The presence of the anti-inflammatory protein MAM, from Faecalibacterium prausnitzii, in the intestinal ecosystem.

International audienceFaecalibacterium prausnitzii strain A2-165 was previously reported to have anti-inflammatory properties and prevent colitis in a TNBS model. We compared the immunomodulatory properties of strain A2-165 to four different F. prausnitzii isolates and eight abundant intestinal commensals using human dendritic cells (DCs) and mouse BMDCs in vitro. Principal component analysis revealed that the cytokine response to F. prausnitzii A2-165 is distinct from the other strains in eliciting high amounts of IL-10 secretion. The mouse DNBS model of relapsing IBD was used to compare the protective effects of F. prausnitzii A2-165 and Clostridium hathewayi, a low secretor of IL-10, on the Th1-driven inflammatory response to DNBS; attenuation of disease parameters was only observed with F. prausnitzii. In an in vivo mouse model of nasal tolerance to ovalbumin, F. prausnitzii A2-165 enhanced ovalbumin-specific T cell proliferation and reduced the proportion of IFN-γ(+) T cells in CLNs. Similarly, in vitro F. prausnitzii A2-165 stimulated BMDCs increased ovalbumin-specific T cell proliferation and reduced the number of IFN-γ(+) T cells. These mechanisms may contribute to the anti-inflammatory effects of F. prausnitzii in colitis and support the notion that this abundant bacterium might contribute to immune homeostasis in the intestine via its anti-inflammatory properties

High-resolution mass spectrometry and partial de novo sequencing constitute a useful approach for determining the profile of chemokine secretion following the stimulation of human intestinal epithelial cells.

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Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

<div><h3>Background</h3><p>Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.</p> <h3>Methodology/Principal findings</h3><p>Potential PLD substrates and products were studied in <em>Arabidopsis thaliana</em> suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of <em>n</em>-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in <em>in vitro</em> assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the <em>in vivo</em> products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.</p> <h3>Conclusions</h3><p>MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products <em>per se</em> is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.</p> </div

Profiles of PBut produced <i>in vitro</i> by Arabidopsis microsomal PLDs.

<p>Microsomes were used in an enzymatic assay on lipid vesicles. The substrate used was PE. The reaction assay was defined as α-type, β/γ-type or δ-type. The reactions were performed at 37°C for 20 min in the presence of 0.6% (v/v) <i>n-</i>butanol. White bars, substrate; black bars, PBut.</p

Mass scan of the main glycerophospholipids in suspension cells.

<p>Mass spectra of parent ions of phosphatidylethanolamine (A), phosphatidylcholine (B) and phosphatidylinositol (C) were recorded by the neutral loss of fragment 141 amu (from positive ions), the neutral loss of fragment 60 amu (from negative ions) and the precursors of fragment <i>m/z</i> 241 (from negative ions), respectively. Mass spectra correspond to the [M+H]⁺ ions for PE, [M+HCOO]⁻ for PC and [M−H]⁻ ions for PI. The glycerophospholipid fatty acid composition was determined in the negative mode by CID as shown (insert) for 16∶0/18∶2-molecular species.</p

PBut profiles analyzed before and after SA addition.

<p>Cell medium was supplemented with 0.1% (v/v) <i>n</i>-butanol. After 45 minutes, 750 µM SA was added, and lipids were extracted 100 min later. (A) Black bars, PBut in untreated cells; white bars, PBut in SA treated cells; striped bars, PC; grey bars, PE. (B) PBut in the presence of SA compared to PC. Black bars, PBut in presence of SA; white bars, PC. Insert: for molecular species representing more than 1% of the species of PBut and PC, we calculated the ratio of the value obtained in PBut to the value obtained in PC. Results are represented on a <i>log2</i> scale. (C) PBut in the presence of SA compared to PE. Black bars, PBut in the presence of SA; white bars, PE. Insert: for molecular species representing more than 1% of the species of PBut and PE, we calculated the ratio of the value obtained in PBut to the value obtained in PE. Results are represented on a <i>log2</i> scale.</p