Bacterial skin infection imprints bone marrow eosinophils to promote allergic skin-lung crosstalk

Description

Methods (English)

Cell extraction

Cell extraction and metabolomic analysis was performed by the CeMM Molecular Discovery Platform – Metabolomics. Extraction was performed from 2 x 10⁵ BMDEos in sterile 1.5 ml microcentrifuge tubes (Eppendorf) by adding 500 µl of ice-cold 80:20 (v/v) MeOH:H₂O solution to the cell pellet and vigorous vortexing. Samples were centrifuged at 10000 g for 10 minutes at 4 C before transferring the cell extract supernatant into 1.5 ml HPLC vials. Extraction of cell pellets was repeated and supernatants of the same samples were combined. Cell extracts were dried using a nitrogen evaporator. The dried residue was reconstituted in 50 µl water. An aliquot of 10 µl reconstituted extract was mixed with 10 µl of isotopically labelled internal standard mixture in a HPLC vial, vortexed and used for the metabolite analysis.

Metabolite analysis 

The LC-MS/MS analysis relied on a 1290 Infinity II UHPLC system (Agilent Technologies) coupled with a 6470 triple quadrupole mass spectrometer. The samples’ chromatographic separation was performed on a ZORBAX RRHD Extend-C18, 2.1 x 150 mm, and 1.8 µm analytical column (Agilent Technologies). The column was maintained at a temperature of 40°C while 4 µl of each sample was injected in each run. The mobile phase A consisted of 3% methanol (v/v), 10 mM tributylamine, 15 mM acetic acid in water and the mobile phase B of 10 mM tributylamine, 15 mM acetic acid in methanol. The gradient elution was carried out at a flow rate of 0.25 ml/min for a total duration of 24 minutes. Subsequently, the column was back flushed for 8 min using acetonitrile with a 6port/2-position divert valve, followed by column equilibration with 100% mobile phase A for another 8 min. The triple quadrupole mass spectrometer was operated in electrospray ionization negative mode with a spray voltage of 2 kV, gas temperature of 150°C, gas flow of 1.3 L/min, nebulizer pressure of 45 psi, sheath gas temperature of 325°C, and sheath gas flow of 12 L/min. Metabolite detected was performed with a dynamic MRM mode. Data processing was performed using the MassHunter 10.0 software (Agilent Technologies). Finally, ten-point linear calibration curves using internal standardization were generated for metabolite quantification. 

Abstract (English)

Related publication Abstract: 

Microbial exposure at barrier interfaces drives development and balance of the immune system but the consequences of local infections for systemic immunity and secondary inflammation are unclear. Here, we show that epicutaneous exposure to the bacterium Staphylococcus aureus persistently shapes the immune system of mice with specific impact on progenitor and mature bone marrow neutrophil and eosinophil populations. The infection-imposed changes in eosinophils were long-lasting and associated with functional as well as imprinted epigenetic and metabolic changes. Bacterial exposure enhanced skin allergic sensitization and resulted in exacerbated allergen-induced lung inflammation. S. aureus-mediated functional bone marrow eosinophil reprogramming and pulmonary allergen responses were driven by the alarmin interleukin 33 and the complement cleavage fragment C5a. Our study highlights the systemic impact of skin inflammation and reveals eosinophil progenitor training and organ-crosstalk mechanisms that modulate systemic responses to allergens.

Notes (English)