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 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 colonystimulating 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 MCP1, granulocyte–macrophage colony-stimulating factor, interleukin-1a, interleukin-6 and the anti-inflammatory cytokine transforming growth factor-b in Int407 cells with V cholerae culture supernatant, cholera toxin, lipopolysaccharide 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 interleukin-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 elementbinding 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 mediated by cholera toxin-independent factors as well We conclude that V cholerae elicits a proinflammatory response in Int407 cells that is mediated by activation of nuclear factor-jB and cAMP response element-binding protein by cholera toxin, lipopolysaccharide and ⁄ or other secreted products of V cholerae The acute diarrheal disease cholera remains a significant 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 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 Cytokine response in infected epithelial cells A Bandyopadhaya et al inflammatory mediators The response of the intestine to infection by pathogens represents a complex interaction between nonspecific inflammatory mechanisms and immunologically specific adaptive events Although cholera has been traditionally classified as a noninflammatory diarrheal disease [2], various reports point towards an inflammatory component in the pathogenesis of the disease [3,4] Lymphocytes and mononuclear cells have been observed in the intestinal lamina propria 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 production 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 intestinal 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 vaccine strains in T84 cells have been shown [11,13] Moreover, 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 proinflammatory mediators, such as cytokines [14] However, 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 proteins 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 development 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 4632 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 therefore 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 coactivators 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 different functional groups in three different intestinal epithelial cell lines upon V cholerae infection These cytokines are not induced upon incubation with nonpathogenic 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 cytokine 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 provide the underlying mucosa with the first signals of an infection [16] As the intestinal mucosal epithelial surface 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 proinflammatory cytokines IL-1a, IL-1b, IL-6, IL-8, tumor necrosis factor-a (TNF-a), granulocyte–macrophage colonystimulating 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 proinflammatory cytokines also showed increased expression in the FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS A Bandyopadhaya et al Cytokine response in infected epithelial cells Table Cytokines studied in human intestinal epithelial cells after V cholerae infection ++ ⁄ +++, upregulated; –, downregulated; NA, no expression available; NC, no significant upregulation; ND, not determined Differential expression of cytokines after V cholerae infection Cytokine name IL-1a IL-1b TNF-a IL-6 GM-CSF IL-8 MCP-1 TGF-b IL-2 IL-4 IL-5 IL-10 IL-12p40 IFN-c Nature of cytokines Int407 T84 Caco-2 Proinflammatory Proinflammatory Proinflammatory Proinflammatory Proinflammatory C-X-C chemokine C–C chemokine Anti-inflammatory Acquired specific immune response (ASIR) Anti-inflammatory, ASIR ASIR Anti-inflammatory ASIR Immunoregulatory, ASIR ++ +++ +++ ++ ++ ++ ++ – NA +++ ++ ++ NC + +++ +++ + NA NC – NA – ND NC NC + NA NA NA NA NA ++ NA NA NA NA ND NA – NA NA ND V cholerae-infected T84 cell line, except for IL-6, which did not show upregulation of expression following infection (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 intestinal epithelial cells upon infection Comparison of the status of induction of cytokines by quantitative realtime 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 detectable in both uninfected and infected Int407 cells, protein secretion was measured by ELISA IFN-c was detected at h following infection; the level increased to about 23.7 pgỈmL)1 at 3.5 h, and to 33.86 pgỈmL)1 at 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 Realtime 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 compared 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 serovar 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 inflammatory 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 observation of cytokine induction following bacterial infection 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 bactericidal activity of leukocytes, GM-CSF is a strong chemoattractant 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, particularly neutrophils, and MCP-1, which belongs to the C–C family of chemokines, can variably act as chemoattractants for monocytes ⁄ macrophages, eosinophils, and subpopulations of T cells [23] In contrast to the proinflammatory cytokines, downregulation of the FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4633 Cytokine response in infected epithelial cells A Bandyopadhaya et al Fig Induction of cytokine expression in intestinal epithelial cell lines by V cholerae (A) Int407 and T84 cells were infected with V cholerae 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 values 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 Ct (threshold value) of GAPDH subtracted from the Ct 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, and 24 h Values are mean and SD from two independent experiments **Significant difference from values at h (P < 0.05) RT-PCR amplification of (D) GM-CSF (upper panel), (E) ENA-78 (upper panel) and internal 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 experiments were performed by omitting RNA from the cDNA synthesis *Significant difference from uninfected cells (P < 0.05) anti-inflammatory cytokine TGF-b and the absence of expression of another major anti-inflammatory cytokine, IL-10, following V cholerae infection in Int407 cells, account for a predominant proinflammatory cytokine response in this system The findings of this investigation 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 choleraeinduced cytokine network in epithelial cells, the induction of MCP-1 (C–C chemokine), GM-CSF 4634 (proinflammatory), IL-1a and IL-6 (proinflammatory) and the anti-inflammatory cytokine TGF-b, belonging to various functional categories, was investigated further 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 cytokines is a general phenomenon among V cholerae isolates or there is some variability related to the FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS A Bandyopadhaya et al Cytokine response in infected epithelial cells pathogenicity of these strains, the expression of different 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 : 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 expression 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 variable induction of proinflammatory cytokines, irrespective 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 infection (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 infection 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 suggest 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 determinants; 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 mannose sensitive hemagglutinin and is also a hapR mutant, VCE232 is an environmental CT-producing isolate, and GP-7 and VCE309 are naturally occurring nontoxinogenic strains As all V cholerae strains, irrespective of Table Bacterial strains used in this study Strains Vibrio cholerae O395 569B N16961 SG24 VCE 232 VCE 309 GP-7 O395CTXAN Escherichia coli DH5a Relevant genotype or phenotype Reference O1 serotype Ogawa, biotype Classic, streptomycin resistant, CT+ O1 serotype Inaba, biotype Classic, CT+ O1 serotype Inaba, biotype ElTor, CT+ O139, CT+ Non-O1 environmental, CT+ Non-O1 environmental, CT– O1 serotype Ogawa, biotype ElTor, naturally occurring CT– strain O395 insertion in ctxA gene Laboratory collection [36] [37] [38] [26] [26] [39] 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 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS [40] 4635 Cytokine response in infected epithelial cells A Bandyopadhaya et al Fig (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 secretion 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 cytokine mRNAs in Int407 cells by different strains of V cholerae after incubation for 3.5 h determined by RT-PCR The lanes indicate uninfected 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 experiments *Significant difference from uninfected cells (P < 0.05) 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 4636 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 · A were incubated with Int407 cells, and cytokine mRNA expression was determined IL-1a expression was upregulated in Int407 cells when incubated with · A culture supernatant, and this increased to 2.4-fold with · A supernatant; this is, however, lower than the expression of IL-1a in Int407 cells treated with whole live V cholerae (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 · A supernatant (fourfold) 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 expression 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 supernatant-treated cells, being downregulated as compared to untreated control (Fig 3A) These results clearly indicate 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 proteinase 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, suggesting the involvement of nonproteinaceous components 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 classically considered to be predominantly responsible for cytokine induction by Gram-negative bacteria [30] FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS A Bandyopadhaya et al Cytokine response in infected epithelial cells Fig 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 · 100 and · 100 MOI, respectively, for 3.5 h (1A, 5A), LPS at lgỈmL)1, incubated for h (LPS), and CT at 4.5 and ngỈmL)1, incubated for 3.5 h (CT5 and CT9) (B) The lanes indicate Int407 cells treated with filter-sterilized culture supernatants from V cholerae O395 at · 100 and · 100 MOI, respectively, for 3.5 h (1A, 5A), and pretreated supernatant from · 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) Although LPS (1 lgỈmL)1 for 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 on epithelial cells [31] To determine whether CT is a potent inducer of cytokines, Int407 cells were incubated with commercial CT at 4.5 ngỈmL)1 and ngỈmL)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 · 10)7, one-way ANOVA), being higher at both concentrations (Fig 3A) TGF-b expression also differed following CT treatment (P ẳ 0.00048, one-way ANOVA), although treatment with ngặmL)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 · 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 culture 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 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4637 Cytokine response in infected epithelial cells A Bandyopadhaya et al Fig 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) (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, suggesting 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 reduction 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, indicating 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 4638 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-treated or LPS-treated intestinal epithelial cells Activation of NF-jB p65 and CREB was observed in Int407 cells at and 30 min, respectively, following infection with wild-type V cholerae (Fig 5A) Delayed activation was, however, observed following LPS treatment LPStreated (1 lgỈmL)1) Int407 cells showed p65 and p50 activation as compared to untreated control at h (Fig 5B), whereas the active form of CREB was observed at 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 determined with induction by CT at 4.5 ngỈmL)1 and ngỈmL)1 for 1, and h Although the dominant transcription factor NF-jB p65 could not be activated by CT at 4.5 ngỈmL)1, activation was observed at h of incubation with ngỈmL)1 CT as determined by western blot analysis (Fig 5C,D) Activation of NF-jB p50 was observed with CT-treated (4.5 ngỈmL)1) Int407 cells at and h, respectively, which gradually declined at h (Fig 5C), whereas ngỈmL)1 of CT caused activation of p50 at h (Fig 5D), as compared to uninfected Int407 cells The phosphorylated CREB was found to be induced at h by CT (4.5 ngỈmL)1) or at h by ngỈmL)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 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 fractionation 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 genespecific 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 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS A Bandyopadhaya et al Cytokine response in infected epithelial cells Fig 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 · 106 Int407 cells treated with (A) wild-type V cholerae, (B) ctxA mutant of V cholerae at · 100 MOI for different times (0, 5, 10, 15, 30, 60, 120 and 180 min), (C) LPS at lgỈmL)1 for 0, and h, (D) CT at 4.5 ngỈmL)1 for 0, 1, and h, and (E) CT at ngỈmL)1 for 0, 1, and 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) 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 activation Such responses are essential components of the inflammatory immune response to enteric pathogens; additionally, these data provide insights into the mechanisms of tissue damage by V cholerae that could contribute to a proinflammatory response These findings further support the premise that inflammation plays FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS 4639 Cytokine response in infected epithelial cells A Bandyopadhaya et al 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 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 mgỈmL)1 and 15 lgỈmL)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 (GibcoBRL) containing penicillin ⁄ streptomycin and gentamicin in the presence of 5% CO2 at 37 °C Caco-2 cells, in addition, were supplemented with mm l-glutamine and 1% nonessential 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 supernatants (equivalent to an MOI of 100 bacteria per cell and · 100 MOI) were centrifuged at 6000 g for (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 37 °C under 5% CO2 for 3.5 h In some experiments, supernatant was heat treated (30 min, 95 °C), trypsin treated (2 h, 40 lgỈmL)1; Invitrogen, Life Technologies, Carlsbad, CA, USA) or proteinase K treated (2 h, 200 lgỈmL)1; Invitrogen) before incubation Stimulation with commercial CT (Sigma-Aldrich) was done at concentrations of 4.5 ngỈmL)1 and ngỈmL)1 for 3.5 h, and LPS of V cholerae (isolated from V cholerae O139AP-1) was used at a concentration of lgỈmL)1 for 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 Synthesis System (Invitrogen) as described previously [12] 4640 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 previously [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 uninfected Int407, T84 or Caco-2 control samples Semiquantitative RT-PCR In semiquantitative RT-PCR reactions for determining cytokine mRNA expression, about 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, 0.26 mm each dNTP, and 25 pmol of each primer [12,33,34] Hot start PCR was used to increase the specificity 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-acetatestimulated and ionomycin-stimulated peripheral blood mononuclear cells for IL-2, IL-4, IL-5, IFN-c, and LPSstimulated 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 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 · 106) were incubated for 5, 10, 15, 30, 60, 120 and 180 with wild-type or ctxA mutant at 100 · MOI or LPS (1 lgỈmL)1, and h) or CT (4.5 and FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS A Bandyopadhaya et al ngỈmL)1) for 1, and 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 analyzed by western blotting using rabbit phospho-NF-jB p65(Ser536) (Cell Signaling Technology, Danver, MA, USA), rabbit polyclonal NF-jB-p50 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phosphoCREB(Ser133) at a dilution of : 1000 or mouse b-actin (Sigma-Aldrich) at : 2000 dilutions in NaCl ⁄ TrisT buffer ⁄ 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 : 2000 dilution in NaCl ⁄ TrisT buffer ⁄ 5% BSA The alkaline phosphatase-positive bands were visualized in a 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Bandyopadhaya A, Ray K & Chaudhuri K (2005) Upregulation of human mitochondrial NADH dehydrogenase subunit in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis FEBS Lett 579, 3449–3460 FEBS Journal 274 (2007) 4631–4642 ª 2007 The Authors Journal compilation ª 2007 FEBS ... of cytokines upon V cholerae infection in intestinal epithelial cells Reports have shown the induction of IL-8 in intestinal epithelial cells upon V cholerae infection [11–13] The induction of. .. upregulation of many proinflammatory mediators, such as cytokines [14] However, little is known about the role of V cholerae in initiating and sustaining the innate in? ??ammatory response in intestinal epithelial. .. another intestinal epithelial cell line, T84, substantiate the fact that V cholerae does indeed induce a range of cytokine expression in intestinal epithelial cells upon infection Comparison of the