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: keyachaudhuri@yahoo.com o
Trang 1human 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: keyachaudhuri@yahoo.com or
kchaudhuri@iicb.res.in
*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 choleraeelicits 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.
Trang 2inflammatory 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 choleraeinfection 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 choleraefactors 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 difdif-ferent 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
Trang 3V 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)1at 3.5 h, and to 33.86 pgÆmL)1at 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-2 Acquired specific
immune response (ASIR)
Trang 4anti-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 choleraein 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 Ctof 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 (http://rsb.info.nih.gov/ij/index.html), 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).
Trang 5pathogenicity 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.
Vibrio cholerae
streptomycin resistant, CT +
Laboratory collection
naturally occurring CT – strain
[39]
Escherichia coli
DH5a F – f80d ⁄ lacZ DM15 D(lacZYA argF) U169 rec A1 end A1
hsdR17(rk,mk) supE441-thi-1 gyrA relA1
Bethesda Research Laboratories, MD, USA
Trang 6CT 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 choleraesupernatants showed higher mRNA expres-sion as compared to untreated cells, and the expresexpres-sion 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 choleraein 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 inunin-fected 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).
Trang 7Although 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 (P¼ 7 · 10)7, one-way ANOVA),
being higher at both concentrations (Fig 3A) TGF-b
expression also differed following CT treatment (P¼
0.00048, one-way ANOVA), although treatment with
9 ngÆmL)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 (P¼ 4.7 · 10)8and
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 choleraeculture 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).
Trang 8(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-treaCT-trea-ted 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
9 ngÆmL)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)1CT 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)1of 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) or at
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 choleraefactors 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).
Trang 9an 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)1for 0, 4 and 8 h, (D) CT at 4.5 ngÆmL)1for 0, 1, 2 and 3 h, and (E) CT at 9 ngÆmL)1for 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).
Trang 10a 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 choleraecells 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% CO2at 37C Caco-2 cells, in addition,
were supplemented with 2 mm l-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 lm), added to Int407 cells,
and incubated at 37C under 5% CO2for 3.5 h In some
experiments, supernatant was heat treated (30 min, 95C),
trypsin treated (2 h, 40 lgÆmL)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)1for 3.5 h, and LPS
of V cholerae (isolated from V cholerae O139AP-1) was
used at a concentration of 1 lgÆmL)1for 8 h
RNA extraction and cDNA preparation
Both uninfected and V cholerae-infected Int407, T84 or
Caco-2 cells were washed with NaCl⁄ Pi, 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 (Ct) 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 MgCl2,
[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 lL of 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 37C 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· 106
) 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