Induction of TLR-2 and TLR-5 Expression by Helicobacter pylori Switches cagPAI-Dependent Signalling Leading to the Secretion of IL-8 and TNF-α
Backert S (2011) Induction of TLR-2 and TLR-5 Expression by Helicobacter pylori Switches cagPAI-
Dependent Signalling Leading to the Secretion of IL-8 and TNF-a. PLoS ONE 6(5): e19614. doi:10.1371/journal.pone.0019614
Induction of TLR-2 and TLR-5 Expression by Helicobacter pylori Switches cag PAI-Dependent Signalling Leading to the Secretion of IL-8 and TNF-a
Suneesh Kumar Pachathundikandi 0
Sabine Brandt 0
Joseph Madassery 0
Steffen Backert 0
Yoshio Yamaoka, Veterans Affairs Medical Center (111D), United States of America
0 1 Institute for Medical Microbiology, Otto von Guericke University , Magdeburg, Germany , 2 Department of Biotechnology, University of Calicut, Calicut University (PO) , Kerala , India
Helicobacter pylori is the causative agent for developing gastritis, gastric ulcer, and even gastric cancer. Virulent strains carry the cag pathogenicity island (cagPAI) encoding a type-IV secretion system (T4SS) for injecting the CagA protein. However, mechanisms of sensing this pathogen through Toll-like receptors (TLRs) and downstream signalling pathways in the development of different pathologies are widely unclear. Here, we explored the involvement of TLR-2 and TLR-5 in THP-1 cells and HEK293 cell lines (stably transfected with TLR-2 or TLR-5) during infection with wild-type H. pylori and isogenic cagPAI mutants. H. pylori triggered enhanced TLR-2 and TLR-5 expression in THP-1, HEK293-TLR2 and HEK293-TLR5 cells, but not in the HEK293 control. In addition, IL-8 and TNF-a cytokine secretion in THP-1 cells was induced in a cagPAI-dependent manner. Furthermore, we show that HEK293 cells are not competent for the uptake of T4SS-delivered CagA, and are therefore ideally suited for studying TLR signalling in the absence of T4SS functions. HEK293 control cells, which do not induce TLR-2 and TLR-5 expression during infection, only secreted cytokines in small amounts, in agreement with T4SS functions being absent. In contrast, HEK293-TLR2 and HEK293-TLR5 cells were highly competent for inducing the secretion of IL-8 and TNF-a cytokines in a cagPAI-independent manner, suggesting that the expression of TLR-2 or TLR-5 has profoundly changed the capability to trigger pro-inflammatory signalling upon infection. Using phospho-specific antibodies and luciferase reporter assays, we further demonstrate that H. pylori induces IRAK-1 and IkB phosphorylation in a TLR-dependent manner, and this was required for activation of transcription factor NF-kB. Finally, NF-kB activation in HEK293-TLR2 and HEK293-TLR5 cells was confirmed by expressing p65-GFP which was translocated from the cytoplasm into the nucleus. These data indicate that H. pylori-induced expression of TLR-2 and TLR-5 can qualitatively shift cagPAI-dependent to cagPAI-independent pro-inflammatory signalling pathways with possible impact on the outcome of H. pylori-associated diseases.
Funding: The work of SKP was supported by a DAAD Fellowship. The work of SBA is supported through grants from the Deutsche Forschungsgemeinschaft DFG
grant (Ba1671/8-1), from the National Institute of Diabetes and Digestive and Kidney Diseases (R56DK064371) and from University College Dublin (R11408). 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.
The innate immune system is evolutionarily conserved in
higher eukaryotes and is the first line of defence for protecting
hosts from invading microbial pathogens . Toll-like
receptors (TLRs) are surface-exposed pattern recognition
receptors which can recognize molecular structures on pathogenic
microbes associated molecular patterns (PAMPs). Bacterial
molecules like lipopolysaccharides (LPS), lipoprotein,
lipotheichoic acid, peptidoglycan, lipoarabinomannan (LAM), flagellin
and CpG containing DNA are well-known examples of PAMPs
. TLRs recognize these compounds in the extracellular space
and subsequently transduce signals through downstream effectors
to mount innate immune responses against infections and pave
way for successful adaptive immunity [3,5]. Currently, eleven
members of the TLR family have been identified in mammals.
TLRs are type I integral membrane glycoproteins and on the
basis of cytoplasmic homologous regions, they are included in the
interleukin-1 receptor superfamily . Two additional families of
sensing receptors have also been discovered. Sensing of
microorganisms intracellularly can be achieved by nucleotide
oligomerization domain (NOD)-like receptors (NLRs) and
Retinoic acid inducible gene-1 (RIG-1)-like receptors (RLRs).
These two families comprise the intracellular sensors, of which
NLRs recognize primarily molecules of bacterial origin while
RLRs are involved in antiviral responses [7,8]. Individual TLRs
interact with different combinations of adapter proteins and
activate various transcription factors such as nuclear factor
(NF)kB, activator protein-1 (AP-1) and interferon regulatory factors
(IRF), driving a specific immune response . TLRs trigger
intracellular signalling pathways that result in the induction of
inflammatory cytokines, type-I interferon (IFN) and chemokines.
Microbial pattern recognition by TLRs in dendritic cells
upregulate the expression of co-stimulatory molecules, which is
essential for the initiation of adaptive immune responses in the
host, thus linking innate and adaptive immunity [2,10].
TLR receptor-ligand binding activates the interaction with
MyD-88 (myeloid differentiation primary response protein-88)
through the TIR domain in its cytoplasmic tail which in turn
recruits IRAK-4 (Interleukin-1 receptor associated kinase-4) and
thereby induces the association of another kinase member,
IRAK1 . IRAK-1 is phosphorylated at threonine residue 209
(T209), which results in a conformational change of the kinase
domain and subsequent phosphorylation at threonine residue 387
(T-387) and other residues in the activation loop. This results in
autophosphorylation and full enzymatic activity of IRAK-1.
IRAK-1 hyperphosphorylation triggers its dissociation from
MyD-88 without affecting the association with TRAF-6 .
Subsequently, phosphorylated IRAK-1 and TRAF-6 dissociate
from the complex and bind to cell membrane protein TAB-1
(TAK-1 binding protein-1) followed by binding of TAK-1
(transforming growth factor-b-activated kinase) and TAB-2.
Finally, IRAK-1 ubiquitinylation and degradation are rapidly
induced and the remaining complex translocates into the
cytoplasm. The latter complex associates with ubiquitin ligases
such as UBC-13 (ubiquitin conjugating enzyme-13) and UEV-1a
(ubiquitin conjugating enzyme E2 variant-1), leading to
ubiquitinylation and degradation of TRAF-6 . This activates TAK-1
and phosphorylation of the IKK (inhibitor of kB kinase) complex
(IKKa, IKKb and IKKc) as well as MAP (mitogen activated
protein) kinases. The IKK complex then phosphorylates IkB
(inhibitor of kB) which leads to its ubiquitinylation and
degradation. This ultimately releases NF-kB, which enables to
translocate to the nucleus for transcription of pro-inflammatory
Helicobacter pylori is a major human pathogen colonizing the
stomach of about half the world population and is the causative
agent of chronic gastritis, gastric ulcer disease, and even gastric
cancer . Thus, the World Health Organization (WHO)
classified H. pylori as a type-1 carcinogen. Colonization of the
gastric epithelium by this bacterium and subsequent host/
pathogen interactions lead to strong inflammatory changes in
many cases and that paves way for the development of gastritis,
ulceration and metaplasia . Although the gastric
epithelial cells represent a barrier against natural infections,
immune cells are the real mediators of inflammation to ward off
invading pathogens . Infection of epithelial cells with H.
pylori activates different molecular signalling mechanism
including NF-kB, AP-1 and MAP kinases, leading to the expression
and secretion of pro-inflammatory cytokines . These
mediators of inflammation include interleukins such as IL-1,
IL-6, IL-8, IL-18 as well as tumour necrosis factor alpha (TNF-a)
and others which change the microenvironment and even
regulate deleteriously host cellular mechanisms at the site of
infection . The cytotoxin-associated genes pathogenicity
island (cagPAI), a 40 kb stretch of DNA, encodes orthologs of
components of a type IV secretion system (T4SS), and
T4SSpositive H. pylori strains are associated with more severe disease
[22,23]. This T4SS forms a pilus, capable of injecting the CagA
protein, peptidoglycan, and possibly other factors into host cells
using integrin b1 as a receptor [24,25]. Once delivered, CagA
can be phosphorylated by tyrosine kinases Src and Abl and
interacts with a large number of cellular proteins to trigger
multiple effects on host signal transduction pathways to the
nucleus, cytoskeleton and cell junctions . Injected factors and
structural components of the T4SS have been shown to influence
membrane dynamics, actin-cytoskeletal rearrangements and the
disruption of cell-to-cell junctions as well as proliferative,
proinflammatory and anti-apoptotic nuclear responses in the host
The role of innate immune responses by sensing receptors in
response to H. pylori infection is not yet completely understood and
sometimes controverse in the literature. Numerous previous
studies have reported the involvement of some TLRs and NOD
proteins in the detection of H. pylori, and induction of
proinflammatory and other responses . For example, a
landmark paper has shown that peptidogycan can be injected by
the H. pylori T4SS to stimulate Nod1 and subsequently NF-kB
, while other factors such as injected CagA may also play a
role in stimulating NF-kB and IL-8 [30,31]. However, it was also
shown that H. pylori induces Nod1, which activates a RICKR
TRAF3RTBK1RIKKRIRF7 pathway leading to the synthesis
of type-I interferon, but not NF-kB . In addition, there are
numerous studies on the putative role of TLRs in H. pylori
infection. Gastric epithelial cells are reported to be not sufficient to
provide all the TLR molecules expressed on its surface for
detection of H. pylori. Smith and co-workers reported that gastric
epithelial cells recognize and respond to H. pylori infection at least
in part through TLR-2 and TLR-5 . TLR-4 mediated
recognition of LPS from many bacteria is a key activator of the
innate immune response in epithelial cells, while the purified form
of H. pylori LPS is a relatively weak inducer. However, H. pylori
LPS does activate NF-kB, but this was achieved via TLR-2 rather
than TLR-4 . In contrast to this, H. pylori LPS was
reported to activate NF-kB in association with the expression of
mitogen oxidase-1 (MOX-1), cyclooxygenase-II (COX II) and
TNF-a transcripts in gastric pit cells, which express more TLR-4
but no TLR-2 . Immunocytochemical studies using gastric
mucosal biopsies have revealed that TLR-5 and TLR-9 expression
on the gastric epithelium changed to an exclusive basolateral
localization without detectable expression at the apical pole in H.
pylori gastritis, however, TLR-4 expression was highly polarized in
an apical and a basolateral compartment identical to the
noninflamed mucosa . Interestingly, Mandell and co-workers
reported that H. pylori LPS induced cytokine production was
mediated through TLR-4, but the response to infecting bacteria
such as H. pylori, H. hepaticus or H. felis were mediated through
TLR-2 . H. pylori infection of HEK-293 cells stably transfected
with TLR-2 revealed many differently regulated genes as
compared to HEK293 control cells, and eight of them showed
changing expression patterns in infected epithelial cell lines .
Finally, it was also shown that chemically synthesized lipid-A
(mimicking the natural lipid-A portion of LPS from H. pylori) has a
low endotoxic potency and immunobiological activities, and is
recognized by TLR-4 . A study using recombinant FlaA
protein and DflaA mutants of H. pylori revealed the less potent
activity of TLR-5 mediated IL-8 secretion in epithelial cells . A
recent report attributed the FlaA evasion of TLR-5 is due to
amino acids 8996 of the N-terminal D1 domain and that may be
responsible for low TLR-5 mediated activity on IL-8 secretion
The above discussed studies indicate a highly complex scenario
of possible involvement of TLRs in H. pylori infection, but this is
still not fully understood. Our group is interested in the
characterisation of H. pylori signalling in innate immune responses.
Here we show that H. pylori infection of THP-1 monocytes induced
not only the secretion of pro-inflammatory cytokines such as IL-8
and TNF-a in a cagPAI-dependent manner, but also upregulation
of TLR-2 and TLR-5 expression. To investigate the role of TLR-2
and TLR-5 during infection in more detail and to clearly
distinguish this from signalling induced by the cagPAI-dependent
injection of peptidoglycan and CagA, we were screening for a cell
model system in which T4SS effectors cannot be injected. We
demonstrate that HEK293 is such useful system. Infected
HEK293 wild-type cells show no cagPAI-dependent upregulation
of cytokines and CagA cannot be injected and phosphorylated in
these cells at all. By contrast, stable expression of TLR-2 or TLR-5
in HEK293 cells dramatically changes the capability of H. pylori to
activate NF-kB, IL-8 and TNF-a as shown by luciferase reporter,
ELISA and immunofluorescence assays. We also demonstrate that
H. pylori infection of either TLR-expressing cell line induces the
phosphorylation of IRAK-1 and IkB followed by NF-kB
activation, which is in agreement with the hypothesis that
upregulation of both TLR-2 and TLR-5 by H. pylori can switch
cagPAI-dependent signalling to cagPAI-independent TLR
signalling, thus may changing the outcome of infections substantially.
Results and Discussion
H. pylori infection of THP-1 cells upregulates TLR-2 and
TLR-5 expression and cytokine secretion in a
TLR-2 and TLR-5 are two candidates of sensing receptors that
maybe involved in interactions of host cells with H. pylori .
To investigate if the expression of both receptors can be regulated
during infection, monocytic THP-1 cells were co-cultured with
wild-type H. pylori for 24 hours followed by preparation of mRNA.
The quantification of TLR-2 and TLR-5 mRNA expression was
performed using Taqman Real Time PCR. As shown in Figure 1,
H. pylori induced a more than 3- and 11-fold increase in TLR
expression as compared to expression of the house keeping gene
GAPDH, respectively. The protein expression of TLR-2 and
TLR-5 in THP-1 cells in Western blots were found to be similarly
up-regulated during infection with H. pylori (data not shown). We
have then analyzed the secretion of IL-8 and TNF-a from THP-1
cells during infection with wild-type H. pylori and an isogenic
DcagPAI deletion mutant by ELISA. The results indicate the
induction of IL-8 and TNF-a secretion from THP-1 cells during
infection with wild-type H. pylori (Figure 2). The induction of IL-8
and TNF-a, however, was not observed in infections with the
DcagPAI mutant (Figure 2), which is in agreement with earlier
studies indicating that T4SS-dependent activities trigger
proinflammatory signalling .
HEK293 cells are a useful system to study TLR-2 and
TLR5 signalling during H. pylori infection
To investigate the role of TLR-2 and TLR-5 in greater detail
and to exclude signalling induced by the cagPAI-dependent
injection of bacterial effectors, we next tested several cell lines
for their susceptibility for injection and phosphorylation of CagA.
Among the cell lines tested, CagA was strongly phosphorylated in
infected AGS, MKN-28, MKN-45, KATO-III, THP-1, J774.A or
HT-29 cell lines after 6 hours (Figure 3A, and data not shown),
but not in wild-type HEK293 cells, or HEK293 cells stably
transfected with expression constructs for human TLR-2 and
TLR-5, respectively (Figure 3BD). The experiments were
performed under identical conditions. We also confirmed by
phase microscopy the motility of each of the different bacterial
strains and that they can bind to the cells with high efficiency
(.80%), thus excluding the possibility that altered bacterial fitness
or reduced host cell binding accounts for the observed CagA
translocation defect. In addition, an initial centrifugation step (at
2,000 rpm for 10 minutes) of wild-type H. pylori onto HEK293
cells followed by 6 hours infection did not reveal any detectable
signal for phosphorylated CagA (data not shown). Thus, the
different HEK293 cell lines are not competent for the injection of
CagA, and are therefore ideally suited for investigation of TLR
signalling in the absence of T4SS functions.
Role of cagPAI status on induction of TLRs and
proinflammatory cytokines in HEK293-TLR2 and
HEK293TLR5 cells during H. pylori infection
To further confirm the usefulness of the HEK system, we have
infected HEK293 wild-type, HEK293-TLR2 and HEK293-TLR5
cells with H. pylori for 8 hours followed by protein preparation for
Western blotting or mRNA isolation for Taqman Real Time
PCR. We found significant up-regulation of TLR-2 in
HEK293TLR2 cells and TLR-5 in HEK293-TLR5 cells both at protein
level (Figure 4A) and mRNA level (Figure 4B), which was similar
Figure 3. CagA injection by H. pylori cannot be achieved in infected HEK293 cell lines but in AGS gastric epithelial cells. Western blot
analysis of (A) AGS, (B) HEK293-TLR2, (C) HEK293 and (D) HEK293-TLR5 cells infected with H. pylori wild-type strains P1, P12, P310 or 26695 for 6 hours.
Phosphorylation of injected CagA was monitored using phosphotyrosine a-PY-99 and a-CagA antibodies. Red arrows indicate the position of
phosphorylated CagA on the blot. Western blots for the house keeping gene GAPDH served as loading control.
to the elevated TLR expression pattern observed in infected
THP1 cells (Figure 1). In addition, we performed cytokine ELISAs of
cell supernatants for secreted IL-8 and TNF-a. We only found
very small amounts of induced IL-8 or TNF-a in supernatants of
infected HEK293 control cells, which is in agreement with the
observation that T4SS functions are absent (Figure 5A, B). In
contrast, infection of HEK293-TLR2 and HEK293-TLR5 cell
lines with wild-type H. pylori strongly induced the secretion of a few
thousand-fold IL-8 and several-fold TNF-a (Figure 5A, B).
Interestingly, a parallel infection experiment of HEK293-TLR2
and HEK293-TLR5 cell lines with the isogenic DcagPAI deletion
mutant also induced very high amounts of IL-8 and TNF-a similar
to wild-type bacteria (Figure 5A, B), suggesting that the expression
of either TLR-2 or TLR-5 has changed the capability of HEK293
cells to trigger H. pylori-induced pro-inflammatory signalling in a
H. pylori induces IRAK-1 and IkB phosphorylation during
infection with HEK293-TLR2 and HEK293-TLR5 cell lines
but not in HEK293 wild-type cells
To investigate the functional status of TLR-2 and TLR-5 in
HEK293 cells, we next analyzed the initiation of classical TLR
signalling through IRAK-1 phosphorylation. IRAK-1 is
commonly phosphorylated at several threonine and serine residues which
are involved in its activation as described above.
Activationspecific phospho antibodies are available for the phosphorylated
serine residue 376 (S-376) in IRAK-1. For this purpose, HEK293
wild-type, HEK293-TLR2 and HEK293-TLR5 cells were
infected with H. pylori for 2 hours followed by Western blotting
using the a-phospho S-376 IRAK-1 antibody. We found that H.
pylori induced IRAK-1 phosphorylation at S-376 in
HEK293TLR2 and HEK293-TLR5 cell lines but not in the wild-type
HEK293 control cells (Figure 6A, B). Furthermore, the same cell
lysates as prepared above were probed for IkB phosphorylation as
also indicative for the activated NF-kB pathway. The results show
that H. pylori induces the phosphorylation of IkB at serine residue
32 (S-32) in HEK293-TLR2 and HEK293-TLR5 cell lines but not
in the wild-type HEK293 control cells (Figure 7A, B). These
findings suggest that H. pylori are able to induce both activation of
IRAK-1 and IkB in a TLR-dependent manner as required for the
onset of transcription factor NF-kB.
NF-kB and AP-1 activation in HEK293-TLR2, HEK293-TLR5
and HEK293 wild-type cells during infection with H. pylori
As next, NF-kB activity was studied using a luciferase reporter
system with the respective AP-1 construct as control. To this end,
the different HEK293 cell lines were transiently transfected with
NF-kB luciferase and AP-1 luciferase gene constructs for 48 hours,
followed by infection with H. pylori for 5 hours. Cell lysates were
then prepared and the activation of both transcription factors was
quantified by luciferase activity counting. NF-kB or AP-1
luciferase activities were very small both in non-infected and
infected HEK293 wild-type control cells (Figure 8A, B). In
contrast, NF-kB luciferase activity was strongly elevated in
HEK293-TLR2 and HEK293-TLR5 cells infected with H. pylori
(Figure 8A). Interestingly, AP-1 luciferase activity was only
moderately induced in infected HEK293-TLR2 and
HEK293TLR5 cells, and was much lower as compared to NF-kB activity
(Figure 8A, B), Finally, we confirmed NF-kB activation in
HEK293-TLR2 and HEK293-TLR5 cells using a transfected
NF-kB p65-GFP construct. As shown in Figure 9, H. pylori
infection of the HEK293-TLR2 and HEK293-TLR5 cells showed
Mounting evidence supports the view that innate immune
responses to microbes participate in the development of severe
gastrointestinal disorders. With the characterization of the innate
immune system, we have begun to understand the adaptations that
intestine has created to the colonising microbiota. The interaction
between the microbiota and the intestinal mucosa through TLRs
is required to maintain intestinal homeostasis. In particular,
intestinal epithelial cells and lamina propria immune cells must
respond to breaches in the mucosal barrier by activating
TLRdependent signalling pathways that trigger increased epithelial
proliferation, wound healing and recruitment of acute
inflammatory cells. In the setting of chronic inflammation such as H. pylori
infection in the stomach, the process of TLR interaction and
activation is relatively little understood. However, recent reports
showed that TLR-2, TLR-9 (recognising H. pylori DNA) and
RIGI (recognising H. pylori RNA) through the interaction with
respective ligands and downstream signalling were able to activate
dendritic cells leading to adaptive immune responses against H.
pylori infection . Highly purified H. pylori LPS preparations
significantly induced pro-inflammatory reactions via the receptor
complex containing TLR-2/TLR-1 or TLR-2/TLR-6 but not
TLR-4 . In contrast, a study published very recently showed
that H. pylori LPS markedly enhanced IL-8 production induced by
E. coli LPS through upregulating TLR-4 via TLR-2 and the
MEK1/2-ERK1/2 MAP kinase pathway and augmented the
proliferation rate of gastric epithelial cells. This activation was
mediated through LPS carrying a weakly antigenic epitope, which
is frequently found in gastric cancers, than by LPS carrying a
highly antigenic epitope, which is associated with chronic gastritis
. On the other hand, the H. pylori factor activating TLR-5 is
not flagellin and therefore still unknown [41,42].
In the present report we show that H. pylori infection of THP-1
monocytes induced the secretion of IL-8 and TNF-a in a
cagPAIdependent manner, which was associated with enhanced TLR-2
and TLR-5 expression. We investigated the function of TLR-2
and TLR-5 during infection in more detail. We demonstrate that
HEK293 is a very useful cell system in which T4SS effectors
cannot be injected and used it to investigate H. pylori-induced TLR
signalling in the absence of cagPAI-dependent signalling effects.
We could show that expression of TLR-2 or TLR-5 in HEK293
cells dramatically changes the capability of H. pylori to activate
NFkB, IL-8 and TNF-a as demonstrated in different molecular
assays. The H. pylori-induced signalling downstream of TLR-2 and
TLR-5 involves the activation of IRAK-1 and IkB
phosphorylation followed by NF-kB stimulation and cytokine release. Thus,
upregulation of both TLR-2 and TLR-5 during the course of H.
pylori infection can switch cagPAI-dependent signalling to
cagPAIindependent TLR signalling, which may impact disease
development. Our findings are in part supported by an earlier study which
showed that IL-8 secretion induced by H. pylori from HEK293
cells has been augmented by the expression of TLR-2 or TLR-5
and that coincided with increased p38 activation and
phosphorylation of the transcription factor ATF2 . In addition, we have
been able to show that H. pylori induced NF-kB stimulation and
release of IL-8 and TNF-a in a cagPAI-dependent way in THP-1
monocytes and the expression of TLR-2 and TLR-5, as studied in
HEK293 cells, support a model for a switch to
cagPAIindependent signalling at late time points of infection.
The switching of cagPAI-dependent to cagPAI-independent
signalling by induction of TLR-2 and TLR-5 in vitro may have
important implications on H. pylori-associated pathologies in vivo.
For example, in mixed infections of cagPAI-negative and
cagPAIpositive strains the signalling can be severely influenced and
augmented in a mutually supportive way and turn the status to
pro-inflammatory. Interestingly, in biopsies from H. pylori-positive
gastritis patients, expression of TLR-5 and TLR-9 in the gastric
epithelium changed to an exclusive basolateral localization
without detectable expression at the apical pole . This is an
interesting observation because the receptor for the H. pylori
T4SS is integrin b1 and also localised to the basolateral side of
the epithelium . This suggests that several H. pylori receptors
are found both on the apical and basolateral sides of gastric
epithelial cells and there maybe even crosstalk between some of
them . However, the increased production of TNF-a through
TLR-2 and TLR-5 signalling might be a reason for inducing
apoptosis of gastric cells at the colonized area of the stomach.
The apoptotic bodies can also be a source of endogenous ligands
for TLR molecules, which (like TLR-7 and TLR-9) have been
reported for the high proportion of auto-antibodies binding
DNA, RNA or macromolecular complexes that contain DNA or
RNA, and that are commonly associated with systemic
autoimmune diseases such as systemic lupus erythematosus
(SLE), scleroderma and Sjo grens syndrome. H. pylori has been
found to be associated with a number of autoimmune disorders,
such as rheumatoid arthritis, autoimmune thyroiditis, Sjo grens
syndrome, Schonlein-Henoch purpura, and autoimmune
thromFigure 5. IL-8 and TNF-a secretion in HEK293 cell lines during H. pylori infection is mediated in a cagPAI-independent fashion.
Concentrations of IL-8 (A) and TNF-a (B) secreted from HEK293, HEK293-TLR2 and HEK293-TLR5 cell lines after 24 hours of infection with wild-type H.
pylori and an isogenic DcagPAI mutant. TNF-a and IL-8 concentrations in the culture supernatants were analyzed by ELISA.
bocytic purpura . The ability of microbial TLR ligands to
trigger disease onset in experimental models of arthritis, multiple
sclerosis, experimental allergic encephalomyelitis, myocarditis,
diabetes and atherosclerosis have also been reported. Endogenous
ligands for TLR-2 and TLR-4 such as hyaluronate, heparan
sulphate, fibronectin and heat shock proteins have also been
implicated in the pathogenesis of autoimmune diseases such as
rheumatoid arthritis . H. pylori is also reported to provide
epitopes cross-reactive to H+, K+-ATPase and this may lead to
the expansion of cross-reactive and auto-reactive T cells and T
cell-dependent B cell activation, which can be attributed to the
parietal cell loss due to autoimmune gastritis. Autoreactive T cells
produce high concentration of TNF-a and IFN-c, which can in
turn increase the MHC class II and co-stimulatory molecule
expression in gastric epithelial cells and favouring the
presentation of peptides by such non-professional antigen-presenting cells
. A very recent study using mouse models of infection with
Helicobacter felis (a close relative of H. pylori) showed that B cells
activated by Helicobacter TLR-2 ligands induce IL-10-producing
CD4(+)CD25(+) T regulatory-1 (Tr-1)-like cells and these B
cellinduced Tr-1 cells acquire suppressive activities in vitro and
suppress excessive gastric Helicobacter-associated
immunopathology in vivo . These facts along with the ability of H. pylori to
switch signals into a TLR-dependent pathway is showing that it
may influence under certain circumstances not only gastric
pathologies but also a range of other complex responses.
Materials and Methods
Eukaryotic cell culture
The monocytic leukaemia cell line THP-1 (ATCC TIB 202)
was cultured in RPMI-1640 medium supplemented with 10%
(vol/vol) heat-inactivated fetal bovine serum (FBS) (Invitrogen,
Germany) 1% antibiotic and antimycotic solution
(SigmaAldrich, Germany). The transformed human embryonic
kidney cells (HEK293 wild-type, ATCC CRL-1573) were
cultured in Dulbeccos Modified Eagle Medium (DMEM)
containing 4.5 g/L D-glucose, 4 mM L-glutamine, 110 mg/L
sodium pyruvate,10% FBS (Invitrogen, Germany) and was
supplemented with 1% antibiotic and antimycotic solution
(Sigma-Aldrich, Germany). HEK293 cells stably transfected
with pUNOhTLR2 and pUNOhTLR5 constructs (Invivogen,
France), respectively, were cultured in DMEM containing
4.5 g/L D-glucose, 4 mM L-glutamine, 110 mg/L sodium
pyruvate, 10% FBS (Invitrogen, Germany) and was
supplemented with 1% antibiotic and antimycotic solution (Sigma,
Germany) and 10 mg/ml blasticidin (Invivogen, France). Cells
were maintained in 75-cm2 tissue culture flasks
(Greiner-BioOne, Germany) at 37uC in incubators with 5% CO2. All other
cell lines (AGS, MKN-28, MKN-45, KATO-III, J774.A or
HT-29) were cultured as described . Prior to bacterial
infection, all cells were incubated in antibiotic-free medium
Figure 6. H. pylori infection of different HEK293 cell lines induces IRAK-1 phosphorylation at Ser-376 in a TLR-2- or
TLR-5dependent fashion. (A) Western blot analysis of IRAK1 phosphorylation in HEK293, HEK293-TLR2, and HEK293-TLR5 after 2 hours of infection. Blots
for house keeping gene GAPDH were used as loading control. (B) Densitometric measurement of band intensities revealed the percentage of IRAK-1
phosphorylation per sample. The strongest band was set 100% as indicated.
Figure 7. H. pylori infection of different HEK293 cell lines induces IkB phosphorylation at Ser-32 in a TLR2- or TLR-5-dependent
fashion. (A) Western blot analysis of IkB phosphorylation in HEK293, HEK293-TLR2, and HEK293-TLR5 after 2 hours of infection. Blots for house
keeping gene GAPDH were used as loading control. (B) Densitometric measurement of band intensities revealed the percentage of IkB
phosphorylation per sample. The strongest band was set 100% as indicated.
Figure 8. H. pylori infection of different HEK293 cell lines induces NF-kB and AP-1 activation in a TLR2- or TLR-5-dependent fashion.
NF-kB and AP-1 luciferase reporter constructs were transfected into HEK293, HEK293-TLR2, and HEK293-TLR5 cells for 48 hours and followed by
infection with H. pylori for 5 hours. The NF-kB and AP-1 luciferase reporter expression was analyzed as function of activation.
Figure 9. Activation of NF-kB-GFP in HEK293 cell lines is dependent on TLR-2 and TLR-5. Immunofluorescence of HEK293 cell lines
transfected with NF-kB-p65 subunit (p65-GFP, green) for 48 hours followed by infection with H. pylori for 3 hours. Infection of HEK293-TLR2 and
HEK293-TLR5 cells with H. pylori showed a translocation of p65-GFP into the nucleus (arrows), whereas infection of HEK293 control cells did not
induce a nuclear translocation of p65-GFP. Rhodamine-phalloidine (RPH, red) was used to visualize filamentous actin in the cells and DAPI (blue) to
visualize the nucleus and bacteria.
H. pylori growth and conditions for infection
The H. pylori strains employed in this study include the wild-type
strains. P1, P310, 26695, P12 and the isogenic mutant of
P12DcagPAI in which the entire cagPAI was deleted . All
H. pylori strains were grown in thin layers on horse serum agar
plates supplemented with vancomycin (10 mg ml21), nystatin
(1 mg ml21) and trimethoprim (5 mg ml21), and in case of the
mutants with kanamycin (8 mg ml21) or chloramphenicol
(4 mg ml21), respectively. All antibiotics were obtained from
Sigma. Incubation of the bacteria was performed at 37uC for 2
days in an anaerobic jar containing a campygen gas mix of 5% O2,
10% CO2 and 85% N2 (Oxoid, Germany). H. pylori grown on agar
plates was harvested and resuspended in Phosphate Buffered
Saline (PBS, pH 7.4) using a sterile cotton swab (Raucotupf,
Lohmann & Rascher, Germany). The bacterial concentration was
measured as optical density (OD) at 550 nm using an Eppendorf
spectrophotometer. This was also cross checked with
colonyforming units (CFU) grown on horse serum agar plates after serial
dilution of the bacterial suspension. The eukaryotic cells grown in
medium without antibiotics and antimycotics were infected with
H. pylori at a multiplicity of infection (MOI) of 50. The uninfected
cells were incubated with equal amount of PBS as control.
RNA extraction, cDNA preparation and Taqman Real
THP-1 cells and HEK293 cell lines infected with H. pylori were
used for the isolation of mRNA. The THP-1 cells infected with H.
pylori for the required time periods were separated from bacteria
by centrifugation at 1506g for 5 minutes at 4uC. The supernatant
containing the majority of bacteria were removed carefully and the
cell pellet was washed with PBS before adding lysis buffer of the
RNeasy Mini Kit (Qiagen, Germany). In case of HEK293 cell
lines infected with H. pylori, the supernatant was carefully removed
and the monolayer was washed with PBS before adding 300 ml of
lysis buffer directly to the cells. Then, mRNA has been extracted
by the protocol provided by the manufacturer (Qiagen). The
purified mRNAs from H. pylori infected cell lines were used to
prepare cDNA by RT-PCR. The RT-PCR in principle
synthesizing the first strand complimentary DNA from mRNA with the
help of Oligo dT primers, which bind to Poly A tail of all
eukaryotic mRNAs, and Moloney Murine Leukaemia Virus
(MMLV) Reverse Transcriptase (Invitrogen, Germany). The
Taqman Real Time PCR uses the fluorescent oligonucleotide probes
labeled with one reporter dye at 59 end of the probe and quencher
dye at the 39 end along with the usual sense and anti-sense primers
used in PCR . These probes are designed to hybridize to an
internal region of a PCR product. The probe when attached to the
template or unattached, the quencher dye reduces the fluorescence
from reporter dye by the mechanism of Fluorescence Resonance
Energy Transfer (FRET), which is the inhibition of one dye caused
by another without emission of a proton. When the Taqman PCR
proceeds, the probe annealed to the template will be removed by
the exonuclease activity of Taq polymerase and this separates the
reporter dye from the quencher and that increases the emission of
fluorescence. DNase treated mRNA samples isolated from H.
pylori-infected cells were used for first strand cDNA preparation
using reverse transcriptase. Taqman probes and primers prepared
using Primer express software and Taqman gene expression assay
kits (Applied Biosystems, USA) were used for the analysis.
control. The culture supernatants were collected and stored at
280uC until assayed. IL-8 and TNF-a concentration in the
supernatant were determined by standard ELISA with
commercially available assay kits according to manufacturers procedures
(Becton Dickinson, Germany).
SDS-PAGE and immunoblot analysis
Infected and control cells were harvested and mixed with
equal amounts of 26SDS-PAGE buffer and boiled for
5 minutes. Proteins were separated by SDS-PAGE on 612%
polyacrylamide gels and blotted onto PVDF membranes
(Immobilon-P, Millipore, USA) as described [56,57]. Before
addition of the antibodies, membranes were blocked in TBS-T
(140 mM NaCl, 25 mM Tris-HCl pH 7.4, 0.1% Tween-20)
with 3% BSA or 5% skim milk for 1 hour at room temperature.
Phosphorylated and non-phosphorylated CagA proteins were
detected by incubation of the membranes with a mouse
monoclonal a-phosphotyrosine antibody PY99 (Santa Cruz,
USA) and a rabbit polyclonal a-CagA antibody (Austral
Biologicals, USA). Monoclonal antibody recognizing
phosphorylated IkB at S-32 and rabbit polyclonal antibody recognizing
phoshorylated IRAK-1 at S-376 were purchased from NEB cell
signalling (USA). TLR-2, TLR-5 and GAPDH expression was
monitored using antibodies from Santa Cruz (USA). As
secondary antibodies, horseradish peroxidase-conjugated
antimouse or a-rabbit polyvalent rabbit and pig immunoglobulin,
respectively, were used (Dako, Germany). Antibody detection
was performed with the ECL Plus chemoluminescence Western
Blot system for immunostaining (Amersham Pharmacia Biotech,
Transient transfection of HEK293 cells
HEK-cells were transfected with 4 mg of the NF-kB p65-GFP
 or NF-kB luciferase or AP-1 luciferase constructs  for
48 hours with TurboFect reagent according to the manufacturers
instructions (Fermentas, Germany).
Transfected cells were infected with H. pylori for 3 hours. Cells
were fixed with 3.8% PFA and stained with
rhodaminephalloidine to visualize the actin cytoskeleton of the cells and
DAPI to visualize the nucleus. Samples were analyzed using the
fluorescence microscope (Leica DMRE7, Leica, Germany)
equipped with a CCD camera (Spot RT, Diagnostic Instruments,
Burroughs, MI, USA) and a 63/1.4 objective. Separate images
were taken in the corresponding channels, and later merged using
ImageJTM software (NIH, USA).
Quantification of NF-kB or AP-1 activity by luciferase
Transfected cells were infected with H. pylori for 5 hours and
analyzed by luciferase assay using the Dual-Luciferase Reporter
Assay System according the manufactures instruction
(Promega, USA). Briefly, cells were harvested by passive lysis, the
protein concentration was measured with Precision Red
(Cytoskeleton, USA) and the lysates were equalized by adding
passive lysis buffer. The luciferase activity was measured by
using a Plate Luminometer (MITHRAS LB940 from Berthold,
Quantification of cytokines by immunoassay
THP-1 and HEK293 cell lines were incubated for 24 hours
with H. pylori, and PBS-incubated control cells served as a negative
All experiments were done at least three times with similar
results. The data were evaluated using Student t-test with
SigmaStat statistical software (version 2.0). P values = p,0.05 (*)
and p,0.005 (**) were considered as statistically significant.
We are grateful for the technical support of this project by Dr. Roland
Hartig and Corinna Gagell (University of Magdeburg, Germany), and
thank Dr. Richard Peek (Vanderbilt University, USA) and Dr. Anne
Conceived and designed the experiments: SKP S. Brandt S. Backert.
Performed the experiments: SKP S. Brandt. Analyzed the data: SKP
S. Brandt S. Backert JM. Contributed reagents/materials/analysis tools:
JM. Wrote the paper: SKP S. Backert.
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