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

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

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

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

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

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

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

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

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

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

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

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,

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

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

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

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