Tài liệu Báo cáo khoa học: Transcriptional upregulation of inflammatory cytokines in human intestinal epithelial cells following Vibrio cholerae infection pptx

Transcriptional upregulation of inflammatory cytokines in
human intestinal epithelial cells following Vibrio cholerae
infection
Arunava Bandyopadhaya*, Madhubanti Sarkar* and Keya Chaudhuri
Molecular & Human Genetics Division, Indian Institute of Chemical Biology, Kolkata, India
The acute diarrheal disease cholera remains a signifi-
cant public health problem, due to its ability to spread
rapidly and kill a high proportion of those affected.
The etiologic agent of the disease is a highly motile
noninvasive Gram-negative organism Vibrio cholerae,
which colonizes the small intestine and produces a
potent enterotoxin called cholera toxin (CT) ) a major
virulence determinant that is primarily responsible for
the diarrheal syndrome [1]. Although much work has
been done on V. cholerae, very little is known about
the bacterium–host interactions. Epithelial cells are the
first site of entry for intestinal pathogens, and provide
early signals for the acute mucosal inflammatory
response via release of proinflammatory cytokines and
Keywords
cholera toxin; cytokines; intestinal epithelial
cells; nuclear factor-jB; Vibrio cholerae
Correspondence
K. Chaudhuri, Molecular & Human Genetics
Division, Indian Institute of Chemical
Biology, Kolkata-700 032, India
Fax: +91 33 2473 5197
Tel: +91 33 2473 3491
E-mail: or

*These authors contributed equally to this

work
(Received 21 February 2007, revised
31 May 2007, accepted 13 July 2007)
doi:10.1111/j.1742-4658.2007.05991.x
Coordinated expression and upregulation of interleukin-1a, interleukin-1b,
tumor necrosis factor-a, interleukin-6, granulocyte–macrophage colony-
stimulating factor, interleukin-8, monocyte chemotactic protein-1 (MCP-1)
and epithelial cell derived neutrophil activator-78, with chemoattractant
and proinflammatory properties of various cytokine families, were obtained
in the intestinal epithelial cell line Int407 upon Vibrio cholerae infection.
These proinflammatory cytokines also showed increased expression in T84
cells, except for interleukin-6, whereas a striking dissimilarity in cytokine
expression was observed in Caco-2 cells. Gene expression studies of MCP-
1, granulocyte–macrophage colony-stimulating factor, interleukin-1a, inter-
leukin-6 and the anti-inflammatory cytokine transforming growth factor-b
in Int407 cells with V. cholerae culture supernatant, cholera toxin, lipopoly-
saccharide and ctxA mutant demonstrated that, apart from cholera toxin
and lipopolysaccharide, V. cholerae culture supernatant harbors strong
inducer(s) of interleukin-6 and MCP-1 and moderate inducer(s) of inter-
leukin-1a and granulocyte–macrophage colony-stimulating factor. Cholera
toxin- or lipopolysaccharide-induced cytokine expression is facilitated by
activation of nuclear factor-jB (p65 and p50) and cAMP response element-
binding protein in Int407 cells. Studies with ctxA mutants of V. cholerae
revealed that the mutant activates the p65 subunit of nuclear factor-jB and
cAMP response element-binding protein, and as such the activation is med-
iated by cholera toxin-independent factors as well. We conclude that
V. cholerae elicits a proinflammatory response in Int407 cells that is medi-
ated by activation of nuclear factor-jB and cAMP response element-bind-
ing protein by cholera toxin, lipopolysaccharide and ⁄ or other secreted
products of V. cholerae.

Abbreviations
CREB, cAMP response element-binding protein; CT, cholera toxin; ENA-78, epithelial cell derived neutrophil activator-78; GM-CSF,
granulocyte–macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; LPS, lipopolysacccharide; MCP-1, monocyte chemotactic
protein-1; MOI, multiplicity of infection; NF-jB, nuclear factor-jB; PMN, polymorphonuclear neutrophil; TGF-b, transforming growth factor-b;
TNF-a, tumor necrosis factor-a.
FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4631
inflammatory mediators. The response of the intestine
to infection by pathogens represents a complex inter-
action between nonspecific inflammatory mechanisms
and immunologically specific adaptive events. Although
cholera has been traditionally classified as a noninflam-
matory diarrheal disease [2], various reports point
towards an inflammatory component in the pathogene-
sis of the disease [3,4]. Lymphocytes and mononuclear
cells have been observed in the intestinal lamina pro-
pria in biopsy specimens from cholera patients [5,6],
and increased levels of lactoferrin, myeloperoxidase
and prostaglandins have been observed in stool samples
from infected humans [4,7]. The major enterotoxin CT
has been demonstrated to strongly promote the produc-
tion of interleukin (IL)-6 by rat IEC-6 epithelial cells
[8], and CT treatment of rat IEC-17 cells stimulated
both IL-1 and IL-6 secretion [9]. V. cholerae vaccine
strains caused symptoms consistent with inflammation
in human volunteers [10]. Reports suggest that certain
V. cholerae strains, as well as CT, may stimulate a
modest intestinal inflammatory response [3,7].
A few recent reports have also documented the
release of cytokines upon V. cholerae infection in intes-
tinal epithelial cells. Reports have shown the induction

of IL-8 in intestinal epithelial cells upon V. cholerae
infection [11–13]. The induction of IL-8 by reactogenic
V. cholerae strains in the undifferentiated HT29-18N2
cell line model and IL-8 induction by V. cholerae vac-
cine strains in T84 cells have been shown [11,13]. More-
over, our previous reports have shown the association
of adherence and motility with IL-8 induction in Int407
cells [12]. Recent host transcriptional profiling upon
V. cholerae infection in the T84 cell line by microarray
analysis has shown the upregulation of many pro-
inflammatory mediators, such as cytokines [14]. How-
ever, little is known about the role of V. cholerae in
initiating and sustaining the innate inflammatory
response in intestinal epithelial cells, and the potential
contribution of individual V. cholerae components to
cytokine induction. The specific components include
lipopolysacccharide (LPS), any secreted protein of
V. cholerae, including CT, and the major surface pro-
teins of V. cholerae. Moreover, the signaling cascades
involved in the induction and regulation of mucosal
inflammatory responses to infection by V. cholerae are
still largely unknown. Hence, further studies are needed
to elucidate the mechanisms of the V. cholerae-induced
proinflammatory response, as well as to determine the
V. cholerae factors causing inflammation, for the devel-
opment of safe, live attenuated vaccines.
The expression of many cytokine genes is regulated
at both the transcriptional and post-transcriptional
levels. The former is mediated primarily by nuclear
factor-jB (NF-jB), which is an integral part of the

signaling mechanism and is required for maximal
transcription of many proinflammatory cytokines, cell
surface receptors and adhesion molecules, and there-
fore thought to be important in the generation of acute
inflammatory responses [15]. The activities of many
inducible transcription factors, including NF-jB, are
regulated through their association with cellular coacti-
vators. Interaction with coactivators such as cAMP
response element-binding protein (CREB) appears to
be necessary to optimize the transcriptional activity of
NF-jB [15].
This study reports for the first time the coordinated
transcription of a number of cytokines belonging to dif-
ferent functional groups in three different intestinal
epithelial cell lines upon V. cholerae infection. These
cytokines are not induced upon incubation with non-
pathogenic Escherichia coli DH5a. The study further
examines the role of V. cholerae culture supernatant and
major components such as CT and LPS in cytokine
induction, and the results demonstrate the involvement
of CT-dependent and CT-independent factors in cyto-
kine mRNA induction mediated by transcription factor
(NF-jB p50 and p65 subunits, CREB) activation.
Results and Discussion
Identification of differentially expressed cytokines
in intestinal epithelial cells following V. cholerae
infection
Epithelial cells are considered to represent an integral
component of the mucosal immune system, as they pro-
vide the underlying mucosa with the first signals of an

infection [16]. As the intestinal mucosal epithelial sur-
face forms the first barrier encountered by the enteric
pathogen V. cholerae, the intestinal epithelial cell line
Int407 and human colonic epithelial cell lines T84 and
Caco-2 were used to study the activation of cytokine
expression as a response to V. cholerae infection.
Among the 14 cytokines involved in proinflammatory,
anti-inflammatory and antigen-specific immune
responses, the mRNA levels of the eight proinflammato-
ry cytokines IL-1a, IL-1b, IL-6, IL-8, tumor necrosis
factor-a (TNF-a), granulocyte–macrophage colony-
stimulating factor (GM-CSF), monocyte chemotactic
protein-1 (MCP-1) and epithelial cell derived neutrophil
activator-78 (ENA-78) were markedly increased upon
V. cholerae infection in Int407 cells, whereas expression
of the anti-inflammatory cytokine transforming growth
factor-b (TGF-b) was downregulated upon infection
(Table 1; Fig. 1A,B,D,E). All of the above proinflamma-
tory cytokines also showed increased expression in the
Cytokine response in infected epithelial cells A. Bandyopadhaya et al.
4632 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS
V. cholerae-infected T84 cell line, except for IL-6, which
did not show upregulation of expression following infec-
tion (Fig. 1A,B,D,E). Thus, besides Int407 cells, the
similar expression data from another intestinal epithelial
cell line, T84, substantiate the fact that V. cholerae does
indeed induce a range of cytokine expression in intesti-
nal epithelial cells upon infection. Comparison of the
status of induction of cytokines by quantitative real-
time RT-PCR in Int407 and T84 cells showed that all

the proinflammatory cytokines, except IL-1a and IL-8,
were induced to a greater extent in Int407 cells than in
T84 cells upon V. cholerae infection (Fig. 1A). The
major cysteine–cysteine (C–C) chemokine MCP-1
showed upregulation in both Int407 (34-fold) and T84
cells upon infection with V. cholerae; the fold change in
T84 could not be quantitated, due to the absence of
endogenous MCP-1 expression in T84 cells (Fig. 1B).
Another C–C chemokine, regulated upon activation,
normal T cell expressed and secreted (RANTES),
showed no significant alteration in expression in all the
three cell lines studied upon infection (data not shown).
As the mRNA of interferon (IFN)-c was barely detect-
able in both uninfected and infected Int407 cells, protein
secretion was measured by ELISA. IFN-c was detected
at 2 h following infection; the level increased to about
23.7 pgÆmL
)1
at 3.5 h, and to 33.86 pgÆmL
)1
at 8 h, and
declined thereafter (Fig. 1C).
In Caco-2 cells, a colon epithelial cell line often used
to study V. cholerae interactions, constitutive mRNA
expression of IL-1a, IL-8 and MCP-1 was observed
(data not shown). Moreover, significant downregulation
of IL-6 and IL-1b and no detectable level of TNF-a
mRNA was obtained from the Caco-2 cell line. Real-
time RT-PCR showed a 1.5-fold downregulation of the
anti-inflammatory cytokine TGF-b in Int407 cells upon

infection (Fig. 1A). In contrast, upregulation of TGF-b
was obtained in T84 and Caco-2 cells. Thus, a striking
dissimilarity was observed in the nature of the cytokine
expression profile following V. cholerae infection in the
ileocecal epithelial carcinoma cell line Caco-2 as com-
pared to Int407 cells, derived from small intestine or
T84, the colon carcinoma cell line. It is not clear why
Caco-2 cells did not show a significant cytokine response
to V. cholerae. It could be that Caco-2 cells produce few
or no cytokines in the absence of polymorphonuclear
neutrophils (PMNs), supporting previous suggestions
that there is PMN–epithelium crosstalk coordinating
the cytokine response in the gut mucosa [17]. Previous
studies examining the IL-8 response in Caco-2 cells with
Desulfovibrio desulfuricans or Salmonella enterica ser-
ovar Enteritidis have shown that additional stimulation
by natural bacterial products such as butyrate may be
required to cause higher level of cytokine induction
[18,19]. It appears that additional stimulation of Caco-2
cells is required to cause a significant cytokine response.
The expression of several cytokines, such as IL-2,
IL-4, IL-5, IL-10 and IL-12p40, that are commonly
associated with antigen-specific acquired immunity,
could not be detected even upon infection (Table 1).
This indicates that cytokines produced by intestinal
epithelial cells are likely to play a more important role
in initiating and regulating the innate mucosal inflam-
matory response rather than antigen-specific mucosal
immune responses. No change in the mRNA expression
of the studied cytokines was observed in Int407 cells

upon incubation with E. coli DH5a (data not shown),
suggesting that this effect is not a general inflammatory
response but is due to V. cholerae infection. The obser-
vation of cytokine induction following bacterial infec-
tion of epithelial cells has been made in several other
bacteria, e.g. Helicobacter pylori [20], enteropathogenic
E. coli [21], and Campylobacter jejuni [22].
The cytokines TNF-a, IL-6 and IL-1 promote bacteri-
cidal activity of leukocytes, GM-CSF is a strong chemo-
attractant for neutrophils and eosinophils, ENA-78
and IL-8 belong to the C-X-C (where ‘X’ is any amino
acid) family of chemokines, which activate PMNs, par-
ticularly neutrophils, and MCP-1, which belongs to the
C–C family of chemokines, can variably act as chemo-
attractants for monocytes ⁄ macrophages, eosinophils,
and subpopulations of T cells [23]. In contrast to the
proinflammatory cytokines, downregulation of the
Table 1. Cytokines studied in human intestinal epithelial cells after
V. cholerae infection. ++ ⁄ +++, upregulated; –, downregulated; NA,
no expression available; NC, no significant upregulation; ND, not
determined.
Cytokine
name Nature of cytokines
Differential expression
of cytokines after
V. cholerae infection
Int407 T84 Caco-2
IL-1a Proinflammatory ++ +++ NC
IL-1b Proinflammatory +++ ++ –
TNF-a Proinflammatory +++ ++ NA

IL-6 Proinflammatory ++ NC –
GM-CSF Proinflammatory ++ + ND
IL-8 C-X-C chemokine ++ +++ NC
MCP-1 C–C chemokine ++ +++ NC
TGF-b Anti-inflammatory – + +
IL-2 Acquired specific
immune response (ASIR)
NA NA NA
IL-4 Anti-inflammatory, ASIR NA NA NA
IL-5 ASIR NA NA –
IL-10 Anti-inflammatory NA NA NA
IL-12p40 ASIR NA NA NA
IFN-c Immunoregulatory, ASIR ++ ND ND
A. Bandyopadhaya et al. Cytokine response in infected epithelial cells
FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4633
anti-inflammatory cytokine TGF-b and the absence of
expression of another major anti-inflammatory cyto-
kine, IL-10, following V. cholerae infection in Int407
cells, account for a predominant proinflammatory cyto-
kine response in this system. The findings of this investi-
gation eventually support the notion that cholera has an
inflammatory component. Increased TNF-a, IL-6 and
macrophage inhibitory protein-2 concentrations have
also been reported following infection with attenuated
V. cholerae in a murine pulmonary cholera model [24].
To obtain a more detailed view of the V. cholerae-
induced cytokine network in epithelial cells, the
induction of MCP-1 (C–C chemokine), GM-CSF
(proinflammatory), IL-1a and IL-6 (proinflammatory)
and the anti-inflammatory cytokine TGF-b, belonging

to various functional categories, was investigated fur-
ther in Int407 cells following V. cholerae infection, as
V. cholerae is known to adhere to the small intestinal
epithelial layer at the onset of the disease process.
Variability among different isolates of V. cholerae
in cytokine mRNA induction
To determine whether the induction of different cyto-
kines is a general phenomenon among V. cholerae
isolates or there is some variability related to the
Fig. 1. Induction of cytokine expression in intestinal epithelial cell lines by V. cholerae. (A) Int407 and T84 cells were infected with V. chole-
rae O395 (OR), and incubated for 3.5 h, and cytokine expression as indicated was measured by quantitative real-time RT-PCR. Cytokine
expression, shown in the histogram, was estimated as fold change in cells infected with V. cholerae relative to uninfected cells, and the val-
ues were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (control) expression. SD (vertical bars) was calculated
from two to four replicate experiments. (B) MCP-1 expression as detected by real-time RT-PCR is represented as the C
t
(threshold value) of
GAPDH subtracted from the C
t
of MCP both for infected and for uninfected controls in all three cell lines. (C) Kinetics of IFN-c secretion by
Int407 cells following infection with V. cholerae O395 for 2, 3.5, 8 and 24 h. Values are mean and SD from two independent experiments.
**Significant difference from values at 2 h (P<0.05). RT-PCR amplification of (D) GM-CSF (upper panel), (E) ENA-78 (upper panel) and inter-
nal control GAPDH (lower panel) from uninfected and V. cholerae O395-infected Int407 and T84 cells for 3.5 h. Densitometric quantitations
in densitometry units (DU) for each cytokine, determined by
IMAGEJ ( are shown below the representative
agarose gel sections after normalization to GAPDH. The error bars represent SD of three different experiments. Negative control experi-
ments were performed by omitting RNA from the cDNA synthesis. *Significant difference from uninfected cells (P<0.05).
Cytokine response in infected epithelial cells A. Bandyopadhaya et al.
4634 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS
pathogenicity of these strains, the expression of differ-
ent cytokines was determined in several V. cholerae

isolates belonging to different serovars and biovars,
including CT-producing and nontoxinogenic (CT
–
)
strains of both environmental and clinical origin
(Table 2). Cytokine mRNA expression was examined
by semiquantitative RT-PCR following infection of an
Int407 isolate with V. cholerae at a multiplicity of
infection (MOI) of  100 : 1.
To determine CT production under the present
experimental conditions, the expression of CT was
measured by ELISA in tissue culture medium as well
as in the presence of Int407 cells, using CT-producing
and nontoxinogenic strains. Interestingly, CT expres-
sion increased significantly (P<0.05) (Fig. 2A) when
the cells were grown in the presence of Int407 cells as
compared to those grown in tissue culture media in all
toxinogenic strains. The nontoxinogenic strains GP-7
and VCE309 are naturally non-CT strains, and the CT
levels were negligible (comparable to medium-only
control). Thus, the results indicate that CT is expressed
under the present experimental condition (3.5 h of
incubation with Int407 cells). These factors may elicit
some of the host cell responses.
All V. cholerae strains showed significant but vari-
able induction of proinflammatory cytokines, irrespec-
tive of serovar, biovar, and CT production, suggesting
that V. cholerae isolates, in general, are associated with
the inflammatory response (Fig. 2B). It is clear from
the data on the induction of cytokines in Int407 cells

following infection with different V. cholerae isolates
that the naturally occurring nontoxinogenic classic
strain GP-7 could produce a similar proinflammatory
response to that of the toxin-producing strain. Among
the proinflammatory cytokines, GM-CSF expression
was induced substantially in all the strains, the increase
in expression being maximum following N16961 infec-
tion (12.6-fold) and comparatively lower for GP-7
(5.2-fold). IL-1a mRNA expression was maximal in
O395, followed by SG-24. A substantial increment in
expression could also be observed following GP-7 infec-
tion. Interestingly, induction of IL-6 expression was
maximal in the environmental nontoxinogenic strain
VCE309. Similarly, the highest MCP-1 expression
could be observed following infection with another
toxin-producing environmental strain, VCE232. The
expression of TGF-b was downregulated in all clinical
isolates except the atypical hypertoxinogenic strain
569B, which showed unaltered (constitutive) expression.
The two environmental isolates VCE232 (CT
+
) and
VCE309 (CT
–
), however, induced TGF-b mRNA
expression in Int407 cells following infection (Fig. 2B).
No disease association has, however, been reported for
these environmental strains [25].The results thus sug-
gest that although V. cholerae isolates are associated
with proinflammatory responses, these isolates, having

differences in the cell surface architecture and secretory
products [26], may give rise to differential stimulation
of these cytokines, at least under the in vitro conditions
used in the present study. The variability in cytokine
response among different strains could be due to the
differences in the expression of multiple virulence deter-
minants; for example, among the CT-producing classic
strains O395 and 569B, the latter has a deletion in
the rtx locus, eliminating VcRTX [27], and is a poor
producer of HAP, Eltor strain N16961 (CT
+
) has man-
nose sensitive hemagglutinin and is also a hapR mutant,
VCE232 is an environmental CT-producing isolate, and
GP-7 and VCE309 are naturally occurring nontoxino-
genic strains. As all V. cholerae strains, irrespective of
Table 2. Bacterial strains used in this study.
Strains Relevant genotype or phenotype Reference
Vibrio cholerae
O395 O1 serotype Ogawa, biotype Classic,
streptomycin resistant, CT
+
Laboratory
collection
569B O1 serotype Inaba, biotype Classic, CT
+
[36]
N16961 O1 serotype Inaba, biotype ElTor, CT
+
[37]

SG24 O139, CT
+
[38]
VCE 232 Non-O1 environmental, CT
+
[26]
VCE 309 Non-O1 environmental, CT
–
[26]
GP-7 O1 serotype Ogawa, biotype ElTor,
naturally occurring CT
–
strain
[39]
O395CTXAN O395 insertion in ctxA gene [40]
Escherichia coli
DH5a F
–
f80d ⁄ lacZ DM15 D(lacZYA argF) U169 rec A1 end A1
hsdR17(r
k
–
,m
k
–
) supE441-thi-1 gyrA relA1
Bethesda
Research
Laboratories, MD, USA
A. Bandyopadhaya et al. Cytokine response in infected epithelial cells

FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4635
CT production, can cause upregulation of cytokines, it
could be thought that some other factor(s) beside CT
could be responsible for this induction.
Effect of V. cholerae culture supernatant, CT and
LPS on cytokine mRNA expression by Int407 cells
V. cholerae secretes a number of components, both
proteinaceous and nonproteinaceous in nature, in its
culture supernatant. Recent reports have indicated that
secreted factor(s) from V. cholerae can induce IL-8
expression in T84 cells [12,13]. To determine whether
the supernatant of V. cholerae harbors potent inducers
of other cytokine(s), supernatants equivalent to
100 MOI (1 · A) and 5 · A were incubated with
Int407 cells, and cytokine mRNA expression was
determined. IL-1a expression was upregulated in
Int407 cells when incubated with 1 · A culture super-
natant, and this increased to 2.4-fold with 5 · A super-
natant; this is, however, lower than the expression of
IL-1a in Int407 cells treated with whole live V. chole-
rae (3.4-fold) (Fig. 3), suggesting that although the
components present in the supernatant can trigger
expression, other factors are also required for the
expression of IL-1a in this system. Similarly, with
GM-CSF, the whole organism causes a six-fold
increase, which is lower with 5 · A supernatant (four-
fold) in Int407 cells (Fig. 3A). Interestingly, in case of
both MCP-1 and IL-6, Int407 cells treated with
V. cholerae supernatants showed higher mRNA expres-
sion as compared to untreated cells, and the expression

was comparable to that obtained with whole live
V. cholerae (Fig. 3A). TGF-b expression was also
altered significantly (P<0.05) in V. cholerae superna-
tant-treated cells, being downregulated as compared to
untreated control (Fig. 3A). These results clearly indi-
cate the presence of potent stimulators for MCP-1 and
IL-6 and also for IL-1a and GM-CSF, although to a
lesser extent, in V. cholerae culture supernatant.
The inducer of IL-6, MCP-1 and GM-CSF in
V. cholerae supernatant was sensitive to both protein-
ase K and trypsin (Fig. 3B), suggesting that the inducer
is a protein. However, the protein inducer of IL-6 and
GM-CSF is resistant to heat treatment, whereas that of
MCP-1 is heat sensitive (Fig. 3B), suggesting that the
proteins are of a different kind. Heat, proteinase K and
trypsin treatment did not abolish IL-1a expression, sug-
gesting the involvement of nonproteinaceous compo-
nents also. In this context, it is relevant to mention that
both flagellin [28] and lipoprotein [29] are secreted into
the supernatant; whereas flagellin is resistant to heat
treatment but sensitive to proteinase K [28], lipoprotein
is resistant to both proteinase and heat treatment [29].
Therefore, it is possible that lipoprotein and flagellin
may also be stimulators of IL-1a, IL-6 and GM-CSF.
The above facts indicate the existence of more than one
factor(s) that stimulates cytokine expression by
V. cholerae in Int407 cells.
LPS, one of the major components of the outer
membrane of Gram-negative bacteria, has been classi-
cally considered to be predominantly responsible for

cytokine induction by Gram-negative bacteria [30].
Fig. 2. (A) Secretion of CT in tissue culture medium and in the
presence of Int407 cells. Different V. cholerae strains (toxinogenic
and nontoxinogenic) were inoculated in the tissue culture medium
(MEM) in the presence or absence of Int407 cells, and the secre-
tion of CT was measured by ELISA as described in Experimental
procedures. *Significant difference in CT secretion between MEM
and MEM + Int407 cells (P<0.05). (B) Induction of various cyto-
kine mRNAs in Int407 cells by different strains of V. cholerae after
incubation for 3.5 h determined by RT-PCR. The lanes indicate unin-
fected Int407 cells (A), and Int407 cells infected with O395
(B), 569B (C), VCE232 (D), VCE309 (E), N16961 (F), SG24 (G) and
GP-7 (H). The error bars represent SD of three different experi-
ments. *Significant difference from uninfected cells (P<0.05).
Cytokine response in infected epithelial cells A. Bandyopadhaya et al.
4636 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS
Although LPS (1 lgÆmL
)1
for 8 h) caused induction of
2.4-fold and 1.9-fold of IL-1a and IL-6, respectively,
as compared to untreated control, it failed to cause
any significant change in the expression of MCP-1,
GM-CSF or TGF-b (Fig. 3A). Similar results were
also obtained with Salmonella LPS (data not shown).
Such a poor response is in accordance with earlier
studies, which have suggested that LPS effectively
induces cytokine production from macrophages but
only poorly induces epithelial cytokine responses. The
poor response of LPS can be explained by the lack of
CD14 and toll-like receptor 4 on epithelial cells [31].

To determine whether CT is a potent inducer of
cytokines, Int407 cells were incubated with commercial
CT at 4.5 ngÆmL
)1
and 9 ngÆmL
)1
for 3.5 h, and the
cytokine mRNA expression was determined (Fig. 3A).
Following CT treatment, IL-1a mRNA expression
differed significantly (P ¼ 7 · 10
)7
, one-way ANOVA),
being higher at both concentrations (Fig. 3A). TGF-b
expression also differed following CT treatment (P ¼
0.00048, one-way ANOVA), although treatment with
9ngÆmL
)1
CT resulted in TGF-b expression that was
comparable to that of untreated Int407 cells (Fig. 3A).
Both MCP-1 and IL-6 mRNA expression were signifi-
cantly altered upon CT treatment (P ¼ 4.7 · 10
)8
and
7 · 10
)6
, respectively, one-way ANOVA). To our
knowledge, this is the first report of MCP-1 induction
by CT. This is corroborated by our studies showing the
involvement of a heat-sensitive protein component in
V. cholerae culture supernatant in MCP-1 induction. It

is therefore evident that CT is a potent inducer, along
with some other factor(s) in the V. cholerae culture
supernatant in Int407 cells, of most of the cytokines
tested. Previous reports have shown CT-induced
enhancement of IL-1 and IL-6 expression in epithelial
cell lines [9,32]. Our study indicates that V. cholerae cul-
ture supernatant harboring potent inducers, in addition
to CT, could be responsible for the proinflammatory
response in the intestinal epithelial layer, and possibly
contribute to the reactogenecity of the vaccine strains.
Cytokine modulation in a ctxA mutant of
V. cholerae
To substantiate the above observations, a ctxA mutant
of V. cholerae O395, impaired in the major virulence
factor CT, was constructed. Int407 cells were infected
with an insertional mutant in the ctxA gene
Fig. 3. Induction of cytokine mRNAs in Int407 cells by CT, LPS and culture supernatants from V. cholerae O395 under various conditions by
RT-PCR. (A) The lanes indicate uninfected Int407 cells (I), Int407 cells infected with live V. cholerae O395 for 3.5 h (IOR), Int407 cells treated
with filter-sterilized culture supernatants from V. cholerae at 1 · 100 and 5 · 100 MOI, respectively, for 3.5 h (1A,5A), LPS at 1 lgÆmL
)1
,
incubated for 8 h (LPS), and CT at 4.5 and 9 ngÆmL
)1
, incubated for 3.5 h (CT5 and CT9). (B) The lanes indicate Int407 cells treated with fil-
ter-sterilized culture supernatants from V. cholerae O395 at 1 · 100 and 5 · 100 MOI, respectively, for 3.5 h (1A,5A), and pretreated super-
natant from 5 · 100 MOI V. cholerae with heat (95 °C for 30 min) (HT), trypsin (TRP) and proteinase K (PNK), before stimulating Int407
cells. *Significant difference from uninfected cells (P<0.05). **Significant difference from 5A supernatant-treated cells (P<0.05).
A. Bandyopadhaya et al. Cytokine response in infected epithelial cells
FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4637
(O395CTXAN), and IL-1a, IL-6, MCP-1 and TGF-b

mRNA expression levels were determined (Fig. 4). As
compared to O395-infected cells, the expression of
IL-1a and IL-6 was reduced by 2.6-fold and 1.4-fold,
respectively, upon infection with O395CTXAN, suggest-
ing that CT is one of the factors responsible for IL-1a
and IL-6 induction in Int407 cells, which is in good
agreement with our previous observation obtained with
commercial CT. No change in TGF- b expression was
observed when Int407 cells were infected with
O395CTXAN as compared to an uninfected control.
Moreover, O395CTXAN caused no significant reduc-
tion in MCP-1 expression as compared to cells infected
with live V. cholerae, suggesting the presence of some
other heat-sensitive potent stimulator of a proteinaceous
nature in V. cholerae culture supernatant. Hence CT,
alongwith other CT-independent factors, might be an
important determinant of IL-1a, IL-6 and MCP-1 gene
expression modulation. These findings make it clear that
the V. cholerae culture supernatant harbors a potent
inducer of cytokine expression to a varying degree, indi-
cating the multifactorial nature of V. cholerae infection.
Differential activation of transcription factors
NF-jB and CREB by CT, LPS and a ctxA mutant
of V. cholerae
The NF-jB family of transcription factors is known to
play a role in promoting the expression of cytokines
through interaction with other cofactors such as CREB
within the gene promoter regions [15]. To determine
whether the upregulation of proinflammatory cytokines
by wild-type V. cholerae O395 and its components such

as CT or LPS could be mediated by NF-jB and CREB,
activation of NF-jB and CREB was assayed in CT-trea-
ted or LPS-treated intestinal epithelial cells. Activation
of NF-jB p65 and CREB was observed in Int407 cells
at 5 min and 30 min, respectively, following infection
with wild-type V. cholerae (Fig. 5A). Delayed activation
was, however, observed following LPS treatment. LPS-
treated (1 lgÆmL
)1
) Int407 cells showed p65 and p50
activation as compared to untreated control at 8 h
(Fig. 5B), whereas the active form of CREB was
observed at 4 h of LPS treatment. Such facts suggest an
optimal association of transcription factors at the site of
transcription of IL-1a and IL-6 by LPS.
The activation of transcription factors was deter-
mined with induction by CT at 4.5 ngÆmL
)1
and
9ngÆmL
)1
for 1, 2 and 3 h. Although the dominant
transcription factor NF-jB p65 could not be activated
by CT at 4.5 ngÆmL
)1
, activation was observed at 3 h
of incubation with 9 ngÆmL
)1
CT as determined by wes-
tern blot analysis (Fig. 5C,D). Activation of NF-jB

p50 was observed with CT-treated (4.5 ngÆmL
)1
) Int407
cells at 1 and 2 h, respectively, which gradually declined
at 3 h (Fig. 5C), whereas 9 ngÆmL
)1
of CT caused acti-
vation of p50 at 3 h (Fig. 5D), as compared to un-
infected Int407 cells. The phosphorylated CREB was
found to be induced at 2 h by CT (4.5 ngÆmL
)1
)orat
3 h by 9 ngÆmL
)1
of CT (Fig. 5C,D). To substantiate
the above finding, Int407 cells were incubated with a
V. cholerae ctxA mutant, which caused activation of
both p65 and CREB at an early time point of infection
(within 30 min) in Int407 cells (comparable to wild-type
infection); this declined thereafter (observed up to 3 h),
showing the transient nature of activation (Fig. 5E). As
O395CTXAN was impaired in CT secretion (data not
shown), it is evident from the above results that, besides
CT, other secretory factors of V. cholerae are also
responsible for NF-jB and CREB activation. The frac-
tionation of V. cholerae culture supernatant followed
by the induction of epithelial cells with each of these
fractions could identify the probable combination of
V. cholerae factors involved in cytokine induction. Such
studies are being initiated in our laboratory. Moreover,

we are in the process of constructing several gene-
specific insertion mutants impaired in virulence, with
the goal of identifying the ligands responsible for
stimulation of proinflammatory cytokines, and to
understand the mechanism of reactogenicity caused by
V. cholerae.
In summary, the present study demonstrates that the
induction of proinflammatory cytokines appears to be
Fig. 4. Modulation of various cytokine mRNAs in Int407 cells by a
ctxA mutant of V. cholerae. RT-PCR of the respective cytokines in
Int407 cells (1), followed by infection with wild-type V. cholerae
O395 (2) and O395CTXAN (3) for 3.5 h. A closed circle indicates a
significant difference from V. cholerae-infected cells (P<0.05).
Cytokine response in infected epithelial cells A. Bandyopadhaya et al.
4638 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS
an important component of the cellular responses to
V. cholerae infection. Experiments with V. cholerae
strains of varied pathogenicity suggest that the inducer
of cytokines is not evenly distributed or secreted among
different V. cholerae strains and could be multifactorial,
at least under the in vitro culture conditions studied
here. Studies on IL-1, IL-6, GM-CSF, MCP-1 and
TGF-b have documented that, apart from CT and LPS,
V. cholerae culture supernatant harbors strong indu-
cer(s) of IL-6 and MCP-1 and moderate inducer(S) of
IL-1a and GM-CSF. These transcriptional responses
were apparently mediated by NF-jB and CREB activa-
tion. Such responses are essential components of the
inflammatory immune response to enteric pathogens;
additionally, these data provide insights into the mecha-

nisms of tissue damage by V. cholerae that could
contribute to a proinflammatory response. These find-
ings further support the premise that inflammation plays
Fig. 5. Differential activation of NF-jB and CREB in Int407 cells by wild-type V. cholerae, ctxA mutant, LPS and CT. A total of 3.2 · 10
6
Int407 cells treated with (A) wild-type V. cholerae, (B) ctxA mutant of V. cholerae at 1 · 100 MOI for different times (0, 5, 10, 15, 30, 60,
120 and 180 min), (C) LPS at 1 lgÆmL
)1
for 0, 4 and 8 h, (D) CT at 4.5 ngÆmL
)1
for 0, 1, 2 and 3 h, and (E) CT at 9 ngÆmL
)1
for 0, 1, 2 and
3 h. The problem of equal loading here was solved by normalization with human b-actin control. These experiments were performed three
times, and the figure shows representative data from a single experiment. *Significant difference from uninfected cells (P<0.05).
A. Bandyopadhaya et al. Cytokine response in infected epithelial cells
FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4639
a significant role in V. cholerae pathogenesis, and that
the intestinal epithelial tissue probably plays significant
roles in initiating the inflammatory response.
Experimental procedures
Bacterial strains and plasmids
The bacterial strains used in this study are listed in Table 2.
All V. cholerae and E. coli strains were maintained at ) 70 °C
in LB medium containing 20% (v ⁄ v) glycerol. E. coli and
V. cholerae cells were grown in LB medium. Streptomycin and
ampicillin concentrations were 1 mgÆmL
)1
and 15 lgÆmL
)1

,
respectively, for V. cholerae wherever appropriate.
Cell culture, infection and stimulation
The human intestinal epithelial cell lines Int407 and Caco-2
from NCCS, Pune, India were grown and maintained in
MEM (Gibco-BRL, Gaithersburg, MD, USA), and T84 cells
(a gift from S Visyeswariah, IISc, Bangalore, India) were
grown in DMEM and Ham’s F-12 medium (Gibco-BRL) at
pH 7.4, supplemented with 10% fetal bovine serum (Gibco-
BRL) containing penicillin ⁄ streptomycin and gentamicin in
the presence of 5% CO
2
at 37 °C. Caco-2 cells, in addition,
were supplemented with 2 mml-glutamine and 1% nones-
sential amino acids (Sigma-Aldrich, St Louis, MO, USA).
Cells were seeded in T-75 tissue culture flasks (Falcon, San
Jose, CA, USA). Bacteria from overnight culture suspended
in fresh medium without antibiotics were added at 100 MOI.
For stimulation by supernatant, bacterial culture super-
natants (equivalent to an MOI of 100 bacteria per cell and
5 · 100 MOI) were centrifuged at 6000 g for 5 min (Model
Z200 M/H, rotor type 220.95 V01/V02, Hermle, Gosheim,
Germany), filter sterilized (0.2 l m), added to Int407 cells,
and incubated at 37 °C under 5% CO
2
for 3.5 h. In some
experiments, supernatant was heat treated (30 min, 95 °C),
trypsin treated (2 h, 40 lgÆ mL
)1
; Invitrogen, Life Technolo-

gies, Carlsbad, CA, USA) or proteinase K treated (2 h,
200 lgÆmL
)1
; Invitrogen) before incubation. Stimulation
with commercial CT (Sigma-Aldrich) was done at concen-
trations of 4.5 ngÆmL
)1
and 9 ngÆmL
)1
for 3.5 h, and LPS
of V. cholerae (isolated from V. cholerae O139AP-1) was
used at a concentration of 1 lgÆmL
)1
for 8 h.
RNA extraction and cDNA preparation
Both uninfected and V. cholerae-infected Int407, T84 or
Caco-2 cells were washed with NaCl ⁄ P
i
, infected cells being
washed vigorously to remove nonadherent bacteria. Total
RNA was extracted from each with the Rneasy Mini Kit
(Qiagen Inc., Valencia, CA, USA). cDNA preparation was
carried out using the SUPERSCRIPT First-Strand Synthe-
sis System (Invitrogen) as described previously [12].
Quantitative real-time RT-PCR
Cytokine mRNA expression was determined by real-time
quantitative RT-PCR using relative quantitation by the
comparative threshold cycle number (C
t
) method using

iCycler (Bio-Rad, Hercules, CA, USA) and SYBR Green
Jump Start TaqReadymix (Sigma-Aldrich) as described pre-
viously [12]. Cytokine and the internal control gene
GAPDH (primers described in Sarkar & Chaudhuri [12],
Jung et al. [33] and Yang et al. [34]) were amplified in each
tube. The calibrator used in our experiments was the unin-
fected Int407, T84 or Caco-2 control samples.
Semiquantitative RT-PCR
In semiquantitative RT-PCR reactions for determining
cytokine mRNA expression, about 2 lL of cDNA was
PCR amplified in a 30 lL reaction volume containing
10 mm Tris ⁄ HCl (pH 8.3), 50 mm KCl, 2.0 mm MgCl
2
,
0.26 mm each dNTP, and 25 pmol of each primer
[12,33,34]. Hot start PCR was used to increase the specific-
ity of amplification. Multiplex RT-PCR was performed
wherever possible.
For specific PCR amplification of positive controls, RNA
from cells known to abundantly express the respective
mRNA were used: 4b-phorbol 12-myristate 13-acetate-
stimulated and ionomycin-stimulated peripheral blood
mononuclear cells for IL-2, IL-4, IL-5, IFN-c, and LPS-
stimulated peripheral blood mononuclear cells for IL-10,
IL-12p40, ENA-78 and Caco-2 cells for MCP-1.
Determination of IFN-c secretion by ELISA
The level of IFN-c protein in the culture supernatant of
infected or uninfected Int407 cells was measured by ELISA.
For ELISA, the OptEIA human IFN-c ELISA KITII (BD
Biosciences Pharmingen, San Diego, CA, USA) was used,

following the manufacturer’s instructions [12].
GM1-ganglioside dependent ELISA
V. cholerae strains were grown overnight, and 50 lLof
sample from culture was added in 1 mL portions of MEM
in the presence of Int407 cells (equivalent to  100 · MOI)
or MEM alone at 37 °C for 3.5 h. Samples of the cultures
were removed, and concentrations of CT were measured by
ELISA as described previously [35].
Western blot analysis of NF-jB p65 and p50
subunits and CREB
Int407 cells (3.2 · 10
6
) were incubated for 5, 10, 15, 30, 60,
120 and 180 min with wild-type or ctxA mutant at
100 · MOI or LPS (1 lgÆmL
)1
, 4 and 8 h) or CT (4.5 and
Cytokine response in infected epithelial cells A. Bandyopadhaya et al.
4640 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS
9ngÆmL
)1
) for 1, 2 and 3 h. The untreated and treated cells
were lysed with lysis buffer (200 lL; 1.5 mm Tris ⁄ HCl,
pH 6.8, 10% SDS, 10% glycerol, 1% bromophenol blue).
Before loading, samples were boiled (100 °C, 10 min) with
b-mercaptoethanol (Sigma-Aldrich) and cooled on ice;
equal amounts of (30 lL per lane) samples were subjected
to 12% SDS ⁄ PAGE with prestained protein molecular
weight marker (10 lL; GENEI, Bangalore, India) and ana-
lyzed by western blotting using rabbit phospho-NF-jB

p65(Ser536) (Cell Signaling Technology, Danver, MA,
USA), rabbit polyclonal NF-jB-p50 (Santa Cruz Bio-
technology, Inc., Santa Cruz, CA, USA), phospho-
CREB(Ser133) at a dilution of 1 : 1000 or mouse b-actin
(Sigma-Aldrich) at 1 : 2000 dilutions in NaCl ⁄ TrisT buf-
fer ⁄ 5% BSA; this was followed by incubation with alkaline
phosphatase-conjugated goat anti-(rabbit IgG) (GENEI) or
rabbit anti-(mouse IgG) (GENEI), which was added at a
1 : 2000 dilution in NaCl ⁄ TrisT buffer ⁄ 5% BSA. The alka-
line phosphatase-positive bands were visualized in a devel-
oping solution containing 1 · 5-bromo-4-chloroindol-2-yl
phosphate ⁄ Nitro Blue tetrazolium (GENEI), 1.5 mm
Tris ⁄ HCl (pH 8.8) and water in the dark at room tempera-
ture for 10 min and quantitated.
Statistical analysis
The data on semiquantitative RT-PCR, ELISA and densi-
tometric scanning of western blots were recorded as
mean ± standard deviation (SD) from at least three inde-
pendent experiments. Comparison between two groups was
done by Student’s t-test. For comparing more than one
treatment, one-way ANOVA was used. Differences were
considered significant at P < 0.05.
Acknowledgements
The study was supported by the Council of Scientific
and Industrial Research (CSIR), Government of India.
A. Bandyopadhaya and M. Sarkar are the recipients
of the CSIR fellowship. We are grateful to Pratim
Chaudhuri for experimental help.
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