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

Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells

Commensal bacteria are crucial for the development and maintenance of a healthy immune system therefore contributing to the global well-being of their host. A wide variety of metabolites produced by commensal bacteria are influencing host health but the characterization of the multiple molecular mechanisms involved in host-microbiota interactions is still only partially unraveled. The intestinal epithelial cells (IECs) take a central part in the host-microbiota dialogue by inducing the first microbial-derived immune signals. Amongst the numerous effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) is essential for gut homeostasis. IDO-1 expression is dependent on the microbiota and despites its central role, how the commensal bacteria impacts its expression is still unclear. Therefore, we investigated the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression and found that the short chain fatty acid (SCFA) butyrate was the main metabolite controlling IDO-1 expression in human primary IECs and IEC cell-lines. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43, and GPR109a and of the transcription factors SP1, AP1, and PPARγ for which binding sites were reported in the IDO-1 promoter. We demonstrated for the first time that butyrate represses IDO-1 expression by two distinct mechanisms. Firstly, butyrate decreases STAT1 expression leading to the inhibition of the IFNγ-dependent and phosphoSTAT1-driven transcription of IDO-1. In addition, we described a second mechanism by which butyrate impairs IDO-1 transcription in a STAT1-independent manner that could be attributed to its histone deacetylase (HDAC) inhibitor property. In conclusion, our results showed that IDO-1 expression is down-regulated by butyrate via a dual mechanism: the reduction of STAT1 level and the HDAC inhibitor property of SCFAs

Commensal gut bacteria modulate phosphorylation-dependent PPAR gamma 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 PPAR gamma, 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 PPAR gamma using a reporter cell line. Activation of PPAR gamma 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 upregulate two PPAR gamma-target genes, ANGPTL4 and ADRP. The molecular mechanisms of these activations involved the phosphorylation of PPAR gamma 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

Isolates from normal human intestinal flora but not lactic acid bacteria exhibit 7α- and 7β-hydroxysteroid dehydrogenase activities

Ursodeoxycholic acid (UDCA)-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. UDCA is the 7 b -OH epimer of the primary (i.e. synthesized by the liver) bile acid chenodeoxycholic acid (CDCA). Epimerization proceeds in two subsequent and reversible steps, catalysed by a 7 a - and a 7 b -hydroxysteroid dehydrogenase (7 a - and 7 b - HSDH), with 7oxo-lithocholic acid (7oxo-LCA) as the intermediate product. The aim of this study was to test the 7 a - and 7 b -HSDH activities of anaerobic whole cell cultures of a number of lactic acid bacteria and human intestinal isolates, using CDCA, UDCA and 7oxo- LCA as the substrates. Among 140 strains tested, 21 exhibited at least one of both 7-HSDH activities. 7 a -HSDH activity was detected in six strains, 7 b -HSDH in nine strains, and both activities in six other strains. All active strains were isolated from normal human and infant faeces. They belonged to the genera Clostridium , Eubacterium and Ruminococcus , whereas no strain of Lactobacillus , Bifidobacterium or Streptococcus was found to be active under our study conditions. The present study therefore revealed, for the first time, a number of normal human intestinal isolates supporting the epimerization of CDCA to UDCA, and further extended our knowledge of those intestinal bacteria which are responsible for 7 a -or7 b -HSDH activity. Key words: screening, bile acids, epimerization, intestinal microflora, lactic acid bacteria, probiotics

Identification of the novel role of butyrate as AhR ligand in human intestinal epithelial cells

Abstract The ligand activated transcription factor, aryl hydrocarbon receptor (AhR) emerged as a critical regulator of immune and metabolic processes in the gastrointestinal tract. In the gut, a main source of AhR ligands derives from commensal bacteria. However, many of the reported microbiota-derived ligands have been restricted to indolyl metabolites. Here, by screening commensal bacteria supernatants on an AhR reporter system expressed in human intestinal epithelial cell line (IEC), we found that the short chain fatty acid (SCFA) butyrate induced AhR activity and the transcription of AhR-dependent genes in IECs. We showed that AhR ligand antagonists reduced the effects of butyrate on IEC suggesting that butyrate could act as a ligand of AhR, which was supported by the nuclear translocation of AhR induced by butyrate and in silico structural modelling. In conclusion, our findings suggest that (i) butyrate activates AhR pathway and AhR-dependent genes in human intestinal epithelial cell-lines (ii) butyrate is a potential ligand for AhR which is an original mechanism of gene regulation by SCFA

Influence of bile salt molecular species on cholesterol crystallization from supersaturated model biles

Time-sequential enzymatic determination of cholesterol (CH) crystals harvested by ultrafiltration, and concomitant polarizing light microscopy observations corroborated the striking importance of the bile salts (BS) species in determining CH crystals formation rate from supersaturated model biles incubated in vitro. The more hydrophilic tauroursodeoxycholate, taurohyocholate, glycohyocholate, taurohyodeoxycholate, glycohyodeoxycholate and glyco-3α, hydroxy-6 oxo-5ß-cholanate inhibited CH precipitation through the formation of a stabilized liquid-crystalline phase. In contrast, in all hydrophobic systems (taurine (T) and glycine (G) conjugates of cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC)), CH crystals precipitated with time. When crystallized CH concentrations were plotted vs. time, the figures showed a sigmoidal pattern, consistent with the transition from metastable systems to stable equilibrium states. Over the equilibration period, the nucleation kinetics (as inferred from enzymatic measurements) and all crystallization events (as microscopically observed) were both shifted in time, depending on the BS species: they were earliest in CDC systems, then in DC systems, and finally in C systems. In the latter, the delay was clearly due to the formation of a transient labile liquid-crystalline phase. G-conjugation also induced a significant delay in CH precipitation, compared to T-conjugation. At last, maximum crystallized CH concentrations at equilibrium were in the decreasing order: C > CDC > DC and T-conjugates > G-homologues. All data are discussed in connection with BS hydrophobicities, with predictions from the phase equilibria of aqueous biliary lipid systems and with new insights into CH crystal habits

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

Butyrate Produced by Commensal Bacteria Down-Regulates Indolamine 2,3-Dioxygenase 1 (IDO-1) Expression via a Dual Mechanism in Human Intestinal Epithelial Cells.

Commensal bacteria are crucial for the development and maintenance of a healthy immune system therefore contributing to the global well-being of their host. A wide variety of metabolites produced by commensal bacteria are influencing host health but the characterization of the multiple molecular mechanisms involved in host-microbiota interactions is still only partially unraveled. The intestinal epithelial cells (IECs) take a central part in the host-microbiota dialogue by inducing the first microbial-derived immune signals. Amongst the numerous effector molecules modulating the immune responses produced by IECs, indoleamine 2,3-dioxygenase-1 (IDO-1) is essential for gut homeostasis. IDO-1 expression is dependent on the microbiota and despites its central role, how the commensal bacteria impacts its expression is still unclear. Therefore, we investigated the impact of individual cultivable commensal bacteria on IDO-1 transcriptional expression and found that the short chain fatty acid (SCFA) butyrate was the main metabolite controlling IDO-1 expression in human primary IECs and IEC cell-lines. This butyrate-driven effect was independent of the G-protein coupled receptors GPR41, GPR43, and GPR109a and of the transcription factors SP1, AP1, and PPARγ for which binding sites were reported in the IDO-1 promoter. We demonstrated for the first time that butyrate represses IDO-1 expression by two distinct mechanisms. Firstly, butyrate decreases STAT1 expression leading to the inhibition of the IFNγ-dependent and phosphoSTAT1-driven transcription of IDO-1. In addition, we described a second mechanism by which butyrate impairs IDO-1 transcription in a STAT1-independent manner that could be attributed to its histone deacetylase (HDAC) inhibitor property. In conclusion, our results showed that IDO-1 expression is down-regulated by butyrate via a dual mechanism: the reduction of STAT1 level and the HDAC inhibitor property of SCFAs

Figure 4

<p>c-Fos A) and Cyclin D1 B) gene expression determined by Quantitative real-time PCR on total RNA extracted from cells exposed to PMA (0.1 µM), Na-Butyrate (2 mM), or both of them, for 1 h and 6 h, respectively. A positive, synergistic effect is observed on c-Fos expression for cells co-stimulated with PMA/Na-butyrate, while, in the same conditions, an opposite effect is observed for cycline D1. Data are mean + standard error of the mean (SEM) of (triplicate) measurement of a representative of three independent experiments. Different letters indicate statistically different results (p<0.05).</p

C-fos time course upon PMA induced activation.

<p>Cells were treated with PMA (0,1 µM), butyrate (2 mM), TSA 500 nM, or association of PMA with butyrate or TSA for 0,5, 1 h A), 6 and 24 h B) and total proteins were Western blotted for c-fos. A synergistic effect following PMA/Butyrate co-stimulation is observed after 6 h of stimulation and 24 h. Western blot of GAPDH (lower panel) was shown as loading control. Data are representative of three independent experiments.</p