Transcriptomic response of immune signalling pathways in intestinal epithelial cells exposed to lipopolysaccharides, Gram-negative bacteria or potentially probiotic microbes

Abstract

In order to understand the appropriate use of potentially probiotic Gram-positive microbes through their introduction in the gut microbiome, it is necessary to understand the influence of individual bacteria on the host-response system at a cellular level. In the present study, we have shown that lipopolysaccharides, flagellated Gram-negative bacteria, potentially probiotic Gram-positive bacteria and yeast interact differently with human intestinal epithelial cells with a custom-designed expression microarray evaluating 17 specific host-response pathways. Only lipopolysaccharides and flagellated Gram-negative bacteria induced inflammatory response, while a subset of Gram-positive microbes had anti-inflammatory potential. The main outcome from the study was the differential regulation of the central mitogen-activated protein kinase signalling pathway by these Gram-positive microbes versus commensal/pathogenic Gram-negative bacteria. The microarray was efficient to highlight the impact of individual bacteria on the response of intestinal epithelial cells, but quantitative real-time polymerase chain reaction validation demonstrated some underestimation for down-regulated genes by the microarray. This immune array will allow us to better understand the mechanisms underlying microbe-induced host immune responses.

Multistrain Probiotic Modulation of Intestinal Epithelial Cells' Immune Response to a Double-Stranded RNA Ligand, Poly(I·C)

“…Cell challenges were performed as explained previously by Audy et al (18). Briefly, HT-29 cells were incubated for 3 h with poly(I·C) or PC alone or PC in combination with poly(I·C) at 10 g/ml.…”

“…Fifteen micrograms of control and treated total RNA was used for RT and the direct method of dye labeling as previously described by Audy et al (18). Briefly, RNA was aliquoted and concentrated with a SpeedVac apparatus for about 45 min at medium temperature.…”

“…Measurement of the transcript abundance of differentially expressed and reference genes was confirmed by comparative qRT-PCR of cDNA as described previously by Audy et al (18) according to MIQE guidelines (23). Briefly, 4 g of total RNA was treated with RQ1 DNase (Promega, Madison, WI) per the manufacturer's instructions.…”

“…As previously described by Audy et al (18), the immune array was a custom-designed two-color, long oligonucleotide (70-mer) DNA microarray detecting 1,354 human genes belonging to 17 pathways connected to innate immunity and barrier defense. Genes were selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and belonged to the following pathways: cell adhesion molecules (CAMs), defensins, mucus production, gap junction, tight junction, apoptosis, adherence, cytokine-cytokine receptor interaction, chemokine signaling, B cell receptor signaling, JAK-STAT signaling, transforming growth factor beta (TGF-␤) signaling, T cell receptor signaling, MAPK signaling, TLR signaling, NOD-like receptor (NLR) signal-ing, and natural killer cell-mediated cytotoxicity (19).…”

“…The intention was to evaluate at the transcriptional level whether the combination of these three bacteria, here, the probiotic combination (PC), has the capacity to immune modulate an in vitro model of IECs cochallenged with a dsRNA ligand, poly(I·C). To this end, we used a custom-designed, two-color expression microarray targeting genes of the human immune system, here referred to as the immune array (18). The aim of this study was to evaluate by which pathways this combination of bacteria could modulate the host cell response to a dsRNA ligand, poly(I·C), which could potentially help in the design of future clinical trials based on potential biomarkers confirmed in this study.…”