytokines, but did not reach
significance (23% decrease after IL-4 stimulation, p ⫽ 0.3, vs 29%
decrease after priming with IL-13, p ⫽ 0.3). IL-4 and IL-13 also
minimally down-regulated TLR5 mRNA expression (15% decrease after stimulation with IL-4, p ⫽ 0.6, vs 19% decrease after
stimulation with IL-13, p ⫽ 0.7). In contrast, IFN-␥ priming significantly enhanced mRNA expression of TLR2 (4.2-fold increase;
p ⬍ 0.01), TLR3 (7.9-fold increase; p ⬍ 0.01), TLR4 (5.7-fold
increase; p ⬍ 0.01) and TLR5 (6.7-fold increase; p ⫽ 0.03). Comparable data were obtained in HT29 cells (data not shown).
To determine whether IL-4 counteracted the up-regulatory effect
of IFN-␥ on TLR expression in IECs, T84 cells were concomitantly stimulated with IL-4 and IFN-␥. Fig. 2 shows that IL-4
attenuated IFN-␥-dependent enhancement of TLR mRNA, but
failed to completely neutralize the up-regulatory effects of IFN-␥.
Th2 cytokines decrease mRNA of MD-2, a coreceptor molecule
necessary for TLR4 signaling, and attenuate MD-2 mRNA
induction in IFN-␥-stimulated IECs
Real-time RT-PCR in Th2 cytokine-primed T84 cells was performed to assess concomitant changes in the mRNA expression of
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Recent data have demonstrated that differentiated T84 cells constitutively express different TLR family members and their associated signaling molecules, suggesting that they are suitable models for in vitro IEC studies. To evaluate possible effects of Th2
cytokines on TLR mRNA expression, polarized T84 cell monolayers were stimulated from the basolateral compartment with 50
ng/ml IL-4 or 50 ng/ml IFN-␥ overnight. Northern blotting showed
decreased TLR3 and TLR4 mRNA expression in T84 cells after
priming with IL-4, while IFN-␥ enhanced TLR3 and TLR4 mRNA
expression (Fig. 1A). In contrast, IL-4 stimulation did not markedly affect TLR2 and TLR5 mRNA expression in T84 cells (data
not shown). Comparable results were obtained in HT29 cells, an
undifferentiated colonic cancer cell line which has recently been
shown to be susceptible to Th1 cytokine-mediated changes in TLR
signaling (18, 19), and SW480 and Colo205 cells (data not shown).
IL-4-mediated down-regulation of constitutive TLR4 mRNA
expression in T84 cells was concentration-dependent. The representative Northern blot in Fig. 1B demonstrates detectable downregulation of TLR4 mRNA expression at 10 ng/ml IL-4 (15% decrease as determined by real-time RT-PCR, data not shown),
which became more apparent after stimulation with 25 ng/ml IL-4
(25–30% decrease) and was significant after stimulation with 50
ng/ml or 100 ng/ml IL-4 (55– 65% decrease). Similar data were
obtained from IL-13-stimulated IECs (data not shown). The decrease in TLR4 mRNA expression was first apparent after 6 h of
cytokine stimulation, was clearly marked after 12 h and was sustained throughout the 24-h observation period (Fig. 1C). In subsequent experiments, IECs were primed with 50 ng/ml IL-4 for
18 h.
5807
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Th2 CYTOKINES DECREASE TCR SIGNALING IN HUMAN IECs
MD-2, the requisite TLR4 coreceptor. As shown in Fig. 3A, left
panel, priming with IL-4 markedly decreased MD-2 mRNA expression in T84 cells to 47% of MD-2 mRNA expression in unstimulated T84 cells ( p ⫽ 0.02). IL-13 priming similarly downregulated MD-2 mRNA to 53% of MD-2 expression in
unstimulated T84 cells ( p ⬍ 0.04). Moreover, concomitant priming with IL-4 also significantly attenuated up-regulation of MD-2
mRNA expression in IFN-␥-stimulated T84 cells. Although IFN-␥
stimulation alone increased MD-2 mRNA expression in T84 cells
⬍7-fold ( p ⬍ 0.01), IL-4/IFN-␥-primed T84 cells showed only
1.5-fold enhanced MD-2 mRNA expression.
Recent studies demonstrated that HT29 cells exhibit increased
sensitivity to LPS after IFN-␥ stimulation, which has been largely
attributed to increased MD-2 expression (18, 19). We examined
the effect of Th2 cytokines on MD-2 expression in HT29 cells and
were able to demonstrate that IL-4 and IL-13 markedly decreased
MD-2 mRNA expression in unstimulated and IFN-␥-primed HT29
cells (Fig. 3A, right panel). Moreover, real-time RT-PCR and RTPCR (Fig. 3B) clearly depicted that concomitant IL-4 priming significantly attenuated enhanced MD-2 mRNA expression in IFN␥-primed HT29 cells.
Taken together, these data indicated that signature Th2 cytokines appeared to exert a broad down-regulatory effect on mRNA
expression for both TLR4 and its important coreceptor MD-2
in IECs.
Diminished TLR4 protein expression reflects down-regulated
TLR4 mRNA expression in Th2 cytokine-primed IECs
IP-Western blotting was performed to determine whether decreased TLR4 mRNA expression translated into diminished TLR4
protein expression in Th2 cytokine-treated T84 cells. Fig. 4A, left
panel, shows that IL-4 and IL-13, but not IL-5, markedly decreased TLR4 protein expression. Comparable data were obtained
in HT29 cells (data not shown). Densitometric analysis of IPWestern blots from a series of four independent experiments
showed up to 50% decreased TLR4 protein in Th2 cytokineprimed T84 cells ( p ⫽ 0.01; Fig. 4B, left panel).
Moreover, the representative IP Western blot of Fig. 4A, right
panel, shows that concomitant IL-4 priming partly reversed the
up-regulatory effects IFN-␥ on TLR4 protein expression in IECs
(4, 19). Fig. 4B, right panel, summarizes a series of four separate
experiments, showing a 3.7-fold increase in TLR4 protein expression after IFN-␥ stimulation compared with a moderate 1.6-fold
increase after concomitant IL-4- and IFN-␥-priming of T84 cells.
Comparable data were obtained from HT29 cells (data not shown).
Taken together, these data demonstrated that Th2 cytokine-mediated down-regulation of TLR4 mRNA expression in resting and
Th1 cytokine-primed IECs led to a significant reduction in TLR4
protein expression.
Th2 cytokines reduce intracytoplasmic TLR3 and TLR4 protein
expression in IECs
Membranous TLR4 expression has been located on the cell surface
and in the cytoplasm of IECs. We performed flow cytometry to
determine whether Th2 cytokines changed the surface and/or the
cytoplasmic TLR expression of SW480 cells. SW480 cells have
been validated as a model IEC line for studying TLR4 protein
expression by flow cytometry because they exhibit detectable surface TLR4 expression, which distinguishes them from T84 cells or
HT29 cells (5, 19). Moreover, similar to our findings in T84 and
HT29 cells, IP-Western blots from SW480 cells confirmed TLR4
down-regulation after Th2 cytokine stimulation (data not shown).
Immunocytochemistry demonstrated TLR4 surface protein on the
cell membrane and to a lesser extent in the cytoplasm of SW480
cells (Fig. 5A). Intact SW480 cells were stained with PE-labeled
anti-human TLR Abs to examine Th2 cytokine-mediated changes
in TLR surface expression, while fixed and permeabilized SW480
cells were used to evaluate changes in cytoplasmic TLR protein
expression. The abundant TLR surface expression in human U937
monocytes served as a positive control. As shown in Fig. 5, IL-4
(blue graphs) and IL-13 (red graphs) only slightly decreased constitutive surface TLR4 expression (green graphs) in SW480 cells
(Fig. 5B), but markedly diminished cytoplasmic TLR4 protein expression (Fig. 5C). In contrast, U937 monocytes exhibited a
marked decrease in constitutive surface and cytoplasmic TLR4
protein expression after IL-4 and IL-13 stimulation (Fig. 5, D and
E). These data extended earlier studies examining the effect of Th2
cytokines on TLR expression in human PBMC (21).
SW480 and U937 monocytes cells did not show surface TLR3
protein expression (data not shown), but priming with IL-4 (blue
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FIGURE 2. IL-4 and IL-13 decrease TLR3 and TLR4 mRNA expression in T84 cells without significant effect on TLR2 and TLR5
mRNA expression. Real-time RTPCR analysis was performed to quantify changes in TLR mRNA expression in T84 cells after 18 h of
stimulation with 50 ng/ml IL-4, 50
ng/ml IL-13, or concomitant stimulation with 50 ng/ml IL-4 and 50 ng/ml
IFN-␥. Results are shown as TLR
mRNA expression in relation to
mRNA expression for the housekeeping gene GAPDH and are presented
as fold decrease or increase compared
with TLR:GAPDH mRNA ratio in
unstimulated T84 cells (set as 1 arbitrary unit) and graphed on a log scale.
Comparable data were generated in
HT29 cells (data not shown). Data are
mean ⫾ SE of five separate experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
The Journal of Immunology
5809
graph) and IL-13 (red graph) significantly diminished constitutive
cytoplasmic TLR3 expression in both cell lines (green graph, Fig.
5, F and G). Taken together, FACS analysis corroborated our IPWestern blot findings and attributed decreased TLR4 protein expression in Th2 cytokine-primed IECs to diminished cytoplasmic
TLR4 expression. Our flow cytometry data also suggested that Th2
cytokine-induced down-regulation of TLR3 mRNA expression in
IECs and monocytes led to diminished cytoplasmic TLR3 protein
expression in both cell types.
Th2 cytokine-primed IECs incorporate less LPS
Next, we attempted to determine whether our TLR expression data
correlated with changes in the TLR function in IECs. We hypothesized that the simultaneous decrease of TLR4/MD-2 expression
in IECs after Th2 cytokine-priming might diminish LPS signaling
triggered by the TLR4/MD-2/CD14 complex. We performed functional LPS uptake studies in several IEC lines known to respond to
IL-4. Among these cell lines, earlier experiments by our group had
demonstrated that Colo205 cells and HT29 cells appeared to be
most suitable for LPS internalization studies (19). We used flow
cytometry to detect incorporated fluorescent LPS particles in
Colo205 cells and evaluated the mean fluorescence intensity (MFI)
of unstimulated and Th2 cytokine-primed Colo205 cells after incubation with fluorescent LPS. As shown in a representative experiment in Fig. 6A, IL-4- and IL-13-primed Colo205 cells incorporated significantly less fluorescent LPS than unstimulated cells
(Fig. 6A). In a series of four independent experiments, IL-4 and
IL-13 decreased LPS uptake over the complete observation period
(Fig. 6B). Similar data were obtained in HT29 and SW480 cells
(data not shown). Diminished LPS uptake seemed to be independent
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FIGURE 3. IL-4 and IL-13 down-regulate MD-2 mRNA expression in T84 and HT29 cells. A, Real-time RT-PCR analysis was performed to quantify
changes in MD-2 mRNA expression in T84 cells and HT29 cells after priming with 50 ng/ml IL-4, 50 ng/ml IL-13 alone (18 h), or after concomitant
stimulation with 50 ng/ml IL-4 and 50 ng/ml IFN-␥. Results are shown as MD-2 mRNA expression in relation to GAPDH mRNA expression and are
presented as fold decrease or increase compared with the MD-2:GAPDH ratio in unstimulated T84 cells and HT29 cells and graphed on a log scale. Data
are mean ⫾ SE of four independent experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01. B, IL-4 attenuates MD-2 mRNA induction in IFN-␥ stimulated HT29 cells.
HT29 cells were treated for 18 h with 50 ng/ml IFN-␥ in the presence or absence of 50 ng/ml IL-4. Comparable data were obtained in T84 cells (data not
shown). The results are representative of four independent experiments with similar results.
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Th2 CYTOKINES DECREASE TCR SIGNALING IN HUMAN IECs
of CD14 expression, as flow cytometry detected no changes in CD14
expression after Th2 cytokine stimulation (data not shown).
Th2 cytokine-primed IECs show functionally impaired TLR
signaling
Both HT29 cells and T84 cells were used for additional functional
studies. HT29 cells have been reported to respond to all major TLR
ligands, including the TLR5 ligand flagellin (22). Moreover, as
noted above, recent studies also used HT29 cells to show increased
LPS responsiveness after IFN-␥ priming (4, 18, 19).
We first examined the influence of IL-4 on LPS-induced phosphorylation of inhibitory I-␬B␣ in unstimulated and IFN-␥-primed
HT29 cells, which would eventually lead to the activation of the
NF-␬B signaling complex. As shown in Fig. 7, concomitant IL-4
stimulation prevented the increase in I-␬B␣ phosphorylation following LPS stimulation in IFN-␥-primed HT29 cells, suggesting
that Th2 cytokines attenuated the increased LPS responsiveness in
IFN-␥-primed IECs.
We then examined the effects of Th2 cytokines on IL-8 secretion
from HT29 cells and T84 cells following stimulation with specific
TLR ligands. To exclude direct Th2 or Th1 cytokine-mediated
effects, increasing concentrations of IL-4, IL-13 and IFN-␥ were
tested in HT29 and T84 cells for their potential influence on spontaneous IL-8 secretion (data not shown). We found that preincubation with up to 20 ng/ml IL-4 or IL-13 and 10 ng/ml IFN-␥ did
not change subsequent spontaneous IL-8 secretion from HT29
cells during the observation period (Fig. 8A). HT29 cells secreted
varying amounts of IL-8 after stimulation with the specific TLR
ligands (Fig. 8, B–E). IL-4 and IL-13 priming significantly decreased IL-8 secretion triggered by the TLR3 ligand poly(I:C)
(Fig. 8C) and the TLR4 ligand LPS (Fig. 8D), but did not affect
IL-8 secretion after stimulation with the TLR2 ligand Pam3CSK4
(Fig. 8B) or the TLR5 ligand flagellin (Fig. 8E). In contrast, IFN␥-primed HT29 cells secreted significantly more IL-8 after stimulation with these ligands (Fig. 8, B–E). In conclusion, these data
suggested that the down-regulatory effect of Th2 cytokines on TLR
expression in IECs led to a functional impairment of TLR3- and
TLR4-mediated signaling in IECs.
Discussion
Human IECs express TLRs and their associated signaling molecules, which serve important immune and nonimmune functions in
the intestinal mucosa. Because of the intimate contact between the
IEC monolayer and adjacent intraepithelial and lamina propria
lymphocytes, pro- and anti-inflammatory cytokines secreted by the
latter (23, 24) may influence TLR-mediated innate immune responses of IECs. Recent in vitro studies demonstrated that the
signature proinflammatory Th1 cytokine IFN-␥ enhanced TLR4/
MD-2 expression and LPS sensitivity of human IECs and monocytes (4, 18, 19, 25). In contrast, the key Th2 cytokines IL-4 and
IL-13 have been shown to down-regulate TLR4 surface expression
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FIGURE 4. IL-4 and IL-13 decrease TLR4 protein expression in T84 cells. T84 cells were incubated for 18 h with medium alone, medium containing
50 ng/ml IL-4, IL-5, IL-13, or IFN-␥ or medium with 50 ng/ml IL-4 plus 50 ng/ml IFN-␥. TLR4 protein expression was determined by IP followed by
Western blotting. T84 cell lysates containing 4 mg of total protein were immunoprecipitated with monoclonal mouse anti-human TLR4 Ab. Immunoprecipitates were subjected to Western blot analysis using monoclonal mouse anti-human TLR4 Ab. A, A representative IP-Western blot for TLR4 protein
expression in T84 cells after stimulation with different Th2 cytokines is shown on the left panel. A representative IP-Western blot for TLR4 protein
expression in T84 cells after stimulation with 50 ng/ml IFN-␥ in the absence or presence of 50 ng/ml IL-4 is shown on the right panel. B, Densitometric
analysis of TLR4 protein expression in Th2 cytokine-primed T84 cells normalized to unstimulated T84 cells in the absence (left panel) or presence (right
panel) of IFN-␥. Comparable data were obtained in HT29 cells (data not shown). Data are mean ⫾ SE of four separate experiments; ⴱ, p ⬍ 0.05.
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FIGURE 5. Th2 cytokines diminish cytoplasmic TLR3 and TLR4 protein expression in SW480 cells and human U937 monocytes. Cell surface and
cytoplasmic TLR3 and TLR4 protein expression in unstimulated and Th2 cytokine-primed SW480 cells and human U937 monocytes was determined by
immunocytochemistry (A) or flow cytometry (B–G). A, Endogenous TLR4 expression in SW480 cells detected by monoclonal mouse anti-human TLR4
Ab and FITC-conjugated anti-mouse IgG secondary Ab. DAPI was used for nuclear staining. A representative example of five experiments is shown. B–G,
Surface and cytoplasmic expression of TLR3 and TLR4 in SW480 cells and human U937 monocytes visualized by flow cytometry using PE-labeled mouse
anti-human TLR3 and PE-labeled mouse anti-human TLR4 Abs. TLR3 and TLR4 expression in unstimulated SW480 cells and monocytes (green graphs)
was compared with IL-4-stimulated (blue graphs) and IL-13-stimulated (red graphs) SW480 cells and monocytes. Gray graphs depict corresponding
PE-labeled mouse IgG isotype controls for each experiment. The results are representative of six independent experiments with comparable results.
in human peripheral blood monocytes (21) and inhibited the secretion of proinflammatory cytokines after LPS stimulation (26).
We speculated that Th2 cytokines may also exert broad antiinflammatory effects on human IECs and potentially impair their
capacity to initiate appropriate TLR-mediated immune responses.
In the present study, we examined whether key Th2 cytokines
influenced TLR expression and function in validated model IEC
lines. Incubation with IL-4 and IL-13 (but not IL-5) markedly decreased TLR3 and TLR4 mRNA and protein expression. More-
over, reduced I-␬B␣ phosphorylation and diminished IL-8 secretion after stimulation with specific TLR ligands showed that TLR
signaling in Th2 cytokine-primed IECs was functionally impaired.
The lack of response to IL-5 may be explained by the absence of
IL-5Rs in IECs, which contrasts the abundant expression of IL-4
and IL-13Rs in primary and model IECs (20, 27, 28).
Increased LPS responsiveness in Th1 cytokine-primed IECs has
not only been attributed to increased TLR4 expression but also to
the enhanced expression of MD-2, the important TLR4 coreceptor,
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Th2 CYTOKINES DECREASE TCR SIGNALING IN HUMAN IECs
FIGURE 7. IL-4 attenuates IFN-␥-induced enhancement of LPS responsiveness in HT29 cells. HT29 cells were primed for 18 h with medium
alone or medium containing 50 ng/ml IFN-␥ in the absence or presence of
increasing concentrations of IL-4 before incubation with 100 ng/ml LPS
for 2 h. Phosphorylation of I-␬B␣ was analyzed by immunoblotting. To
confirm equal protein loading, blots were reprobed with Ab against total
I-␬B␣ Ab. Comparable data were obtained in T84 cells (data not shown).
Results are representative of three independent experiments with similar
results.
which might also influence TLR4 expression patterns in IECs (18,
19, 29). We were able to demonstrate that Th2 cytokines downregulated both constitutive and IFN-␥-induced TLR4 and MD-2
expression. These data suggested that Th2 cytokines may dampen
the increase in IEC immune reactivity after Th1 cytokine stimulation by counteracting both the up-regulation of TLR4 and the
increase of MD-2 expression.
IL-4 and IFN-␥ have been shown to activate STAT6 and
STAT1, respectively, leading to the promotion of target gene transcription (30). In mouse macrophages, IL-4-induced STAT6 activation directly suppressed IFN-␥-triggered, STAT1-dependent
gene transcription by competing with STAT1 for the same regulatory STAT binding element (31). Transcriptional repression of
MD-2 and select TLRs by STAT6 may also explain our observations in Th2 cytokine-stimulated IECs. We were able to demonstrate STAT6 phosphorylation in HT29 cells after IL-4 stimulation
(data not shown), and another study showed STAT6 phosphorylation in HT29/B6 cells after stimulation with IL-13 (28). Further
analysis of the promoter regions of MD-2, TLR3 and TLR4 in
IECs will clarify how these two factors may compete for regulatory regions with opposite functional consequences.
Taken together, key Th2 and Th1 cytokines appeared to have
opposite regulatory effects on TLR expression and function in hu-
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FIGURE 6. Th2 cytokine-priming decreases uptake of fluorescent LPS
by Colo205 cells. Colo205 cells were primed for 18 h with 50 ng/ml IL-4
or 50 ng/ml IL-13 before incubation with 2 ␮g/ml fluorescent LPS. Uptake
of fluorescent LPS was determined using FACS. A, A representative experiment comparing fluorescent LPS uptake in unstimulated Colo205 cells
and Colo205 cells primed with IL-4 or IL-13 before incubation with fluorescent LPS is shown. Comparable data were obtained from HT29 cells
(data not shown). B, Colo205 cells were incubated with 2 ␮g/ml fluorescent LPS for the indicated time periods. MFI values (⌬MFI) were obtained
by subtracting the MFI values of fluorescent LPS incorporated into
Colo205 cells from the corresponding background value. Data are mean ⫾
SE of four separate experiments. ⴱ, p ⬍ 0.05. Comparable data were obtained from HT29 cells (data not shown).
man IECs: IFN-␥ enhanced TLR-mediated IEC immune responses
by increasing key TLR signaling, while IL-4 and IL-13 appeared
to impair IEC immune defense by down-regulating TLR3 and
TLR4/MD-2 expression and function. A similar negative correlation between IL-4 and TLR4 expression has been described in the
lungs of patients with tuberculosis, another organ with abundant
TLR expression and constant exposure to a vast array of Ags (32).
Although direct anti-inflammatory effects of Th2 cytokines may
be beneficial for the host by restricting ongoing inflammation
(likely linked with a Th1 cytokine profile leading to increased TLR
signaling), down-regulated TLR-expression and function can also
paradoxically impair the host innate immune response. In this context, the down-regulated TLR4 expression after Th2 cytokine
priming has been shown to reduce the ability of human mononuclear
phagocytes to eliminate Listeria monocytogenes (33). Furthermore,
Th2 cytokines impair IEC barrier function (28, 34), which likely facilitate nonphysiological encounters of the TLR ligand-bearing intestinal microflora with the subsequently “unprotected” IECs, rendering
the host susceptible to chronic inflammation in response to the apparently normal intestinal microenvironment.
The IECs used in our study represent validated models for
studying molecular mechanisms in the human intestine in vitro,
and TLR down-regulation after Th2 cytokine priming was consistently found in different IEC lines, suggesting that these effects do
not reflect idiosyncratic features of a single cell line. However,
model IEC lines also have inherent limitations and may not entirely address the functional relevance of Th2 cytokine-mediated
changes in TLR signaling in vivo. Models IECs may fail to address
the complex interaction between primary IECs and the host intestinal microenvironment with its heterogeneous mucosal cytokine
milieu including the presence of abundant commensals and potential pathogens which likely stimulate multiple TLRs. It will be
important to define how Th2 cytokine-mediated effects on TLR
signaling may be modulated by products of the luminal flora,
which have been shown to induce selective TLR trafficking (35)
and may also directly regulate TLR expression in IECs (36). In our
experiments, highly purified TLR ligands were used to characterize TLR function in IECs in a homogenous Th2 and/or Th1 cytokine environment.
The influence of the intestinal microenvironment may also explain the discrepancy between our present findings and earlier immunohistochemical studies showing down-regulated TLR3 expression in the mucosa of patients with Crohn’s disease (17),
usually considered a prototypic Th1 cytokine disease. This study
also showed up-regulated mucosal TLR4 expression in patients
with ulcerative colitis, which has recently been linked to atypical
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FIGURE 8. Th2 cytokines impair TLR ligand-induced IL-8 secretion in HT29 cells. HT29 cells were primed with medium alone, medium containing
20 ng/ml IL-4 or 20 ng/ml IL-13 or 10 ng/ml IFN-␥ before stimulation with specific TLR ligands. Supernatants were assayed for IL-8 secretion. A,
Spontaneous IL-8 secretion does not change after stimulation with Th cytokines. B–D, Comparison of IL-8 secretion from unstimulated or Th2 or Th1
cytokine-primed HT29 cells after stimulation with TLR2 ligand Pam3CSK4 (B), TLR3 ligand poly(I:C) (C), TLR4 ligand LPS (D), and TLR5 ligand
flagellin (E). Comparable data were obtained in T84 cells (data not shown). Data are mean ⫾ SE of three separate experiments. ⴱ, p ⬍ 0.05.
mucosal IL-13 expression (28, 37). However, other studies described different mucosal Th cytokine profiles in patients with ulcerative colitis, ranging from diminished mucosal IL-13 expression (38, 39) to increased IL-5 expression (40).
Murine models of chronic intestinal inflammation underscore
the alternative proinflammatory potential of Th2 cytokines within
the intestinal microflora, exemplified by the induction of potentially fatal colitis in healthy mice after colonic (but not systemic)
adenoviral IL-4 overexpression (41). A mucosal IL-4 predominance within the intestinal microenvironment may play an immunopathogenic role in the development and/or maintenance of both
hapten-induced (42) and spontaneous chronic colonic inflammation in mice (43). Moreover, IL-4 producing Th2-type T cells appear to mediate the development of chronic inflammation and ep-
ithelial damage in a recently described murine model showing
spontaneous Crohn’s-like ileitis, where immunobacterial interactions may be an important trigger for the onset of chronic inflammation (44, 45). Our study may be helpful in understanding increased IEC damage in these models, as down-regulated TLR
signaling in IECs likely weakens the host innate immune defense
against the apparently normal microflora.
In conclusion, the present study showed that the key Th2 cytokines IL-4 and IL-13 down-regulated TLR3 and TLR4/MD-2 expression and function in validated model IECs. Further in vivo
studies are needed to determine whether the Th2 cytokine-induced
attenuation of TLR-mediated innate immune signaling in IECs
protects the host from excessive innate immune responses or
whether impaired TLR signaling in IECs may render the host more
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Th2 CYTOKINES DECREASE TCR SIGNALING IN HUMAN IECs
susceptible for the development of chronic intestinal inflammation
in response to the intestinal microenvironment.
Acknowledgments
We are grateful to Dr. Hans-Christian Reinecker, Dr. Nicolas Barnich, Dr.
Jiri Kalabis, Dr. Jesus Yamamoto-Furusho, Dr. Emiko Mizoguchi, and
Kathryn Devaney for helpful discussions and critical reading of the
manuscript.
Disclosures
The authors have no financial conflict of interest.
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