Induction of a chemoattractant transcriptional response by a Campylobacter jejuniboiled cell extract in colonocytes
© Mellits et al; licensee BioMed Central Ltd. 2009
Received: 26 June 2008
Accepted: 04 February 2009
Published: 04 February 2009
Campylobacter jejuni, the commonest cause of bacterial diarrhoea worldwide, can also induce colonic inflammation. To understand how a previously identified heat stable component contributes to pro-inflammatory responses we used microarray and real-time quantitative PCR to investigate the transcriptional response to a boiled cell extract of Campylobacter jejuni NCTC 11168.
RNA was extracted from the human colonocyte line HCA-7 (clone 29) after incubation for 6 hours with Campylobacter jejuni boiled cell extract and was used to probe the Affymetrix Human Genome U133A array. Genes differentially affected by Campylobacter jejuni boiled cell extract were identified using the Significance Score algorithm of the Bioconductor software suite and further analyzed using the Ingenuity Pathway Analysis program. The chemokines CCL20, CXCL3, CXCL2, Interleukin 8, CXCL1 and CXCL6 comprised 6 of the 10 most highly up-regulated genes, all with Significance Scores ≥ 10. Members of the Tumor Necrosis Factor α/Nuclear Factor-κB super-family were also significantly up-regulated and involved in the most significantly regulated signalling pathways (Death receptor, Interleukin 6, Interleukin 10, Toll like receptor, Peroxisome Proliferator Activated Receptor-γ and apoptosis). Ingenuity Pathway Analysis also identified the most affected functional gene networks such as cell movement, gene expression and cell death. In contrast, down-regulated genes were predominantly concerned with structural and metabolic functions.
A boiled cell extract of Campylobacter jejuni has components that can directly switch the phenotype of colonic epithelial cells from one of resting metabolism to a pro-inflammatory one, particularly characterized by increased expression of genes for leukocyte chemoattractant molecules.
Campylobacter jejuni (C. jejuni) is a gram-negative micro-aerophilic bacterium responsible for the majority of human bacterial enteric infections worldwide [1, 2]. C. jejuni is commonly found as a commensal organism in the intestinal tracts of a wide range of wild and domestic animals, including commercial poultry . Cross-contamination from raw poultry or insufficient cooking of poultry meat are common sources of infection. Enteric infections by this pathogen are often associated with a potent localized inflammatory response. Symptoms arising from infection include watery or bloody diarrhoea with abdominal cramping and fever. In addition, C. jejuni can be invasive and is associated with septicaemia, meningitis, Guillain-Barré syndrome  and more recently with immuno-proliferative disease .
C. jejuni virulence factors for human disease include flagella based chemotaxis, adhesin-based cellular adherence, host cell invasion and the elaboration of a heat labile cytolethal distending toxin (CLDT) [2, 6, 7] In previous studies we have additionally shown that a heat stable C. jejuni boiled cell extract (BCE) is able to activate the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) . This signalling molecule is responsible for inducing the expression of a number of genes involved in inflammation and cell mediated immunity , including chemokines capable of attracting leukocytes, resulting in inflammation. NF-κB is held inactive in the cytoplasm of a cell, whilst its nuclear localization domain is masked by inhibitory IκB proteins. If IκB is phosphorylated, leading to ubiquitin-mediated proteolysis, then NF-κB is released to transport to the nucleus of the cell, where it affects transcription of κB-responsive promoters. Therefore products that activate NF-κB can be presumed to have a strong role in triggering inflammation. Previous work has shown that live C. jejuni and a BCE can induce both NF-κB, and the synthesis and release of the chemokine interleukin-8 .
In order to identify a wider range of genes affected by C. jejuni products and assess the relative importance of the NF-κB response we used microarray technologies to identify genes that were both up and down-regulated in HCA-7 cells after exposure to a C. jejuni BCE [8, 10]. Use of the Ingenuity Pathway Analysis (IPA) program suite enabled us to group co-regulated genes in order to identify the cellular signalling pathways activated in HCA-7 cells in response to C. jejuni BCE. The transcriptomic data were confirmed by real time quantitative PCR (RQ-PCR).
C. jejuniculture and preparation of BCE
The type strain C. jejuni National Collection of Type Cultures (NCTC) 11168 was used throughout these experiments, since it was originally isolated from a patient with diarrhoea, its genome sequence is available and it has a well-characterized pathological phenotype . It was incubated on blood-agar plates (Blood Agar Base CM0271 from Oxoid, Basingstoke, UK with 5%, v/v defibrinated horse blood) under micro-aerobic conditions for 24 h. and used to inoculate Nutrient Broth no. 2 (Oxoid CM0067, 600 ml in 1000 ml flask). Inoculated flasks were shaken at 140 rpm at 42°C for 16 h. under micro-aerobic conditions. Culture purity was determined by plating samples from each overnight culture onto blood plates and incubating for 24 h., 42°C in micro-aerobic conditions. Bacteria were collected by centrifugation at 10,000 g for 15 min. The cell pellet was washed three times in Phosphate Buffered Saline (PBS), weighed and re-suspended in PBS to achieve a 10% (w/v) suspension, which was boiled for 10 min., cooled on ice for 5 min. before being centrifuged at 10, 000 g for 10 min. The supernatant was collected, passed through a 0.2 μm filter to remove residual bacteria and stored at -20°C until required.
HCA-7 cell culture and treatment with C. jejuniBCE
The human colonocyte line HCA-7 , clone 29, was grown to confluence in a 5% CO2 atmosphere in monolayer cultures on monolayer dishes in Dulbecco's Modified Eagle's Medium supplemented (DMEM) with 100 μg/ml penicillin, 100 μg/ml streptomycin and fetal calf serum at 10% (v/v, Fisher Scientific, Loughborough, UK) at 37°C. Twenty-four hours prior to induction by BCE, HCA-7 cells were transferred to serum-free DMEM. HCA-7 cells were then incubated for 6 h. with 25 μl BCE or PBS control in a total volume of 1 ml of DMEM. The BCE preparation was determined in parallel to induce NF-κB 300-fold using a reporter cell assay . At 6 h. post induction total RNAs were extracted using RNAeasy columns (Qiagen, West Sussex, UK). Total RNA yields and purity were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies UK Limited, Stockport, UK).
Approximately 10 μg of total RNA was reverse transcribed at 42°C for 1 h. to generate first strand DNA using 100 pmol oligo dT(24) primer containing a 5'-T7 RNA polymerase promoter sequence (5'-GCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3'), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT), 10 mM dNTPs and 200 units SuperScript II reverse transcriptase (Invitrogen Life Technologies, Strathclyde, UK). Second strand DNA synthesis was carried out at 16°C for 2 h., using 10 units of E. coli polymerase I, 10 units of E. coli DNA ligase and 2 units of RNase H in a reaction containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 10 mM (NH4)SO4, 0.15 mM β-NAD+ and 10 mM dNTPs. 10 units of T4 DNA polymerase were added and the reaction allowed to proceed for a further 5 min. before termination with 0.5 M EDTA. Double stranded cDNA products were purified using the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA, USA).
The synthetic cDNAs were in vitro transcribed using T7 RNA polymerase (ENZO BioArray High Yield RNA Transcript Labeling Kit, Affymetrix, Santa Clara, CA, USA) with biotinylated ribonucleotides to generated biotinylated complementary RNAs (cRNAs). The cRNAs were purified using the GeneChip Sample Cleanup Module before random fragmentation at 94°C for 35 min. in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate to generate molecules of approximately 35 to 200 bases long.
Changes in gene transcription were analyzed by hybridization to Affymetrix Human Genome U133A array (HG-U133A) which contains probes for over 22,000 transcripts, including representation of the RefSeq database sequences and probe sets http://www.affymetrix.com/products_services/arrays/specific/hgu133.affx. The fragmented cRNAs were mixed with 0.1 mg/ml of sonicated herring sperm DNA in a hybridization buffer containing 100 mM 2-N-morpholino-ethane-sulfonic acid (MES), 1 M NaCl, 20 mM EDTA and 10% Tween 20 to make the hybridization mixture. The hybridization mixture containing the fragmented cRNA was denatured at 99°C for 5 min. and equilibrated for a further 5 min. at 45°C before centrifugation at 10,000 g for 5 min. to remove any insoluble material from the hybridization mixture. The hybridization mix was transferred to the ATH1-121501 genome array (Affymetrix, Santa Clara, CA, USA) cartridge and hybridized at 45°C for 16 h. on a rotisserie at 60 rpm.
After a 16 h. hybridization period the arrays were washed and stained in a Fluidics station (Affymetrix, Santa Clara, USA). The arrays were initially washed in a low stringency buffer A (6 × SSPE [0.9 M NaCl, 0.06 M NaH2PO4, 0.006 M EDTA], 10% Tween 20) at 25°C for 10 min. and then incubated with a high stringency buffer B (100 mM MES, 0.1 M NaCl, 10% Tween 20) at 50°C for 20 min. and stained with 10 mg/ml of streptavidin phycoerythrin (SAPE), in stain buffer containing 100 mM MES, 1 M NaCl, 0.05% Tween 20 and 2 mg/ml BSA at 25°C for 10 min. After a further wash in wash buffer A at 25°C for 20 min. they were stained with biotinylated anti-streptavidin antibody at 25°C for 10 min. After antibody staining the arrays were stained again with SAPE for signal amplification and washed with buffer A at 30°C for 30 min. The arrays were finally scanned and the intensities averaged with the Agilent GeneArray Scanner (Agilent Technology UK, West Lothian, UK).
Statistical analysis of Array data and Generation of Networks and Canonical Pathways
In order to identify genes of interest we used the S Score (Significance Score) algorithm as implemented in the Bioconductor software suite http://www.bioconductor.org based on the R package http://www.r-project.org that takes advantage of the fact that most genes are unchanged and calculates an S score (SD from the mean). The S score threshold of +/- 2.5 and an alpha value of P = 0.005 was used to define gene changes of interest. Data listing all genes that satisfied these criteria were analyzed by Ingenuity Pathway Analysis, Ingenuity® Systems, http://www.ingenuity.com. This generated functional networks and canonical pathways that connect the differentially expressed genes, using the IPA Knowledge base, where the interactions are supported by peer reviewed publications and which contains over 1.4 million interactions between genes, proteins, and drugs. Scores were assigned allowing ranking of the networks, using a Fisher's right tailed exact test.
Analysis of microarray data by real time quantitative PCR
Primers and probes used in the study
ATPase, Na+/K+ transporting, Beta1 polypeptide
Retinoic acid receptor responder (tazarotene induced) 1
tumor necrosis factor, alpha-induced protein 3
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
matrix metallo-peptidase 7
Up-regulated genes. Functional classes of genes shown are ordered by the S score of the most highly regulated examples in the class with S score ≥ 5.
Chemokine (C-C Motif) Ligand 20
Chemokine (C-X-C Motif) Ligand 3
Chemokine (C-X-C Motif) Ligand 2
Chemokine (C-X-C Motif) Ligand 1
Chemokine (C-X-C Motif) Ligand 6
Chemokine (C-C Motif) Ligand 2
Tumor Necrosis Factor, Alpha-Induced Protein 3
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
TNFAIP3 Interacting Protein 1
Tumor Necrosis Factor, Alpha-Induced Protein 2
Nuclear Factor Kappa B (P105)
Baculoviral IAP Repeat-Containing 3
CASP8 And FADD-Like Apoptosis Regulator
Serum/Glucocorticoid Regulated Kinase
Interferon Stimulated Exonuclease Gene 20 kda
Matrix Metallopeptidase 7 (Matrilysin, Uterine)
Syndecan 4 (Amphiglycan, Ryudocan)
Laminin, Alpha 3
Laminin, Gamma 2
Folate Receptor 1 (Adult)
Superoxide Dismutase 2, Mitochondrial
Thioredoxin Reductase 1
Intercellular Adhesion Molecule 1
Fibronectin Type III Domain Containing 3B
Interferon Gamma Receptor 1
Colony Stimulating Factor 2
Plasminogen Activator, Tissue
Serpin Peptidase Inhibitor 2
Atpase, Na+/K+ Transporting, Beta 1 Peptide
CCAAT/Enhancer Binding Protein Delta
Retinoic Acid Receptor Responder
Peptidase Inhibitor 3 (Elafin)
Cell Division Cycle 42
Dual Specificity Phosphatase 5
Sphingosine-1-Phosphate Lyase 1
PDZ And LIM Domain 5
Transcription, protein synthesis and export
Splicing Factor 3b, Subunit 1,
UDP-Glucose Ceramide Glucosyltransferase
Polo-Like Kinase 2
Transporter 1, ATP-Binding Cassette
Chemokine and cytokine analyses
Cultured cells were prepared and induced as described above. After 6 h. incubation, the media was removed and stored at -20°C until examined using a Coulter-Alter Flow Cytometer in conjunction with a BD cytometric bead array human inflammation kit according to manufacturer's instructions (BD Biosciences, Oxford, UK). IL8 and CCL20 (MIP-3α) were specifically measured using a sandwich ELISA, by capture with a murine anti-human IL8 or CCL20 and detected using biotinylated goat anti-human IL8 using streptavidin-coupled horseradish-peroxidase, according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).
The Bioconductor and IPA programs identified 356 genes that changed with a positive or negative S score of 2.5 or greater (maximum 13.54). Three hundred were up-regulated and 56 were down-regulated (Additional file 1).
Table 2 shows 48 genes that were up-regulated with an S score of 5 or greater. These were grouped by class and ordered by the highest S score in each class. Chemokines dominate the most highly up-regulated genes with six of the ten highest S scores. Members of the TNFα-NF-κB super family were also highly up-regulated (Table 2). Other highly up-regulated genes were those involved in apoptosis and ubiquitination, extra-cellular matrix proteins, the folate receptor, superoxide dismutase, thioredoxin reductase, Intercellular Adhesion Molecule (ICAM) 1 and cytokines or their receptors (Colony Stimulating Factor [CSF] 2 and interferon-γ receptor 1).
Down-regulated genes Functional classes of genes shown are ordered by the S score of the most highly regulated examples in the class with S score ≤ -2.6.
Cell cycle, DNA replication and Mitosis
Inhibitor Of DNA Binding 1
Inhibitor Of DNA Binding 3
Inhibitor Of DNA Binding 2
LIM Homeobox 3
Kruppel-Like Factor 1
Forkhead Box F2
Fibroblast Growth Factor Binding Protein 1
S-Phase Kinase-Associated Protein 2 (P45)
Replication Protein A3
Replication Factor C 4
Spindle Pole Body Component 25 Homolog
Regenerating Islet-Derived 1 Alpha
Collagen, Type IV, Alpha 5
Outer Dense Fiber Of Sperm Tails 1
CD248 Molecule, Endosialin
Solute Carrier Family 2, Member 1
Cysteine-Rich Protein 1 (Intestinal)
Sodium Channel, Nonvoltage-Gated 1 Alpha
Transcription, protein synthesis and export
Chromatin Modifying Protein 6
RAN Binding Protein 1
EH Domain Binding Protein 1
Ribonucleotide Reductase M2 Polypeptide
Small Carboxy-Terminal Domain Phosphatase
Aspartyl-Trna Synthetase 2 (Mitochondrial)
Polymerase (RNA) Subunit K
ATPase Family, AAA Domain Containing 4
Cytochrome C Oxidase Subunit 7B
Myosin Phosphatase-Rho Interacting Protein
Mal, T-Cell Differentiation Protein-Like
Rho Gtpase Activating Protein 29
Rho-Associated, Coiled-Coil Containing Protein Kinase 2
Transforming Growth Factor, Beta 2
C1q And TNF Related Protein 3
Serine Peptidase Inhibitor, Kazal Type 1
Thioredoxin Interacting Protein
HS1-Binding Protein 3
Annexin A1 (Lipocortin 1)
Laminin, Beta 1
The antigen presentation pathway was identified through up-regulation of the Large Multifunctional Protease (LMP)-7, Transporter Associated with Antigen Processing (TAP) 1, TAP-binding protein (TAPBP), Calreticulin (CALR) and the Major Histocompatibility Complex (MHC)1-α.
Activation of the interferon-γ receptor defence signalling pathway was noted through up-regulation of both components of interferon-γ receptor, Janus kinase (JAK) 1 and Tyrosine Kinase (TYK) 2.
Activation of the ephrin signalling pathway, indicating activation of actin-based cytokinesis and repulsion. The pathway included up-regulation of ephrin receptor sub components, RHO family, GTP binding protein (Rac1), Cell Division Cycle (CDC) 42, Wiskott-Aldrich syndrome protein (WASP), actin-related protein 2 (ARP2), V-crk homologue (CRK) and Ras oncogene family member (RAP)1B with rho-associated coiled-coil containing protein kinase (ROCK) 2.
Finally, up-regulation of most components of the PI3K-phosphatase signalling pathway were noted, including phosphatase and tensin homology (PTEN) pathway indicating possible effects on the cell cycle, including Cell Division Cycle (CDC) 37, Forkhead Box (FOX)O1A and Cyclin Dependent Kinase Inhibitor (CDKN)1a (P21). SFN (Stratifin or 14-3-3σ) however, was down-regulated.
Predicted functional effects
The IPA program constructed 16 interconnected gene networks that were significantly altered as a result of treatment of HCA-7 cells with C. jejuni BCE, all with network scores of ≥ 8. The network score is the probability that a network would be assembled by chance where a level of > 3 is statistically significant, at p < 0.001. In the four most significantly regulated all 35 focus genes of the network were affected, all giving an identical score of 52 (P < 1E-52).
The first network (Figure 3) contains genes concerned with cellular movement, particularly chemotaxis. NF-κB occupies a central position in the network and includes a number of genes which are known to up-regulate including a number of chemokines.
The second network (Additional file 2) likewise contains genes associated with cellular movement, including cytokinesis and inflammatory responses. Up-regulated genes include Ephrin Receptor B2 (EPHB2), PTGS2 (COX-2), ICAM1, both components of interferon-γ receptor, IL23A, IL27RA, JAK1, JUNB proto oncogene, Mitogen Activated Protein Kinase Kinase Kinase Kinase (MAP4K4), TYK2, Mothers Against DPP homologues (SMAD) 3, with 2 genes shown to be significantly down-regulated (SH2B and Transforming Growth Factor [TGF] β2).
MYC occupies a central position in the third network (Additional file 3), which contains genes concerned with the regulation of the cell cycle. Up-regulated genes include MYC as well as FAS, folate receptor (FOLR1), HLA molecules E, F and G, laminins β3, α3 (LAM-B3, A3) and γ2 (LAMC2), Matrix Metallo Proteinase (MMP)7, and SOD2. Down-regulated were Laminin β1 (LAMB1), RAN Binding Protein 1 (RANBP1) Thioredoxin Interacting Protein (TXNIP) and Thymidylate Synthetase (TYMS).
Finally, a network (Additional file 4) contains genes affecting cell death and gene expression. The network contains 25 genes that were up-regulated, including Activating Transcription Factor (ATF) 3, cellular Inhibitor of Apoptosis Proteins (cIAP) 1 and 2 (BIRC 2 and 3), cyclin dependent kinase (CDK) 7, cyclin dependant kinase inhibitor (CDKN) 1A, GATA binding protein (GATA) 6, TNFα-Induced Protein (TNFAIP) 2, the TNF-Related Apoptosis-Inducing Ligand (TRAIL or TNFSF10), its receptor TRAILR2 (TNFRSF10B or Death receptor [DR] 5) and TNF Receptor Associated Factor (TRAF) 2. Whilst CDKN1A is up-regulated, CDKN3 is down-regulated, as are the Inhibitors of DNA Binding (ID)1,2 and 3, Mini-Chromosome Maintenance homologue (MCM) 6, RCF4, rho-associated, coiled-coil containing protein kinase (ROCK) 2 and S-Phase Kinase-Associated Protein (SKP) 2.
Validation of Microarray data
Comparison of results for selected up-regulated genes determined by Affymetrix/S score and RQ-PCR.
Affymetrix Probe Set
59.4 ± 15.5
See Figure 3
ATPase, Na+/K+ transporting, Beta 1 polypeptide
4.5 ± 1.8
4.0 ± 0.84
Retinoic acid receptor responder (tazarotene induced) 1
tumor necrosis factor, alpha-induced protein 3
2.0 ± 0.2
See Figure 3
nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha
4.0 ± 1.2.
See Figure 3
Matrix Metallo-peptidase 7
2.1 ± 4.2
9 & See Additional file 3
Chemokine and cytokine responses
Cytokine and chemokine levels (pg/ml) pre- and post-induction of HCA-7 cells with C. jejuni BCE for 6 h.
12 (± 2)
15 (± 3)
30 (± 3)
150 (± 5)
20 (± 4)
30 (± 6)
40 (± 16)
18,400 (± 400)
30 (± 6)
380 (± 40)
Understanding the pathogenesis of C. jejuni enteric disease is important both because C. jejuni is a major cause of diarrhoeal illness worldwide and because it may serve as a model for ulcerative colitis, the pathology of which it closely resembles . Previous work has shown that direct interaction between C. jejuni and epithelial cells is capable of inducing pro-inflammatory and pro-secretory processes [8, 16]. These are associated with cellular invasion  and secretion of IL8 by CLDT dependent and independent mechanisms [16, 18]. Direct use of a BCE has allowed us to use a reductionist approach to investigate effects of C. jejuni that are not dominated by these linked processes of cellular invasion by live bacteria and by toxin based cell lysis. BCE has been determined to contain polysaccharide and protein components of the cell. As demonstrated previously the NF-κB inducing activity of C. jejuni BCE is relatively insensitive to digestion by protease K . However the protein content has been determined using tryptic digests of SDS-polyacryamide extracted protein bands using MALDI-TOF mass spectrometry as flagellin (Cj1339c), trigger factor (Cj0193c), lipoprotein (Cj0983), major outer membrane protein (Cj0599), cytochrome-c peroxidase (Cj0358), bacterioferritin (Cj1534c), cell binding factor PEB4A (Cj0496), hypothetical protein (Cj0706), periplasmic protein (Cj0772c), fibronectin binding protein (Cj1478c), non-heme iron protein (Cj0012c), periplasmic protein (Cj1380), periplasmic protein (Cj0420), periplasmic protein (Cj0998c), DNA-binding protein HU (Cj0913c), periplasmic cytochrome C (Cj1153) and thioredoxin (Cj0147c) . The polysaccharide component features α-glucan oligomers. The C. jejuni extract is notably devoid of the dominating heat-labile effects of the CLDT. C. jejuni BCE, like infection with live C. jejuni, has been shown to be a potent inducer of NF-κB using either luciferase based reporter assays, western blots with antibodies against IκB or electrophoretic mobility shift assays in epithelial cells  but, unlike treatment with live C. jejuni, this does not lead to host cell lysis. These observations are consistent with the hypothesis that a heat stable component plays a significant role in the pro-inflammatory response upon exposure to C. jejuni.
We hypothesize that NF-κB modulation is central to the response of enterocytes to C. jejuni BCE; to study this we determined the global changes in gene expression induced by C. jejuni BCE treatment of the well-differentiated human colonocyte line HCA-7, clone 29. In order to ensure the relevance of our results we have adopted stringent criteria for the identification of significantly affected genes and used the IPA program to determine the functional links between these gene products, identify the signalling pathways and networks to which they belong. These changes were validated by showing similar affects on mRNA levels when genes of interest were investigated by real-time quantitative PCR.
Consistent with the initial hypothesis that NF-κB plays a major role in the response of HCA-7 cells to C. jejuni BCE, and features in 8 of the 11 designated signalling pathways identified by IPA as up-regulated. Moreover, all genes in the NF-κB associated network (Figure 3) were up-regulated by C. jejuni BCE. The dominant component of this response concerned up regulation of chemokines that would act to induce the influx of acute inflammatory cells that characterize Campylobacter colitis. Our data are remarkably similar to transcriptomic data reported by Hinata et al., who activated NF-κB by transfecting clones expressing subunits of NF-κB to show up-regulation of the chemokines CXCL3 (GRO3) IL8, CXCL6, CXCL2 (GRO2), CXCL20 (SCYA20), CXCL1 (GRO1), CCL2 (CXYA2) as well as IL1α and CSF2, all of which were also significantly up-regulated in our study . The NFKB1, NFKB2 and RELB components of NF-κB are also similarly up-regulated in our study. Other changes that are likely to be of functional importance and are the up-regulation of COX2 (PTGS2), TNIP2, MYC, SOD2, ELF3 and ICAM1 (Additional file 1), where all of these processes are also downstream targets of NF-κB  and mediators of feedback inhibition of NF-κB activation such as NFKBIA (IκB) , TNIP1  and TNIP2 (Figure 3) . A central role for NF-κB is also supported by data using the monocytic cell line THP-1 . Studies in which Caco-2 cells were incubated with live bacteria resulted in expression of many genes similar to those reported here, including chemokines, but additionally, the NF-κB inhibitor NFKBIZ . This difference may reflect the ability of live bacteria to invade cells and/or elaborate a CLDT with DNase activity .
The pattern of significantly down-regulated genes (Table 3) is remarkably different with a reduction in expression in constitutively expressed genes concerned with nucleotide synthesis, transcription, DNA replication, mitosis, structural protein synthesis, membrane transport and energy metabolism. These changes likely reflect the reprioritization of cellular metabolism in response to pro-inflammatory products.
Whether the changes caused by the C. jejuni BCE would lead to increased or reduced apoptosis is difficult to predict, especially as HCA-7 lack a functional TP53 protein, although these cells are capable of apoptosis given the appropriate signal . Invasive C. jejuni infection can cause cell death in HCA-7 cells , although we did not see this with the addition of BCE . Increased expression of members of the death receptor pathway, the TNFα superfamily and their receptors, but also of TNFα agonists may imply regulated activation of pro-apoptotic activity [26–30]. Up-regulation of TRAIL, DR5, and FAS ligand acting via FADD, the universal adaptor protein known domain-containing members of the TNF receptor superfamily, would successively activate caspases 8, 10 and 3 as well as possible G1-S cell cycle progression . However, the antagonists TNFAIP3, FLIP and cIAP, which respectively inhibit apoptosis via TRAF6, caspases 8, 9, 10 and TRAF-2 directly or indirectly are also prominent amongst the up-regulated genes [29–32].
Moreover, several other key proteins for the cell cycle and apoptosis are affected. Thus CDKN1A (P21, WAF, WAF1 or CIP1) which plays a pivotal role in inhibiting cell cycle progression at several points in response to DNA damage , is up-regulated, as are FOXO1A and SMAD 2 (Additional file 1) and 3 (Additional file 2), which act together to increase CDKN1A activity [34, 35]. Conversely, other genes that inhibit cell cycle progression are down-regulated. These include SKP2, the F-box receptor that interacts with p19 and the CDK2/cyclin A to prevent entry into G1  and SFN (stratifin or 14-3-3σ) a key target of the tumour suppressor gene TP53 which acts to cause G2 arrest .
Five other changes of potential functional importance are of note. Firstly, a number of potentially antibacterial agents are highly induced, including LCN2 (lipocalin-2) [38, 39] and PI3 (peptidase inhibitor 3, aka ELAFIN) , whilst MMP7 is thought to activate defensins . Secondly, five key molecules involved in antigen processing and presentation (Figure 1, 2)  were also up-regulated and could play a role in the development of immune responses to C. jejuni. Thirdly, alterations in matrix metalloproteinases and leukocyte receptors would influence the inflammatory response, with MMP9 acting to facilitate neutrophil transfer by activating interleukin-8  and MMP7 acting to localize them to sites of tissue damage . Fourthly, the ephrin pathway (Figure 2), including Ephrin A2 and B2 receptors (EPHA2, EPHB2) and Ephrin A1 (EFNA1, Figure 3), rho kinase (ROCK2), Rac, ARP2/3, CDC42 and WASP appeared to be strongly up-regulated. This pathway is concerned with activation of cytokinetic changes that may potentially play a role in rapid restitution [45, 46]. Finally, up-regulation of the folate receptor (FOLR1) may reflect preparation for reparative nucleotide synthesis dependent upon one-carbon transfer activity .
The data we have generated using a BCE of C. jejuni represents a reductionist approach to determine some of the cellular responses associated with C. jejuni infection. However, because C. jejuni BCE represents a robust NF-κB inducing activity that is not only heat-stable but resistant to protease and acidic pH (pH 3) , these may indeed be of clinical significance if these products are shed upon C. jejuni infection or co-delivered through the diet. C. jejuni has been detected in many commercially available chicken portions  and clinical cases of Campylobacter enterocolitis are frequently associated with ingestion of partially cooked poultry meat .
Changes in host gene expression following C. jejuni BCE interestingly reflects some of the changes that are known to occur in inflammatory bowel diseases (IBD) such as ulcerative colitis, for which C. jejuni colitis can be considered a model, and may therefore indicate other potential targets for investigation of epithelial-derived mediators of inflammation in ulcerative colitis/IBD. Up-regulation of NF-κB is well recognized and considered a possible target of mesalazine [49, 50]. Genes up-regulated by C. jejuni that have been associated with active ulcerative colitis/IBD include chemokines , such as IL8 and CCL20 (macrophage inflammatory protein 3α) [52–54] cytokines, including TNFα , eicosanoids  and elafin . IL23, IL32 [57–59] and receptors such as interferon-γ receptor, and TLR2  have all been demonstrated to be altered here (Table 2, Additional file 1). Activation of pro-apoptotic pathways involving the TNF superfamily and death domain signalling pathway have been reported to be up-regulated in colonic enterocytes isolated from patients with ulcerative colitis, from which C-IAP2 (BIRC3) has been proposed as a disease marker , whilst the leukocytes serine anti-proteinase elafin has recently been identified as a candidate biomarker for ulcerative colitis but with attenuated induction in Crohn's disease . Thus, the data we report here include a number of pathways and mediators that may be realistic anti-inflammatory therapeutic targets to prevent or reduce the activity of C. jejuni colitis or ulcerative colitis. These targets include mechanisms for chemoattraction of inflammatory cells, cellular processes associated with repair and the processes associated with apoptosis, as well as NF-κB itself, the utilization of which can be investigated by intervention studies in model systems and humans.
We acknowledge the assistance if Drs. Janet Higgins and John Okyere, at NASC array service, University of Nottingham and Mr. Lyndon Cochrane for help with illustrations. IFC and KHM were the recipients of a grant award from the Biotechnology and Biological Research Council, UK
- Snelling WJ, Matsuda M, Moore JE, Dooley JS: Campylobacter jejuni. Lett Appl Microbiol. 2005, 41 (4): 297-302. 10.1111/j.1472-765X.2005.01788.x.PubMedView ArticleGoogle Scholar
- Young KT, Davis LM, Dirita VJ: Campylobacter jejuni: molecular biology and pathogenesis. Nat Rev Microbiol. 2007, 5 (9): 665-679. 10.1038/nrmicro1718.PubMedView ArticleGoogle Scholar
- Jorgensen F, Bailey R, Williams S, Henderson P, Wareing DR, Bolton FJ, Frost JA, Ward L, Humphrey TJ: Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods. Int J Food Microbiol. 2002, 76 (1–2): 151-164. 10.1016/S0168-1605(02)00027-2.PubMedView ArticleGoogle Scholar
- Hughes RA, Cornblath DR: Guillain-Barre syndrome. Lancet. 2005, 366 (9497): 1653-1666. 10.1016/S0140-6736(05)67665-9.PubMedView ArticleGoogle Scholar
- Lecuit M, Abachin E, Martin A, Poyart C, Pochart P, Suarez F, Bengoufa D, Feuillard J, Lavergne A, Gordon JI, et al: Immunoproliferative small intestinal disease associated with Campylobacter jejuni. N Engl J Med. 2004, 350 (3): 239-248. 10.1056/NEJMoa031887.PubMedView ArticleGoogle Scholar
- Guerry P: Campylobacter flagella: not just for motility. Trends Microbiol. 2007, 15 (10): 456-461. 10.1016/j.tim.2007.09.006.PubMedView ArticleGoogle Scholar
- Smith JL, Bayles DO: The contribution of cytolethal distending toxin to bacterial pathogenesis. Crit Rev Microbiol. 2006, 32 (4): 227-248. 10.1080/10408410601023557.PubMedView ArticleGoogle Scholar
- Mellits KH, Mullen J, Wand M, Armbruster G, Patel A, Connerton PL, Skelly M, Connerton IF: Activation of the transcription factor NF-kappaB by Campylobacter jejuni. Microbiology. 2002, 148 (Pt 9): 2753-2763.PubMedView ArticleGoogle Scholar
- Brasier AR: The NF-kappaB regulatory network. Cardiovasc Toxicol. 2006, 6 (2): 111-130. 10.1385/CT:6:2:111.PubMedView ArticleGoogle Scholar
- Kirkland SC: Dome formation by a human colonic adenocarcinoma cell line (HCA-7). Cancer Res. 1985, 45 (8): 3790-3795.PubMedGoogle Scholar
- Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, Chillingworth T, Davies RM, Feltwell T, Holroyd S, et al: The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature. 2000, 403 (6770): 665-668. 10.1038/35001088.PubMedView ArticleGoogle Scholar
- Kennedy RE, Kerns RT, Kong X, Archer KJ, Miles MF: SScore: an R package for detecting differential gene expression without gene expression summaries. Bioinformatics. 2006, 22 (10): 1272-1274. 10.1093/bioinformatics/btl108.PubMedView ArticleGoogle Scholar
- Zhang J, Carey V, Gentleman R: An extensible application for assembling annotation for genomic data. Bioinformatics. 2003, 19 (1): 155-156. 10.1093/bioinformatics/19.1.155.PubMedView ArticleGoogle Scholar
- Huggett J, Dheda K, Bustin S, Zumla A: Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005, 6 (4): 279-284. 10.1038/sj.gene.6364190.PubMedView ArticleGoogle Scholar
- Colgan T, Lambert JR, Newman A, Luk SC: Campylobacter jejuni enterocolitis. A clinicopathologic study. Arch Pathol Lab Med. 1980, 104 (11): 571-574.PubMedGoogle Scholar
- Beltinger J, Brough J, Skelly MM, Thornley J, Spiller RC, Stack WA, Hawkey CJ: Disruption of colonic barrier function and induction of mediator release by strains of Campylobacter jejuni that invade epithelial cells. 2008, 14 (48): 7345-52.Google Scholar
- Konkel ME, Kim BJ, Rivera-Amill V, Garvis SG: Identification of proteins required for the internalization of Campylobacter jejuni into cultured mammalian cells. Adv Exp Med Biol. 1999, 473: 215-224.PubMedView ArticleGoogle Scholar
- Hickey TE, McVeigh AL, Scott DA, Michielutti RE, Bixby A, Carroll SA, Bourgeois AL, Guerry P: Campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells. Infect Immun. 2000, 68 (12): 6535-6541. 10.1128/IAI.68.12.6535-6541.2000.PubMed CentralPubMedView ArticleGoogle Scholar
- Hinata K, Gervin AM, Jennifer Zhang Y, Khavari PA: Divergent gene regulation and growth effects by NF-kappa B in epithelial and mesenchymal cells of human skin. Oncogene. 2003, 22 (13): 1955-1964. 10.1038/sj.onc.1206198.PubMedView ArticleGoogle Scholar
- Yamamoto Y, Gaynor RB: IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem Sci. 2004, 29 (2): 72-79. 10.1016/j.tibs.2003.12.003.PubMedView ArticleGoogle Scholar
- Heyninck K, Kreike MM, Beyaert R: Structure-function analysis of the A20-binding inhibitor of NF-kappa B activation, ABIN-1. FEBS Lett. 2003, 536 (1–3): 135-140. 10.1016/S0014-5793(03)00041-3.PubMedView ArticleGoogle Scholar
- Van Huffel S, Delaei F, Heyninck K, De Valck D, Beyaert R: Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2. J Biol Chem. 2001, 276 (32): 30216-30223. 10.1074/jbc.M100048200.PubMedView ArticleGoogle Scholar
- Jones MA, Totemeyer S, Maskell DJ, Bryant CE, Barrow PA: Induction of proinflammatory responses in the human monocytic cell line THP-1 by Campylobacter jejuni. Infect Immun. 2003, 71 (5): 2626-2633. 10.1128/IAI.71.5.2626-2633.2003.PubMed CentralPubMedView ArticleGoogle Scholar
- Rinella ES, Eversley CD, Carroll IM, Andrus JM, Threadgill DW, Threadgill DS: Human epithelial-specific response to pathogenic Campylobacter jejuni. FEMS Microbiol Lett. 2006, 262 (2): 236-243.PubMedView ArticleGoogle Scholar
- Huang X, Guo B: Adenomatous polyposis coli determines sensitivity to histone deacetylase inhibitor-induced apoptosis in colon cancer cells. Cancer Res. 2006, 66 (18): 9245-9251. 10.1158/0008-5472.CAN-06-0887.PubMedView ArticleGoogle Scholar
- Yan N, Shi Y: Mechanisms of apoptosis through structural biology. Annu Rev Cell Dev Biol. 2005, 21: 35-56. 10.1146/annurev.cellbio.21.012704.131040.PubMedView ArticleGoogle Scholar
- Werner MH, Wu C, Walsh CM: Emerging roles for the death adaptor FADD in death receptor avidity and cell cycle regulation. Cell Cycle. 2006, 5 (20): 2332-2338.PubMedView ArticleGoogle Scholar
- Wajant H, Scheurich P: Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling. Int J Biochem Cell Biol. 2001, 33 (1): 19-32. 10.1016/S1357-2725(00)00064-9.PubMedView ArticleGoogle Scholar
- Beyaert R, Heyninck K, Van Huffel S: A20 and A20-binding proteins as cellular inhibitors of nuclear factor-kappa B-dependent gene expression and apoptosis. Biochem Pharmacol. 2000, 60 (8): 1143-1151. 10.1016/S0006-2952(00)00404-4.PubMedView ArticleGoogle Scholar
- Liston P, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvat G, Farahani R, McLean M, Ikeda JE, MacKenzie A, et al: Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature. 1996, 379 (6563): 349-353. 10.1038/379349a0.PubMedView ArticleGoogle Scholar
- Kataoka T: The caspase-8 modulator c-FLIP. Crit Rev Immunol. 2005, 25 (1): 31-58. 10.1615/CritRevImmunol.v25.i1.30.PubMedView ArticleGoogle Scholar
- Li X, Yang Y, Ashwell JD: TNF-RII and c-IAP1 mediate ubiquitination and degradation of TRAF2. Nature. 2002, 416 (6878): 345-347. 10.1038/416345a.PubMedView ArticleGoogle Scholar
- Sherr CJ, Roberts JM: CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999, 13 (12): 1501-1512. 10.1101/gad.13.12.1501.PubMedView ArticleGoogle Scholar
- Arden KC: FoxO: linking new signaling pathways. Mol Cell. 2004, 14 (4): 416-418. 10.1016/S1097-2765(04)00213-8.PubMedView ArticleGoogle Scholar
- Seoane J, Le HV, Shen L, Anderson SA, Massague J: Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell. 2004, 117 (2): 211-223. 10.1016/S0092-8674(04)00298-3.PubMedView ArticleGoogle Scholar
- Pagano M: Control of DNA synthesis and mitosis by the Skp2-p27-Cdk1/2 axis. Mol Cell. 2004, 14 (4): 414-416. 10.1016/S1097-2765(04)00268-0.PubMedView ArticleGoogle Scholar
- Wilker EW, van Vugt MA, Artim SA, Huang PH, Petersen CP, Reinhardt HC, Feng Y, Sharp PA, Sonenberg N, White FM, et al: 14-3-3sigma controls mitotic translation to facilitate cytokinesis. Nature. 2007, 446 (7133): 329-332. 10.1038/nature05584.PubMedView ArticleGoogle Scholar
- Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, Aderem A: Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature. 2004, 432 (7019): 917-921. 10.1038/nature03104.PubMedView ArticleGoogle Scholar
- Borregaard N, Cowland JB: Neutrophil gelatinase-associated lipocalin, a siderophore-binding eukaryotic protein. Biometals. 2006, 19 (2): 211-215. 10.1007/s10534-005-3251-7.PubMedView ArticleGoogle Scholar
- Sallenave JM: The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease. Respir Res. 2000, 1 (2): 87-92. 10.1186/rr18.PubMed CentralPubMedView ArticleGoogle Scholar
- Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, Lopez-Boado YS, Stratman JL, Hultgren SJ, Matrisian LM, Parks WC: Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense. Science. 1999, 286 (5437): 113-117. 10.1126/science.286.5437.113.PubMedView ArticleGoogle Scholar
- Kloetzel PM: Generation of major histocompatibility complex class I antigens: functional interplay between proteasomes and TPPII. Nat Immunol. 2004, 5 (7): 661-669. 10.1038/ni1090.PubMedView ArticleGoogle Scholar
- Steen Van den PE, Proost P, Wuyts A, Van Damme J, Opdenakker G: Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminoterminal processing, whereas it degrades CTAP-III, PF-4, and GRO-alpha and leaves RANTES and MCP-2 intact. Blood. 2000, 96 (8): 2673-2681.PubMedGoogle Scholar
- Li Q, Park PW, Wilson CL, Parks WC: Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell. 2002, 111 (5): 635-646. 10.1016/S0092-8674(02)01079-6.PubMedView ArticleGoogle Scholar
- Kullander K, Klein R: Mechanisms and functions of Eph and ephrin signalling. Nat Rev Mol Cell Biol. 2002, 3 (7): 475-486. 10.1038/nrm856.PubMedView ArticleGoogle Scholar
- Anton IM, Jones GE, Wandosell F, Geha R, Ramesh N: WASP-interacting protein (WIP): working in polymerisation and much more. Trends Cell Biol. 2007, 17 (11): 555-562. 10.1016/j.tcb.2007.08.005.PubMedView ArticleGoogle Scholar
- Stanger O: Physiology of folic acid in health and disease. Curr Drug Metab. 2002, 3 (2): 211-223. 10.2174/1389200024605163.PubMedView ArticleGoogle Scholar
- Friedman CR, Neimann J, Wegener HC, Tauxe RV: Epidemiology of Campylobacter jejuni infections in the United States and other industrialized nations. Campylobacter. Edited by: Nachamkin I, Blaser MJ. 2000, Washington, DC ASM Press, 121-138. 2Google Scholar
- Zhang SZ, Zhao XH, Zhang DC: Cellular and molecular immunopathogenesis of ulcerative colitis. Cell Mol Immunol. 2006, 3 (1): 35-40.PubMedGoogle Scholar
- Bantel H, Berg C, Vieth M, Stolte M, Kruis W, Schulze-Osthoff K: Mesalazine inhibits activation of transcription factor NF-kappaB in inflamed mucosa of patients with ulcerative colitis. Am J Gastroenterol. 2000, 95 (12): 3452-3457.PubMedGoogle Scholar
- Papadakis KA: Chemokines in inflammatory bowel disease. Curr Allergy Asthma Rep. 2004, 4 (1): 83-89. 10.1007/s11882-004-0048-7.PubMedView ArticleGoogle Scholar
- Mahida YR, Ceska M, Effenberger F, Kurlak L, Lindley I, Hawkey CJ: Enhanced synthesis of neutrophil-activating peptide-I/interleukin-8 in active ulcerative colitis. Clinical Science. 1992, 82 (3): 273-275.PubMedView ArticleGoogle Scholar
- Cole AT, Pilkington BJ, McLaughlan J, Smith C, Balsitis M, Hawkey CJ: Mucosal factors inducing neutrophil movement in ulcerative colitis: the role of interleukin 8 and leukotriene B4. Gut. 1996, 39 (2): 248-254. 10.1136/gut.39.2.248.PubMed CentralPubMedView ArticleGoogle Scholar
- Watanabe S, Yamakawa M, Hiroaki T, Kawata S, Kimura O: Correlation of dendritic cell infiltration with active crypt inflammation in ulcerative colitis. Clin Immunol. 2007, 122 (3): 288-297. 10.1016/j.clim.2006.10.013.PubMedView ArticleGoogle Scholar
- Fujino S, Andoh A, Bamba S, Ogawa A, Hata K, Araki Y, Bamba T, Fujiyama Y: Increased expression of interleukin 17 in inflammatory bowel disease. Gut. 2003, 52 (1): 65-70. 10.1136/gut.52.1.65.PubMed CentralPubMedView ArticleGoogle Scholar
- Flach CF, Eriksson A, Jennische E, Lange S, Gunnerek C, Lonnroth I: Detection of elafin as a candidate biomarker for ulcerative colitis by whole-genome microarray screening. Inflamm Bowel Dis. 2006, 12 (9): 837-842. 10.1097/01.mib.0000232469.23574.11.PubMedView ArticleGoogle Scholar
- McGovern D, Powrie F: The IL23 axis plays a key role in the pathogenesis of IBD. Gut. 2007, 56 (10): 1333-1336. 10.1136/gut.2006.115402.PubMed CentralPubMedView ArticleGoogle Scholar
- Lakatos PL, Szamosi T, Szilvasi A, Molnar E, Lakatos L, Kovacs A, Molnar T, Altorjay I, Papp M, Tulassay Z, et al: ATG16L1 and IL23 receptor (IL23R) genes are associated with disease susceptibility in Hungarian CD patients. 2008, 40 (11): 867-73.Google Scholar
- Shioya M, Nishida A, Yagi Y, Ogawa A, Tsujikawa T, Kim-Mitsuyama S, Takayanagi A, Shimizu N, Fujiyama Y, Andoh A: Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease. Clin Exp Immunol. 2007, 149 (3): 480-486.PubMed CentralPubMedView ArticleGoogle Scholar
- Rodriguez-Bores L, Fonseca GC, Villeda MA, Yamamoto-Furusho JK: Novel genetic markers in inflammatory bowel disease. World J Gastroenterol. 2007, 13 (42): 5560-5570.PubMed CentralPubMedView ArticleGoogle Scholar
- Seidelin JB, Nielsen OH: Expression profiling of apoptosis-related genes in enterocytes isolated from patients with ulcerative colitis. Apmis. 2006, 114 (7–8): 508-517. 10.1111/j.1600-0463.2006.apm_116.x.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.