Patient groups

A total of 202 volunteers were recruited for this study—obese asthma (n = 50), obese non-asthma (n = 51), non-obese asthma (n = 53), and non-obese non-asthma healthy controls (n = 48). Asthma patients had a physician diagnosis of asthma. Severe asthma was defined according to the American Thoracic Society (ATS) Workshop on Refractory Asthma 2000 report and by the 2013 European Respiratory Society (ERS)/ATS guidelines22,41. Obesity was defined as having a body mass index (BMI) greater than 30 kg/m2. Non-obese individuals had a BMI of 20–25 kg/m2. All relevant ethical regulations for work with human participants were complied with, and informed consent was obtained from all participants. The human biological samples were sourced ethically and their research use was in accordance with the terms of the informed consents. Patients were recruited under informed consent at two centers, ALL-MED Medical Research Institute, Wroclaw, Poland and the Pulmonary Division, University Hospital of Zurich, Switzerland. Ethical approval was granted at both sites from the local ethical committee for all study procedures (KEK-ZH-Nr. 2012–0443—Kantonale Ethik-Kommission Zürich; KB-70/2013 and KB-567/2014—Bioethical Committee, Wroclaw Medical University). Patient demographic details and comorbidities are detailed in Table 1 and Supplementary Table 1, respectively.

Measurement of inflammatory mediators and cells

Serum was obtained by allowing blood collection tubes (with no anti-coagulant) to rest at room temperature for 1 h and then tubes were centrifuged at 800×g for 10 min. Serum was removed, aliquoted in 500 μl quantities and stored at −80 °C for later analysis. BAL was obtained and filtered through a 70 μm filter into sterile tubes. Aliquots of 500 μl were stored at −80 °C for later analysis. BAL cells were centrifuged onto slides and following air drying were stained using the Diff-Quik stain or remained unstained. Fixed slides were stored at −20 °C and differential cell counts were performed by two independent histopathologists. All soluble mediators were measured using the mesoscale discovery platform (MSD) kits according to manufacturer’s instructions. Data were analyzed using one-way ANOVA (correcting for gender) and Tukey’s correction.

Gene expression analysis

Peripheral blood was collected in Paxgene tubes and immediately frozen at −80 °C until later analysis. Total mRNA was extracted from peripheral blood using Qiagen RNeasy kit (Qiagen, Valencia, CA) and quantified by NanoDrop (Thermo Fisher Scientific, Waltham, MA). RNA integrity was confirmed using the Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA). Samples were normalized to the lowest concentration sample, and cDNA was made using Superscript Vilo cDNA synthesis Master Mix (Invitrogen Life Technologies, Grand Island, NY). The cDNA samples were then labeled with biotin with the FL-Ovation cDNA Biotin Module V2 and hybridized to a Human Genome U133 Plus 2.0 Array using a Genechip Hybridization Kit. The microarray chips were washed and stained using a Genechip Hybridization Wash and Stain Kit and then scanned using a Genechip Scanner. All reagents and readers were used according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA).

The microarray gene expression data were analyzed using ArrayStudio 7.0 (OmicSoft, Cary, NC). Data from.CEL files from Human Genome U133 Plus 2.0 Affymetrix chips were normalized using robust multiarray averaging and scaled to a mean target intensity of 150.

BAL cells and bronchial biopsies were immediately placed in RNAlater and stored frozen until RNA extraction was performed. Total RNA from BAL cells and bronchial biopsies was extracted using the RNeasy Mini kit (Qiagen) and RNA integrity was confirmed using the Agilent 2100 BioAnalyzer (Agilent, Palo Alto, CA). Samples were required to have a RIN of at least 7.0. Sequencing libraries were prepared from RNA from each sample using the Illumina TruSeq Stranded RNA HT sample preparation kit with RiboZeroTMGold. Libraries were barcoded and then sequenced using an Illumina HiSeq 2000 with 51 bp paired-end reads for targeted coverage of 50 million paired reads per sample. Quality control and trimming of the unaligned FASTQ files was performed using the FASTX-Toolkit (version 0.0.14). Read pairs for each biological replicate were aligned to the human genome version hg38 using STAR (version 2.3.0e_r291) with a STAR index using the default parameters. Samtools (version 0.1.18) was used to convert the aligned SAM files to BAM files42,43. Read counts were determined using HTSeq (version 0.6.0) using the htseq-count command with the default parameters (–m union –r pos –s reverse) to define gene-levels counts from the Ensembl v85 annotated.gtf file44.

Microbiota analysis

Fecal samples were collected by volunteers at their home, immediately chilled using ice-packs and delivered to the lab within 4 h. Upon receipt in the lab, fecal samples were separated into 1 g aliquots and stored at −80 °C until analysis. Pre-procedure bronchoscope wash fluid (10 ml), BAL (10 ml), and mouth wash fluid (2 ml) were centrifuged at high speed (22,500×g) for 30 min (Hermle Z 231 M microcentrifuge; Hermle Labortechnik GmbH, Wehingen, Germany) in dolphin-nosed eppendorf tubes and the pellets stored at −80 °C until the time of DNA extraction. Nasal swabs of the middle meatus were stored in preservative (Norgen Biotek, Thorold, ON) and immediately frozen. DNA was isolated using PSP Spin Stool DNA Plus Kit (Cat#10381102, Invitek, Berlin, Germany) according to the manufacturer’s instructions. Each DNA sample was quantified by spectrophotometry. PCR amplification of the 16S rRNA V4 region was conducted with primers, 515f (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806r (5’-GGACTACHVGGGTWTCTAAT-3’) including the Illumina sequencing adapters and a 12 bp error-correcting Golay barcode sequence45,46. Each 50 µl PCR reaction contained 100 ng of genomic DNA, 2× Phusion High-Fidelity PCR Master Mix with HF Buffer (Cat# M0531L, New England Biolabs, Inc., Ipswich, MA), and 0.2 µM of each primer (Integrated DNA Technologies, Coralville, IA). PCR was performed on an ABI 9700 thermocycler and included the following cycling steps: Initial denaturing at 98 °C for 5 min followed by 40 cycles of 98 °C ×30 s, 60 °C ×45 s, and 72 °C ×1 min ending with a 72 °C 1× min extension. PCR products from the extracted DNA sample were run on a 2.0% TAE agarose gel, excised and purified using QIAquick Gel Extraction Kit (Cat# 28704, Qiagen, Valencia, CA). PCR products were quantitated using Quant-iT PicoGreen dsDNA reagent (Cat # P7589, Invitrogen, Eugene, OR).

To check for proper cluster density and sample normalization, an Illumina MiSeq single-end 26 bp + 12 bp index sequencing run was performed using the MiSeq instrument. The pool was mixed with a PhiX library (Illumina, San Diego CA) at a ratio of 1:9 in order to increase the entropy of the library. A final MiSeq 2 × 150 bp + 12 bp index sequencing run was performed on the pooled samples. Reads were first filtered to remove the PhiX library reads. All reads mapping to the Enterobacteria phage PhiX 174 reference genome (GenBank: NC_001422.1) using the software Bowtie (v1.0.1) were removed from the analysis47. The paired reads were next merged with the software PEAR (v0.9.5)48. Successfully assembled reads were analyzed using the QIIME software package (v1.8) with the default quality control parameters49. Chimeric sequences were identified and removed from the dataset using the UCHIME (v6.1) method50. The closed-reference QIIME protocol was used with the UCLUST method to select operational taxonomic units (OTUs)51. Sequences with at least 97% identity were clustered together. A representative sequence from each cluster was used to identify bacterial taxa from the May 2013 edition of the Greengenes 16S rRNA database (13_8)52,53.

In order to confirm the 16s sequencing data, A. muciniphila levels in fecal samples were also quantified using PCR. Quantitative PCR analysis was performed using an Applied Biosystems 7900 HT Fast Real-Time PCR system and the A. muciniphila-specific primer sequences used were AM1 5’CAG CAC GTG AAG GTG3‘ and AM2 5’CCT TGC GGT TGG CTT CAG3‘. The experimental cycling conditions were: 40 cycles of 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, 60 °C for 1 min.

Because the airway microbiome samples (nasal, oral, and BAL) began with lower biomass starting material, they were subjected to additional quality control using the negative bronchoscope wash to detect possible contamination. The predominant 16S rRNA V4 amplicons of pre-procedure bronchoscope control wash sample (while of low read count) aligned predominantly (84%) to the families Verrucomicrobiaceae and Enterobacteracaea. These families were assumed to be contaminants and were subtracted from the analysis of the airway microbiome samples.

Animal models

Both acute and chronic models of airway inflammation were performed. Female and male wild-type and MyD88−/− BALB/c mice aged 6–8 weeks were obtained from Charles River (Sulzfeld, Germany) and housed at AO Research Institute Davos for the acute ovalbumin (OVA) and acute house dust mite (HDM) models. Mice were housed 4–6 animals per cage in individually ventilated cages in a 12/12 h light/dark cycle with food and water available ad libitum. All experimental procedures were carried out in accordance with Swiss law and ethical approval was obtained from Amt fur Lebensmittelsicherheit und Tiergesundheit, Chur. Female wild-type BALB/c mice for the chronic HDM model were bred and housed at GlaxoSmithKline (GSK), Stevenage. Animal studies carried out at GSK were ethically reviewed by the GSK ethics committee and studies were carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. All studies were performed using littermates.

Either ovalbumin (OVA) or house dust mite (HDM) extract were used in acute models of airway inflammation. In the OVA model, mice were sensitized by intraperitoneal (i.p.) injection of 20 µg of OVA grade VI (Sigma-Aldrich, Buchs, Switzerland) emulsified in 500 µg Alum (Pierce, Rockford, IL, USA) in 200 µl sterile 0.9% isotonic Sodium chloride (NaCl) on days 0, 7, and 21, followed by 20 min 1% OVA grade V (Sigma-Aldrich, Buchs, Switzerland) aerosol exposures on days 26, 27, and 28. Negative control animals received NaCl and alum injections and were exposed to the OVA aerosolization. Analysis of mice occurred 24 h after the last aerosol challenge. In the acute HDM model, HDM extract (Greer labs, USA) was administered intra-nasally (i.n.) on day 0 (1 μg), followed by higher dose i.n. administrations on days 7, 8, 9, 10, and 11 (10 μg each day). Negative control animals received saline i.n. on the same days as the positive control animals received HDM extracts i.n. Animals were euthanized on day 12 for analysis. In both acute models, A. muciniphila and media control was administered daily (~1 × 108 cells per dose) by oral gavage, beginning at day −5 until the end of the study. A. muciniphila was pre-grown in fresh cultures every day in 10 ml aliquots of anaerobic Mucin v3 media (10% inoculation) at 37 °C. Nitrogen (and boiling) was used to remove the presence of oxygen from Mucin v3 media. Mucin v3 media contains peptone (Fluka), yeast extract (Roth), KH 2 PO 4 (Fluka), NaCl (Fluka), (NH 4 )SO 4 (Acros organics), MgSO 4 (Acros organics), CaCl 2 (Acros), NaHCO 3 (Fluka), D-glucose (Fischer chemical), porcine mucin type II (Sigma), porcine hemin (Acros organics), L-cysteine (Sigma), and water. For the indicated experiments, freshly cultured A. muciniphila was heat killed by boiling at 100 °C for 15 min. Cell free supernatants were generated by filtering the culture broth, following A. muciniphila 24 h culture at 37 °C, through a 0.22 µm syringe filter (Sarstedt). Two hundred microliter of the filtered supernatants were orally gavaged daily to the mice. Fecal and BAL levels of A. muciniphila were determined using quantitative PCR. Identical primers (i.e., AM1 5’CAG CAC GTG AAG GTG3‘ and AM2 5’CCT TGC GGT TGG CTT CAG3‘) and cycling conditions were used as described above for A. muciniphila quantification in human samples.

BALs were performed with 1 ml of PBS containing 1× protease inhibitor cocktail (Roche, Mannheim, Germany). Red blood cells were removed using red blood cell lysis buffer (Sigma-Aldrich). The total number of leukocytes was counted with a Neubauer counting chamber. For differential cell counts, cytospin preparations were fixed and stained with Diff-Quick (Merz & Dade Ag, Dudingen, Switzerland). Neutrophils, eosinophils, lymphocytes and macrophages were identified using standard morphologic criteria and at least 200 cells were counted per cytospin preparation54. Single cell suspensions were obtained from lung tissue using GentleMACS (Miltenyi Biotec). Lung-derived single-cell suspensions were plated at a concentration of 1 × 106 cells/ml in complete RPMI (Sigma-Aldrich) and were re-stimulated with 50 µg/ml grade VI OVA (Sigma-Aldrich) for 48 h at 37 °C. Culture supernatants were assayed for cytokine levels by Bio-Plex Multiplex Immunoassay System (Bio-Rad). For lung function measurements, mice were intubated under anesthesia and airway resistance was assessed using the forced oscillation technique (FlexiVent system, SCIREQ). Airway resistance was measured in response to increasing concentrations of methacholine from 0, 0.1, 0.3, 1, 3, 10, to 30 mg/ml (Sigma-Aldrich)55.

For the chronic HDM models, female BALB/c mice were challenged i.n. with either sterile saline or 25 µg of HDM extract for 5 days a week, over 3 weeks (sensitization period, days 1–19). The resulting pulmonary inflammation in the HDM sensitized mice was then allowed to resolve for a period of 2 weeks (days 20 to 33). At the end of the resolution period (day 34), mice were intranasally challenged with either saline or rechallenged with a high dose of HDM (100 μg). HDM rechallenge in this model has been shown to induce granulocytic and lymphocytic infiltration into the airways over a 7 day period (days 34 to 40). Mice were orally gavaged with either mucin v3 media control or A. muciniphila, once a day, starting on day 20 (post cessation of HDM sensitization). Oral dosing continued throughout the resolution period and post-HDM rechallenge until day 39. Groups of mice were sacrificed at pre-determined time-points during the resolution phase of the experiment (day 28) and post HDM rechallenge at 4 h (day 34), 24 h (day 35), and day 7 (day 40).

Flow cytometry

Cells present in the broncho-alveolar lavage fluid (BALF) and lung tissue were quantified using multi-color flow cytometry. Analysis of myeloid and lymphoid subsets was performed on a FACSCantoII machine (Becton Dickinson) running FACSDiva software version 8. Data analysis was performed using FlowJo version 7.6.5 (FlowJo LLC, US). Red blood cells of single cell suspensions containing 1 × 106 cells were lysed using a 1/10 dilution in sterile distilled water of IO Test 3 solution (Beckman Coulter, UK) for 5 min at room temperature. The lysing solution was then washed twice by centrifugation at 370×g for 5 min at 4 oC. Supernatants were discarded and cell pellets were resuspended in 20 μl Fc−BlockTM to block non-specific Fcγ binding sites for 10 min at 4 oC. Without washing off Fc−BlockTM, cells were stained with 50 μl surface marker antibody cocktails for 10 min at 4 oC in the dark after which cells were washed twice to remove any unbound antibodies. Cell pellets were then resuspended in 200 μl of FACS buffer (PBS containing 1% Fetal Calf Serum). For intracellular cytokine staining, freshly isolated cells were stimulated with PMA/Ionomycin (50 ng/ml and 500 ng/ml, respectively) for 4 h at 37 °C and 5% CO 2 in the presence of Brefeldin (eBioscience, Vienna, Austria). Cells were permeabilized with reagents from eBioscience (Vienna, Austria). Data was acquired on a calibrated FACSCantoII flow cytometer (Becton Dickinson, UK) using fluorochrome compensated protocols. Gating of intact and single cells (forward scatter Height versus Area) was based on forward and side scatter characteristics. Leukocyte populations were identified as CD45+ in the single cell gate. Cell subsets were identified as lung resident eosinophils (CD11c-CD11b+MCHIIloCD24+Siglec-Fint), inflammatory eosinophils (CD11c−CD11b+MHCIIloCD24+Siglec-Fhi), monocytes (CD11c-CD11b+CD24loMHCIIloCD64loLy6-C+), neutrophils (CD11c−CD11b+MHCIIloCD24+Ly6-G+), CD8 + T cells (TCRβ+CD8+), B cells (CD45+CD19+), NK cells (CD45+CD49b+), T cell subsets (TCRβ+CD4+CD44+CD62L−CD183+/− CD196+/−, IL-4+/−, IFN-γ+/−), or Treg cells (CD3+, CD4+, CD25+, Foxp3+, IL10+). Flow antibodies to CD45 (30-F11, 1/200 dilution), CD183 (CXCR3–173, 1/200 dilution), CD196 (140706, 1/200 dilution), TCRβ (H57–597, 1/200 dilution), CD44 (IM7, 1/200 dilution) and CD62L (MEL-14, 1/200 dilution) were obtained from Becton Dickinson Biosciences, UK; Biolegend, UK CD11b (M1/70, 1/200 dilution), CD64 (X54–5/7.1, 1/200 dilution), MHCII (M5/114.15–2, 1/200 dilution), CD11c (N418, 1/400 dilution), Ly6-G (1A8, 1/400 dilution), Ly6-C (HK1.4, 1/400 dilution), CD8 (53–6.7, 1/200 dilution), CD49b (DX5, 1/200 dilution), CD3 (145–2C11, 1/100 dilution) and CD4 (RM4–5, 1/200 dilution) were obtained from Biolegend, UK; eBioscience, Austria CD24 (M1/69, 1/200 dilution), CD25 (PC61.5, 1/200 dilution), Foxp3 (FJK-16s, 1/50 dilution), IL-10 (JES5–16E3, 1/200 dilution), IL-4 (11B11, 1/100 dilution), and IFN-γ (XMG1.2, 1/200 dilution) were obtained from eBioscience, Austria; Siglec−F (ES22–10DB, 1/50 dilution) and CD19 (6D5, 1/30 dilution) were obtained from Miltenyi Biotech, UK.

Statistical analysis

16S rRNA gene sequencing data analyses were performed using QIIME or the R language and environment (version 3.3.2). Samples were rarefied to 24,725 reads, which corresponded to the minimum number of aligned reads to a sample passing quality standards. The Shannon diversity, inverse Simpson, or Chao1 indices and weighted and unweighted UniFrac distances were computed at this rarefaction level. Differential abundance for comparisons of taxa between groups was performed with a one-way ANOVA after correcting for known covariates, age and gender and a 10% FDR correction for comparisons of taxa, which were present in at least 10% of all samples for that body site.

Normalization and differential expression analysis of the transcriptomics data was performed with DESeq2 (version 1.12.4)56 and custom R scripts (version 3.3.2). Differentially expressed transcripts were identified using a one-way ANOVA, correcting for age, gender and collection site, followed by Tukey’s multiple comparison test to compare each group to the non-obese non-asthmatic group. Significance thresholds were set at a p-value equal to or less than 0.01 and at least a 1.2-fold change difference. Pathway analysis was performed using MetaCore software (Thomson Reuters, Rochester, NY).

Unless otherwise indicated, data are presented as box-and-whisker plots with the median value and 10–90 percentile illustrated. For analysis of more than two groups, statistical significance was determined using one-way ANOVA and Tukey’s multiple comparison test.