Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface epithelial cells
Hou et al. Eye and Vision
Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface epithelial cells
Aihua Hou 1
Min Qi Tin 1
Louis Tong 0 1 2 3
0 Duke-NUS Graduate Medical School, Singapore , Singapore
1 Ocular Surface Research Group, Singapore Eye Research Institute, Singapore , Singapore
2 Singapore National Eye Center, Singapore , Singapore
3 Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore
Background: Gram-positive bacteria stimulate Toll-like receptor (TLR) 2 and then activate the pro-inflammatory nuclear factor-kappa B (NF-κB) pathway. As the human ocular surface is heavily colonised by gram-positive cocci bacteria, a balance of activation/repression of NF-κB target genes is essential to avoid uncontrolled infection or autoimmune-related inflammation. It is advantageous to test NF-κB targeting molecules in an ocular surface culture system that allows assessment of temporal NF-κB activation in a longitudinal fashion without destruction of cells. Such initial testing under standardised conditions should reduce the number of molecules that progress to further evaluation in animal models. This study aims to establish an in-vitro cell culture system to assess NF-κB activation in the context of ocular surface cells. Methods: NF-κB activity was evaluated through a secretory alkaline phosphatase reporter assay (SEAP). Immunoblots and immunofluorescence were used to examine IκBα phosphorylation and p65/p50 nuclear localization. Monocyte chemoattractant protein-1 (MCP-1) transcripts were evaluated by real time PCR and protein levels were measured by ELISA. Results: NF-κB activity in HCE-T cells treated with TLR2 activator Pam3CSK4 was higher than control cells at both 6 and 24 h. Pam3CSK4-stimulated NF-κB activation was inhibited by IκK inhibitors, Wedelolactone and BMS-345541. In Pam3CSK4 treated cells, active NF-κB subunits p50 and p65 increased in cell nuclear fractions as early as 1.5 h. Although the level of total IκB-α remained constant, phospho-IκB-α increased with treatment over time. In the culture media of Pam3CSK4stimulated cells, MCP-1 protein level was increased, which was suppressed in the presence of IκK inhibitors. Conclusion: NF-κB pathway can be activated by the TLR2 ligand and inhibited by IκK inhibitors in the ocular surface cell culture system. This cell culture system may be used to evaluate TLR-related innate defences in ocular surface diseases.
TLR2; NF-κB pathway; Activation; Ocular surface cells
The ocular surface is constantly exposed to the
colonization of various commensal microbes, playing an
important role in defence against microbes and other
inflammatory insults [
]. The nuclear factor kappa
(NFκ) B transcription pathway is the central regulator of
ocular surface inflammation and disease [
receptors (TLRs) are a family of transmembrane
receptors that recognize microbial pathogens and trigger early
innate immune responses leading to inflammation in
mammalian cells [
]. TLRs present on the
membrane of ocular surface cells can recognize and bind to a
variety of microbial components including bacterial
lipopeptides, lipopolysaccharide (LPS), flagellin, viral
dsRNA, ssRNA and other ligands [
]. On activation
of TLR, subsequent signalling of the cells can activate
the master kinase called the Ikappa-B kinase (IκK),
which phosphorylates the Ikappa-B alpha (IκBα) subunit
of NF-κB. The phosphorylated IκBα undergoes
degradation, releasing the p50 and p65 subunits of NF-κB,
which translocate to the cellular nuclei to bind to the
promoter of target genes [
3, 8, 14
upregulation, and less often, down-regulation of target
genes, this signalling pathway has a major influence on
the local immune defence and may be critical in control
of ocular infections.
The regulation of NF-κB is highly context- and
tissuedependent, so it is important to define the triggers and
targets of this pathway in healthy ocular surface and in
disease. TLR2 is reported to function as a Gram-positive
bacteria sensor in the cornea [
]. In Gram-positive
bacterial infections, pro-inflammatory mediators such as
TNF-alpha, interleukin (IL)-6, IL-8 and the adhesion
molecule ICAM-1 were secreted by human cornea
epithelial cells through activation of NF-κB [
propagation of inflammation by these molecules can
increase the severity of infection. On the other hand,
NF-κB-mediated expression of anti-microbial molecules
human defensin (hBD)-2 and homeostatic molecules
manganese superoxide dismutase may help to control
the pathogenicity of microbes or increase defences
against oxidative stress [
]. It is therefore important
to understand how NF-κB is finely tuned in health and
disease involving ocular surface cells, so that appropriate
therapeutic strategies can be implemented to protect the
eye against ocular infections .
We aim to describe the activation of NF-κB in an
invitro system of ocular surface cells using promoter assay
and other approaches, induced by the presence of a
Materials and methods
Human SV-40 immortalized corneal epithelial cell line
(HCE-T) cell line was obtained from the Riken Cell Bank
]. Cells were cultured with DMEM/F-12
(Life Technologies, CA, USA) medium supplemented
with 5% foetal bovine serum (FBS) (Life Technologies,
CA, USA) and maintained in a 37 °C incubator with 5%
CO2. Medium was changed every 2 days. Cells with
passage no. 69–73 were used in this study.
Transfection, IκK inhibitor treatment and Pam3CSK4
HCE-T cells were cultured to 90% confluent in 6-well
plates and changed to fresh medium before transfection.
Six microliters of Fugene 6 Transfection Reagent
(Promega, Madison, USA) and 200 μl of Epilife medium
(Life Technologies, CA, USA) were mixed and incubated
at room temperature for 5 min. Subsequently, vectors
(2 μg) pSEAP-basic or pSEAP-NF-κB (Clontech
Laboratories, Mountain View, CA) were added to the
mixture and incubated at room temperature for 15 min.
The mixture was then added to the cells and incubated
in a 37 °C incubator with 5% CO2 overnight.
IκK inhibitor Wedelolactone (Calbiochem, San Diego,
CA) or BMS-345541 (Calbiochem, San Diego, CA) was
added to select wells, which were transfected with
pSEAP-NF-κB at a concentration of 10 μM or 5 μM,
respectively. Pam3CSK4 is a well-established TLR2 specific
]. Depending on the experiment group
assignment, Pam3CSK4 was added to selected wells at a final
concentration of 250 ng/mL an hour later. The
assignment of cell transfection and treatment groups is
depicted in Fig. 1a.
NF-κB transcriptional activity detected by secreted
alkaline phosphatase (SEAP) assay
NF-κB transcriptional activity was detected using the
Great EscAPe SEAP detection kit (Clontech Laboratories,
Mountain View, CA) following the manufacturer’s
instruction. Briefly, culture media (400 μl) were collected
from cells transfected with different pSEAP vectors with
or without Pam3CSK4 stimulation and IκK inhibitors
treatment. The culture media were centrifuged at
12,000 rpm for 10 s before adding to a 96-well black
walled plate (12 μl per well). Fresh media was added
(48 μl per well) to the culture medium. Serial dilutions of
the positive control alkaline phosphatase were also added
to the same 96-well plate. The plate was sealed with
adhesive aluminium foil, incubated at 65 °C for 30 min, and
then cooled on ice for 2–3 min. After equilibrium to room
temperature, 60 μl of assay buffer was added to each well
and the plate was incubated for 5 min at room
temperature. Subsequently, 60 μl of substrate working
solution was added and the plate was incubated at room
temperature for 10 min. Chemiluminescence was
measured by Tecan GENios (Tecan, Männedorf, Switzerland).
Nuclear extraction from HCE-T cells stimulated with
Pam3CSK4 was performed using the nuclear extraction
kit (Active Motif, Carlsbad, CA) following the
manufacturer’s instruction. Briefly, HCE-T cells were stimulated
with Pam3CSK4 (250 ng/ml) for 0, 1, 1.5 and 2 h in
100 mm culture plates, washed with 10 ml of cold PBS,
and scrapped off from culture plates with 400 μl of cold
PBS containing protease and phosphatase inhibitors
(Roche Applied Science, Penzburg, Germany). Scrapped
cells were transferred to pre-chilled tubes and
centrifuged for 5 min at 500 rpm at 4 °C. Cell pellet was
re-suspended in 500 μl of 1X hypotonic buffer and
incubated on ice for 15 min. Twenty-five microliters of
detergent was added to the cell pellet suspension and
centrifuged for 30 s at 14,000 rpm at 4 °C. The pellet
was re-suspended in 50 μl of complete lysis buffer and
incubated for 30 min on ice with shaking at 150 rpm,
and then centrifuged at 14,000 rpm for 10 min at 4 °C.
The nuclear containing supernatant was transferred into
pre-chilled 1.5 ml tubes and stored at −80 °C until use.
Protein concentration was determined by BCA method
following manufacturer’s instruction [
Western blots were performed as described previously
]. Briefly, protein samples (30 μg per lane) were
separated in 12% SDS-PAGE gel and transferred to PVDF
membranes. The membranes were then blocked with 5%
BSA in TBS-Tween 20 (TBS-T) for 1 h at room
temperature and incubated with primary antibodies for
2 h at room temperature. After that, the membranes were
incubated with horseradish peroxidase conjugated
secondary antibodies for 1 h at room temperature, washed with
TBS-T for three times (5 min each time), incubated with
SuperSignal West Pico chemiluminescent substrates
(Pierce Biotechnology, Rockford, USA) and signals were
visualized on X-ray films. Primary and secondary
antibodies used in this study are listed in Table 1.
Immunofluorescent staining of nuclear translocation of
NF-κB active subunits, p50 and p65, were performed
according to a previous protocol [
]. In summary, cells
seeded in chamber slides treated with or without
Pam3CSK4 were fixed with 100% methanol for 10 min
at room temperature, washed with PBS, permeated in
PBS containing 0.15% Triton X-100 for 15 min, blocked
with 4% BSA in PBS containing 0.1% Triton X-100
(Sigma) for 1 h, then incubated with primary antibodies
at 4 °C overnight. After washing with PBS containing
0.1% Tween-20, cells were incubated with Alexa Fluor
488-conjugated secondary antibodies at room temperature
for 45 min. Subsequently, chamber walls were
removed and slides were mounted with VectaShield
mounting medium with DAPI (Vector Lab, Burlingame,
USA). Slides were observed and imaged using a Zeiss
Axioplan 2 fluorescence microscope (Zeiss, Oberkochen,
Germany). Primary and secondary antibodies used are
listed in Table 1.
Enzyme-linked immunosorbent assay (ELISA)
MCP-1 protein in the culture media was quantified by
ELISA (R&D system) following the manufacturer’s
instruction. Briefly, 200 μl of standards and culture
medium were added to microplates coated with MCP-1
antibody and incubated for 2 h at room temperature.
Solution from each well was aspirated and 400 μl of
1:20 for IF, 1:1000
1:20 for IF 1:1000
washing buffer added. The wash step was repeated two
more times. MCP-1 conjugates (200 μl) were added to
each well and incubated for 1 h at room temperature.
Subsequently, the microplates were washed three times
with washing buffer, incubated with substrate solution
(200 μl/well) for 30 min followed by the stop solution
(50 μl/well). The optical density of each well was
determined by Tecan GENios Pro microplate reader at
450 nm. Actual MCP-1 concentrations were determined
using the standard curve generated with MCP-1
standards of known concentrations.
Real time quantification PCR
Real time PCR was performed as described previously
according to the manufacturer’s instructions [
summary, total RNA was extracted from HCE-T cells using
the RNeasy Mini Kit (Qiagen, Hilden Germany) and RNA
concentration determined by the nanodrop method [
One microgram of RNA for each condition was used to
synthesize cDNA. First strand synthesis was performed
using Superscript II Reverse Transcriptase (Life
Technologies, CA, USA) and real time PCR reaction was performed
with Roche UPL Mastermix and an appropriate probe from
the human library (Roche Applied Science, Penzburg,
Germany). Forward and reverse primers used are:
5’TTCTGTGCCTGCTGCTCAT3’ and 5’GGGGCATTG
ATTGCATCT3’ respectively. GAPDH was used as the
endogenous control, forward and reverse primers are
5’AGCCACATCGCTGAGACA3’ and 5’GCCCAATACG
ACCAAATCC3’ respectively. PCR cycles were performed
on Roche LightCycler 480 (Roche Diagnostics, Basel,
Switzerland) with the following conditions:
denaturation at 95 °C for 10 min, followed by 24 cycles of
denaturation at 95 °C for 10 s, annealing at 54 °C for
10 s and extension at 72 °C for 30 s. Delta-Delta Ct
method was used to analyse data and fold change was
expressed relative to GAPDH levels.
In the NF-кB activity assay, we performed the cell
cultures in 3 independent wells and repeated 3 further wells
on a different occasion. From each of the wells, the
culture media were removed and split into 4 replicates for
evaluation of the luminescence. The mean of the 4
replicates (which originated from a single well of cells) was
evaluated. The means were used to calculate overall
mean of the wells that had the same experimental
treatment. To demonstrate overall findings in a graphical
format, the means over the 2 occasions (which had 6
independent wells) were then calculated. The same
calculation method was used for MCP-1 real time PCR and
ELISA assay. All results are expressed as the
mean ± standard error of the mean. Statistical analyses
were performed using student’s t-test for statistical
comparison between different conditions. P values less than
0.05 were considered statistically significant.
NF-κB was activated through TLR2 in human corneal
The NF-κB activity of HCE-T cells stimulated with
Pam3CSK4 for 6 and 24 h was compared to the
unstimulated cells using SEAP assay. At both 6 and 24 h,
Pam3CSK4 treated HCE-T cells showed significantly
higher levels of NF-κB activity compared to controls
(Fig. 1a, column 3 compared to columns 1 and 2).
Protein level of NF-κB active subunit p50 increased in
the nuclear fractions of Pam3CSK4 treated cells as early
as 1.5 h (Fig. 1b). At the same time, cytosolic p50 and
p65 decreased over time (data not shown).
Immunostaining of the HCE-T cells treated with Pam3CSK4 over
the time intervals 0, 1, 2 and 4 h showed increase of p50
and p65 in the nuclei (data for 4 h shown in Fig. 1c).
There was an increase of phospho-IκB-α overtime with
Pam3CSK4 treatment, while the level of total IκB-α
protein remained relatively constant (Fig. 1d).
suppressible with BMS-345541 (Fig. 2b) as in the case of
the transcript levels.
Pam3CSK4-stimulated NF-κB activation was mediated by IκK
To investigate whether Pam3CSK4-stimulated NF-κB
activation is mediated by IκK, IκK inhibitors
Wedelolactone and BMS-345541 were added to cells 1 h
before stimulating cells with Pam3CSK4. SEAP assay
showed that both Wedelolactone and BMS-345541
could significantly inhibit Pam3CSK4-induced NF-κB
activity (Fig. 1a, column 3 compared to columns 5
and 7). Pam3CSK4-induced phosphorylation of IκB-α,
the inhibitory subunit of NF-κB, was also suppressed
by these inhibitors (Fig. 1d), indicating that
Pam3CSK4 stimulated NF-κB activity was mediated
MCP-1 was up-regulated by TLR2-NF-κB activation
MCP-1 also known as chemokine (C-C motif ) ligand 2
(CCL2) is one of the critical regulators for leukocyte
recruitment during cornea infection [
levels of MCP-1 were evaluated by qPCR in Pam3CSK4
treated and un-treated cells. Consistent with the NF-κB
activity SEAP assay, at both 6 and 24 h,
Pam3CSK4treated HCE-T cells showed higher levels of MCP-1
compared to controls (Fig. 2a, column 3 compared to
columns 1 and 2). MCP-1 up-regulation was similarly
inhibited with IκK inhibitors Wedelolactone and
BMS345541 (Fig. 2a, column 3 compared to columns 5 and
7). ELISA was used to examine MCP-1 protein levels in
the culture media. Results showed that after 24 h
treatment, MCP-1 protein in the medium was also
up-regulated after Pam3CSK4 stimulation and it was
In this study, we found that in HCE-T cells, NF-κB
activity was increased with TLR2 ligand Pam3CSK4
treatment, and the phosphorylation level of IκB-α was also
increased with the addition of Pam3CSK4, while total
IκB-α protein level remained the same. NF-κB active
subunit p50 translocated to the cell nuclei as early as
1.5 h after Pam3CSK4 treatment; NF-κB activation was
mediated by IκK. The chemokine MCP-1 was
upregulated by the TLR2 NF-κB pathway at both the
transcript and protein levels, and this was also mediated by
IκK. Four previous studies evaluated the activation of
NF-κB downstream of TLR-2 in cornea epithelial cells.
One study investigated NF-κB using the detection of p65
in the nuclear fractions of cells [
], another study
investigated NF-κB activity in cell lysate with anti-NF-κB
antibodies, whereas two other studies examined the
levels of pIκB-α [
]. Neither of these examined the
expression of MCP-1 as a possible target, and in
addition, the previous studies in corneal epithelial cells
did not employ NF-κB promoter assays. In our current
report, we examined the activation of NF-κB using a fine
tune promoter SEAP assay and reported the regulation
of MCP-1 through TLR2 NF-κB pathway. The advantage
of using SEAP assay is that the culture medium can be
analysed at more than one time point in the experiment
without destroying the cells.
Two studies have shown apparently contradictory
results when using peptidoglycan (PGN) to treat human
corneal epithelial cells, one group found activation of
NF-κB as well as increases in IL-6 and IL-8 expressions
], but not the other [
]. The controversy in this area
cannot be easily resolved unless researchers agree to use
a common testing strategy for screening potential drugs.
We hope that the methodology we propose will be
adopted by others and this should help to prevent
confusing results from such studies. The strength of this
study is the use of more than one approach to
investigate the TLR2-NF-κB pathway. The limitation of the
study is that in-vivo animal models of Gram-positive
infection was not investigated and compared to our
In many experimental scenarios, it is valuable to
screen a variety of potential new therapeutic agents that
may treat Gram-positive eye infections. It is too
expensive and resource intensive to investigate all these agents
in animal studies in the first instance. It is therefore
advantageous to be able to rapidly investigate NF-κB
agents, especially those suspected to act via the IκK
kinases, in a cell culture system such as the one in our
current study to shortlist more promising candidates
and reduce the number of molecules that progress to
further evaluation in animal models.
In conclusion, the NF-κB pathway can be activated by
the TLR2 ligand and inhibited by IκK inhibitors in the
ocular surface cell culture system. This cell culture
system may be used to evaluate TLR-related innate
defences in ocular surface diseases.
The authors thank Peggy Teng Wan Peng for help in performing the NF-κB
activity assay and measuring MCP-1 levels.
Availability of data and materials
Data sharing is not applicable to this article as no datasets were generated
or analysed during the current study.
This research is supported by the Singapore National Research Foundation
(NMRC/CSA/045/2012) and administered by the Singapore Ministry of
Health’s National Medical Research Council. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of
AH designed and performed experiments, analysed data and wrote the
manuscript. TMQ planned and conducted experiments and contributed to
discussion. LT conceived the study, analysed data and wrote the manuscript.
All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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