Metabolomics dataset - Microbial metabolism of L-tyrosine protects against allergic inflammation

Published: 18 September 2020| Version 1 | DOI: 10.17632/z2knkcmntc.1


The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This led to increased levels of p-cresol sulfate (PCS) that protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells, due to an uncoupling of EGFR and TLR4 signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.


Steps to reproduce

25 µL of plasma samples were extracted with 100 µL of chilled methanol containing internal standard (PCS-d4 at 500 ng/ml plasma concentration), shaken on ice for 30 min and centrifuged at 4 °C for 10 min. 100 µL of supernatant was diluted with 100 µL of 0.1% FA in water. Frozen fecal samples were weighted (3 - 5 mg) and extracted with 20 µL/mg of 80% chilled methanol containing internal standard PCS-d4, vortexed at 4°C for 15 minutes, shaken at RT 60 minutes and centrifuged at 4 °C at 14800g for 30 min. Supernatant was collected and diluted 2.5x with 0.1% FA. PCS-d4 concentration is 50 ng/ml in the samples which is equal to 2.5 ng/1mg feaces). 50 µL of BAL fluid were extracted with 200 µL of cold methanol, mixed on ice for 30 min and centrifuged at 4°C at 14800g for 10 min. 200 µL of the supernatant was transferred to new Eppendorf tubes and evaporated under nitrogen stream for 60 min at 20 °C. Samples are resolubilized in 100 µL 0.1% FA in water, mixed for 15 min at RT, sonicated with ice for 15 min, centrifuged at 4°C and transferred to vials. Samples were analysed on the same day as prepared injecting 6 µL and using the following LCMS acquisition method: LCMS data was acquired on Q-Exactive mass spectrometer coupled with Dionex Ultimate 3000 RSLC separation system (Thermo Scientific, Waltham, Massachusetts, USA). Ascentis Express C8 (100 x 2.1 mm, 2.7 µm, Supelco) column protected with a guard column (C8, 2x2mm, Phenomenex) was used for separation. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in acetonitrile. Gradient elution was achieved starting at 10% B concentration and increased to 95% B in 3.5 min, kept at 95% B until 4.5 min, reduced to 10% B at 5 min and equilibrated at that ratio until 7 min. Autosampler temperature was kept at 4°C and column oven at 40°C. HESI source spray voltage was set to 4 kV, capillary temperature 300°C, auxiliary gas temperature 120°C, sheath gas flow rate to 50, auxiliary gas to 20, sweep gas to 2 arbitrary units and S-lens RF level 50. Mass spectrometer operated in PRM acquisition mode in negative ion polarity using inclusion list for PCS and PCS-d4 m/z (m/z 187.0071 and 191.0321, respectively) with specified HCD collision energy NE = 50 and retention time between 2 - 3.5 min. Other PRM parameters were as follows: 1 microscan, 17.5 k resolution, AGC target 2e5, maximum IT 100 ms, isolation window 2 m/z, loop count 4, MSX count 1. Peak integration and quantitation were performed using Tracefinder application (Thermo Scientific). Measured peak areas were used for relative quantification in different sample groups.


Monash University