Study design and sampling
The aim of this study was to characterise the human DC response to Bt OMVs and determine whether it is altered in IBD. Healthy donors (age 17-86) undergoing investigative colonoscopy and individuals diagnosed with CD (age 21-80) and UC (age 24-70) were recruited from outpatient clinics at St Marks Hospital, London North West University Healthcare NHS Trust. Clinical characteristics of patients and controls are in Tables 1 and 2. Active disease for CD and UC was defined as having macroscopic inflammation (rated from mild (1) to severe (3); see Table 1) from colonoscopy findings and/or presence of blood inflammatory markers C-reactive protein (CRP) >5 and erythrocyte sedimentation rate (ESR) between 1 and 20 or faecal calprotectin >55 (See Table 2). Inactive disease was defined as no inflammation from rectum to terminal ileum (TI) according to colonoscopy findings and/or no more than one marker of inflammation in blood (CRP >5 or ESR between 1 and 20) and normal faecal calprotectin (<55).
Patients were recruited over a fixed period determined by ethical permission, and no data were excluded at the end of the study. Additional healthy blood volunteers were recruited from hospital staff and visitors. Ethics approval was obtained from the Health Research Authority UK and London Brent Research Ethics Committee. Written informed consent was received from participants prior to inclusion in the study.
For whole biopsy culture experiments, healthy donors were recruited from endoscopy clinics at Norfolk and Norwich University Hospital following informed consent (see Table 3 for demographics data). Ethics approval was obtained from the University of East Anglia Faculty of Medicine and Heath Sciences Ethics Committee and Human Tissue Act Subcommittee ref 20152016-39HT and the Norfolk and Norwich University Hospital Research and Development Committee ref 20-01-16.
Preparation of bacteria stocks
Bacteroides thetaiotaomicron VPI-5482 (Bt) (DSMZ 2079) and Prevotella nanciensis (Pn) (DSMZ 19126) (both from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) were grown under anaerobic conditions at 37 °C in brain heart infusion (BHI) medium (Oxoid/Thermo Fisher, Basingstoke, UK) supplemented with 0.5 mg/L haemin (Sigma-Aldrich, St Louis, MO, USA) (BHI–haemin). Aliquots (1 mL) were centrifuged (13,000 rpm for 10 min), supernatants removed and cell pellets were killed by snap-freezing before storage at -80 °C. Pellets were resuspended in phosphate-buffered saline (PBS) for use. Other commensal bacteria were isolated from the caecum of healthy donors with the exception of Collinsella aerofaciens, which was from faeces74–76. Strains were grown anaerobically in Hungate tubes containing Wilkins-Chalgren broth at 37 °C for 24 h. Cell pellets were killed by snap-freezing at -80 °C and enumerated by flow cytometry following SYBR Green staining.
Preparation of bacterial OMVs
The isolation of bacterial vesicles was done as previously described25. Briefly, Bt or Pn were grown under anaerobic conditions at 37 °C in an anaerobic cabinet. Bacterial starter-cultures were grown overnight in 20 mL BHI medium supplemented with 15 µM haemin (Sigma-Aldrich) (BHIH). An aliquot (0.5 mL) of the starter-culture was used to inoculate 500 mL BHI supplemented with 0.75 µM haemin. Cells were harvested after 16 h at an approximate OD (600 nm) of 4.0. The cells were centrifuged at 5500 g for 45 min at 4 °C and the supernatants filtered through polyethersulfone (PES) membranes (0.22 μm pore-size) (Sartorius) to remove debris and cells. Supernatants were concentrated by ultrafiltration (100 kDa molecular weight cut-off, Vivaspin 50R, Sartorius), the retentate was rinsed once with 500 mL of PBS (pH 7.4) and concentrated to 0.5 mL.
Further purification was performed by fractionation of the OMV suspension by size-exclusion chromatography using a CL2-B sepharose (Sigma-Aldrich) column (120 cm x 1 cm) in PBS. The absorbance of the fractions was measured at 280 nm and the first fractions displaying an absorbance peak were pooled and concentrated down to 1 mL with a Vivaspin 20 centrifugal concentrator (100 kDa molecular weight cut-off, Sartorius) and filtered through a 0.22 µm PES membrane (Sartorius). Concentration of vesicles was determined using nanoparticle tracking analysis as described previously29. Bt OMVs were at 1011 vesicles/mL of PBS and Pn OMVs were at 2 x1010 vesicles/mL of PBS.
Transmission Electron Microscopy
Bt OMVs were observed using negative staining with transmission electron microscopy (TEM) as previously described53. Briefly, isolated Bt OMVs were adsorbed to carbon–formvar-coated copper EM grids (Agar Scientific) for 1 min before wicking off with filter paper and negatively staining with 2% uranyl acetate solution (BDH) in water for 1 min. Grids were air-dried before analysis using a Tecnai G2 20 Twin TEM (FEI) at 29,000× magnification.
Polarised in vitro organ culture of colonic biopsies
Colonic biopsies were taken from the rectosigmoid junction (around 18 cm from rectum) of macroscopically normal patients following informed consent. The polarised in vitro culture (pIVOC) of colonic biopsies used was adapted from a previous study77. Briefly, five colonic biopsies were collected in IVOC medium (Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) (45 %) and distilled water (45 %) containing 0.47 g NCTC-135 (Sigma-Aldrich), 0.11 g sodium bicarbonate (Sigma-Aldrich), and 10 % newborn calf serum (NCS, Sigma-Aldrich). Each biopsy was orientated with the mucosal side uppermost on a cellulose nitrate filter within a snapwell support and mounted within a well of a six-well plate containing 3 mL of IVOC medium. Once mounted, 200 uL of IVOC medium was added apically with or without 108-109 Bt OMVs/ml. The plate was incubated on a rotor (12 rpm) at 37 °C for 6 h, after which the biopsy was removed intact from each support, flash-frozen in liquid nitrogen and stored at -80 °C for later protein extraction and cytokine analysis.
Biopsy tissue lysate extraction
Biopsies were thawed on ice and a mixture of 122ul CellLytic MT (Sigma C3228) and 3ul protease inhibitor cocktail (Sigma P2714-1BTL) was added with 5 acid-washed glass beads (3mm diameter). Homogenisation was performed using a MP Biomedical Fastprep-24 instrument (Fisher Scientific) at 4m/s for 30 seconds. Samples were centrifuged at 10,000rpm at 4° C for 2 min. The tissue lysate was then transferred to a 1.5ml pre-cooled tube and centrifuged at 10,000rpm for 10min at 4° C. The supernatant was carefully removed and stored at -80° C until use for cytokine bead array (see below).
Isolation of colonic lamina propria cells
Five proximal and five distal colon biopsies (10 mg tissue each) were obtained from macroscopically non‐lesional tissue sites at routine colonoscopy in all patients as previously described55. Biopsies were washed in HBSS containing 1 mM DTT and 1 mM EDTA in a shaking incubator at 37 °C for 30 min to remove the epithelial layer. Supernatants were discarded and wash was repeated for a second 30 min with HBSS/DTT/EDTA. Following discard of supernatants, biopsies were further digested in RPMI medium containing collagenase D (1 mg/mL) and Liberase TL (0.1 mg/mL) for 1 h shaking at 37 °C to release the lamina propria (LP) cells. LP cells were then filtered through a 100 µM strainer, washed with PBS and centrifuged at 600 g for 5 min before proceeding to either culture or FACS staining.
Isolation of peripheral blood mononuclear cells (PBMC)
Blood obtained by venepuncture was diluted 1:1 (vol:vol) in PBS and layered over Ficoll-Paque Plus (Amersham Biosciences, Chalfont St. Giles, UK). After centrifugation at 800 g for 30 min at 18 °C, PBMC were collected at the interface. PBMC were resuspended in complete medium (Dutch modified RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 100 U/mL penicillin/streptomycin, 2 mM L-glutamine, 50 μg/mL gentamicin (Sigma-Aldrich) and 10% faetal calf serum (TCS cell works, Buckingham, UK)) for culture or in PBS for FACS analysis.
Bacterial stimulation of blood DC or LP cells
Per condition, 5 x105 PBMC were plated in 96-well U-bottom plates. PBMC were incubated with either 106/mL freeze-killed commensal bacteria or OMVs at 10-fold incremental concentrations from 1010 to 10 OMVs/mL at 37 °C, 5 % CO2 for 20 h. LP cells were isolated as described above and resuspended in complete medium as described for PBMC. LP cells (1-2 x 105 per condition) were plated in 96-well U-bottomed plates. LP cells were incubated with either 107/mL freeze-killed Bt, 1010/mL of Bt OMVs or complete medium only at 37 °C, 5 % CO2 for 20 h. For intracellular cytokine responses, 2 µM monensin (Biolegend) was added to wells during incubation.
Surface marker and intracellular cytokine profiling of DC
Following incubation, PBMC and LP cells were washed with PBS and viability was determined by labelling cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (ThermoFischer Scientific) according to the manufacturer’s instructions. Cells were washed with FACS Buffer (1x PBS containing 2 % FCS, 1 mM ETDA and 0.02 % sodium azide) and then labelled with antibodies to identify the dendritic cells (Table 4). Cells were fixed in 1 % paraformaldehyde (PFA) if only surface markers were examined.
For intracellular cytokine analysis, cells were fixed using Leucoperm A buffer (Bio-Rad) and permeabilised using Leucoperm B (Bio-Rad) and then labelled with antibodies to interleukin (IL)-10 and IL-6 (Table 4). Finally, cells were fixed again in 1 % PFA and stored at 4°C.
Single cell suspensions were acquired on the BD FACSCanto II (BD Biosciences) or the BD LSR Fortessa (BD Biosciences). Compensation was carried out on FACS Diva software using Anti-Mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences) conjugated to antibodies used in above labelling experiments. The ArC™ Amine Reactive Compensation Bead Kit (ThermoFischer Scientific) was used for compensation of the LIVE/DEAD™ Fixable Near-IR Dead Cell Stain according to kit instructions. Data analysis was done using FlowJo_v.10 software.
Cytokine bead array
The LEGENDplex Human Inflammation Panel I (Biolegend, London, UK ) was used to simultaneously quantify 13 human cytokines/chemokines (IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33) in biopsy tissue lysates according to the manufacturer’s instructions and analysed on a BD LSR Fortessa using the PE and APC channels. Data were analysed using the LEGENDplex data analysis software (Biolegend, London, UK). All cytokines/chemokines were detectable apart from IFN-α2 which was below detection limits.
PBMC were cultured with Bt or Bt OMVs as described above and cell supernatants were taken at 20 h post-stimulation and stored at -80 °C. Amounts of cytokines (IL-6 and IL-10) were measured using Human DuoSet ELISA kits (R and D systems) according to the manufacturer’s instructions. Plates were read on the Tecan Infinite F50 plate reader and data were analysed using MagellanTM software (Tecan Group Ltd, Mannedorf, Switzerland).
Statistical analysis was carried out using GraphPad Prism software version 8. For in vitro experiments, data were analysed using nonparametric Mann Whitney U-tests (Figure 1b and Supplementary Figure 1a); unpaired t-tests (Figure 3); ordinary one-way ANOVA with Dunnett’s multiple comparisons test (Figure 1D, Figure 2B and D (IL-10), Figure 5B and Supplementary Figure 2B), one-way ANOVA with Brown-Forsyth and Welch corrections for unequal variance with Dunnett’s T3 multiple comparisons test (Figure 2C and Figure 4A and B) or non-parametric Kruskal Wallis ANOVA with Dunn’s multiple comparisons test (Figure 1E, Figure 2D (IL-6) and Figure 4C) when making comparisons between experimental conditions within single group; or ordinary two-way ANOVA with Holm’s Sidak’s multiple comparisons test (Figures 4 and 5) for making comparisons between groups for different experimental conditions. Colonic LP DC data from HC, CD and UC patients (Supplemental Figure 3) were analysed using non-parametric Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test; *p<0.05.