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Lactobacillus rhamnosus CNCMI-4317 Modulates Fiaf/Angptl4 in Intestinal Epithelial Cells and Circulating Level in Mice.

Jacouton E, Mach N, Cadiou J, Lapaque N, Clément K, Doré J, van Hylckama Vlieg JE, Smokvina T, Blottière HM - PLoS ONE (2015)

Bottom Line: Lactobacilli are often considered to display beneficial effect for their hosts, acting on different regulatory pathways.We then validated in vivo bacterial effects using C57BL/6 mono-colonized mice fed with normal chow.Moreover, this effect was accompanied by transcriptome modulation of several pathways including immune response and metabolism in vitro.

View Article: PubMed Central - PubMed

Affiliation: Danone Nutricia Research, Palaiseau, France; INRA, UMR 1319 Micalis, Jouy en Josas, France; AgroParistech, UMR Micalis, Jouy en Josas, France.

ABSTRACT

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-γ independent but PPAR-α 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.

No MeSH data available.


Related in: MedlinePlus

IECs transcriptome analysis in presence of L. rhamnosus CNCMI–4317 and rosiglitazone; (a) Venn diagram, (b) IPA networks detected when comparing L. rhamnosus CNCMI–4317 to negative control or rosiglitazone treatment (c) to negative control in IECs, (d) validation of microarray modulated genes by RT-qPCR.(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 β-Actin and Gapdh as control genes.
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pone.0138880.g004: IECs transcriptome analysis in presence of L. rhamnosus CNCMI–4317 and rosiglitazone; (a) Venn diagram, (b) IPA networks detected when comparing L. rhamnosus CNCMI–4317 to negative control or rosiglitazone treatment (c) to negative control in IECs, (d) validation of microarray modulated genes by RT-qPCR.(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 β-Actin and Gapdh as control genes.

Mentions: We performed a whole genome transcriptome analysis of IECs in response to bacterial strains. HT–29 cells were incubated for 6 hours either with the bacterial strain of interest (L. rhamnosus CNCMI–4317), a control bacterium that did not induce Fiaf gene expression (L. rhamnosus CNCMI–2493), a culture medium as negative control, or rosiglitazone. We performed eight independent cultures of HT–29 cells at different passage number for L. rhamnosus CNCMI–4317, negative and rosiglitazone controls and four replicates for L. rhamnosus CNCMI–2493. In view of the strong effect of the cell culture (S1 Fig), we decided to include it as a covariable in the statistical model. We failed to detect genes significantly differentially expressed (DE) between L. rhamnosus CNCMI–2493 and the negative control (without bacterial strain), and we also hardly detected significant differences between the two bacteria L. rhamnosus CNCMI–4317 and CNCMI–2493 (data not shown). However, when comparing L. rhamnosus CNCMI–4317 strain and rosiglitazone to the negative control, respectively 63 and 21 genes were modulated (P<0.05). An Euler diagram visualization approach of these results highlighted that only Fiaf gene was commonly expressed (Fig 4a), strongly supporting the hypothesis that bacterial strain CNCMI–4317 acted in a PPAR-γ independent manner. As presented in Table 1, the most activated genes by L. rhamnosus CNCMI–4317 were Ddit4 (DNA damage inducible transcript 4, fold change (FC) = 2.70, qvalue = 0.003), Bhlbh2 (Basic helix loop helix family member 40, FC = 1.96, qvalue = 0.0005), Adm (Adrenomedullin, FC = 1.66, qvalue = 0.025) and Fiaf (FC = 1.63, qvalue<0.00089). To explore the molecular functions modified in response to L. rhamnosus CNCMI–4317, we measured the subsets of DE genes between treatments by using the core analysis function included in IPA software. Most biological functions found to be significantly enriched (P<0.05), by L. rhamnosus CNCMI–4317 were related to gene expression machinery, cell death/survival, cellular growth/proliferation, cell-mediated immune response and lipid metabolism categories (Table 1). Interestingly, those functions included canonical pathways associated with PPAR signalling, and HIF1α signalling (P<0.05) (S2 Fig). Four networks were identified with scores ranging from 41 to 19. The Fiaf gene was found to play a role in the regulatory network involved in putative functions such as neurological disease, cell cycle and cell development (Fig 4b). On the contrary, most of the genes regulated by rosiglitazone were involved in lipid or carbohydrate metabolism functions (Table 1). In this context, it is no surprisingly that the Fiaf gene was found to play a role in the regulatory network involved in energy production and lipid metabolism putative functions (Fig 4c). To validate technically the microarray gene expression data, IECs RNA in response to L. rhamnosus CNCMI–4317 were analysed by RT-qPCR for 12 genes (Table B in S1 File). RT-qPCR results confirmed the microarray expression levels with most genes having high r2 values (Fig 4d).


Lactobacillus rhamnosus CNCMI-4317 Modulates Fiaf/Angptl4 in Intestinal Epithelial Cells and Circulating Level in Mice.

Jacouton E, Mach N, Cadiou J, Lapaque N, Clément K, Doré J, van Hylckama Vlieg JE, Smokvina T, Blottière HM - PLoS ONE (2015)

IECs transcriptome analysis in presence of L. rhamnosus CNCMI–4317 and rosiglitazone; (a) Venn diagram, (b) IPA networks detected when comparing L. rhamnosus CNCMI–4317 to negative control or rosiglitazone treatment (c) to negative control in IECs, (d) validation of microarray modulated genes by RT-qPCR.(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 β-Actin and Gapdh as control genes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4595210&req=5

pone.0138880.g004: IECs transcriptome analysis in presence of L. rhamnosus CNCMI–4317 and rosiglitazone; (a) Venn diagram, (b) IPA networks detected when comparing L. rhamnosus CNCMI–4317 to negative control or rosiglitazone treatment (c) to negative control in IECs, (d) validation of microarray modulated genes by RT-qPCR.(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 β-Actin and Gapdh as control genes.
Mentions: We performed a whole genome transcriptome analysis of IECs in response to bacterial strains. HT–29 cells were incubated for 6 hours either with the bacterial strain of interest (L. rhamnosus CNCMI–4317), a control bacterium that did not induce Fiaf gene expression (L. rhamnosus CNCMI–2493), a culture medium as negative control, or rosiglitazone. We performed eight independent cultures of HT–29 cells at different passage number for L. rhamnosus CNCMI–4317, negative and rosiglitazone controls and four replicates for L. rhamnosus CNCMI–2493. In view of the strong effect of the cell culture (S1 Fig), we decided to include it as a covariable in the statistical model. We failed to detect genes significantly differentially expressed (DE) between L. rhamnosus CNCMI–2493 and the negative control (without bacterial strain), and we also hardly detected significant differences between the two bacteria L. rhamnosus CNCMI–4317 and CNCMI–2493 (data not shown). However, when comparing L. rhamnosus CNCMI–4317 strain and rosiglitazone to the negative control, respectively 63 and 21 genes were modulated (P<0.05). An Euler diagram visualization approach of these results highlighted that only Fiaf gene was commonly expressed (Fig 4a), strongly supporting the hypothesis that bacterial strain CNCMI–4317 acted in a PPAR-γ independent manner. As presented in Table 1, the most activated genes by L. rhamnosus CNCMI–4317 were Ddit4 (DNA damage inducible transcript 4, fold change (FC) = 2.70, qvalue = 0.003), Bhlbh2 (Basic helix loop helix family member 40, FC = 1.96, qvalue = 0.0005), Adm (Adrenomedullin, FC = 1.66, qvalue = 0.025) and Fiaf (FC = 1.63, qvalue<0.00089). To explore the molecular functions modified in response to L. rhamnosus CNCMI–4317, we measured the subsets of DE genes between treatments by using the core analysis function included in IPA software. Most biological functions found to be significantly enriched (P<0.05), by L. rhamnosus CNCMI–4317 were related to gene expression machinery, cell death/survival, cellular growth/proliferation, cell-mediated immune response and lipid metabolism categories (Table 1). Interestingly, those functions included canonical pathways associated with PPAR signalling, and HIF1α signalling (P<0.05) (S2 Fig). Four networks were identified with scores ranging from 41 to 19. The Fiaf gene was found to play a role in the regulatory network involved in putative functions such as neurological disease, cell cycle and cell development (Fig 4b). On the contrary, most of the genes regulated by rosiglitazone were involved in lipid or carbohydrate metabolism functions (Table 1). In this context, it is no surprisingly that the Fiaf gene was found to play a role in the regulatory network involved in energy production and lipid metabolism putative functions (Fig 4c). To validate technically the microarray gene expression data, IECs RNA in response to L. rhamnosus CNCMI–4317 were analysed by RT-qPCR for 12 genes (Table B in S1 File). RT-qPCR results confirmed the microarray expression levels with most genes having high r2 values (Fig 4d).

Bottom Line: Lactobacilli are often considered to display beneficial effect for their hosts, acting on different regulatory pathways.We then validated in vivo bacterial effects using C57BL/6 mono-colonized mice fed with normal chow.Moreover, this effect was accompanied by transcriptome modulation of several pathways including immune response and metabolism in vitro.

View Article: PubMed Central - PubMed

Affiliation: Danone Nutricia Research, Palaiseau, France; INRA, UMR 1319 Micalis, Jouy en Josas, France; AgroParistech, UMR Micalis, Jouy en Josas, France.

ABSTRACT

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-γ independent but PPAR-α 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.

No MeSH data available.


Related in: MedlinePlus