5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein-coupled receptor D.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca2+ imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)- and pertussis toxin-dependent mechanism, suggesting that the effect was mediated by a G protein-coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.BBSRC BB/R006210/1 to James R F Hockley and Ewan St John Smith Rosetrees 834 Postdoctoral Grant (A1296) awarded to James R F Hockley and Ewan St John Smit

A fermented milk concentrate and a combination of short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides/pectin-derived acidic oligosaccharides protect suckling rats from rotavirus gastroenteritis

Human milk contains bioactive compounds that confer a protective role against gastrointestinal infections. In order to find supplements for an infant formula able to mimic these benefits of breast-feeding, two different concepts were tested. The products consisted of the following: (1) a Bifidobacterium breve- and Streptococcus thermophilus-fermented formula and (2) a combination of short-chain galacto-oligosaccharides/ long-chain fructo-oligosaccharides with pectin-derived acidic oligosaccharides. A rotavirus infection suckling rat model was used to evaluate improvements in the infectious process and in the immune response of supplemented animals. Both nutritional concepts caused amelioration of the clinical symptoms, even though this was sometimes hidden by softer stool consistency in the supplemented groups. Both products also showed certain modulation of immune response, which seemed to be enhanced earlier and was accompanied by a faster resolution of the process. The viral shedding and the in vitro blocking assay suggest that these products are able to bind the viral particles, which can result in a milder infection. In conclusion, both concepts evaluated in this study showed interesting protective properties against rotavirus infection, which deserve to be investigated further

Bacteria-derived long chain fatty acid exhibits anti-inflammatory properties in colitis.

OBJECTIVE: Data from clinical research suggest that certain probiotic bacterial strains have the potential to modulate colonic inflammation. Nonetheless, these data differ between studies due to the probiotic bacterial strains used and the poor knowledge of their mechanisms of action. DESIGN: By mass-spectrometry, we identified and quantified free long chain fatty acids (LCFAs) in probiotics and assessed the effect of one of them in mouse colitis. RESULTS: Among all the LCFAs quantified by mass spectrometry in Escherichia coli Nissle 1917 (EcN), a probiotic used for the treatment of multiple intestinal disorders, the concentration of 3-hydroxyoctadecaenoic acid (C18-3OH) was increased in EcN compared with other E. coli strains tested. Oral administration of C18-3OH decreased colitis induced by dextran sulfate sodium in mice. To determine whether other bacteria composing the microbiota are able to produce C18-3OH, we targeted the gut microbiota of mice with prebiotic fructooligosaccharides (FOS). The anti-inflammatory properties of FOS were associated with an increase in colonic C18-3OH concentration. Microbiota analyses revealed that the concentration of C18-3OH was correlated with an increase in the abundance in Allobaculum, Holdemanella and Parabacteroides. In culture, Holdemanella biformis produced high concentration of C18-3OH. Finally, using TR-FRET binding assay and gene expression analysis, we demonstrated that the C18-3OH is an agonist of peroxisome proliferator activated receptor gamma. CONCLUSION: The production of C18-3OH by bacteria could be one of the mechanisms implicated in the anti-inflammatory properties of probiotics. The production of LCFA-3OH by bacteria could be implicated in the microbiota/host interactions

5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein-coupled receptor D

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca2+ imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)- and pertussis toxin-dependent mechanism, suggesting that the effect was mediated by a G protein-coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.status: publishe

PPARγ Is Activated during Congenital Cytomegalovirus Infection and Inhibits Neuronogenesis from Human Neural Stem Cells

<div><p>Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1% of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses and investigated the outcomes of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARγ), a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARγ levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARγ agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARγ activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARγ was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV-infected NSCs with the PPARγ inhibitor T0070907 restored a normal rate of differentiation. The role of PPARγ in the disease phenotype was strongly supported by the immunodetection of nuclear PPARγ in brain germinative zones of congenitally infected fetuses (N = 20), but not in control samples. Altogether, our findings reveal a key role for PPARγ in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARγ gene targets in the infected brain.</p></div

Nuclear PPARγ expression in germinative zone of HMV-infected human fetal brains.

<p>Shown are representative results of immunohistological staining of brain sections from fetuses infected by HCMV (A-G) or from controls (H, I) using antibodies against PPARγ (A-E; F, left; G-I) or IE (F, right). The reference number of each donor is indicated at the bottom left of each panel. Clinical details are summarized in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005547#ppat.1005547.t001" target="_blank">Table 1</a>. PPARγ positive cells (arrows) are detected in the germinative, periventricular, areas and in ependyma (double arrow) in cases, but not in controls. Insets show the localization of the optical field within the brain sections (arrowheads). Note the nuclear localization of PPARγ (A-G), the presence of PPARγ positive cell islets surrounding one IE positive cell in two fields from serial sections (F) and clusters of PPARγ immunoreactive cells around lesional tissue (G). Scale bar: 50 μm.</p

Proposed model for the role of PPARγ during HCMV infection of NSCs.

<p>HCMV particles (HCMV) carry onboarded a cell-derived packaged cPLA2 (oPLA2, dots), which catalyzes the release of linoleic acid (LA) from host membrane phospholipids (PL) upon infection. LA undergoes oxidization driven by 15-lipoxygenase (LOX), which generates 9-HODE. 9-HODE, in turn, increases PPARγ levels. Activated PPARγ dimerizes with RXR to regulate the expression of host and viral genomes, resulting in impaired neuronogenesis in vitro and enhanced viral replication. M: cell membrane, C: cytoplasm, N: nucleus.</p

NSCs are permissive to HCMV infection.

<p>(A) Immunofluorescence analysis of NSCs infected by live (HCMV) or UV-irradiated (HCMV+UV) HCMV, or uninfected (NI), showing nuclear staining to the HCMV Immediate Early antigen (IE) two days post infection (dpi) at a multiplicity of infection (MOI) of 10. DAPI staining and merged pictures are shown. Scale bar: 50 μm. (B) Top: automated counting of immunofluorescence data showing increasing numbers of IE-positive NSCs over time in cultures infected by live HCMV at a MOI of 1 or 10, but not in cultures infected by UV-irradiated HCMV or in uninfected cultures. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. Bottom: western blot analysis showing increasing levels along time of the 72 and 86 kDa isoforms of IE in infected NSCs (MOI 10). (C) Western blot analysis showing production of the early and late HCMV antigens UL44 and pp28, respectively, in infected NSCs (MOI 10), at 8 days pi. (D) Top: transmission electron microscopy of NSC cultures infected by HCMV (MOI 10), showing a cytomegalic NSC (arrowhead) and lipid vesicles (arrows), close to two morphologically normal NSCs (NI), and HCMV particles adsorbed onto the cell surface (inset). Scale bar: 5μm or 0.2 μm (inset). Bottom: transmission electron microscopy of the cytoplasm of an infected NSC, revealing mature viral particles (arrowheads) and dense bodies (asterisks). Pictures were taken 6 days after infection. Scale bar: 0.5μm. (E) Titration of viral particles present in the supernatants of infected NSCs (MOI 10). Supernatants were harvested at different times pi (horizontal axis) and were titrated on MRC5 fibroblasts. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. Virus strain was AD169 except for panel A (VHL/E).</p

HCMV infection triggers the expression and activity of PPARγ in NSCs.

<p>(A) Immunofluorescence analysis using antibodies specific to IE or PPARγ showing strong nuclear staining of PPARγ in NSCs infected by HCMV at a MOI of 10 (HCMV), as compared to non infected NSC cultures (NI) or NSCs infected with UV-irradiated HCMV (HCMV+UV). In merged pictures, double stained nuclei appear cyan (PPARγ and DAPI) or magenta (IE and DAPI); nuclei stained by DAPI and PPARγ and IE antibodies appear purple. (B) Western blot analysis showing increased levels of PPARγ polypeptide in NSCs infected by HCMV (MOI 10) (HCMV) as compared to the uninfected control (NI), at 2 days post infection (dpi). (C) Q-RTPCR analysis showing increased levels of PPARγ transcript in NSCs infected by HCMV (MOI 10) (HCMV) as compared to the uninfected control (NI, value set to 1), or NSCs infected with UV-irradiated HCMV (HCMV+UV) at 2 dpi. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. (D) Luciferase reporter assays showing non specific (pGL4) or PPARγ dependent (pGL4-PPRE) luciferase activity in uninfected NSCs (NI), uninfected NSCs treated with rosiglitazone (NI+rosi), NSCs infected by live HCMV at a MOI of 10, 2 days pi (HCMV), and NSCs infected in the presence of T0070907 (HCMV+T0). Data represent means ± CI of 3 independent experiments, each being performed in triplicate. (E) Chromatin immunoprecipitation assays using an antibody against K4-trimethylated histone 3 (H3K4triMe) as the positive control or two different antibodies against PPARγ (H100 and A3409A), showing increased occupancy by PPARγ of PPREs within the <i>DLK1</i> gene in NSCs infected by HCMV, as compared with uninfected NSCs. Shown are the fold change ratio from infected versus uninfected (NI) cells. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. (F) Oil red O staining showing numerous lipid vesicles in infected NSC cultures (MOI 10) (HCMV) as compared to uninfected NSCs (NI). Virus strain was AD169. Scale bar: 50 μm. *: p<0.05; ***: p<0.005.</p

HCMV infection of NSCs impairs neuronogenic differentiation in vitro.

<p>(A) Representative immunofluorescence analysis of NSCs infected by HCMV at a MOI of 10 (HCMV) and uninfected (NI) NSC cultures, 6 days after the onset of differentiation (i.e., 5 days post infection [dpi]) using a βIII tubulin antibody. Scale bar: 50μm. (B) Western blot analysis of whole lysates from differentiating NSC infected by live HCMV at a MOI of 10 (HCMV) or by UV-irradiated HCMV (UV), or uninfected controls, showing decreased levels of βIII tubulin (βIII tub) in the infected cultures. (C) Automated immunofluorescence analysis of differentiating NSC cultures infected or not by HCMV with an HUC/D antibody (HUC/D+). Data represent means ± CI of 3 independent experiments, each being performed in triplicate. (D) Automated immunofluorescence analysis of differentiating NSC cultures infected or not by HCMV using the cell death marker Image-It Dead (ID). Inset: immunofluorescence analysis using an antibody specific to activated (cleaved) caspase 3 (casp3) of infected (MOI 1, 2 dpi) or uninfected (NI) NSC cultures. HCMV strain was AD169. Data represent means ± CI of 2 independent experiments, each being performed in triplicate. **: p<0.01, ***: p<0.005.</p