159 Microbiome modulation by antibiotics in patients with asthma bacteria during the bronchoscopy (cultures remained negative) and the participant was discharged without complaints. Bronchoalveolar lavage handling, assays and flow cytometry Both the saline-challenged subsegment of the lung and the HDM/LPS challenged subsegment of the lung were lavaged by the pulmonologist with eight successive 20 ml aliquots of saline. BALF was immediately pooled per lung subsegment and centrifuged at 4C° and 400g for 10 minutes. The cell-free supernatant of the BALF was stored at -80°C until analysis by Luminex assay (Bio-Techne Ltd, Abingdon, United Kingdom). BALF cells were stained with CD4 Alexa fluor 700 (eBiosciences, San Diego, CA), CD45 phycoerythrin (PE)-CF594, CD71 Brilliant Violet (BV)421, CD11b BB515, viability dye APCCy7 (all BD Biosciences), CD16 BV650, Siglec-8 Allophycocyanin (APC) (Biolegend, San Diego, CA). All singlet cells were selected for CD45+ and viability dye-. Subsequently, eosinophils (CD71-CD4-CD16-Siglec-8+), neutrophils (CD71-CD4-CD16+Siglec-8-), alveolar macrophages (CD71+) and CD4 T-cells (CD71-CD4+) were identified. Data were collected on a BD FACSAria™ III flow cytometer and analyzed using FlowJo software (Treestar, Palo Alto, CA). We used Luminex beads assays (Bio-Techne Ltd, Abingdon, United Kingdom) to measure the following markers of systemic inflammation: interleukin (IL)-1ß, IL-1 receptor antagonist, IL-2, IL-6, IL-8, IL-33, periostin, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-a, regulated upon activation, normal T-cell expressed and presumably secreted (RANTES), myeloperoxidase (MPO), eotaxin-1. Assays were used in accordance with the manufacturer’s instructions. Microbiome analysis Repeated bead-beating of 250 mg feces was performed as described elsewhere but with STAR (Stool transport and recovery) buffer (Roche, Basel, Switzerland)[5]. Following centrifugation, 250 µl supernatant was used with the Maxwell® RSC Blood Deoxyribonucleic acid (DNA) Kit (Promega, Madison), and the DNA was eluted in 50 µl DNAse free water. Twenty nanograms of DNA were used for the amplification of the V3-V4 region of the 16S ribosomal ribonucleic acid (rRNA) gene as described, with barcoded 341 forward and 805 reverse primers for 25 cycles [6]. For the purification of the amplified product, the AMPure XP beads (Beckman Coulter, Indianapolis) were used according to manufacturer’s guidelines on a Beckman Coulter Biomex FX. The purified product was equimolar mixed and load for sequencing on the Illumina MiSeq with the MiSeq V3 - 600 cycle kit, as instructed by Illumina. Statistical analysis Patient characteristics were compared using a T-test or Mann-Whitney U-test. Comparisons of BALF measurements were done between samples obtained from the saline and HDM/LPS challenged lung in each group (antibiotics group and control group) using paired Wilcoxon ranked sum test. A P-value of <0.05 was considered statistically significant. The microbiome sequence reads were analysed as follows. Read pairs with perfect matching forward and reverse barcodes were assigned to their corresponding samples. The forwards and reverse reads were length trimmed at 240 and 210 respectively, which were inferred and merged with ASVs using DADA2 7
RkJQdWJsaXNoZXIy MTk4NDMw