The host response to the probiotic Escherichia coli strain Nissle 1917: Specific up-regulation of the proinflammatory chemokine MCP-1
© Ukena et al; licensee BioMed Central Ltd. 2005
Received: 27 July 2005
Accepted: 13 December 2005
Published: 13 December 2005
The use of live microorganisms to influence positively the course of intestinal disorders such as infectious diarrhea or chronic inflammatory conditions has recently gained increasing interest as a therapeutic alternative. In vitro and in vivo investigations have demonstrated that probiotic-host eukaryotic cell interactions evoke a large number of responses potentially responsible for the effects of probiotics. The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells.
Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal cell line and also pieces of small intestine from BALB/c mice were used to confirm regulatory data of selected genes by real-time RT-PCR and cytometric bead array (CBA) to detect secretion of corresponding proteins.
Whole genome expression analysis revealed 126 genes specifically regulated after treatment of confluent Caco-2 cells with E. coli Nissle 1917. Among others, expression of genes encoding the proinflammatory molecules monocyte chemoattractant protein-1 ligand 2 (MCP-1), macrophage inflammatory protein-2 alpha (MIP-2α) and macrophage inflammatory protein-2 beta (MIP-2β) was increased up to 10 fold. Caco-2 cells cocultured with E. coli Nissle 1917 also secreted high amounts of MCP-1 protein. Elevated levels of MCP-1 and MIP-2α mRNA could be confirmed with Lovo cells. MCP-1 gene expression was also up-regulated in mouse intestinal tissue.
Thus, probiotic E. coli Nissle 1917 specifically upregulates expression of proinflammatory genes and proteins in human and mouse intestinal epithelial cells.
Probiotic microorganisms have traditionally been characterized as viable nutritional agents conferring benefits to the health of the human host . The definition of the term "probiotic" has evolved through the years. The latest and most appropriate definition describes probiotics as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" . Beneficial activities of probiotics most likely result from complex interactions of the microorganisms with the intestinal microflora and the gut epithelium of the individual . A proposed mechanism by which probiotics mediate their effects is the modulation of the innate immune response both to antiinflammatory  and proinflammatory directions. Furthermore, probiotic bacteria have been shown to enhance the adaptive immune response and antibody formation [7, 8]. Inhibition of adherence of attaching and effacing organisms , modulation of the mucosal barrier function [10, 11] as well as inhibition of neutrophil migration  may also be important mechanisms whereby probiotics can impact in intestinal diseases.
Next to lactic acid bacteria and the probiotic yeast S. boulardii, the non-pathogenic E. coli strain Nissle 1917 (EcN) of serotype O6:K5:H1 is one of the best characterized probiotics. EcN was originally isolated by the army surgeon Dr. Alfred Nissle in 1917 from the feces of a soldier who did not develop diarrhea during a severe outbreak of shigellosis . A controlled clinical trial published recently implies probiotic EcN being as effective as standard medication with a dose of 1,5 g/day mesalazine in remission maintenance of ulcerative colitis. In this study recurrance rates were 33.9% for mesalazine treatment compared to 36.4% for treatment with EcN (Mutaflor®) . Recently, we have demonstrated, that recombinant EcN had no effect on migration, clonal expansion and activation status of specific CD4+ T cells, neither in healthy mice nor in animals with acute colitis . Despite the successful therapeutic applications of EcN, only limited information is available about the beneficial traits contributing to the strains' probiotic character. Several strain specific characteristics have been detected so far including expression of two microcins , presence of six iron-uptake systems or lack of defined virulence factors . Moreover, EcN exhibits a unique semirough lipopolysaccharide phenotype, responsible for its serum sensitivity [18, 19]. All these properties might be advantageous for EcN in competing with other colonic bacteria or adapting to the intestinal situation. However, the mechanisms underlying the probiotic nature, especially at the molecular level, yet have to be elucidated.
In this study we aimed to analyze the genomic expression program initiated by the interaction of probiotic EcN with human intestinal epithelial cells. Our results demonstrate a transient proinflammatory signaling of human and mouse intestinal epithelial cells illustrated by induced gene expression of MCP-1, MIP-2α and MIP-2β after treatment with EcN.
Cell culture conditions
The human colon adenocarcinoma cell lines Caco-2  and Lovo  were maintained in IMDM cell culture medium (Invitrogen, Karlsruhe, Germany) containing 10% fetal calf serum (PAA Laboratories, Cölbe, Germany) and 250 μg/ml penicillin/streptomycin (Invitrogen) at 37°C in a cell culture incubator. Caco-2 cells were used from passage 12 – 26. Cells were split twice a week at a ratio of 1:3. 4 – 8 × 105 cells per well were seeded in six well plates (Nunc, Wiesbaden, Germany) and cultured for approximately four days until confluence.
BALB/c mice were obtained from Harlan (Borchen, Germany). The animal experiments reported here were permitted from the district authority of Braunschweig and were conducted according to the German animal protection law.
Preparation of bacteria
E. coli strain Nissle 1917 was isolated from a tablet of Mutaflor® (Ardeypharm, Herdecke, Germany) cultured on MacConkey plates (Oxoid, Wesel, Germany). The isolate was serotyped and confirmed by EcN specific PCR . E. coli K12 laboratory strain MG1655 was kindly provided by Ulrich Dobrindt (Institute for Molecular Infection Biology, Würzburg, Germany). Both strains were grown overnight in Luria Bertani (LB) medium (Invitrogen) at 37°C on a shaker. The cultures were then diluted 1:100 in 50 ml prewarmed IMDM medium containing 10% fetal calf serum and grown at 37°C. Bacteria were harvested in late logarithmic phase, after reaching an OD600 = 1.
Coculture of cell lines
Confluent Caco-2 and Lovo cells (0.4 – 1 × 107 cells per well) were washed with phosphate buffered saline (PBS) and cocultured with a low bacterial MOI (multiplicity of infection) of 1 (1.3 – 3.3 × 106 CFU/ml) in IMDM medium containing 10% fetal calf serum at 37°C in a cell culture incubator for 6 hours. For time-dependent expression experiments conditioned media was collected by centrifugation after coincubation for 6 hours, redissolved in 3 ml IMDM medium with 10% fetal calf serum containing 200 μg/ml gentamicin (Sigma-Aldrich, Taufkirchen, Germany) and 250 μg/ml penicillin/streptomycin (Invitrogen), and used to incubate the Caco-2 cells for another 42 hours. Prior to RNA isolation cells were washed twice with PBS.
Experimental setup was performed following a modified protocol described by Cima et al. . In brief, small intestine was dissected, washed and opened logitudinally, cut into 5 mm long pieces and washed with PBS/DTT for 15 min shaking at 37°C to remove the excess mucus. Tissue pieces were then washed with PBS followed by two washing steps with HBSS/2% fetal calf serum. 3 – 5 tissue pieces per well were placed in a 24 well plate in IMDM containing 10% fetal calf serum without antibiotics. Bacteria were applied at 5 × 105 CFU/ml.
Cocultivation of Caco-2 cells with inactivated bacterial pellets or bacteria conditioned media
Bacteria were grown overnight as described above. Overnight culture was diluted in IMDM with 10% fetal calf serum. For coincubation experiments with conditioned media (CM), bacterial suspensions were again incubated for 5 hours to an OD600 = 1 at 37°C on a shaker and diluted to 1 × 108 CFU/ml in IMDM medium containing 10% fetal calf serum. CM was centrifuged and supernatants were sterile filtered. Caco-2 cells were incubated with inactivated bacteria or CM for 6 hours. For experiments with inactivated bacteria, an aliquot of the bacterial suspension was taken at an OD600 = 0.5 and centrifuged. The bacterial pellet was fixed in 4% paraformaldehyde for one hour at room temperature, washed four times with PBS, redissolved in IMDM medium with 10% fetal calf serum and adjusted to an MOI of 2. Aliquots of the fixed bacteria were plated onto Müller-Hinton-agar to test for sterility.
Primers used for real-time RT-PCR.
5' Sequence 3'
Product length, bp
AGC CCC AAG AGC AAC TGT GAT T
AGT CCC GAG AAC CTA CCC TGA G
GTC AGC AAG CCA GCC CCT ACC AC
GGA TCC CCC TTC TTG CAG TCA CGA
GCG AGA ATG GCT GGC TGA AG
GGA TCT CCT CGG CGT GAA TG
GTC TCT GCC GCC CTT CTG TG
AGG TGA CTG GGG CAT TGA TTG
TTT TAG GTC AAA CCC AAG TTA GTT
TTC TTG GAT TCC TCA GCC TCT ATC
AAG AAG CTT ATC AGC GTA TCA T
AAT AAG TAG AAC CCT CGT AAG AAA
CGC CCA AGC ACC CGG ATA CAG C
TTC AGC CCC TTT GCA CTC ATA ACG
CGC AGG CGC AGA CGG TGG AAG C
CGA AGG GTC TCC GCG GGG TCA CAT
ATT GGC CTC TTT GAT ACA CTT TTG
CTC ATC CCT GCT CCT TCC CTA TCT
AAG GAG GAG GGC AGA ATC ATC ACG
CAC ACT CCA GGC CCT CGT CAT TG
DNA microarray hybridization
Quality and integrity of the total RNA isolated from 1 × 106 cells was controlled by running all samples on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). For biotinylated target synthesis, RNA was labeled using standard protocols supplied by the manufacturer (Affymetrix, Santa Clara, CA). Briefly, 5 μg total RNA was converted to dsDNA using 100 pmol of a T7T23V primer (Eurogentec, Seraing, Belgium) containing a T7 promoter. The cDNA was then used directly in an in vitro transcription reaction in the presence of biotinylated nucleotides. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 μg of each biotinylated cRNA preparation were fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix HG_U133A arrays for 16 hours. After hybridization, the GeneChips were washed, stained with strepavidin-phycoerythrin and read using an Affymetrix GeneChip fluidic station and scanner.
Analysis of microarray data was performed using Affymetrix Microarray Suite 5.0, Affymetrix MicroDB 3.0 and the Affymetrix Data Mining Tool 3.0. For normalization all array experiments were scaled to a target intensity of 150, otherwise using the default values of the Microarray Suite. Results were filtered as follows: Genes are considered strongly regulated when their fold change is greater than or equal 2 or less than or equal -2, the statistical parameter for a significant change is less than 0.01 (change p-value for changes called increased) or greater than 0.99 (change p-value for changes called decreased). Additionally, the signal difference of a certain gene should be greater than 100. Genes are considered as weakly regulated when their fold change is greater than or equal 1.5 or less than or equal -1.5, the statistical parameter for a significant change is less than 0.001 or greater than 0.999 and the signal difference of a certain gene should be greater than 40.
Total RNA preparation and cDNA synthesis were performed as described for RT-PCR. Quantitative real-time RT-PCR was done with an ABI PRISM cycler (Applied Biosystems, Foster City, CA) using SYBR Green PCR kit from Stratagene and specific primers optimized to amplify 90 – 250 bp fragments from the genes under investigation (Tab. 1). A threshold was set in the linear part of the amplification curve, and the number of cycles needed to reach it was calculated for every gene. Relative mRNA levels were determined by using included standard curves for each individual gene and further normalization to the housekeeping gene RPS9. Melting curves established the purity of the amplified band.
Cytokine analysis by CBA
Quantification of cytokines was performed using the BD® Cytometric Bead Array Human Chemokine Kit I with antibodies specific for IL-8, RANTES, MIG, MCP-1 and IP-10 (BD Biosciences, Heidelberg, Germany). Supernatants of 3–5 tissue culture wells were pooled and data were acquired as described previously  by flow cytometry with a FACSCalibur® flow cytometer (BD Biosciences) using 2-color detection. Data analysis was performed by BD Bioscience Cytometric Bead Array software.
Field emission scanning electron microscopy
Caco-2 cells were seeded on 22 mm Biocoat Collagen I coated coverslips (BD Biosciences) in six well plates and grown until confluence as described above. After coincubation, cells were washed three times with 1 × PBS and fixed with a fixation solution containing 5% formaldehyde and 2% glutaraldehyde in cacodylate buffer (0.1 M cacodylate, 0.01 M CaCl2, 0.01 M MgCl2, 0.09 M sucrose, pH 6.9), washed with cacodylate buffer and then with TE-buffer (10 mM TRIS, 2 mM EDTA, pH 6.9). Finally, cells were scratched from the wells, pelleted and embedded in 2% aqueous agar before dehydrating with a graded series of acetone (10, 30, 50, 70, 90, 100%) on ice for 30 min at each step. Samples in 100% acetone were allowed to reach room temperature before another change of 100% acetone. They were then subjected to critical-point drying with liquid CO2 (CPD 030, BAL-TEC, Schalksmühle, Germany). The dried cells were covered with a gold film of about 10 nm by sputter coating (SCD040, BAL-TEC) before examination in a field emission scanning electron microscope Zeiss DSM 982 Gemini (Carl Zeiss, Oberkochen, Germany) using the Everhart Thornley SE detector and the inlens detector in a 50:50 ratio at an acceleration voltage of 5 kV. Data were stored digitally on MO-disks.
Genome-wide expression analysis of Caco-2 cells treated with E. coliNissle 1917
Differential gene expression of Caco-2 cells cocultured with EcN.
GB Acc. No.b
Description and/or putative function
Enhances the inflammatory response, upregulated by IL1α and TNFα
Involved in platelet activation and aggregation; expressed in hematopoetic and epithelial cells
Produced by activated monocytes, expressed at inflammation sites, up-regulated by IL1α and TNFα
Plays a role in inflammation, autocrine effect on endothelial cells
May play a role in lymphocyte acitivation, angiogenesis inhibitor, putative therapeutic agent for gastric cancer
Serine protease inhibitor
EGF-signaling in oocyte
Hydrolase, negative feedback role in IL-2 signaling, deactivation of mitogen- or stress-activated protein kinases
Essential for constitution of the intestinal mucosal barrier, is downregulated by TGFβ
Downregulation of NF-κB activity, contributed in Crohn's disease
Regulates the activation of NF-κB by modulation of IκBα phosphorylation
HIF-1α responsive proapoptotic protein
Interacts with NAF1 and inhibits TNF-induced NF-κB dependent gene expression
Exonuclease specific for small oligoribonucleotides, sensitive epithelial marker
Transcription and translation
Epithelial specific ets transcription factor, regulates MIP3α expression which is NF-κB dependent
May faciliate ds RNA-regulated gene expression
Krüppel-like transcription factor, up-regulated by TGFβ
May act as transcription factor in B-cells
Apoptosis inducing protein, binding to BCL2
Growth inhibitory role, induced app. 20 × during in vitro differentiation of Caco-2
Probably involved in cell proliferation
Induced by mek1, role of vascular endothelial growth factor in IBD
Real-time RT-PCR to confirm data obtained with gene expression analysis arrays
Detection of MCP-1 in Caco-2 coculture supernatants by CBA analysis
EcN specific up-regulation of MCP-1 and MIP-2α is not a Caco-2 cell specific phenomenon
Gene expression of MCP-1, MIP-2α and MIP-2β is time-dependent
EcN induced expression of MIP-2α and MIP-2β is not dependent on viable bacteria
EcN specific expression of selected genes in Caco-2 cells cocultured with inactivated bacteria or bacteria conditioned media for 6 hours.
no EcN specific gene regulation
no EcN specific gene regulation
no EcN specific gene regulation
no EcN specific gene regulation
no EcN specific gene regulation
no EcN specific gene regulation
no EcN specific gene regulation
no EcN specific gene regulation
MCP-1 is up-regulated in small intestine after EcN treatment
Our results demonstrate that EcN up-regulates gene expression of molecules involved in pro- as well as antiinflammatory processes. A proinflammatory response of intestinal epithelial cells to EcN was mainly demonstrated by the temporary up-regulation of MCP-1 gene expression in confluent human cell lines and in primary mouse tissue culture as well as by an increased secretion of this chemokine. This EcN specific change of MCP-1 gene expression could neither be induced with bacteria conditioned media nor with formalin inactivated organisms suggesting that for EcN mediated MCP-1 gene expression viable bacteria are necessary. MCP-1 is produced by many cells, including epithelial, endothelial and mast cells as well as tumor cells, and shows chemotactic activity for monocytes, basophils, natural killer cells and T lymphocytes during inflammation. Among other things, MCP-1 influences the release of specific enzymes of these target cells [27–30]. MCP-1 is expressed under many pathological conditions, including asthma and inflammatory bowel diseases [31, 32]. Infection of human macrophages, which have a central role in innate immune response to bacteria, with non-pathogenic Lactobacillus rhamnosus GG, results in an enhanced MCP-1 expression and an induction of T helper cell type 1 (Th1) cell migration . Earlier this year, Lan et al. have shown that exposure of primary murine colonic epithelial cells to the same Lactobacillus strain also resulted in an exclusive induction of MCP-1 and MIP-2α expression . Moreover, it has been reported, that lactobacilli are able to activate myeloid dendritic cells that skew CD4+ and CD8+ T cells to Th1 and Tc1 polarization . Earlier studies have shown that MCP-1 expression also shifts the immune response towards release of Th2 cytokines  and thus can potentially affect antibacterial defenses. Although it seems unexpected at first sight that the probiotic EcN up-regulates MCP-1 mRNA expression and protein secretion, an important role of this proinflammatory molecule might be established in protecting the host from bacterial infection through induction of a strong T cell immune response, thus providing a reasonable explanation for the EcN induced expression of this chemokine. The neutrophil attracting chemokines MIP-2α and MIP-2β represent two additional genes involved in inflammatory processes and are exhibiting a number of immunoregulatory activities. Both of which showed an increased expression in our coculture experiments after 6 hours, reaching peak levels after 24 hours. Subcutaneous injection of MIP-2 into footpads of C3H/HeJ mice elicited an inflammatory response characterized by neutrophil infiltration, suggesting MIP-2 to be an endogenous mediator playing a role in the host response during inflammatory processes . The MIP-2 expression of lipopolysaccharide (LPS) or IL-1β stimulated intestinal epithelial cells is amplified by butyrate, a metabolite of nonpathogenic, resident bacteria. This is a potential mechanism by which resident bacteria may regulate inflammatory processes in the small intestine . In contrast to MCP-1, EcN mediated expression of MIP-2α and MIP-2β seemed to be independent on the viability of the probiotic in our experiments, as inactivated bacteria induced gene expression as well. Thus, it appears that EcN can induce a potent proinflammatory cytokine response which is in part dependent on the viability of the bacteria. It can be speculated that the local induction of proinflammatory immune responses within the intestinal immune system characterized by up-regulation of MCP-1, MIP-2α and MIP-2β upon contact with probiotic EcN might reflect being part of the host defense process against pathogenic bacteria through the establishment of a protective immunological barrier. However, further in vitro and especially in vivo experiments are needed to provide evidence that such mechanisms are part of the repertoire of protective responses exhibited by EcN in the intestine.
The transcription factor NF-κB and the corresponding pathway play an important role in proinflammatory signaling [39, 40]. Prevention of the NF-κB translocation into the nucleus by the IκB proteins NFκBIA and NFκBIB leads to inhibition of NF-κB, followed by suppression of proinflammatory genes. In our experiments up-regulated gene expression of the transcription inhibitor NFκBIA could be observed. It can be assumed that the observed EcN specific chemokine expression is induced via a NF-κB independent pathway. The JNK/AP-1 signaling cascade is a likely candidate, since transcription and secretion of MCP-1 can also be activated through this pathway triggered by TLR4 signaling . It is a well-known fact that toll-like receptor 4 (TLR4) and 5 (TLR5) recognize LPS  and bacterial flagellin [43, 44], respectively. Both receptors induce among others the transcription of MCP-1, MIP-2α and MIP-2β . Induction of DSS colitis in TLR4-/- mice led to an altered neutrophil recruitment due to diminished MIP-2 expression by lamina propria macrophages. Thus, TLR4 participates in the intestinal immune response to luminal bacteria and the development of colitis . Since both, EcN and E. coli MG1655 possess flagellin and LPS, it can be speculated that an additional component of EcN not yet identified is able to induce MCP-1 gene expression. However, the semirough O6 lipopolysaccharide phenotype of EcN, which is different from O6 LPS of uropathogenic E. coli and which is responsible for serum sensitivity of EcN , might also play a role in this context.
Even though the proinflammatory activity of EcN, manifested by MCP-1 gene expression and protein secretion, appears surprising at first sight, the genome content of this probiotic strain offers explanations for its proinflammatory properties. Genome comparisons of probiotic EcN and the uropathogenic E. coli (UPEC) strain CFT073 as recently published by our laboratory  and Grozdanov et al.  revealed a high degree of homology between these two strains. The existence of about 130 "common virulence factors" in probiotic EcN, as defined by the PRINTS protein fingerprint database, suggests that these proteins cannot be considered as virulence factors per se, but may also contribute to the general fitness of EcN .
The results of our study revealed that EcN specifically upregulated genes in confluent human intestinal epithelial cell culture. The majorities of these genes were proinflammatory cytokines or belonged to inflammatory pathways. These surrogate in vitro markers of probiotic activity of EcN need to be corroborated in other model systems to test their in vivo relevance. Additionally, the cell culture model described here will serve as a readout, to evaluate the impact of potential probiotic genes and gene products of EcN, which will consecutively be identified, when our genome sequencing project is finished.
List of abbreviations used
colony forming units
dual specificity phosphatase 5
- E. coli Nissle 1917:
E74-like factor 3, ets domain transcription factor, epithelial-specific
epithelial membrane protein 3
chemoattractant protein-1 ligand 2
macrophage inflammatory protein-2 alpha
macrophage inflammatory protein-2 beta
multiplicity of infection
nuclear factor of kappa light polypeptide gene enhancer in B cell inhibitor, alpha
T helper cell type 1
tumor necrosis factor, alpha-induced protein 3
tumor necrosis factor
vascular growth factor.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 621) to S. S, J. B. and F. G. We thank Tanja Toepfer for excellent technical assistance and Hartmut Steinrück for serotyping E. coli Nissle 1917.
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