Early inflammation precedes cardiac fibrosis and heart failure in desmoglein 2 murine model of arrhythmogenic cardiomyopathy.

The study of a desmoglein 2 murine model of arrhythmogenic cardiomyopathy revealed cardiac inflammation as a key early event leading to fibrosis. Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure due to abnormalities in the cardiac desmosome. We examined how loss of desmoglein 2 (Dsg2) in the young murine heart leads to development of AC. Apoptosis was an early cellular phenotype, and RNA sequencing analysis revealed early activation of inflammatory-associated pathways in Dsg2-null (Dsg2-/-) hearts at postnatal day 14 (2 weeks) that were absent in the fibrotic heart of adult mice (10 weeks). This included upregulation of iRhom2/ADAM17 and its associated pro-inflammatory cytokines and receptors such as TNFα, IL6R and IL-6. Furthermore, genes linked to specific macrophage populations were also upregulated. This suggests cardiomyocyte stress triggers an early immune response to clear apoptotic cells allowing tissue remodelling later on in the fibrotic heart. Our analysis at the early disease stage suggests cardiac inflammation is an important response and may be one of the mechanisms responsible for AC disease progression

P053 3-Oxo-C12:2, a quorum sensing molecule from the human gut, exerts anti-inflammatory effects through a bitter taste receptor

Abstract Background Acyl-homoserine lactones (AHLs) are quorum sensing molecules involved in bacterial communication network and can also have an impact on host’s cells. We recently showed the presence of AHLs in the gut ecosystem and identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in faecal samples of inflammatory bowel disease (IBD) patients, especially during flare, and its absence was correlated to dysbiosis. 3-Oxo-C12:2 is structurally close to an AHL well described and synthesised by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identify which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesised that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor, called T2R138, which is a GPCR expressed by immune and epithelial gut cells. Methods To test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-γ (IFN-γ; 20 U/ml) and lipopolysaccharide (LPS; 10 ng/ml). Inflammatory response was monitored by measuring cytokine secretion (TFNα, IL-6 and IL-10) via ELISA. Protein expression was assessed by Western blot. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. Cytotoxicity was checked by measuring LDH release. Results In steady state, challenged by 3-oxo-C12:2, RAW264.7 macrophages showed no change in cytokine expression (TNFα, IL-6 and IL-10). After LPS/IFN-γ activation, we observed a decrease in the ratio of secreted TNFα/IL-10 when cells are exposed to 3-oxo-C12:2, in a dose-dependent manner: 15 μM (−30%, p &amp;lt; 0.05), 25 μM (−50%, p &amp;lt; 0.001) and 50 μM (−65%, p &amp;lt; 0.0001). This reflects an anti-inflammatory effect, without increasing cytotoxicity. Those immune-modulatory effects were lost in presence of Probenecid. Moreover, the amount of T2R138 protein was not changed in the presence of Probenecid and/or 3-oxo-C12:2. Therefore, the loss of anti-inflammatory effects with the inhibitor was not due to a decrease of the receptor expression in our experimental conditions. Conclusion 3-Oxo-C12:2 exerts a dose-dependent anti-inflammatory effect on murine immune cells. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL. We are currently studying via a multiplex assay the cytokine response after exposing cells to 3-oxo-C12:2. Studying the signalling between the receptor and the anti-inflammatory response would allow us to understand better the inter-kingdom dialogue between microbiota and the host and to what extend AHLs are involved. We are grateful for the FRM for the financial support (ECO201806006843) </jats:sec

P043 3-oxo-C12:2, a Quorum Sensing molecule from the gut, exerts anti-inflammatory effects through a bitter taste receptor

Abstract Background Acyl-Homoserine Lactones (AHLs) are Quorum Sensing molecules involved in the communication network of bacteria and can also have an impact on the host’s cells. We recently showed, in the human gut ecosystem, the presence of AHLs. Among them, we identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in Inflammatory Bowel Disease (IBD) patients, and its presence was correlated to normobiosis. Interestingly, 3-oxo-C12:2 is structurally close to an AHL well described and synthesized by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identified which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesized that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor (BTR), called T2R138, which is a GPCR expressed by immune and epithelial gut cells. Methods To test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-γ (IFN-γ, 20U/mL) and lipopolysaccharide (LPS, 10ng/mL). Inflammatory response was monitored by measuring cytokine secretion via ELISA We performed a transcriptome analysis to identify inflammatory pathways involved in the effects and analyse pathways by capillary Western blot. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. BTR screening assay was performed to extend search for 3-oxo-C12:2 receptors. Results After LPS/IFN-γ activation, we observed a decrease of secreted TNFα when cells were exposed to 3-oxo-C12:2, in a dose dependent manner: 15μM (-30%, p&amp;lt;0.05), 25μM (-50%, p&amp;lt;0.001) et 50μM (-65%, p&amp;lt;0.0001) while no change were observed in steady state. This reflects an anti-inflammatory effect, in absence of cytotoxicity. By transcriptomic analysis, we identified the JAK-STAT pathway as differentially down-regulated. Exposing cells to 3-oxo-C12:2 prevented JAK1 and STAT1 protein phosphorylation. In addition, the observed anti-inflammatory effects were lost in presence of Probenecid, a T2R138 inhibitor. In a BTR screening assay, we confirmed that 3-oxo-C12:2 activates T2R38, but also five other BTR (T2R13, T2R8, T2R14, T2R1, T2R10). Conclusion 3-oxo-C12:2 exerts a dose dependent anti-inflammatory effect on murine immune cells by preventing the activation of the JAK-STAT pathway. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL of interest. Studying the signalling between the receptor and the anti-inflammatory response would allow us to better understand the inter-kingdom dialogue between microbiota involving AHL in IBD. </jats:sec

P064 3-oxo-C12:2, a Quorum Sensing molecule from the gut, exerts anti-inflammatory effects through a bitter taste receptor

Abstract Background Acyl-Homoserine Lactones (AHLs) are Quorum Sensing molecules involved in the communication network of bacteria and can also have an impact on the host’s cells. We recently showed, in the gut ecosystems, the presence AHLs and among them we identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in Inflammatory Bowel Disease (IBD) patients, especially during flare, and its presence was correlated to normobiosis. Interestingly, 3-oxo-C12:2 is structurally close to an AHL well described and synthesized by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identified which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesized that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor (BTR), called T2R138, which is a GPCR expressed by immune and epithelial gut cells. Methods To test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-ɣ (IFN-g, 20U/mL) and lipopolysaccharide (LPS, 10ng/mL). We performed a transcriptome analysis using RNAseq to identify inflammatory pathways involved in the effects. Inflammatory response was monitored by measuring cytokine secretion TFNα via ELISA. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. BTR screening assay was performed to extend search for 3-oxo-C12:2 receptors. Cytotoxicity was measured via Lactate Dehydrogenase release. Results After LPS/IFN-γ activation, we observed a decrease of secreted TNFα when cells are exposed to 3-oxo-C12:2, in a dose dependent manner: 15μM (-30%, p&amp;lt;0.05), 25μM (-50%, p&amp;lt;0.001) et 50μM (-65%, p&amp;lt;0.0001), no change were observed in steady state. Itreflects an anti-inflammatory effect, without increasing cytotoxicity. To identify mechanisms behind those effects, we analysed the transcriptome of RAW264.7 cells exposed to AHL. JAK-STAT and NF-κB pathways were differentially down-regulated in presence of 3-oxo-C12:2. In addition, the anti-inflammatory effects were lost in presence of Probenecid, a T2R138 inhibitor. In a BTR screening assay, we confirmed that 3-oxo-C12:2 activates T2R38, but also five other BTR (T2R13, T2R8, T2R14, T2R1, T2R10). Conclusion 3-oxo-C12:2 exerts a dose dependent anti-inflammatory effect on murine immune cells. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL of interest and we are currently studying the inflammatory pathways involved behind those effects. Studying the signalling between the receptor and the anti-inflammatory response would allow us to better understand the inter-kingdom dialogue between microbiota involving AHL in IBD. </jats:sec