Probiotics are live microorganisms known to have beneficial effects on the health of humans or animals. Although the use of probiotics has a long tradition in medicine, the molecular mechanisms behind the probiotic-host interaction remain unclear. We here report the results of a study, aimed to characterize the host gene expression program initiated by the probiotic E. coli strain Nissle 1917 (EcN) in human intestinal epithelial cells. Confluent Caco-2 cells were therefore cocultured with EcN and the E. coli K12 laboratory isolate MG1655 for 6 hours. Analysis of microarray data revealed selective expression of 126 genes after EcN treatment in comparison with the control strain. For further studies, 10 genes of major interest were chosen to confirm regulatory data by real-time RT-PCR and, if possible, by cytometric bead array (CBA) at protein level. Interestingly, treatment of Caco-2 cells with EcN significantly up-regulated 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), and led to expression of more than 300 pg/ml MCP-1 protein into tissue culture media. EcN-mediated gene expression of MCP-1 peaked at 6 hours. In Caco-2 cells protected from bacterial overgrowth with gentamycin, maximum gene expression of MIP-2α and MIP-2β was reached after 24 hours. We here describe a proinflammatory signaling of Caco-2 cells after EcN treatment which adds novel facts to the multifaceted probiotic-host interaction of EcN and will direct further research to fully elucidate the molecular basis of the probiotic nature of this strain. Cell culture conditions. Caco-2 cells were maintained in IMDM cell culture medium (Invitrogen, Karlsruhe, Germany) containing 10 % fetal calf serum (PAA Laboratories, Cölbe, Germany) and 5 % penicillin/streptomycin at 37°C in a cell culture incubator. They were split twice a week at a ratio of 1:3. 4 x 105 cells per well were seeded in six well plates (Nunc, Wiesbaden, Germany) and cultured for approximately four days until confluence.
Preparation of bacteria. E. coli strain Nissle 1917 was cultured from a tablet of Mutaflor® (Ardeypharm, Herdecke, Germany), isolated from MacConkey plates (Oxoid, Wesel, Germany), serotyped and confirmed by EcN specific PCR (6). E. coli K12 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 with 10 % fetal calf serum and grown at 37 °C. Bacteria were harvested in late logarithmic phase, after about 5 hours at an OD600 = 1.
Cocultivation of Caco-2 cells with EcN. Confluent Caco-2 cells (~ 0.4 x 107 cells per well) were washed with phosphate buffered saline (PBS) and cultured in IMDM cell culture medium containing 10 % fetal calf serum at 37 °C in a cell culture incubator for 6 hours. Prior to RNA isolation Caco-2 cells were washed twice with PBS.
RT-PCR. RNA was extracted using TriFast FL (PEQLAB, Erlangen, Germany) according to the manufacturer’s protocol, following DNAse I digestion with DNA-free® (Ambion, Huntingdon Cambridgeshire, UK) and reversely transcribed with 200 U Superscript II® (Invitrogen) for 45 minutes at 42 °C in 20 µl assays containing 1 µl Oligo dT-primers and 10 mM deoxynucleoside triphosphates (dNTPs). Conventional PCR was performed with 250 U AmpliTaq Gold® DNA polymerase (Applied Biosystems, Darmstadt, Germany) in 20 µl assays containing 0.25 pmol primers and 0.5 mM dNTP using the following cycling conditions: activation of Taq polymerase for 10 minutes at 95 °C, followed by a first round of 10 cycles each consisting of a 30 second denaturing interval at 95 °C, a 90 second annealing step at the respective primer Tm and a primer extension at 72 °C for again 90 seconds. This first amplification step was followed by a second round of 27 cycles, consisting of 15 seconds denaturation, 45 seconds annealing and 90 seconds extension. PCR products were separated in 2 % agarose gels and visualized by ethidium-bromide staining.
DNA microarray hybridization. Quality and integrity of the total RNA isolated from approximately 1 x 106 cells was controlled by running all samples on an Agilent Technologies 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.
Data analysis. 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 (I)) or greater than 0.99 (change p-Value for changes called decreased (D)). 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.
