Activation of Epidermal Growth Factor Receptor Is
Required for NTHi-Induced NF-kB-Dependent
Xiangbin Xu1*, Rachel R. Steere1, Christine A. Fedorchuk1, Jinjiang Pang2, Ji-Yun Lee1,3, Jae Hyang
Lim1,3, Haidong Xu1,3, Zhixing K. Pan4, Sanjay B. Maggirwar1, Jian-Dong Li1,3*
1Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America, 2Aab Cardiovascular Research
Institute, University of Rochester Medical Center, Rochester, New York, United States of America, 3Department of Biology, Center for Inflammation, Immunity and
Infection, Georgia State University, Atlanta, Georgia, United States of America, 4Department of Medical Microbiology and Immunology, University of Toledo Medical
Center, Toledo, Ohio, United States of America
Background: Inflammation is a hallmark of many serious human diseases. Nontypeable Haemophilus influenzae (NTHi) is an
important human pathogen causing respiratory tract infections in both adults and children. NTHi infections are
characterized by inflammation, which is mainly mediated by nuclear transcription factor-kappa B (NF-kB)-dependent
production of proinflammatory mediators. Epidermal growth factor receptor (EGFR) has been shown to play important roles
in regulating diverse biological processes, including cell growth, differentiation, apoptosis, adhesion, and migration. Its role
in regulating NF-kB activation and inflammation, however, remains largely unknown.
Methodology/Principal Findings: In the present study, we demonstrate that EGFR plays a vital role in NTHi-induced
NF-kB activation and the subsequent induction of proinflammatory mediators in human middle ear epithelial cells and
other cell types. Importantly, we found that AG1478, a specific tyrosine kinase inhibitor of EGFR potently inhibited NTHi-
induced inflammatory responses in the middle ears and lungs of mice in vivo. Moreover, we found that MKK3/6-p38 and
PI3K/Akt signaling pathways are required for mediating EGFR-dependent NF-kB activation and inflammatory responses by
Conclusions/Significance: Here, we provide direct evidence that EGFR plays a critical role in mediating NTHi-induced NF-kB
activation and inflammation in vitro and in vivo. Given that EGFR inhibitors have been approved in clinical use for the
treatment of cancers, current studies will not only provide novel insights into the molecular mechanisms underlying the
regulation of inflammation, but may also lead to the development of novel therapeutic strategies for the treatment of
respiratory inflammatory diseases and other inflammatory diseases.
Citation: Xu X, Steere RR, Fedorchuk CA, Pang J, Lee J-Y, et al. (2011) Activation of Epidermal Growth Factor Receptor Is Required for NTHi-Induced NF-kB-
Dependent Inflammation. PLoS ONE 6(11): e28216. doi:10.1371/journal.pone.0028216
Editor: Ulrike Gertrud Munderloh, University of Minnesota, United States of America
Received February 8, 2011; Accepted November 3, 2011; Published November 23, 2011
Copyright: ? 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from National Institutes of Health DC010048 (to X.X.), DC005843, DC004562 and AI073374 (to J.D.L), NS054578,
NS066801 (to S.B.M) and AHA 10SDG2630077 (to J.H.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Xiangbin_Xu@urmc.rochester.edu (XX); firstname.lastname@example.org (JDL)
Inflammation is a hallmark of many serious human diseases.
Appropriate inflammation is a protective host defense response to
remove the injurious stimuli and initiate tissue healing and repair.
However, overactive inflammation is detrimental to the host,
leading to inflammatory diseases. Thus, inflammation must be
tightly regulated. The molecular mechanisms underlying tight
regulation of inflammation remain largely unknown. Steroids and
cyclooxygenase inhibitors have long been used as the main thera-
peutic anti-inflammatory agents, but they are frequently associated
with significant detrimental effects in patients. In addition, inap-
propriate antibiotic treatment for bacterial infection contributes
significantly to the worldwide emergence of antibiotic resistance.
Thus, there is an urgent need for the development of novel anti-
Nontypeable Haemophilus influenzae (NTHi), a gram-negative
bacterium, is an important human pathogen in both children and
adults . In children, it causes otitis media (OM), the most
common childhood infection and the leading cause of conductive
hearing loss [2,3]. In adults, it exacerbates chronic obstructive
pulmonary disease (COPD)[4,5], animportant lung diseaseand the
fourth leading cause of death in the United States . Like most
bacterial infections, NTHi infection is characterized by inflamma-
tion, which is mainly mediated by nuclear factor-kappa B (NF-kB)-
dependent production of proinflammatory mediators [7,8]. NF-kB
is a transcription factor consisting of homo- or heterodimers of Rel-
related proteins . It has five members in mammalian cells: RelA
PLoS ONE | www.plosone.org1November 2011 | Volume 6 | Issue 11 | e28216
(p65), RelB, c-Rel, p50/p105, and p52/p100. The heterodimer
consisting of two subunits, p65 and p50, is most commonly involved
in the regulation of a variety of physiologic processes, including
inflammation, differentiation, proliferation, and survival, among
others . In its inactive state, NF-kB resides in the cytoplasm and
forms a multiprotein complex with an inhibitory subunit, inhibitor
of NF-kB (IkB). Upon activation by external stimuli, the inflam-
matory signal converges on and activates a set of IkB kinases known
as the IkB kinase (IKK) complex, which are composed of three
subunits: IKKa, IKKb, and IKKc. IkBa is phosphorylated by
IKKs and this phosphorylation results in the degradation and
dissociation of IkBa from NF-kB. Once released from the complex
involving IkBa, NF-kB translocates to the nucleus, where it binds to
DNA and promotes the transcription of target genes. NF-kB is
activated by inflammatory stimuli and involved in regulating
expression of proinflammatory mediators, including cytokines,
chemokines, and adhesion molecules, thereby playing a critical
role in mediating inflammatory responses .
Toll-like receptor 2 (TLR2) plays a crucial role in mediating
NTHi-induced inflammatory response. However, directly blocking
TLR2 signaling may result in some unwanted detrimental side
effects because appropriate immune response mediated by TLR2
signaling is also required for host defense against invading
bacterial pathogens. For instance, uncontrolled bacterial growth,
decreased bacteria clearance and increased susceptibility to
bacterial infection was observed in TLR2 KO mice [11,12,13]
and impairment of TLR2 signaling due to genetic mutations in
human populations closely correlates with increased susceptibility
to bacterial pathogens [14,15]. Thus, identifying a non-TLR2
therapeutic target for NTHi infection is in high demand.
The epidermal growth factor receptor (EGFR) is a member of
the HER family composed of four distinct receptors: EGFR/
ErbB1, Her-2/ErbB2/c-neu, Her-3/ErbB3, and Her-4/ErbB4,
which are predominantly located at the basolateral surface
of polarized epithelial cells. EGFR is traditionally known as a
growth factor receptor that mediates cell differentiation and
proliferation. Elevated levels of EGFR and/or its cognate ligands
have been shown to be involved in tumor growth . In
addition, EGFR is activated by multiple TLRs to produce innate
immune response in airway epithelium . Activation of EGFR
plays an important role in recruiting leukocytes , inducing
mucins and antimicrobial peptides to clear pathogens [19,20],
and increasing wound repair . Recent studies from our group
suggested that EGFR is at least in part activated by NTHi via
NTHi-derived EGF-like growth factor and plays an important
role in negatively regulating TLR2 induction during bacterial
infections . In addition, exogenous EGF increases NTHi
invasion of host epithelial cells, demonstrating the biological
significance of TLR2 regulation by EGFR signaling .
However, the role of EGFR in regulating NTHi-induced NF-
kB signaling and inflammatory response in airway inflammatory
diseases has yet to be fully explored.
Based on the essential role of TLR2 in NTHi-induced NF-kB
signaling and inflammatory responses and the role of EGFR in
controlling TLR2 induction, we hypothesized that EGFR may
regulate NTHi-induced NF-kB activation and inflammation in the
middle ear and lung. Here, we provide direct evidence for the
critical role of EGFR signaling in regulating NTHi-induced
inflammation in human middle ear and airway epithelial cells in
vitro, and in mouse middle ear and lung in vivo. Our studies will not
only provide novel insights into the molecular mechanisms
underlying the regulation of inflammation, but will also facilitate
translational research toward novel therapeutic strategies for the
treatment of respiratory and other inflammatory diseases.
Materials and Methods
AG1478, wortmannin and SB2030580 were purchased from
Calbiochem (PA,USA). Polyclonal antibodyagainst phospho-IkBa,
IkBa, phospho-IKKa/b, IKKa, IKKb, phospho-p38, p38, phos-
pho-MKK3/6, MKK3, phospho-Akt, Akt, phospho-EGFR and
EGFR were purchased from Cell Signaling (MA, USA). Antibody
against actin was purchased from Santa Cruz (CA, USA).
Bacteria Strain and Culture
Clinical isolate of NTHi wild-type strain 12 was used in in vitro
cell culture experiments and in vivo animal experiments [22,23].
Bacteria was grown on chocolate agar at 37uC in an atmosphere of
5% CO2overnight and inoculated in brain heart infusion broth
supplemented with 3.5 mg of NAD per mL. For in vitro experiments,
the epithelial cells were treated with NTHi at a multiplicity of
infection (MOI) of 1:25 for various times as indicated in figures. For
invivoanimal experiments,NTHiwasinoculatedinto the middleear
for the OM model and the lung for the pneumonia model as
described below in animal experiments.
Human middle ear epithelial cell line (HMEEC-1) [7,22,24],
a commonly used middle ear cell line, was derived by human
papilloma virus immortalization of primary human middle ear
epithelial cells, and was maintained in a 1:1 mixture of Bronchial
Epithelial Basal Medium (BEBM) and Dulbecco’s modified Eagle’s
medium (DMEM) as described [7,22]. Human airway epithelial
cell line (A549), human cervix epithelial cell line (HeLa) and
mouse macrophage cell line (RAW 264.7, American Type Culture
Collection, Manassas, VA) were maintained as described [22,25].
MDA-MB453 (hereafter MB453), a breast cancer epithelial cell line
in which the level of EGFR expression is not detectable with anti-
EGFR antibody, and MDA-MB468 (hereafter MB468), a breast
cancer epithelial cell line in which the level of EGFR expression is
readily detectable with the same anti-EGFR antibody were main-
tained as described [21,26]. All cells were cultured under standard
conditions (5% CO2in air in a humidified environment at 37uC).
Plasmids, Transfections and Luciferase Reporter Assay
The EGFR dominant-negative mutant (DN) and NF-kB-
luciferase reporter plasmids were described previously [21,25].
Cells were co-transfected with NF-kB-luciferase reporter plasmid
together with or without EGFR DN expression plasmids. Empty
vector was used as a control. All transient transfections were
carried out in triplicate using a TransIT-LT1 reagent (Mirus Co.)
following the manufacturer’s instructions. At 40 hours after the
start of transfection, cells were inoculated with NTHi for 5 hours
before cell lysis for luciferase assay as described previously.
RNA-mediated interference for down-regulating EGFR expres-
sion was carried out using EGFR siRNA as described previously
using Lipofectamine 2000 (Invitrogen) . EGFR small interfer-
ing RNA oligonucleotide was purchased from Dharmacon. Forty
hours after the start of transfection, cells were treated with NTHi
for the indicated time before being lysed for luciferase assay.
RNA Isolation and Real-time Quantitative PCR (Q-PCR)
Total RNA was isolated with TRIzol reagent (Invitrogen) by
following the manufacturer’s instructions. For the reverse transcrip-
tion reaction, TaqMan reverse transcription reagents (Applied
EGFR Regulates Inflammation
PLoS ONE | www.plosone.org2November 2011 | Volume 6 | Issue 11 | e28216
Biosystems) were used. Briefly, the reverse transcription reaction
was performed for 60 min at 37uC, followed by 60 min at 42uC by
using oligo (dT) and random hexamers. PCR amplifications were
performed by using SYBR Green Universal Master Mix. In brief,
reactions were performed in duplicate containing 2X Universal
Master Mix, 1 mL of template cDNA and 100 nM primers in a
final volume of 12.5 mL, and they were analyzed in a 96-well
optical reaction plate (Applied Biosystems). The relative quantities
of mRNAs were obtained by using the comparative Ct method and
were normalized with pre-developed Taqman assay reagent mouse
GAPDH or human cyclophilin as an endogenous control (Applied
Biosystems). The primers for human TNF-a, IL-1b, IL-8, cyclo-
philin, and mouse TNF-a, IL-1b, MIP-2, EGFR, and GAPDH
were described previously .
Western Blot Analysis
Cell lysates were prepared in the buffer containing 20 mM Tris-
HCl (pH 8.0), 0.5 M NaCl, 0.25% Triton X-100, 1 mM EDTA,
1 mM EGTA, 10 mM glycerophosphate, 10 mM NaF, 300 mM
Na3VO4, 1 mM benzamidine, 2 mM PMSF, 1 mM DTT and
protease inhibitor cocktail (Sigma, MO, USA) by scraping,
incubating on ice for 30 min, and centrifugation at 12,000 g for
15 min. Supernatant was collected and then subjected to SDS-
PAGE, and transferred to poly-vinylidine difluoride membranes.
The membrane was blocked with 5% nonfat milk, incubated in a
1:1,000 dilution of a primary antibody, and incubated with
1:2,000 dilution of the corresponding secondary antibody. The
membrane was reacted with chemiluminescence reagent ECL to
visualize the blots.
C57 BL/6 mice were purchased from National Cancer Institute
(NCI, NIH), and eight week old male mice were used in this study.
For the NTHi-induced OM model, anaesthetized mice were
transtympanically inoculated with NTHi under the surgical
microscope, and saline was inoculated as control. AG1478
(10 mg/kg of body weight) or an equal volume of vehicle control
was administered via an intraperitoneal route 2 hours before the
transtympanic inoculation of NTHi. Animals were then sacrificed
by intraperitoneal inoculation of 100 mg/kg sodium pentobarbital
at 9 and 24 hours after NTHi inoculation. To assess the mRNA
expression of proinflammatory mediators, total RNA was
extracted from the bullae of NTHi- or saline-inoculated ears at
the time points indicated above. For histological analysis, dissected
temporal bones were fixed with 10% buffered formaldehyde
overnight with rocking, decalcified with CalEX, embedded in
paraffin, and sectioned at 5-mM thickness. Sections were then
stained with hematoxylin and eosin (H&E) to visualize inflamma-
tory response and pathological changes in the middle ear. H&E-
stained middle ear sections were then evaluated using Axiovert
40 CFL (Carl Zeiss), and images were recorded with an AxioCam
MRC (Carl Zeiss).
For NTHi-induced pneumonia model, anaesthetized mice were
intratracheally inoculated with NTHi, and saline was inoculated as
control. AG1478 (10 mg/kg of body weight) or an equal volume of
vehicle control was administered via an intraperitoneal route
2 hours before the intratracheal inoculation of NTHi. Animals
were then sacrificed by intraperitoneal inoculation of 100 mg/kg
sodium pentobarbital at 9 and 24 hours after NTHi inoculation.
For histological analysis, dissected lung was inflated and fixed with
10% buffered formaldehyde, embedded in paraffin, and sectioned
at 5-mM thickness. Sections were then stained and inspected as
described above. For polymorphonuclear neutrophil (PMN) analysis,
bronchoalveolar lavage (BAL) was performed by cannulating the
trachea with sterilized PBS. Cells from BAL fluid were stained with
Hemacolor (EM Science) after cytocentrifugation (Thermo Elec-
tronic Co.). To assess the mRNA expression of proinflammatory
mediators, total RNA was extracted from the lungs of NTHi- and
experiments were approved by the Institutional Animal Care and
2007-058 and 2005-209).
Data are shown as mean6S.D. Statistical evaluation was done
by unpaired Student’s t test and p,0.05 was taken as a significant
EGFR plays a critical role in mediating NTHi-induced
NF-kB activation and subsequent inflammatory
response in vitro
EGFR represents one of the important tyrosine kinases and can
be activated via phosphorylation by many stimuli. We first
evaluated if EGFR is phosphorylated by NTHi. As shown in Fig 1,
NTHi induced EGFR phosphorylation in a time-dependent
manner in HMEEC-1 cells.
Because NTHi infection is mainly characterized by inflamma-
tion, it is likely that EGFR may play an important role in
mediating NTHi-induced inflammatory response, which is mainly
mediated by NF-kB. We thus determined if EGFR is involved in
NTHi-induced NF-kB activation by using multiple approaches to
assess the effects on NTHi-induced NF-kB activation of AG1478 (a
chemical inhibitor for EGFR), EGFR DN, EGFR siRNA, and
EGFR-deficient MB-453 cells. As shown in Fig. 2, NTHi greatly
induces NF-kB activation in HMEEC-1, A549, HeLa, RAW 264.7
and MB-468 cells. Interestingly, AG1478 markedly inhibited
NTHi-induced NF-kB activation in a dose-dependent manner
(Fig. 2A–D). Please note that no significant effect of AG1478 on cell
morphology and viability was observed at the concentration used in
Figure 1. EGFR is activated by NTHi in middle ear epithelial
cells. HMEEC-1 cells were treated with NTHi for the time indicated, then
were lysed and blotted with anti-phospho-EGFR and EGFR antibody.
Data are representative of three or more independent experiments.
EGFR Regulates Inflammation
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the experiments (data not shown). Moreover, overexpression of
EGFR DN (Fig. 2E) and knockdown of EGFR using EGFR siRNA
(Fig. 2F) greatly inhibited the NTHi-induced NF-kB activation.
Furthermore, NTHi-induced NF-kB luciferase activity was much
lower in MB453 cells compared to that in MB468 cells (Fig. 2G).
Taken together, these data indicate that EGFR plays a critical role
in mediating NTHi-induced NF-kB activation in vitro.
We further determined the role of EGFR in mediating NTHi-
induced NF-kB-dependent up-regulation of proinflammatory
mediators. As shown in Fig. 3A–C, AG1478 greatly inhibited
NTHi–induced up-regulation of TNF-a, IL-1b and IL-8 mRNA
in a dose-dependent manner, thereby demonstrating the critical
role for EGFR in NTHi-induced pro-inflammatory responses in
Figure 2. EGFR mediates NTHi-induced NF-kB transcriptional activity in a variety of cell types. (A–D) The effect of AG1478 on NTHi-
induced NF-kB activation was evaluated by performing luciferase assay in HMEEC-1 (A), A549 (B), HeLa (C) and RAW 264.7(D). (E–F) Overexpression of
EGFR DN (E) or EGFR knockdown using EGFR siRNA (F), and NTHi-induced NF-kB activation was measured by performing luciferase assay. (G) NTHi-
induced NF-kB activation was measured by luciferase assay in EGFR-competent MB468 and EGFR-deficient MB453 cells. Data represent the mean6SD
of at least three independent experiments, and each experiment was performed in triplicate. *p ,0.05 vs. control; #p ,0.05 vs. NTHi alone.
Figure 3. EGFR mediates NTHi-induced upregulation of proinflammatory mediators in middle ear epithelial cells. (A–C) Cells were
treated with EGFR inhibitor AG1478 for 1 hour followed by 5 hour NTHi treatment. The mRNA expression of TNF-a, IL-1b and IL-8 was measured by
performing Q-PCR analysis. Data represent the mean 6 SD of at least three independent experiments, and each experiment was performed in
triplicate. *p,0.05 vs. control; #p,0.05 vs. NTHi alone.
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EGFR is also crucial for mediating NTHi-induced
inflammation in the middle ear and lung tissues of mice
To further confirm the involvement of EGFR in NTHi-induced
inflammation, we determined if EGFR inhibitor AG1478 inhibits
inflammation in the middle ear and lung tissues of mice in vivo.
As shown in Fig. 4A and 4B, intraperitoneal administration of
AG1478 significantly inhibited mRNA expression of TNF-a, IL-
1b, and MIP-2 in the ears and lungs of mice after transtympanic or
intratracheal inoculation of NTHi. Consistent with this result,
histological analysis of the ear and lung tissues of NTHi-inoculated
mice showed that AG1478 markedly inhibited leukocyte infil-
tration (Fig. 4C and 4D). Also in agreement with these results,
AG1478 significantly inhibited polymorphonuclear neutrophil
(PMN) infiltration in bronchoalveolar lavage (BAL) fluids (Fig. 4E
and 4F). These results demonstrate that EGFR is critical for
mediating NTHi-induced inflammatory responses in vivo.
EGFR mediates NTHi-induced NF-kB activation via an
IKKa/b-IkBa-dependent pathway in middle ear epithelial
IKKa/b plays a key role in NTHi-induced NF-kB activation by
inducing phosphorylation of IkBa. Thus, we determined whether
EGFR mediates NTHi-induced NF-kB activation by regulating
the IKKa/b-IkBa pathway. As shown in Fig. 5A, NTHi induced
phosphorylation of IKKa/b and IkBa in a time-dependent
Figure 4. Inhibition of EGFR inhibited NTHi-induced inflammation in the middle ear and lung tissues of mice in vivo. (A & B) Animals
were intraperitoneally inoculated with AG1478 or vehicle control. Two hours after AG1478 inoculation, NTHi was inoculated into middle ear via
tympanic membrane (A) or into trachea (B). The mRNA expression of TNF-a, IL-1b, and MIP-2 was measured in the middle ear (A) or lung tissues (B) of
mice inoculated with NTHi or saline as a control. *p,0.05 vs. untreated group; #p,0.05 vs. NTHi alone. (C & D) Middle ear (C) and lung tissues (D) of
mice inoculated with NTHi with or without AG1478 were stained with Hematoxylin and Eosin for histological analysis (H&E stain, magnification6200).
(E & F) Bronchoalveolar lavage (BAL) was performed in NTHi-inoculated mice with or without AG1478, and total and polymorphonuclear (PMN)
neutrophils were counted (E) and cytocentrifuged to stain with Hemacolor (F).
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Figure 5. EGFR mediates NTHi-induced NF-kB activation by inducing IKKa/b and IkBa activation in middle ear epithelial cells. (A)
Cells were treated with NTHi at various time points as indicated in the figure, and whole cell protein was collected and blotted against total- and
phospho-IKKa/b and IkBa. (B) Cells were treated with NTHi with or without AG1478, and whole cell protein was collected and blotted against total-
and phospho-IKKa/b and IkBa. Data are representative of three or more independent experiments.
Figure 6. MKK3/6-p38 mediates EGFR-dependent inflammation induced by NTHi in middle ear epithelial cells. (A) Cells were incubated
with NTHi with or without SB203580, and NTHi-induced NF-kB activation was measured by performing luciferase assay. (B) Cells were incubated with
NTHi with or without SB203580, and mRNA expression of TNF-a, IL-1b and IL-8 was measured by performing Q-PCR analysis. (C) Cells were incubated
with NTHi with or without AG1478, and whole cell protein was collected and blotted against total- and phospho-p38 and MKK3/6. Data represent the
mean6SD of at least three independent experiments, and each experiment was performed in triplicate. *p,0.05 vs. control; #p,0.05 vs. NTHi alone.
EGFR Regulates Inflammation
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manner. Furthermore, inhibition of EGFR using AG1478 signifi-
cantly inhibited NTHi-induced IKKa/b andIkBa phosphorylation
(Fig. 5B). These data indicate that EGFR acts upstream of IKKa/b
in mediating NTHi-induced NF-kB activation.
MKK3/6-p38 MAPK also mediates EGFR-dependent
inflammation induced by NTHi in middle ear epithelial
Having demonstrated that EGFR mediates NTHi-induced NF-
kB activation and the subsequent inflammatory responses, we
sought to determine which signaling pathways are involved in
EGFR-mediated NF-kB-dependent inflammatory responses in-
duced by NTHi. The p38 mitogen-activated protein kinase (MAPK)
signaling pathway plays an important role in NTHi-induced NF-kB
signaling pathway . We thus determined if p38 MAPK is
involved in EGFR-mediated NF-kB activation and inflammatory
responses induced by NTHi. As shown in Fig. 6, p38-specific
inhibitor SB203580 inhibited NTHi-induced NF-kB activation
(Fig. 6A) and also mRNA expression of proinflammatory mediators
(TNF-a, IL-1b and IL-8) (Fig. 6B) in HMEEC-1. It should be noted
that SB203580 exhibited no effect on NTHi-induced IKKa/b and
IKBa phosphorylation and nuclear translocation of NF-kB, thereby
indicating that p38 mediates NTHi-induced NF-kB activation
independently of IKKa/b and IKBa as well as nuclear translocation
of NF-kB (data not shown). Next, we determined if EGFR is
involved in NTHi-induced activation of MKK3/6 and p38 MAPK
6 and p38 phosphorylation. As shown in Fig. 6C, AG1478 inhibited
NTHi-induced MKK3/6 and p38 phosphorylation in HMEEC-1
cells. Together, these data suggest that the MKK3/6-p38 signaling
pathway also mediates EGFR-dependent NF-kB activation and
inflammatory responses induced by NTHi.
PI3K/Akt mediates EGFR-dependent inflammatory
response induced by NTHi in middle ear epithelial cells
Previously it has been reported that that EGFR acts as the
major upstream activator of phosphatidylinositol 3-kinase (PI3K)/
Akt pathway leading to activation of NF-kB . Thus, we
determined if PI3K/Akt signaling is also involved in EGFR-
dependent NF-kB activation and inflammatory responses in middle
ear epithelial cells . As shown in Fig. 7A and B, wortmannin, a
specific inhibitor for PI3K, inhibited NTHi-induced NF-kB
luciferase activity (Fig. 7A) and phosphorylation of IKKa/b and
IkBa (Fig. 7B). Consistent with these findings, wortmannin
inhibited NTHi-induced mRNA expression of TNF-a, IL-1b and
Figure 7. PI3K/Akt mediates EGFR-dependent inflammation induced by NTHi in middle ear epithelial cells. (A) Cells were incubated
with NTHi with or without wortmannin, and NTHi-induced NF-kB activation was measured by performing luciferase assay. (B) Cells were incubated
with NTHi with or without wortmannin, and whole cell protein was collected and blotted against total- and phospho-IKKa/b and IkBa. (C) Cells were
incubated with NTHi with or without wortmannin, and mRNA expression of TNF-a, IL-1b and IL-8 was measured by performing Q-PCR analysis.
(D) Cells were incubated with NTHi with or without AG1478, and whole cell protein was collected and blotted against total- and phospho-Akt. Data
represent the mean6SD of at least three independent experiments, and each experiment was performed in triplicate. *p,0.05 vs. control; #p,0.05
vs. NTHi alone.
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IL-8 (Fig. 7C) in HMEEC-1, thereby indicating that the PI3K/Akt
signaling pathway mediates NTHi-induced NF-kB activation and
of PI3K/Akt by assessing the effect of AG1478 on NTHi-induced
Akt phosphorylation. As shown in Fig. 7D, AG1478 inhibited
NTHi-induced Akt phosphorylation. Collectively, these data
demonstrate that PI3K/Akt signaling mediates EGFR-dependent
NF-kB activation and inflammatory response induced by NTHi.
In the present study, we provide direct evidence that EGFR me-
diates NTHi-induced NF-kB activation and subsequent inflammation
in vitro and in vivo. Importantly, we demonstrate that AG1478, the
specific tyrosine kinase inhibitor of EGFR, potently inhibited
NTHi-induced inflammatoryresponsesinthemiddleears andlungs
of mice. Moreover, we found that MKK3/6-p38 and PI3K/Akt
signaling pathways mediate EGFR-dependent NF-kB activation
and subsequent inflammatory responses induced by NTHi (Fig. 8).
p38, an important MAPK family member, is activated by multiple
stimuli including bacteria such as NTHi, cytokines (IL-1, TNF-a)
and growth factors . The activated p38 mediates a variety of
cellular responses including inflammation . Its major upstream
kinases include MKK3 and MKK6. MKK3/6-p38 pathway plays
an important role in NTHi-induced NF-kB activation and mucin
production . In addition, p38 is actively involved in mediating
EGFR signaling [21,27,30]. Our data indicate that MKK3/6-p38
mediates EGFR-dependent NF-kB activation and inflammation by
NTHi in middle ear and lung tissues of mice by involving nuclear
events of NF-kB signaling. PI3K facilitates a broad range of cellular
functions in response to extracellular signals. A key downstream
effector of PI3K is the serine-threonine kinase Akt. There is
evidence that EGFR acts as the major upstream activator of the
PI3K/Akt pathway leading to activation of NF-kB in PC-3 cells
. Moreover, NTHi activates the PI3K/Akt pathway in
epithelial cells . Our data suggest that PI3K/Akt may act as
another important signaling transducer mediating EGFR-depen-
dent NF-kB activation and inflammation induced by NTHi in
middle ear and lung inflammation.
Of particular interest in this study is the identification of EGFR
as a critical mediator in NTHi-induced NF-kB activation and
inflammation in vitro and in vivo. Mutations, amplifications or
misregulations of EGFR or family members are implicated in
about 30% of all epithelial cancers. Many anticancer therapeutic
approaches are aimed at EGFR. It has also been reported that
EGFR plays an important role in the pathogenesis of asthma
[32,33,34,35] and inhibitors of tyrosine kinase have been studied
as a novel therapeutic strategy for the treatment of asthma .
Recent studies suggested that EGFR might also play an important
role in inflammation [37,38,39], and Gefinitib, a clinically
approved EGFR inhibitor, has been used for allergic airway
inflammation . We previously showed that NTHi, at least in
part, induces EGFR signaling likely via NTHi-derived EGF-like
factor although our data do not preclude the involvement of other
mechanism . TLRs are critical for detecting invading
microbial pathogens by recognizing pathogen associated molecu-
lar patterns (PAMPs). Among many TLRs, TLR2 detects NTHi
and activates downstream signaling pathways to induce inflam-
matory responses against NTHi. The important role of TLR2 in
bacterial clearance has been reported in both animal studies and
human clinical studies. For instance, deficiency of TLR2 resulted
in uncontrolled bacterial growth and increased susceptibility to
bacterial infection in TLR2 KO mice. Moreover, impairment of
TLR2 signaling due to genetic mutations in human populations
closely correlated with increased susceptibility to bacterial
pathogens. In our previous study, we found that NTHi not only
activates the TLR2 signaling pathway, but also induces TLR2
expression, which may further enhance NTHi clearance depen-
dent on TLR2-mediated inflammatory responses. Besides TLR2,
NTHi also activates EGFR, which negatively regulates TLR2
expression as evidenced by the finding that inhibition of EGFR
using AG1478 enhanced NTHi-induced TLR2 expression. Based
on the finding showing that exogenous EGF enhanced NTHi
invasion and survival in epithelial cells, EGFR-mediated inhibition
of TLR2 expression may exemplify the subversion of the host
signaling pathway by bacteria, as NTHi promotes bacterial survival
in the host by inhibiting TLR2-mediated antibacterial inflamma-
tory response. Thus, enhancing TLR2 expression by inhibiting
EGFR using AG1478 may provide a benefit to the host as it
promotesNTHi clearance.However, itshouldbe noted that uncon-
trolled TLR2 expression may also result in unwanted excess inflam-
matory responses in vivo. In the present study, we found that EGFR
mediates NTHi-induced NF-kB activation and subsequent inflam-
matory responses because inhibition of EGFR via AG1478 inhibits
NTHi-induced NF-kB activation and subsequent inflammatory
responses. This finding is of particular interest because EGFR
inhibition using AG1478 will promote the host’s ability to detect
invading pathogens by enhancing TLR2 expression; on the other
hand, AG1478 will also inhibit NF-kB activation and subsequent
inflammatory responses and thus prevent the uncontrolled
inflammatory response that is caused by enhanced expression of
TLR2. Taking advantage of clinically available EGFR inhibitors
such as gefitinib, lapantinib, erlotinib, cetuximab and panitumu-
mab, findings from this study may not only unveil novel
mechanisms underlying the regulation of inflammation, but may
Figure 8. Schematic representation of EGFR-mediated inflam-
mation. As indicated, EGFR is activated by NTHi, and mediates NTHi-
induced NF-kB activation and inflammation via MKK3/6-p38 and PI3K/
Akt signaling pathways.
EGFR Regulates Inflammation
PLoS ONE | www.plosone.org8November 2011 | Volume 6 | Issue 11 | e28216
also facilitate translational research toward developing novel Download full-text
therapeutic strategies for the treatment of respiratory and other
Conceived and designed the experiments: XX J-DL. Performed the
experiments: XX RRS CAF JP JYL JHL HX. Analyzed the data: XX
RRS CAF JP JYL JHL ZKP SBM J-DL. Wrote the paper: XX J-DL.
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