Lactobacillus rhamnosus CNCMI-4317 modulates Fiaf/Angptl4 in intestinal epithelial cells and circulating level in mice

Background and Objectives Identification of new targets for metabolic diseases treatment or prevention is required. In this context, FIAF/ANGPTL4 appears as a crucial regulator of energy homeostasis. Lactobacilli are often considered to display beneficial effect for their hosts, acting on different regulatory pathways. The aim of the present work was to study the effect of several lactobacilli strains on Fiaf gene expression in human intestinal epithelial cells (IECs) and on mice tissues to decipher the underlying mechanisms. Subjects and Methods Nineteen lactobacilli strains have been tested on HT-29 human intestinal epithelial cells for their ability to regulate Fiaf gene expression by RT-qPCR. In order to determine regulated pathways, we analysed the whole genome transcriptome of IECs. We then validated in vivo bacterial effects using C57BL/6 mono-colonized mice fed with normal chow. Results We identified one strain (Lactobacillus rhamnosus CNCMI-4317) that modulated Fiaf expression in IECs. This regulation relied potentially on bacterial surface-exposed molecules and seemed to be PPAR-gamma independent but PPAR-alpha dependent. Transcriptome functional analysis revealed that multiple pathways including cellular function and maintenance, lymphoid tissue structure and development, as well as lipid metabolism were regulated by this strain. The regulation of immune system and lipid and carbohydrate metabolism was also confirmed by overrepresentation of Gene Ontology terms analysis. In vivo, circulating FIAF protein was increased by the strain but this phenomenon was not correlated with modulation Fiaf expression in tissues (except a trend in distal small intestine). Conclusion We showed that Lactobacillus rhamnosus CNCMI-4317 induced Fiaf expression in human IECs, and increased circulating FIAF protein level in mice. Moreover, this effect was accompanied by transcriptome modulation of several pathways including immune response and metabolism in vitro

Supplementary materials for Beneficial Microbes: <b>Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using </b><b><i>in vitro</i></b><b> and </b><b><i>in vivo</i></b><b> approaches</b>

Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable in vitro experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the Lactobacillus and Bifidobacterium genera, were screened using three different sets of in vitro experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: Lactobacillus gasseri PI41, Lacticaseibacillus rhamnosus PI48 and Bifidobacterium animalis subsp. lactis PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, L. gasseri PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of in vitro experiments combined with in vivo assessments allowed us to identify a promising probiotic candidate strain, L. gasseri PI41, in the context of IBS.</p

List of regulated genes revealed by transcriptomic analysis after rosiglitazone or <i>L</i>. <i>rhamnosus</i> treatment of HT–29 cells.

<p>Fold change (FC) are expressed in comparison with negative control (DMEM treatment), ns means that gene was not statistically significantly regulated by the treatment</p><p>List of regulated genes revealed by transcriptomic analysis after rosiglitazone or <i>L</i>. <i>rhamnosus</i> treatment of HT–29 cells.</p

Effect of <i>L</i>. <i>rhamnosus</i> and <i>L</i>. <i>paracasei</i> on <i>Fiaf</i> expression in IECs. Cells were stimulated 6h with 20% of final volume of bacterial cultures.

<p>Bars represent mean of <i>Fiaf</i> relative expression (percentage of rosiglitazone) from two to seven independent experiments performed in triplicates. Clear bars correspond to <i>L</i>. <i>paracasei</i> strains and dark bars correspond to <i>L</i>. <i>rhamnosus</i> strains. Data are normalized using β-<i>Actin</i> as control gene. Stars represent p<0.001 (***) in comparison with negative control (DMEM).</p

IECs transcriptome analysis in presence of <i>L</i>. <i>rhamnosus</i> CNCMI–4317 and rosiglitazone; (a) Venn diagram, (b) IPA networks detected when comparing <i>L</i>. <i>rhamnosus</i> CNCMI–4317 to negative control or rosiglitazone treatment (c) to negative control in IECs, (d) validation of microarray modulated genes by RT-qPCR.

<p>(b) FC are expressed in comparison with negative control (DMEM treatment), ns means that gene was not statistically significantly regulated by the treatment. Up-regulated genes are represented in grey shade except DKK1, which is down-regulated. (c) The networks included genes involved in neurological disease, cell cycle and cell development or Energy production, Lipid metabolism and small molecule biochemistry presented a score of 41 and 28 respectively (few genes are deleted to network for better view). The network displayed graphically as nodes (gene/gene products) and edges (the biological relationship between nodes). The node grey intensity indicates the expression of genes: black and bold: up-regulated, grey: down-regulated in intestinal tissues. The shapes of nodes indicate the functional class of the gene product. The log fold change values are indicated under each node. PPAR signalling canonical pathway was added. CP mean canonical pathway. (d) RT-qPCR data are normalized using geometrical mean of <i>β-Actin</i> and <i>Gapdh</i> as control genes.</p

<i>L</i>. <i>rhamnosus</i> CNCMI–4317 may induce <i>F</i>iaf in a PPAR-α independent (a) but PPAR-γ dependent (b) manner. The antagonists (GW7647 and GW9662) were respectively added at 1 and 10μM 1h before challenging with agonists (GW6471 and rosiglitazone) during 6h.

<p>Bars represent means of <i>Fiaf</i> relative expression (percentage of rosiglitazone and GW7647 respectively) from three independent experiments performed in triplicates. Data are normalized using β-<i>Actin</i> as control gene and by GW7647 (a) or rosiglitazone (b). Stars represent p<0.05 (*), p< 0.01 (**) and p<0.001 (***) in comparison with negative control (DMEM). ns represent a non significant difference between <i>L</i>. <i>rhamnosus</i> CNCMI- 4317 versus <i>L</i>. <i>rhamnosus</i> CNCMI—4317 supplemented with GW9662.</p

Bacterial effectors characterization. (a) Conditioned media, (b) Heat inactivated bacteria, (c) Transwell. Cells were incubated 6h with 10% of final volume of bacterial fractions (CM, HI) and 20% of bacteria for transwell structure. Transwell prevented contact between cells and bacteria.

<p>Bars represent means of <i>Fiaf</i> relative expression (percentage of rosiglitazone) from three independent experiments performed in triplicates. Data are normalized using β-<i>Actin</i> as control gene. Stars represent p<0.05 (*), p<0.01 (**) and p<0.001 (***) in comparison with negative control (DMEM). ns represents a no significant difference in comparison with negative control.</p

Nature en ville. Desirs & controverses

Il libro descrive numerosi progetti di riqualificazione urbana, volti a migliorare il benessere e la qualità di vita dei cittadini

ErmineJ significant GO pathways modulated by <i>L</i>. <i>rhamnosus</i> CNCMI–4317 in IECs.

<p>ErmineJ significant GO pathways modulated by <i>L</i>. <i>rhamnosus</i> CNCMI–4317 in IECs.</p