Identification of NF-κB Modulation Capabilities within Human Intestinal Commensal Bacteria

The intestinal microbiota plays an important role in modulation of mucosal immune responses. To seek interactions between intestinal epithelial cells (IEC) and commensal bacteria, we screened 49 commensal strains for their capacity to modulate NF-κB. We used HT-29/kb-seap-25 and Caco-2/kb-seap-7 intestinal epithelial cells and monocyte-like THP-1 blue reporter cells to measure effects of commensal bacteria on cellular expression of a reporter system for NF-κB. Bacteria conditioned media (CM) were tested alone or together with an activator of NF-κB to explore its inhibitory potentials. CM from 8 or 10 different commensal species activated NF-κB expression on HT-29 and Caco-2 cells, respectively. On THP-1, CM from all but 5 commensal strains stimulated NF-κB. Upon challenge with TNF-α or IL-1β, some CM prevented induced NF-κB activation, whereas others enhanced it. Interestingly, the enhancing effect of some CM was correlated with the presence of butyrate and propionate. Characterization of the effects of the identified bacteria and their implications in human health awaits further investigations

Commensal gut bacteria modulate phosphorylation-dependent PPARγ transcriptional activity in human intestinal epithelial cells

In healthy subjects, the intestinal microbiota interacts with the host’s epithelium, regulating gene expression to the benefit of both, host and microbiota. The underlying mechanisms remain poorly understood, however. Although many gut bacteria are not yet cultured, constantly growing culture collections have been established. We selected 57 representative commensal bacterial strains to study bacteria-host interactions, focusing on PPARγ, a key nuclear receptor in colonocytes linking metabolism and inflammation to the microbiota. Conditioned media (CM) were harvested from anaerobic cultures and assessed for their ability to modulate PPARγ using a reporter cell line. Activation of PPARγ transcriptional activity was linked to the presence of butyrate and propionate, two of the main metabolites of intestinal bacteria. Interestingly, some stimulatory CMs were devoid of these metabolites. A Prevotella and an Atopobium strain were chosen for further study, and shown to up-regulate two PPARγ-target genes, ANGPTL4 and ADRP. The molecular mechanisms of these activations involved the phosphorylation of PPARγ through ERK1/2. The responsible metabolites were shown to be heat sensitive but markedly diverged in size, emphasizing the diversity of bioactive compounds found in the intestine. Here we describe different mechanisms by which single intestinal bacteria can directly impact their host’s health through transcriptional regulation.ISSN:2045-232

Edge effect reduction on cellular growth applied to the HT-29-PPARγ reporter cell-line.

<p>A Parental HT-29 cells were homogeneously seeded using a pipetting robotic workstation and pre-incubated at room temperature for the indicated time (0, 0.5 or 1 h) prior to 37°C incubation for 24 h. Cellular monolayer homogeneity was monitored using cell staining with crystal violet and quantified by absorption measurement at 595 nm. Left panel is a surface plot of a representative set of plates at increasing times of room temperature pre-incubation. Boxplot representation (right panels) of the OD 595 values for the border compared to the core of the respective representative plates. Mean p-values for different cell-lines tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-t001" target="_blank">Table 1</a>. B HT-29-PPARγ reporter cells were homogeneously seeded using a pipetting robotic workstation and pre-incubated at room temperature for the indicated time (0 or 1 hour) then at 37°C for 24 h prior to sodium butyrate (2 mM) activation. Luciferase activity (RLU) was quantified after 24 h of activation. Graph (left panels) shows two representative plates with different pre-incubation times at room temperature. Boxplots (right panels) represent the border and core values of the surface plots. Mean p-values for different cell-lines tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-t002" target="_blank">Table 2</a>.</p

Different normalizations of assay plates.

<p>Different mathematical approaches for normalization were applied to the dataset from the initial HT-29-PPARγ screening. A shows all data-points as dots ordered by activity-rank whereby the red dots are points on the plate’s cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots. B shows ranked Z-score normalization of the results. (red dots are points on the cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots. C. shows the ranked B-score values for the same data set. (red dots are points on the cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots.</p

Metagenomic clones growth optimization.

<p>Four random plates of EPI300 metagenomic clones were inoculated from their −80°C glycerol stock into 96 well-plates (10% v/v) using a pipetting robotic workstation. After a 24 h pre-culture, they were re-inoculated and cultured overnight (10% v/v) at 37°C. These steps were performed either in static or agitated (700 rpm) culture conditions. Bacterial growth was monitored by optical density (OD 600 nm). A. Border and core wells of bacterial cultures in agitated (left panel) or static culture-conditions (right panel) for thus representative set of 4 plates. p-values indicated on the figure are the result of a unpaired t-test of the core <i>versus</i> border bacterial culture ODs. B. Growth curves of one representative plate of metagenomic clones (OD 600 nm) for agitated and static culture conditions. Red dots: mean OD600, joined grey dots: growth curve of single metagenomic clones.</p

Detection limit on optimized reporter gene assay.

<p>The HT-29-PPARγ cell-line was homogeneously seeded using a pipetting robotic workstation and incubated at room temperature for 1 h prior to 37°C incubation. After 24 h of culture, all wells were activated homogenously with EPI300-Cont lysates for 24 h at 37°C. In randomly chosen wells (50% border and 50% core), three different concentrations of PPARγ activating NaBut (at 500 µM, 160 µM and the sub-activating concentration 80 µM) were randomly added in the 96 well-plates. The graph represents normalized values from 3 independent plates.</p

Cellular growth edge effect reduction.

<p>Cell-lines were processes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g002" target="_blank">Figure 2</a> using crystal violet. Mean p-values +/− SEM comparing borders versus core for different parental cell-lines at 0 or 1 h room temperature incubation are reported.</p

Cellular growth-dependent edge effect reduction applied to three different reporter cell-lines.

<p>Cell-lines were processes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g003" target="_blank">Figure 3</a>. The table represents the mean of three p-values +/− SEM for comparing borders versus core for the different tested reporter cell-lines.</p

PPARγ HTS screening.

<p>Comparison of different data analysis methods of screening data from the screening of 92×96 metagenomic clones on a PPARγ RGA. Figure A represents Z-score normalized data B represents B-score normalized data. The top left picture plots the each readout in function of the growth of the metagenomic clone. The bottom left picture represents the distribution of the readouts over the whole dataset and the large picture on the right part represents all data points ranked by readout. The coloration of red dots for the borders and empty green dots for the core allow to visualize eventual position effects.</p