<i>Bifidobacterium breve</i> MRx0004 protects against airway inflammation in a severe asthma model by suppressing both neutrophil and eosinophil lung infiltration

AnimalsFemale 7-week-old C57BL/6 mice (Charles River Laboratories, France) have been randomly allotted to Sealsafe individually ventilated cages (Indulab AG). Mice have been acclimated to the power for 7 days. Mice have been eight weeks outdated on the initiation of the research. Potable water and meals have been out there advert libitum. Cage enrichment was current and day by day care of the animals was carried out. All experiments have been run at Preclin Biosystems AG, Epalinges, Switzerland. Animal welfare laws have been noticed as dictated by the official authorities of Switzerland below ordinance 455.163 of the FVO (Federal Veterinary Workplace) on laboratory animal husbandry, manufacturing of genetically modified animals, and strategies of animal experimentation. The experimental protocol was authorised by the native authorities “Service de la consommation et des Affaires vétérinaires” (SCAV) and the examine protocol follows the Directive 2010/63/EU revising Directive 86/609/EEC on the safety of animals used for scientific functions. The prophylactic examine involving pre-dosing of micro organism previous to illness induction, was performed as two impartial cohorts with 5 animals per group every. Therapeutic research involving micro organism dosing after illness induction have been performed as a single cohort of 5 animals per group.MRx0004 preparationA cryo-vial of MRx0004 was thawed below sterile circumstances by warming in a gloved hand and ~zero.7 mL of contents injected right into a Hungate tube containing eight mL of anaerobic yeast extract-casein hydrolysate-fatty acids (YCFA) broth. Hungate tubes have been then incubated at 37 °C for 14–16 h to permit the bacterial cultures to achieve late log at which level they have been used as remedies. A Hungate tube containing eight mL of anaerobic YCFA was incubated at 37 °C for 14–16 h to behave as automobile.Extreme neutrophilic bronchial asthma home mud mite (HDM) sensitization and challengeOn day (D) zero and D7, mice within the HDM, HDM + automobile (automobile), HDM + MRx0004 (MRx0004) and HDM + Anti-IL-17 (Anti-IL-17) teams have been sensitized with 50 µg HDM (Greer Laboratories) in phosphate-buffered saline (PBS) emulsified in an equal quantity of full Freund’s adjuvant (CFA, Chondrex Inc) and administered subcutaneously (s.c.) in a quantity of 200 µL, twice over two weeks on reverse flanks. Every week after the second immunisation, mice have been anaesthetized by intraperitoneal (i.p.) injection with 9.75 mg xylasol and 48.75 mg ketasol per kg (Dr. E. Graeub A. G.) after which given intranasal challenges of 15 µg of HDM in a quantity of 30 µL PBS beginning D14 and persevering with on D15, D16 and D17. On D18, mice have been sacrificed by deadly i.p. injection with pentabarbitol (Streuli Pharma AG).Bacterial, automobile and anti-IL-17 dosingPreventative research involving micro organism pre-dosing: 5 mice have been dosed as soon as day by day by oral gavage with MRx0004 (5 × 108 CFU/mouse) and one other 5 mice have been dosed with automobile (YCFA; 400 µL/mouse) from D-14 to D17. 5 mice have been dosed with 12.5 mg/kg of anti-IL-17 antibody (BioX Cell, clone 17F3) on D13, D15 and D17 by i.p. injection. 5 mice have been handled with HDM. Two impartial preventative research have been performed utilizing two cohorts of animals in remedy teams outlined above.Therapeutic research with out micro organism pre-dosing: 5 mice have been dosed as soon as day by day with MRx0004 (5 × 108 CFU/mouse) from D7 to D17. 5 mice have been dosed with 12.5 mg/kg anti-IL-17 neutralizing antibody on D13, D15 and D17 by i.p. injection. Automobile mice have been dosed as soon as day by day with YCFA (400 µl/mouse) from D-14. 5 mice have been handled with HDM.BALFTracheotomy was carried out instantly after euthanasia by inserting an 18 G canula (Tro-Vensite security plus, Troge) into the trachea connected to a 1 ml syringe. BALF was obtained by flushing the lungs twice with zero.5 mL of PBS supplemented with zero.2% (v/v) Bovine serum albumin (BSA, Sigma). BALF was then transferred right into a 1.5 mL Eppendorf tube and saved on ice till additional processing. BALF was centrifuged at 1800 rpm for eight min at four °C. Pelleted BALF cells have been resuspended in PBS/2% BSA and 1 × 105 cells have been transferred to microscope slides utilizing a cytospin centrifuge (800 rpm, 5 min at four °C) and stained with Diff-Quik staining set (Medion Diagnostics). Differential cell counts have been carried out primarily based on morphological and cytochemical standards counting 200 cells per samples in no less than 2 totally different area of view utilizing an Olympus BH-2 microscope.Measurement of inflammatory mediators in lung tissueRight lung lobes remoted for quantification of inflammatory mediators have been weighed and thereafter homogenized in 250 µL PBS supplemented with 1X protease inhibitor cocktail (Roche) utilizing the TissueLyser system (Qiagen). Briefly, one 5 mm chrome steel bead (Qiagen) was added to every pattern. Lungs have been then homogenized for three min at 25 Hz at four °C. Homogenates have been then spun down at 10000 rpm for 20 min at four °C and supernatants have been collected and frozen at −20 °C. Quantification of IFN-γ, IL-1α, IL-1β, CXCL1, CCL3, CXCL2, CCL5, and TNF-α was carried out utilizing the Bio-Plex Professional Cytokine, Chemokine, and Development Issue Assays (Bio-Rad) following producer’s suggestions. Background corrected fluorescence values have been transformed to concentrations (pg/mL) utilizing an ordinary curve. The place fluorescence values have been beneath the detectable restrict, concentrations have been set at zero pg/mL, whereas fluorescence above the detectable restrict have been excluded from the introduced knowledge evaluation. IL-17A and IL-17F ranges fell exterior the detection limits of the assay used however have been supplied in Fig. S2 of the Supplementary Data, introduced utilizing fluorescence depth slightly than pg/ml.Isolation of lung and histological analysisLeft lungs have been mounted in 10% buffered formalin (Baker) in a single day at room temperature. Tissue was processed to paraffin wax within the standard method and 5 µm sections reduce and picked up onto Superfrost Poly-L-Lysine slides (Thermo Fisher Scientific) and dried in a single day at 40 °C. Sections have been dewaxed with xylene (three instances, 15 min every) and rehydrated by a sequence of ethanol washes earlier than performing normal Haemotoxylin and Eosin (H&E) (Sigma Aldrich) staining. Slides have been mounted with Histomount (pH 6.5–7.1, TAAB laboratories) and considered utilizing a Zeiss Axioskop microscope outfitted with x20/zero.5. Plan-NEOFLUAR goal.From every mouse six random fields of view have been digitized (these photos included each bronchioles and blood vessels) utilizing a QIMAGING digital camera managed with Picture Professional Plus software program v6 and saved as JPEGs. Every picture was then assessed for each perivascular and peribronchiolar infiltrate and allotted a single rating primarily based on the next standards the place: zero, no irritation; 1, occasional inflammatory cells; 2, larger accumulations of inflammatory cells across the vessels three, multifocal and even larger irritation round vessels than that seen in grade 2; four, extreme multifocal inflammatory infiltrate62 by two people aware of HDM lung pathology and blinded to remedy teams. The imply rating was then calculated for every particular person mouse.To provide an total view of the lung sections chosen slides have been scanned utilizing an Axioscan Z1 with a Hitachi HV F202scl digital camera outfitted with a Plan Apochromat x10/zero.45. goal. Scaling per pixel was zero.44 × zero.44 µm. Publicity time was 2 µs and depth of focus was 5.43 µm. Zen2 lite software program was used to export photos which have been saved as tiff information.ImmunocytochemistrySerial Formalin mounted paraffin embedded (FFPE) sections ready for histological evaluation have been dewaxed and rehydrated as above earlier than performing antigen retrieval. Antigen retrieval was performed at 90 °C for 25 minutes in Citrate buffer (Abcam). Slides have been then washed twice in PBS and blocked for 30 minutes at room temperature with PBS (pH7.2) 2% BSA (Sigma Aldrich). Extra block was eliminated by capillary motion and the sections have been incubated in a single day at four °C in a humidified chamber with 12.5 µg/mL Rabbit anti-mouse CD4 monoclonal antibody (Abcam) in PBS (pH 7.2) 2% BSA. Subsequently, sections have been equilibrated to room temperature for 1 h, and washed three instances for 10 minutes with PBS (pH 7.2). The secondary antibody was utilized at 2 µg/mL Alexa Fluor™ 488 donkey anti-rabbit IgG(H + L) in PBS (pH 7.2) (Molecular probes) for 30 min at nighttime, at room temperature. Slides have been washed as above and counter stained with DAPI (Sigma) earlier than mounting with Vectashield (Vector laboratories) Slides have been examined utilizing the above microscope, digital camera, software program and x20 goal. Ten randomised fields of view have been imaged utilizing the suitable filter units for Alexa Fluor™ 488 and DAPI. A guide publicity time of two milliseconds was used all through for the Alexa 488™ photos. The digitised photos have been saved as JPEGs. As a result of excessive diploma of auto-fluorescence noticed within the FFPE mounted tissues, the photographs of CD4 constructive cells have been additional processed in Photoshop CC 2015.1.2. The color choose device was used to take away background from the Alexa 488 sign. Every particular person picture was assessed in order that solely clearly labelled cells have been chosen. The choice standards utilizing the color choose device diverse from a degree of 98 to 130. The ‘fuzziness’ setting was saved fixed. The resultant picture was then overlaid on the DAPI picture and flattened in Photoshop.Movement cytometryCell preparationRight lung lobes have been submerged in ice-cold IMDM (Iscove’s Modified Dulbecco’s media, Gibco) and reduce into small items. Tissue was digested for 40 min at 37 °C in IMDM supplemented with 2 mg/mL collagenase sort IV. Following digestion, the tissue was homogenised by 70 µm cell strainers, which have been subsequently washed with 10 mL chilly PBS/zero.2% BSA. Cell suspensions have been centrifuged (1500 rpm, 6 min, four °C) and pelleted cells resuspended in 1 mL PBS/zero.2% BSA at which level cell counts have been made utilizing a Beckman Coulter Z2.Staining for move cytometry100 µL of the above cell suspensions was transferred to a spherical backside 96-well plate and centrifuged (3000 rpm, 1 min, four °C). Cell have been blocked at four °C for 15 min utilizing 24G2 blocking antibody (Preclin Biosystems AG) at 800 µg/mL closing focus. Cells have been washed by centrifugation and resuspension with PBS/zero.2% BSA. Cell have been then centrifuged (3000 rpm, 1 min, four °C) and resuspended in 50 µl of staining answer containing applicable main antibodies (CD11b-PerCp-Cy5.5; CD11c-APC-Cy7; MHCII-AF700; F4/80-AF647; CD40-PE/Cy5; CD86-PE; CD80-PE/Cy7; CD4-Pacific blue; CD8-APC-Cy7; CD25-AF700; CD44-PE; all Biolegend) for 30 min at four °C. After two washes with PBS/zero.2% BSA and one wash in PBS cells have been mounted in 50 µL of BD FACS lysing answer for 15 min at four °C. Cells have been then washed twice in PBS/zero.2% BSA and resuspended in 100 µL of PBS/zero.2% BSA. For intracellular staining cells have been washed as soon as extra in zero.5% saponin. Cells have been then incubated with main antibody (FoxP3-AF647, Biolegend) in a single day at four °C in zero.5% saponin. Cells have been then washed as soon as with zero.5% saponin and twice with PBS/zero.2% BSA earlier than being resuspended in 100 µL of PBS/zero.2% BSA for move cytometry evaluation.Movement cytometry analysisEvents have been captured on a BD LSR Fortessa (BD Biosciences) outfitted with four lasers (405 nm, 488 nm, 561 nm and 640 nm). Compensation was carried out utilizing applicable fluorochromes and BD CompBeads (BD Biosciences). Compensation was enabled earlier than buying samples utilizing the BD FACS Diva software program. The gating methods have been as follows: (a) CD4+ and CD8+ T cells: Reside/lifeless gate, single cell gate, CD4/CD8 gate. The CD44 marker of activation on CD4+ and CD8+ T cells was expressed as MFI (Imply Fluorescence Depth). (b) Tregs: Reside/lifeless gate, single cell gate, CD4 Vs. CD8 gate, CD25+ Vs. FoxP3+ gated on CD4+ cells. (c) Dendritic cells: Reside/lifeless gate, single cell gate, CD11c Vs. FSC-A gate, FITC (autofluorescence) Vs. F4-80 gated on CD11c+ cells, CD11b Vs. MHCII gated on FITC- cells. The activation markers CD40, CD80 and CD86 have been measured.Information analysisAll graphs and statistical analyses have been generated with GraphPad Prism Model 7. Information is introduced as field and whisker plots with minimal and most and particular person knowledge factors. Statistical evaluation was carried out by one-way ANOVA adopted by Tukey’s post-test to appropriate for a number of comparisons, with statistical significance accepted at p < zero.05.Information availabilityThe datasets generated throughout and/or analysed throughout the present examine can be found from the corresponding creator on cheap request.


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