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. 2004 Jun;112(2):310–320. doi: 10.1111/j.1365-2567.2004.01874.x

Differential effect of immune cells on non-pathogenic Gram-negative bacteria-induced nuclear factor-κB activation and pro-inflammatory gene expression in intestinal epithelial cells

D Haller *,‡, L Holt *, A Parlesak †, J Zanga *, A Bäuerlein †, R B Sartor *, C Jobin *
PMCID: PMC1782483  PMID: 15147574

Abstract

We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-κB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism of immune-epithelial cell cross-talk on Gram-negative enteric bacteria-induced NF-κB signalling and pro-inflammatory gene expression in IEC using HT-29/MTX as well as CaCO-2 transwell cultures Interestingly, while differentiated HT-29/MTX cells are unresponsive to non-pathogenic Gram negative bacterial stimulation, interleukin-8 (IL-8) mRNA accumulation is strongly induced in Escherichia coli- but not Bacteroides vulgatus-stimulated IEC cocultured with peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC). The presence of PBMC triggered both E. coli- and B. vulgatus-induced mRNA expression of the Toll-like receptor-4 accessory protein MD-2 as well as endogenous IκBα phosphorylation, demonstrating similar capabilities of these bacteria to induce proximal NF-κB signalling. However, B. vulgatus failed to trigger IκBα degradation and NF-κB transcriptional activity in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation, PBMC from patients with active ulcerative colitis and Crohn's disease differentially trigger epithelial cell activation in response to E. coli and E. coli-derived LPS. In conclusion, this study provides evidence for a differential regulation of non-pathogenic Gram-negative bacteria-induced NF-κB signalling and IL-8 gene expression in IEC cocultured with immune cells and suggests the presence of mechanisms that assure hyporesponsiveness of the intestinal epithelium to certain commensally enteric bacteria.

Keywords: gene expression, gram negative bacteria, inflammatory bowel disease, intestinal epithelial cells, NF-κB, signalling

Introduction

The chronically relapsing inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), appear to be the consequence of overly aggressive immune responses to enteric bacterial components in genetically predisposed individuals.1–3 Duchmann et al. reported a loss of immunologic tolerance in active IBD with mucosal T lymphocytes proliferating in response to enteric bacteria, including the Gram-negative species Bacteroides vulgatus and Escherichia coli.4–6 These results are consistent with a lack of inflammation in bypassed distal ileal or colonic segments after proximal diversion of the faecal stream, but immune activation evident within one week of perfusion of effluent into the bypassed ileum.7–9 In addition, a mechanistic role for the enteric bacterial flora in the pathogenesis of chronic mucosal inflammation is shown in various animal models of experimental colitis.10,11 For example, reconstitution studies of gnotobiotic HLA-B27 transgenic rats12,13 and carrageenan-induced colitis in guinea pigs14 implicate B. vulgatus as particularly important to the induction of colitis in these models. In contrast, Autenrieth and colleagues showed protective effects of B. vulgatus on the development of E. coli-mediated experimental colitis in interleukin (IL)-2–/– mice.15 Despite these findings the mechanisms by which commensal bacteria trigger host mucosal immune response is unclear.

Intestinal epithelial cells (IEC), which isolate the host from the gut luminal environment constitutively express, or can be induced to express Toll-like receptors (TLR), costimulatory molecules, components of the human major histocompatibility complex (MHC) and a wide range of inflammatory and chemoattractive cytokines when activated by enteric pathogens or inflammatory products.16–19 Activated IEC produce a characteristic profile of cytokines, including the chemokine IL-8, which induces neutrophil migration in the gut mucosa.20 Most of the chemokines and other inducible molecules expressed upon exposure to bacterial pathogens or proinflammatory cytokines are transcriptionally regulated by nuclear factor (NF)-κB.16 We have previously shown that the non-pathogenic enteric bacterial strain B. vulgatus induces RelA phosphorylation, NF-κB transcriptional activation and pro-inflammatory gene expression in primary and IEC lines.21,22 This suggests that non-pathogenic as well as pathogenic bacteria have the potential to activate inflammatory processes in the gut epithelium. IEC must adapt to a constant changing luminal environment by processing different biological information through multiple signalling cascades that target a defined set of genes, in order to provide an adequate effector response. In vivo, the response of IEC to the commensal microflora might be regulated by adjacent lamina propria mononuclear cells, which mediate immunologic tolerance and maintain mucosal homeostasis.23 Immune-epithelial cell cross-talk was shown to differentially affect pro-inflammatory gene expression in IEC in response to commensal bacteria.22,24 Importantly, these extracellular signals must be integrated with the action of various cytoplasmic and nuclear signal processes controlling transcription factor activity and gene expression. In the case of NF-κB, this implies activation of the IκB/NF-κB complex which leads to the release of the transcription factor from its cytoplasmic inhibitor IκB, shuttling to the nucleus and access to gene promoter regions.25–28

In this study, we investigated the effect of peripheral blood mononuclear cells (PBMC) from healthy controls as well as from Crohn's disease (CD) and ulcerative colitis (UC) patients with active disease and lamina propria mononuclear cells (LPMC) on Gram-negative enteric E. coli- or B. vulgatus-mediated signalling in IEC. We found that immune cells differentially regulate bacteria-induced NF-κB signalling as well as κB-dependent gene expression in IEC.

Materials and methods

Bacteria and culture conditions

B. vulgatus derived from a guinea pig with carrageenan-induced colitis (a gift from A. B. Onderdonk, Harvard University, Cambridge, MA) was anaerobically grown at 37° in brain–heart-infusion (BHI) broth supplemented with cysteine (0·05%), hemine (5 mg/l) and resazurin. E. coli derived from a patient with active Crohn's disease (provided by the Clinical Microbiology Laboratory of the University of North Carolina Hospitals, Chapel Hill, NC) was aerobically grown in luria broth (LB) containing tryptone (1%), yeast (0·5%) extract and NaCl (0·5%). All bacteria were harvested by centrifugation (3000 g, 15 min) at stationary growth phase, washed three times with phosphate-buffered saline (1×, pH 7·2; Gibco BRL, Carlsbad, CA) and subsequently diluted to obtain final cell densities of 7·5 × 107 colony-forming units (CFU)/ml in Dulbecco's modified Eagle's minimal essential medium (Gibco BRL). For lipopolysaccharide (LPS) purification, B. vulgatus was killed by addition of 1% phenol (Fluka, Heidelberg) and washed thoroughly with bidistilled water. Endotoxins were extracted and purified due to the method of Westphal.29 Purity was checked by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE).30

Transwell IEC-immune cell cultures and bacterial stimulation

The human methotrexate-differentiated colonic epithelial cell line HT-29/MTX (passage 35–60) (provided by Dr Lesuffleur, INSERM, Villejuif, France) and the human adenocarcinoma intestinal epithelial cell line CaCO-2 (passage 30–60) (American Type Culture Collection HTB 38) were grown in 12- or 6-well transwell cell culture inserts (Nunc, Rochester, NJ) to confluency as previously described.22,24,31 Human PBMC from healthy volunteers (n = 9) as well as CD patients with active disease were isolated using Ficoll-Hypaque 1077 (Pharmacia, Piscataway, NJ) gradient centrifugation (500 g, 30 min). The disease activity index of the CD patients (CDAI) and UC (UCSS) was assessed in the IBD clinic at the University Hospital of North Carolina at Chapel Hill. CD and UC patients with CDAI scores >220 (n = 9) and UCSS scores >2 (n = 5), respectively, were randomly selected during regularly scheduled clinic visits and asked for their consent to participate in this study. LPMC were isolated from resected colon of cancer patients (n = 3). Briefly, the intestinal mucosa was separated form the resected tissue, cut in small pieces and sequentially treated with dithiothreitol (1 mm, Sigma) and ethylenediaminetetraacetic acid (1 mm) to remove mucus and epithelial cells to finally digest with collagenase (collagenase type VIII, 1 mg/ml, Sigma, St Louis, MO) and deoxyribonuclease (10 µg/ml, Sigma).32 The resulting cell suspension was purified using Ficoll-Hypaque 1077 (Pharmacia) gradient centrifugation.

PBMC or LPMC were added to the basolateral compartment of transwell IEC cultures (inserts with 0·4 mm pore size) at cell densities of 2 × 106/ml. IEC were stimulated with bacteria by adding 7·5 × 107 CFU/ml B. vulgatus, E. coli, LPS (1 µg/ml; from E. coli serotype O111:B4, Sigma) and tumour necrosis factor (TNF; 5 ng/ml) (both from R & D Systems, Minneapolis, MN) to the apical side of IEC/leucocyte cocultures for various times. In addition, to mimic the effect of translocated bacterial products across the confluent epithelial cell monolayer, PBMC were directly stimulated with LPS at concentrations of 200 pg/ml or 1 µg/ml and HT-29/MTX cells were placed above LPS-activated PBMC. The obligate anaerobic B. vulgatus lost 99·9% of its viability after 1 hr in the transwell system. Gentamicin (100 µg/ml) was added to the cultures to reproduce similar culture conditions between B. vulgatus and E. coli. Where indicated, IEC lines were pretreated with the proteasome inhibitor MG132 (20 µm, Biomol, Plymouth Meeting, PA) to block IκBα degradation and to accumulate phosphorylated IκBα.

Adenoviral infection and reporter gene assay

HT-29/MTX and CaCO-2 cells were infected overnight with Ad5κB-LUC consisting of three consensus NF-κB binding sites linked to luciferase. Ad5GFP containing green fluorescent protein and Ad5LacZ containing the E. coliβ-galactosidase were used as viral negative control.22 The adenovirus was washed off and fresh medium containing serum without antibiotics was added. Cells were stimulated at various time points with B. vulgatus (7·5 × 107 CFU/ml),E. coli (7·5 × 107 CFU/ml), TNF (5 ng/ml) or LPS (1 µg/ml). Cell extracts were prepared using enhanced luciferase assay reagents (Analytical Luminescence, San Diego, CA). Luciferase assay were performed on a Monolight 2010 luminometer for 20 s (Analytical Luminescence), and results were normalized for extract protein concentrations measured with the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

RNA extraction and reverse transcription–polymerase chain reaction (RT–PCR) analysis

RNA was isolated using Trizol (Life Technologies, Grand Island, NY) and 1 µg of total RNA was reverse transcribed and amplified (RT–PCR) using specific primers for rat IL-8, inducible protein-10 (IP-10) and β-actin as described previously. The oligonucleotide primers used were: TLR-4A; (5′) 5-TGT-CCCTGAACCCTATGAAC-3 (position 795–812) and TLR-4B; (3′) 5-ACTCA-AATCTCTCAAAAGGC-3 (position 1211–1230), MD-2A; (5′) 5-GAAGCTCAGAA-GCAGTATTGGGTC-3 (position 174–197) and MD-2B (3′) 5-GGAGTTTGTCATCCT-ACACCAACC-3 (position 572–596), IL-8-F; (5′) 5′-TCTGCAGCTCTGTGTGAAGAAGGT-GCAGTT-3′ (position 120–138) and IL-8-R; (3′) 5′-AACCCTCTGCACCCAGTTTTCCTT-3′ (position 315–338); IP-10-F (5′) 5′-AGTGGCATTCAAGGAGTACC-3′ (position 115–134) and IP-10-R (3′) 5′-ATCCTTGGAAGCACTGCATC-3′ (position 384–403) and β-actin-F; (5′) CCAACCGCGAGAAGATGACC-3′ (position 414–433) and β-actin-R; (3′) 5-GATCTTCATGAGGTAGTCAGT-3′ (position 629–649). The length of the amplified product was of 438, 422, 218, 288 and 235 bp, respectively. The PCR products (5 µl) were subjected to electrophoresis on 2% agarose gels containing gel Star fluorescent dye (FMC, Philadelphia, PA). Fluorescent staining was captured using an AlphaImager 2000 (AlphaInnotech, San Leandro, CA).

To semiquantify the induction of IL-8 mRNA expression in IEC after bacterial stimulation in the presence or absence of immune cells, IL-8 mRNA expression levels were normalized relative to the expression οf β-actin mRNA and IL-8/β-actin ratios were calculated using densitometric analysis. The fold increase in IL-8 mRNA expression was calculated relative to nontreated HT-29/MTX cells.

Western blot analysis

IEC were stimulated for various times (0–12 hr) with bacteria, bacterial products or cytokines. The cells were lysed in 1× Laemmli buffer, and 20 µg of protein was subjected to 10% SDS–PAGE. Anti-phosphoserine IκBα (Cell Signalling, Beverly, MA), anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA), antiphosphoserine RelA (S536, Cell Signalling, Beverly, MA) and antiβ-actin (ICN, Costa Mesa, CA) were used to detect immunoreactive phospho-IκBα, total IκBα, phospho-RelA and β-actin, respectively, using enhanced chemiluminescence light-detecting kit (Amersham, Arlington Heights, IL) as previously described.33

Limulus amoebocyte lysate (LAL) assay

Endotoxin concentrations were measured with a kinetic chromogenic LAL assay delivered by Charles River Endosafe (Charleston, SC) according to the manufacture's instructions.

Enzyme-linked immunosorbent assay (ELISA)

Cell culture supernatants in the basolateral compartment of the IEC transwell cultures were collected after 16 h of stimulation. TNF and IL-10 protein concentrations (pg/ml) were determined using ELISA analysis (R & D Systems).

Statistical analysis

Values are given as means ± standard error of the mean (SEM). Values were transformed logarithmically to obtain normal distribution and homogeneity of variances before being analysed with anova.

Results

Differential immune cell-mediated activation of IL-8 gene expression in HT-29/MTX cells after stimulation with non-pathogenic Gram-negative bacteria

We showed that immune-epithelial cross-talk can modulate non-pathogenic Gram-negative enteric bacteria-induced pro-inflammatory gene expression in IEC.22,24 To further investigate the role of peripheral blood and gut-derived immune cells on pro-inflammatory gene expression in IEC, we cocultured the highly LPS-unresponsive methotrexate differentiated colonic epithelial cell line HT-29/MTX34 zwith PBMC from healthy volunteers and LPMC in the basolateral compartment of a transwell tissue culture system. HT-29/MTX cells were stimulated from the apical surface with non-pathogenic E. coli, B. vulgatus, E. coli-derived LPS or TNF. Figure 1 shows the combined data from nine different experiments including PBMC from healthy volunteers (n = 9) and LPMC from non-inflamed colon cancer patients (n = 3). As shown previously34 IL-8 gene expression is induced in TNF- but not LPS-stimulated HT-29/MTX cells (Fig. 1). Similarly, HT-29/MTX cells remained hyporesponsive to E. coli and B. vulgatus stimulation. Interestingly, HT-29/MTX cocultures with PBMC or LPMC significantly enhanced E. coli and LPS-induced IL-8 mRNA expression, whereas epithelial cells remained hyporesponsive to B. vulgatus. This suggests that immune cells differently impact on IEC responsiveness to these two different gram negative bacterial strains.

Figure 1.

Figure 1

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PBMC and LPMC trigger Gram negative bacteria-induced IL-8 gene expression in HT-29/MTX cocultures. HT-29/MTX cells were stimulated for 16 hr with E. coli (7·5 × 107 CFU/ml), B. vulgatus (7·5 × 107 CFU/ml), E. coli-derived LPS (1 µg/ml) and TNF (5 ng/ml) in the presence or absence of PBMC from healthy volunteers or LPMC from resected, non-inflamed tissue of colon cancer patients. No bacteria were present in the medium control. PBMC and LPMC were isolated as described in Materials and Methods. Total RNA was extracted from HT-29/MTX cells, reverse transcribed and amplified using specific human IL-8. PCR products were run on a 2% agarose gel (representative IL-8 gel is shown) and analysed by densitometry. IL-8 mRNA expression levels were normalized relative to the expression of β-actin mRNA and IL-8/β-actin ratios were calculated. Fold increase was determined relative to non-treated HT-29/MTX cells. The combined results (mean ± SEM) represent PBMC from nine independent experiments and LPMC from three independent experiments, respectively. *P < 0·05 indicates the differences between medium control and the stimulation group.

Gram-negative bacteria trigger host innate immune activation mostly through the cell wall component LPS.28 Although the LAL revealed significantly lower efficiencies to detect B. vulgatus LPS compared to E. coli LPS, we measured endotoxin concentrations in the basolateral compartment of our transwell system. It appears from our studies that LPS from both bacterial species significantly translocate across the epithelial cell monolayer reaching concentrations up to 30 pg/ml LPS in the basolateral compartment (1–3% of the total LPS; data not shown). To investigate whether B. vulgatus- and E. coli-derived LPS differentially induce epithelial cell activation, we stimulated PBMC in the basolateral compartment of HT-29/MTX transwell cultures with B. vulgatus and E. coli LPS (1 µg/ml). As shown in Fig. 2(a)B. vulgatus- and E. coli-derived LPS revealed similar capabilities to induce IL-8 mRNA expression in HT-29/MTX cells. Accordingly, TNF and IL-10 production was significantly induced through B. vulgatus and E. coli LPS (Fig. 2b,c). In conclusion, these results suggest that B. vulgatus and E. coli LPS have comparable capabilities to trigger IEC activation in PBMC transwell cultures.

Figure 2.

Figure 2

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(a–c) Effects of B. vulgatus- and E. coli-derived LPS on the stimulation of HT-29/MTX cells cocultured with PBMC. We stimulated PBMC basolaterally with B. vulgatus- and E. coli-derived LPS (1 µg/ml) and measured IL-8 mRNA expression in HT-29/MTX cells after 16 hr of stimulation. Total RNA was extracted from HT-29/MTX cells after 16 hr of stimulation, reverse transcribed and amplified using specific human IL-8. PCR products were run on a 2% agarose gel and analysed by densitometry. IL-8 mRNA expression levels were normalized relative to the expression οf β-actin mRNA and IL-8/β-actin ratios were calculated. Fold increase was determined relative to non-treated HT-29/MTX cells (a) TNF (b) and IL-10 (c) protein concentrations were measured in the spent culture supernatant of the basolateral compartment of HT-29/MTX transwell cultures after the stimulation of PBMC for 16 hr with B. vulgatus- and E. coli-derived LPS using ELISA. The results represent the mean of triplicate measurements with PBMC from one healthy donor.

PBMC cocultures induce mRNA expression of the TLR-4 accessory protein MD-2 in HT-29/MTX cells

It has been shown that Gram-negative bacteria signal through the TLR-4 cascade to activate NF-κB and pro-inflammatory gene expression28 and that expression of the accessory protein MD-2 is critical in LPS-induced activation of IEC lines.35 Because the presence of PBMC selectively triggered E. coli- but not B. vulgatus-stimulated HT-29/MTX cells, we measured IL-8, MD-2 and TLR-4 mRNA expression in HT-29/MTX cells in the presence and absence of PBMC. Figure 3(a) shows relatively low levels of IL-8 and MD-2 expression in unstimulated HT-29/MTX cells in the absence of PBMC. The stimulation of HT-29/MTX cells cocultured with PBMC with E. coli enhanced IL-8 and MD-2 mRNA expression. Maximal expression levels were reached after 16 hr of stimulation (Fig. 3a). Interestingly, the presence of PBMC alone significantly increased MD-2 mRNA expression in HT-29/MTX cells, whereas TLR-4 mRNA expression remained constant (Fig. 3b). Enhanced MD-2 gene expression correlates with increased E. coli signalling to IL-8 gene expression, but not with the absence of B. vulgatus signalling. Thus, the selective deficiency of B. vulgatus to trigger chemokine expression in HT-29/MTX cells cocultured with immune cells was independent from TLR-4 and MD-2 expression.

Figure 3.

Figure 3

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PBMC trigger IL-8 and MD-2 mRNA expression in HT-29/MTX cells. HT-29/MTX cells were stimulated for 0–16 h with E. coli (7·5 × 107 CFU/ml) in the presence of PBMC from healthy controls (a) HT-29/MTX cells were stimulated with E. coli and B. vulgatus in the presence and absence of PBMC for 16 hr (b) PBMC were isolated as described in Materials and Methods. Total RNA was extracted from HT-29/MTX cells, reverse transcribed and amplified using specific human MD-2, TLR-4, IL-8 and β-actin primers. PCR products were run on a 2% agarose gel and stained with gel star. No bacteria were present in the medium control. These results are representative of three independent experiments.

Differential effect of E. coli and B. vulgatus on IκBα phosphorylation and NF-κB transcriptional activity in IEC cocultured with PBMC

B. vulgatus induced IκBα phosphorylation as well as NF-κB transcriptional activity in primary and IEC lines.22 To investigate the effect of immune cells on B. vulgatus- and E. coli-induced NF-κB signalling in IEC, we measured NF-κB transcriptional activity and endogenous IκBα phosphorylation in HT-29/MTX cells. To assess the effect of immune cells on NF-κB signalling in HT-29/MTX cells, we determined the levels of IκBα phosphorylation following bacteria stimulation in presence and absence of PBMC. To accumulate phospho-IκBα, MG-132 was used to block IκBα degradation. Interestingly, E. coli and B. vulgatus triggered IκBα phosphorylation in HT-29/MTX cells after 6 and 12 hr of stimulation in the presence but not in the absence of PBMC (Fig. 4a). To further investigate the role of PBMC on NF-κB transcriptional activity, HT-29/MTX cells were infected with an adenoviral vector encoding (κB)-luciferase reporter gene (Ad5κB-LUC) and stimulated with B. vulgatus, E. coli, LPS and TNF in the presence or absence of PBMC from healthy controls. Figure 4(b) shows that the presence of PBMC triggered E. coli-, LPS- and TNF- but not B. vulgatus-induced NF-κB transcriptional activity in HT-29/MTX cells.

Figure 4.

Figure 4

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PBMC differentially trigger E. coli and B. vulgatus-induced IκBα phosphorylation and NF-κB transcriptional activity in HT-29/MTX cells. (a) HT-29/MTX cells were stimulated for 6 and 12 hr with E. coli (7·5 × 107 CFU/ml) and B. vulgatus (7·5 × 107 CFU/ml) in the presence or absence of PBMC. Where indicated HT-29/MTX and CaCO-2 cells were pretreated for 1 hr with the proteasome inhibitor MG132 (20 µm) to accumulate phospho-IκBα. Total protein was extracted, and 20 µg of protein was subjected to SDS–PAGE followed by IκBα, phospho-IκBα and β-actin immunoblotting using the ECL technique. (b) HT-29/MTX cells were infected with Ad5NF-κB-LUC (multiplicity of infection of 5) for 12 hr. Cells were then stimulated for an additional 16 hr with E. coli (7·5 × 107 CFU/ml), B. vulgatus (7·5 × 107 CFU/ml), E. coli-derived LPS (1 µg/ml) or TNF (5 ng/ml) in the presence or absence of PBMC. Cell extracts and the luciferase assay were performed as described in Materials and Methods and results were normalized for extract protein concentrations. Luciferase activity is expressed as fold increase over control determined as the mean (± SD) of three independent experiments measured in triplicates. *P < 0·05 indicates the differences in the presence of PBMC relative to the medium control group.

Because B. vulgatus induces IκBα phosphorylation, we next investigated the effect of immune cells on bacteria-induced IκBα degradation in IEC. HT-29/MTX cells show modest IκBα degradation, which is easily replenished through new protein synthesis, leaving the steady-state levels of IκBα almost unaltered. Nevertheless, NF-κB is activated and IL-8 gene expression is induced through a low rate of IκBα degradation in HT-29 cells.33,36 Because HT-29/MTX cells reveal minimal changes in IκBα steady-state levels, we used CaCO-2 cells, which display strong IκBα phosphorylation/degradation in response to B. vulgatus and LPS stimulation.22 As shown in Fig. 5(a)E. coli and B. vulgatus induced IκBα phosphorylation as well as IκBα degradation in CaCO-2 cells. Interestingly, and in contrast to E. coli, the presence of PBMC completely inhibited B. vulgatus-induced IκBα degradation but not IκBα phosphorylation. The presence of PBMC without bacteria did not trigger IκBα phosphorylation or IκBα degradation in CaCO-2 cells at any time point (data not shown). This suggests that immune cells may selectively block B. vulgatus-induced IκBα degradation but allow IκBα phosphorylation in IEC. In addition, we performed NF-κB reporter gene analysis in CaCO-2 cells as described above (see Fig. 4b). Accordingly to our previous findings, CaCO-2 cells are responsive to the stimulation with B. vulgatus- and E. coli-derived LPS even in the absence of PBMC (Fig. 5b). Interestingly, B. vulgatus- but not LPS-induced (E. coli-derived) NF-κB transcriptional activity was significantly inhibited in the presence of PBMC. Of note, B. vulgatus-mediated inhibition of NF-κB transcriptional activity was confirmed in the presence of purified lymphocytes but was reversed in the presence of monocytes. This suggests that different immune cells confer IEC responsiveness to some bacteria through enhanced NF-κB activity.

Figure 5.

Figure 5

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Lymphocytes differentially trigger E. coli and B. vulgatus-induced IκBα phosphorylation/degradation and NF-κB transcriptional activity in CaCO-2 cells. (a) CaCO-2 cells were stimulated for 0–4 hr with E. coli (7·5 × 107 CFU/ml) and B. vulgatus (7·5 × 107 CFU/ml) in the presence or absence of PBMC. Where indicated CaCO-2 cells were pretreated for 1 hr with the proteasome inhibitor MG132 (20 µm) to accumulate phospho-IκBα. Total protein was extracted, and 20 µg of protein was subjected to SDS–PAGE followed by IκBα, phospho-IκBα and β-actin immunoblotting using the ECL technique. (b) CaCO-2 cells were infected with Ad5NF-κB-LUC (multiplicity of infection of 5) for 12 hr. Cells were then stimulated for additional 16 h with B. vulgatus (7·5 × 107 CFU/ml) and E. coli-derived LPS (1 µg/ml) in the presence or absence of PBMC, lymphocytes and monocytes. Cell purification, cell extract preparation and the luciferase assay were performed as described in Materials and Methods and results were normalized for extract protein concentrations. Luciferase activity is expressed as fold increase over control determined as the mean (± SD) of three independent experiments measured in triplicates. *P < 0·05 indicates the differences in the presence of PBMC or lymphocyte relative to the medium control group.

Effect of PBMC from active Crohn's disease and UC patients to induce IL-8 gene expression in HT-29/MTX cells after the stimulation with E. coli, LPS and B. vulgatus

Because luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation, we next investigated the effect of B. vulgatus to trigger epithelial activation in HT-29/MTX cocultured with PBMC from CD patients with active disease. Therefore, we stimulated HT-29/MTX cells apically with B. vulgatus, E. coli and LPS in the presence or absence of PBMC from healthy volunteers as well as PBMC from CD (n = 9, CDAI = 301±16) and UC patients (n = 5, UCSS = 6±0·8). In the CD versus UC group, steroids (one out of nine versus zero out of five patients), purine analogues (four out of nine versus one out of five patients), 5-ASA (four out of nine versus three out of five patients), cyclosporin (zero out of nine versus zero out of five patients) or Remicade (zero out of nine versus zero out of five patients) were received as medical treatment. As shown in Fig. 6(a), while E. coli and E. coli-derived LPS induced significant IL-8 gene expression in HT-29/MTX cocultured with PBMC from healthy volunteers (PBMC control) as well as PBMC from CD patients, B. vulgatus failed to trigger epithelial cell activation in the presence of both control- and CD-derived PBMC. Interestingly, E. coli failed to induce epithelial cell activation in the presence of UC-derived PBMC. The results shown in Fig. 6(b) confirmed the capability of pro-inflammatory stimuli such as TNF to induce IL-8 mRNA expression in the presence of PBMC from UC patients. Of note, PBMC from healthy controls and CD patients increased TNF-induced IL-8 induction in HT-29/MTX cells.

Figure 6.

Figure 6

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E. coli and B. vulgatus differentially induce IL-8 gene expression in HT-29/MTX cells cocultured with PBMC from CD and UC patients with active disease. HT-29/MTX cells were stimulated for 16 hr with E. coli (7·5 × 107 CFU/ml), B. vulgatus (7·5 × 107 CFU/ml) and E. coli-derived LPS (1 µg/ml) in the presence or absence of PBMC from healthy volunteers or PBMC from CD (n = 9, CDAI = 301) and UC patients (n = 5, UCSS = 6) (a) HT-29 MTX cocultures were stimulated apically with TNF and E. coli-derived LPS (1 µg/ml) (b) No bacteria were present in the medium control. PBMC were isolated as described in Materials and Methods. Total RNA was extracted from HT-29/MTX cells, reverse transcribed and amplified using specific human IL-8. PCR products were run on a 2% agarose gel (representative IL-8 gel is shown) and analysed by densitometry. IL-8 mRNA expression levels were normalized relative to the expression οf β-actin mRNA and IL-8/β-actin ratios were calculated. Fold increase was determined relative to non-treated HT-29/MTX cells. The combined results (mean ± SEM) represent PBMC from 5 to nine independent experiments (bar chart). *P < 0·05 indicates the differences between medium control and the stimulation group.

Although the cell culture inserts with a membrane pore size of 0·4 µm are not permeable for intact bacteria, bacterial products such as LPS can translocate across confluent HT-29/MTX monolayers. To confirm the capacity of PBMC from CD patients to induce IL-8 mRNA expression in cocultured IEC, we added 200 pg/ml and 1 µg/ml LPS directly to control PBMC, PBMC from CD and UC patients. As shown in Fig. 7, the direct stimulation of control and CD-derived PBMC with 200 pg/ml and 1 µg/ml triggered a dose-dependent induction of IL-8 mRNA expression in HT-29/MTX cells reaching statistical significance at concentrations of 1 µg/ml. Accordingly to our previous results (see Fig. 6a), the direct stimulation of PBMC from UC patients did not induce IL-8 mRNA expression, although TNF (Fig. 8a) and IL-10 (Fig. 8b) responses were not statistically different to control PBMC. We only detected statistically significant differences in the TNF response between UC and CD PBMC.

Figure 7.

Figure 7

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LPS-stimulated PBMC from healthy volunteers and PBMC from CD as well as UC patients differentially induce IL-8 gene expression in HT-29/MTX cells. HT-29/MTX cells were placed above LPS-stimulated (200 pg/ml and 1 µg/ml) PBMC from healthy volunteers or PBMC from CD (n = 9, CDAI = 301) and UC (n = 5, UCSS = 6) patients with active disease. No LPS was present in the medium control. PBMC were isolated as described in Materials and Methods. Total RNA was extracted from HT-29/MTX cells, reverse transcribed and amplified using specific human IL-8. PCR products were run on a 2% agarose gel and analysed by densitometry. IL-8 mRNA expression levels were normalized relative to the expression οf β-actin mRNA and IL-8/β-actin ratios were calculated. Fold increase was calculated relative to non-treated HT-29/MTX cells. The combined results (mean±SEM) represent PBMC from nine independent experiments (bar chart). *P < 0·05 indicates the differences between medium control and the stimulation group.

Figure 8.

Figure 8

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TNF and IL-10 production in LPS-stimulated PBMC from healthy volunteers and PBMC from CD as well as UC patients. HT-29/MTX cells were placed above LPS-stimulated (200 pg/ml and 1 µg/ml) PBMC from healthy volunteers or PBMC from CD (n = 9, CDAI = 301) and UC (n = 5, UCSS = 6) patients with active disease. No LPS was present in the medium control. PBMC were isolated as described in Materials and Methods. TNF (a) and IL-10(b) protein concentration (pg/ml) in the cell-free supernatant of the basolateral compartment was measured by ELISA. Results are shown as mean±SEM. *P<0·05 indicates the differences between medium control and the stimulation group; #P<0·05 indicates differences between PBMC from CD and UC patients.

In conclusion, E. coli and E. coli-derived LPS induce IL-8 gene expression in HT-29/MTX cells in the presence of immune cells. Although B. vulgatus triggered IκBα phosphorylation in HT-29/MTX and CaCO-2 cells cocultured with PBMC, it failed to induce significant IL-8 mRNA responses in epithelial cell. Interestingly, E. coli-derived LPS differentially induced IL-8 responses in HT-29/MTX cells cocultured with PBMC from patients with chronically active CD und UC.

Discussion

In this study, we used a reductionist approach of IEC/immune cell transwell cultures to mimic the complex network of the gut epithelium with underlying lamina propria immune cells in order to characterize enteric bacteria-induced signalling in IEC. First, we demonstrate that immune cells trigger E. coli-induced IκBα phosphorylation/degradation, NF-κB transcriptional activity and IL-8 gene expression in LPS-unresponsive HT-29/MTX cells. Of note, the physiological relevance of this reductionist ‘lamina propria model’ was confirmed to some extend by using PBMC and LPMC in parallel experiments. Second, we show in the two phenotypically and functionally distinct IEC lines, HT-29/MTX37 and CaCO-238 that non-pathogenic B. vulgatus-induced NF-κB signalling and that pro-inflammatory gene expression is inhibited by the presence of immune cells. Most importantly and in contrast to E. coli, B. vulgatus-induced activation of the IκB/NF-κB system is blocked at the level of IκBα degradation, but allows IκBα phosphorylation in IEC. This suggests that E. coli and B. vulgatus differentially activate IEC in the presence of immune cells.

It is interesting to note that the presence of immune cells modulate responsiveness of differentiated HT-29/MTX cells to E. coli and E. coli-derived LPS but not B. vulgatus stimulation. Since the obligate anaerobic B. vulgatus lost 99·9% of its viability after 1 hr in the transwell system, we added gentamicin to the cultures to reproduce similar culture conditions when B. vulgatus and E. coli bacterial strains were applied. Gram-negative bacteria including E. coli and B. vulgatus signal the IκB/NF-κB system through LPS-mediated activation of the TLR-4 cascade.22 Moreover, Hornef et al.39 showed previously that E. coli-derived LPS was internalized by murine epithelial cells to stimulate the IκB/NF-κB system via intracellular located TLR-4. Since immune cell-derived signals can induce TLR-4 and MD-2 expression40B. vulgatus specific down-regulation of TLR-4 or blockade of PBMC-induced up-regulation of TLR-4 coreceptor MD-2 may also contribute to hyporesponsiveness in IEC-immune cell cocultures.41 However, B. vulgatus and E. coli induced MD-2 gene expression, while TLR-4 remained constant, and both bacteria induced IκBα phosphorylation in IEC cocultures with PBMC, suggesting that immune cells did not prevent initial signalling of B. vulgatus. We showed that LPS (E. coli-derived) concentrations as low as 200 pg/ml in the basolateral PBMC compartment contributed to the induction of IL-8 gene expression in HT-29/MTX cells. Although B. vulgatus-derived LPS can differ from E. coli-derived LPS in its composition and its capacity to stimulate PBMC,42 we showed that B. vulgatus- and E. coli-derived LPS revealed similar capabilities to trigger epithelial cell activation in the presence of immune cells in HT-29/MTX transwell cultures. Considering the fact that E. coli and B. vulgatus initially triggered IκBα phosphorylation and NF-κB activation in IEC in the presence of immune cells, one scenario for the suppressive effect of B. vulgatus might be that further progression of IEC activation requires additional signals from LPS-activated PBMC. However, if the second signal is not provided through translocated LPS, PBMC block NF-κB signalling in IEC.

Non-pathogenic bacteria-mediated regulation of epithelial cell responses by inhibition of IκBα degradation was previously shown by Neish et al.43 The authors demonstrated a specific bacteria-induced blockade of the ubiquitination process necessary to mediate degradation of the cytoplasmic NF-κB inhibitor IκBα through the 26S proteasome complex. We are currently investigating in our model whether the underlying mechanism of this immune cell-mediated blockade of IκBα degradation is due to specific inhibitory effects of certain bacterial cell components of B. vulgatus on the IκB ubiquitin ligase E3-SCFβ–TrCP or the proteasome-specific proteolysis of poly ubiquitinated IκB. Of note, this immune-cell mediated effect was signal-specific, suggesting distinct differences between E. coli and B. vulgatus. The presence of phosphorylated IκBα in the absence of IκBα degradation and activation of κB-dependent gene expression is distinct from reported Yersinia sp.-mediated inhibition of NF-κB blockade. These enteroinvasive organisms inhibit IκB kinase (IKK) activation and subsequent IκBα phosphorylation in IEC through the translocated effector protein YopJ and blockade of the mitogen-activated protein kinase kinase pathway.34,44,45 The presence of additional inhibitory factors specific for B. vulgatus remain to be elucidated; however, various regulatory signalling molecules, including peroxisome proliferator-activated receptor-γ, phosphatidylinositol-3-kinase and signal transducer and activator of transcription-3, which have been shown to negatively control LPS-induced gene expression in monocyte/macrophages and IEC,46–48 may contribute to the differential effects of E. coli and B. vulgatus epithelial cell activation in the presence of immune cells.49

It appears from several studies that luminal enteric LPS poorly translocates across the epithelial barrier under normal conditions in vivo.50,51 However, under the pathological conditions of chronic intestinal inflammation enhanced mucosal permeability is associated with increased interaction of luminal enteric bacteria with the epithelium, particularly the Gram-negative Bacteroides and Enterobacteriaceae species52 as well as increased levels of circulating LPS and LPS antibodies in the serum of IBD patients.53 Interestingly, we could show that PBMC from CD and UC patients dramatically differ in their capability to trigger LPS-induced epithelial cell activation. Consistent with our previous data, B. vulgatus failed to trigger IL-8 gene expression in IEC cocultured with PBMC from active CD and UC patients, suggesting the absence of specific antigen or adjuvant triggers even in the presence of PBMC from chronically inflamed CD patients. It would be interesting to understand the hyporesponsive nature of LPS-activated PBMC from UC patients. Although responsiveness of the intestinal epithelium to B. vulgatus could be observed under specific experimental conditions in vivo21,22 we showed that the initial induction of nuclear phospho-RelA in primary large intestinal epithelial cells was inhibited in both B. vulgatus-monoassociated Fisher wild type and HLA-B27 transgenic rats. A dual function of NF-κB was previously shown in a model of acute inflammation caused by gut ischaemia–reperfusion.54 The authors showed that the selective ablation of IKK-β in IEC prevented from multiple organ dysfunction syndrome in the latter model, but caused severe apoptotic damage to the reperfused intestinal mucosa. Thus, elaborate regulatory mechanisms are present in the intestine to maintain host homeostasis in the face of constant bacterial challenges; however, the effect of different bacteria to induce NF-κB activity in epithelial cells and its role in the development of chronic immune-mediated inflammation remains to be elucidated.

Interestingly, our previous data indicate that lymphocytes negatively regulate B. vulgatus induced proinflammatory gene expression.22 The key role of lymphocytes in controlling the onset of intestinal inflammation has been clearly demonstrated in different severe immune deficiency transfer models of experimental colitis.55 Immunoregulatory T cells arise from antigen-specific antigen-presenting cell stimulation that lead to production/expansion of different effector T cells and regulatory T cells producing transforming growth factor-β or IL-10.56 The activity of the latter cell types is important for the resolution of inflammation and maintenance of host homeostasis. Whether T-cell mediated inhibition of B. vulgatus-induced IL-8 expression is caused by the production of a regulatory protein is unknown. However, PBMC-derived IL-10 was not enhanced in cocultures with IEC after the stimulation with B. vulgatus, nor could we detect phosphorylated Smad2 or Smad3 in IEC as a consequence of the TGF-β signalling cascade.21 Thus, it is unlikely that this classical immunoregulatory cytokines are directly involved in the inhibitory process.

In conclusion, these results demonstrate that immune cells trigger E. coli-induced IκBα phosphorylation/degradation, NF-κB transcriptional activity as well as IL-8 expression in HT-29/MTX cells. Conversely, the same immune cells trigger B. vulgatus-induced IκBα phosphorylation but block NF-κB transcriptional activity as well as IL-8 gene expression. Considering all our results, we propose that bacteria-mediated IEC activation in the presence of immune cells requires two signals (Fig. 9). First, the direct activation of initial NF-κB signalling through bacteria–epithelial cell interaction followed by the second signal resulting from LPS-activated PBMC which further perpetuate IEC activation (Fig. 9b). In the absence of the second signal through translocated LPS, PBMC may rather inhibit epithelial cell activation (Fig. 9a).

Figure 9.

Figure 9

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Differential effects of immune cells on LPS-induced NF-κB activivation in IEC. LPS-mediated IEC activation in the presence of immune cells requires two signals; the direct activation of initial NF-κB signalling through bacteria–epithelial cell interaction followed by the second signal resulting from LPS-activated PBMC which further perpetuate IEC activation (b) In the absence of the second signal through translocated LPS, PBMC may inhibit epithelial cell activation (a).

Acknowledgments

This work was supported by NIH ROI grants DK 47700 to C. Jobin, RO1 DK 40249 and P30 DK34987 to RB. Sartor and by the Crohn's and Colitis Foundation of America to C. Jobin and by Deutsche Forschungsgemeinschaft grant HA 3148/1-2 to D. Haller.

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