Diminished airway host innate response in people with cystic fibrosis who experience frequent pulmonary exacerbations

Rationale Pulmonary exacerbations are clinically impactful events that accelerate cystic fibrosis (CF) lung disease progression. The pathophysiological mechanisms underlying an increased frequency of pulmonary exacerbations have not been explored. Objectives To compare host immune response during intravenous antibiotic treatment of pulmonary exacerbations in people with CF who have a history of frequent versus infrequent exacerbations. Methods Adults with CF were recruited at onset of antibiotic treatment of a pulmonary exacerbation and were categorised as infrequent or frequent exacerbators based on their pulmonary exacerbation frequency in the previous 12 months. Clinical parameters, sputum bacterial load and sputum inflammatory markers were measured on day 0, day 5 and at the end of treatment. Shotgun proteomic analysis was performed on sputum using liquid chromatography-mass spectrometry. Measurements and main results Many sputum proteins were differentially enriched between infrequent and frequent exacerbators (day 0 n=23 and day 5 n=31). The majority of these proteins had a higher abundance in infrequent exacerbators and were secreted innate host defence proteins with antimicrobial, antiprotease and immunomodulatory functions. Several differentially enriched proteins were validated using ELISA and Western blot including secretory leukocyte protease inhibitor (SLPI), lipocalin-1 and cystatin SA. Sputum from frequent exacerbators demonstrated potent ability to cleave exogenous recombinant SLPI in a neutrophil elastase dependent manner. Frequent exacerbators had increased sputum inflammatory markers (interleukin (IL)-1β and IL-8) and total bacterial load compared to infrequent exacerbators. Conclusions A diminished innate host protein defence may play a role in the pathophysiological mechanisms of frequent CF pulmonary exacerbations. Frequent exacerbators may benefit from therapies targeting this dysregulated host immune response.

bacterial qPCR analysis.The remaining sputum was processed to obtain cell-free supernatant which was stored at -80°C.Sputum plugs were weighed and re-suspended in a volume of Dulbecco's Phosphate Buffered Saline (PBS) equal to 8 times the weight of the sputum plugs.
Sputum was homogenised by repeat pipetting with a plastic transfer pipette and vortexed for 15 s.The mixture was placed on a bench rocker for 15 minutes on ice and then centrifuged at 790 x g for 10 min at 4°C.A volume of supernatant equal to four times the weight of the sputum plugs was removed and centrifuged at 1500 x g for 10 min at 4°C.The cell-free supernatant was stored at -80°C.Sputum that remained after the first centrifugation step was re-suspended in a volume of 0.2 % sputolysin (Calbiochem®, Merck) equal to 4 times the weight of sputum plugs and vortexed for 15 secs, then placed on a bench rocker for 15 mins on ice.Sputum was filtered through a 100 μM cell strainer and a total cell count performed on the filtered cell suspension by staining with trypan blue stain by counting on a haemocytometer.Differential cell counts were evaluated following cytospin preparations onto coated cytoslides (Shandon/Thermo Scientific, UK).Cells were stained with Speedy-Diff Complete Kit (CLIN-TECH LTD) and visualised using the Leica DM5500B microscope and images were captured via the Lecia AL software under X 40 objective lens.A minimum of 400 cells were counted using ImageJ software and the relative number of differential white blood cells present calculated.

Blood sample processing
Blood samples were collected at each study visit.Blood was collected in serum vacutainers and allowed to clot at room temperature for 30 mins.Samples were centrifuged at 1500 x g for 10 mins at 4°C and the cell-free supernatant was aliquoted and stored at -80°C prior to analysis.

Protein Analysis
Sputum supernatant SLPI levels were quantified by ELISA (R&D Systems) as per the manufacturer's instructions.Sputum supernatant IL-8 and IL-1β levels were quantified using ELLA Simple Plex assay (R&D systems) as per the manufacturer's instructions.

Neutrophil elastase activity assay
Neutrophil elastase (NE) activity was determined in sputum supernatant using the substrate N-Methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-methylcoumarin (AAPV-AMC; Enzo Life Sciences, Exeter, UK) at a final concentration of 20 uM.Experiments were performed ± NE inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (AAPVCMK) at a final concentration of 1mM.The reaction buffer was 0.1 M Hepes, 0.5 M NaCl, pH 7.5.Samples were incubated in buffer ± inhibitor for 30 min at room temperature before fluorescence (substrate turnover) as determined by excitation at 360 nm and emission at 460 nm was read in a 96-well microplate reader (Synergy HT using Gen5™ software, BioTek UK).Fluorescence emission was read at 1 min intervals for 30 min at 37 °C.Results were expressed as the change in relative fluorescence units (ΔRFU) over time.

Western blotting
Western blots were performed for validation of proteomic data and for visualisation of rSLPI degradation profile.Denatured sputum supernatant samples were separated by electrophoresis on 15% SDS-polyacrylamide gels.Proteins were transferred onto a nitrocellulose membrane and probed using human anti-Cystatin S (R&D systems), human anti-Cystatin SA (R&D systems), human anti-lipocalin-1 (R&D systems), human anti-SPLUNC (R&D systems) and human anti-SLPI (Invitrogen).Binding was detected using the appropriate horseradish peroxidase-conjugated secondary antibodies, visualized by chemiluminescence (GE Healthcare UK, Buckinghamshire) and analysed using the Syngene G:Box and GeneSnap software (SynGene UK, Cambridge).
Sputum was diluted 10-fold in 30 mM tris-buffered saline (TBS) pH 7.5 and incubated with rSLPI for 0 min, 10 min, 1 h, 6 h and 24 h at 37 °C.For inhibition experiments, sputum samples were pre-incubated for 1 h at 37 °C with the following protease inhibitors before incubation with rSLPI: 13 mM EDTA, 1 mM PMSF, 0.4 mM E64, 0.11 mM pepstatin A, 1μM antichymotrypsin (ACT), 1 μM elafin and 0.1 mM Me0Suc-AAPV-CMK.The incubation was terminated at each time-point by boiling the sample in a heat block at 95 °C for 5 min with sample treatment buffer containing reducing agent.Samples were stored at -20 °C until analysed by SDS-PAGE using a 15 % polyacrylamide gel, then blotted onto a nitrocellulose membrane and SLPI detected as previously described.

Sputum microbial DNA extraction
Human DNA depletion: Sputum was thawed and mixed with Sputolysin (Calbiochem®, Merck) in a 1:1 ratio and vortexed.Samples were incubated in a heat block shaker at 37 °C for 30 min at 1000 rpm then centrifuged at 8,000 x g for 5 min.The supernatant was discarded, and the pellet was resuspended in 250 μL sterile PBS and vortexed.A 200 μL volume of 5 % Saponin (Tokyo chemical industry) was added and the sample vortexed again to lyse human cells.Samples were incubated at 21 °C for 10 min.Next, 350 μL of nuclease free water was added and incubated for 30 sec then 12 μL of 5M NaCl was added to create osmotic shock.Samples were vortexed, centrifuged at 6,000 x g for 5 min and the supernatant discarded.Samples were resuspended by vortexing in 100 μL PBS and 100 μL of HL-SAN buffer (5.5 M NaCl, 100 mM MgCl2, filter sterilised) and 10 μL of HL-SANDNase (250,000 units, Articzymes) added to degrade released Human DNA.After incubation at 37 °C for 15 min at 800 rpm, samples were centrifuged at 6,000 x g for 3 min and the supernatant discarded.Pellets were washed twice by vortexing in PBS (800 μL then 1 mL) to resuspend the pellet then centrifuging at 6,000 x g for 3 min.After each wash the supernatant was discarded.Pellets were resuspended in 100 μL PBS and stored at -80°C.
Microbial cell lysis: Pellets were thawed on ice and 300 μL of MagNa Pure 96 Bacterial Lysis Buffer (Roche) containing 0.35 mM lysozyme (Sigma-Aldrich) was added.Samples were vortexed and incubated for 30 min in a heat block shaker at 37 °C for 30 min at 2,000 rpm.The whole sample was transferred to 0.1 mm, 2 mL glass bead tubes (Qiagen) and homogenised in a Fast-Prep-24 rotor stator homogenizer (Mpbio) on speed 6.0 for 40 sec.Samples were incubated at 65 °C for 10 min at 1,500 rpm.A 150 μL aliquot of nuclease-free water was added to tubes before a second homogenisation in the Fast-prep homogeniser as previously described.Samples were incubated at 95 °C for 10 min at 1,000 rpm and centrifuged at 10,000 x g for 10 min at 4 °C.The supernatant was removed and stored at -80 °C for MagNA Pure DNA extraction.
MagNA Pure DNA extraction: Microbial cell lysates were thawed and DNA was extracted from 200 μL of lysate using the Roche MagNA Pure 96 automatic system according to the manufacturer's instructions.DNA amount and purity were verified using the NanoDrop spectrophotometer (Thermo Fisher Scientific).DNA samples were stored at -20°C until use.

Real-time PCR
Total 16S and oprL copy number in sputum was quantified by qPCR using primers and probes detailed in table 1, and validated 16S/oprL standards.The reaction mix comprised of 5 µL Lightcycler 480 Probes Master (Roche), 0.3 µM of each 16S primer or 0.4 µM of each oprL primer (Eurofins), and 0.05 µM hydrolysis probe, and was made up to a final volume of 10 µL with water.
A no template control and a water-only negative control were included.Purified 16S and oprL standards containing 4 x 10 9 copy/μL were serially diluted 10-fold in water, creating eight standards with copies of the target amplicon ranging from 4 x 10 1 to 4 x 10 8 copy/μL.A 7.5 μL volume of reaction mix was added per well of a 96 well PCR plate (Roche) followed by 2.5 μL of standard, sample or control to give a total reaction volume of 10 μL.Each standard, sample or control was with each run in triplicate.Cycling was performed on a LightCycler 480 (Roche).Samples were initially held at 95°C for 10 min, followed by 40 cycles (16S) or 45 cycles (oprL) at 95°C for 10 s, 60°C for 30 s, then 72°C for 1 s.Samples were finally held at 40°C for 30 s.Standard curves with a PCR amplification efficiency of 91 -110 % were considered acceptable.

Table E1. Bacterial qPCR probes and primers (Eurofins
inflammatory biomarker responses to IV-treatment.

Figure E1 .
Figure E1.Cytosolic proteins enriched in frequent exacerbators.Box plots showing Log10 protein abundance of proteins significantly differentially enriched in sputum from frequent exacerbators on D5 of PEx that were annotated with the GO cellular compartment term 'cytosol'.Data are log10 transformed and are presented as median (25th-75th interquartile range).

Figure E2 :
Figure E2: Validation of proteomics results by western blot.A) Western blotting of proteins in D0 sputum supernatants from infrequent and frequent exacerbators and corresponding densitometry of B) lipocalin-1, C) cystatin SA, and D) SPLUNC1.Data are presented as mean ± SEM of n=5.

Figure E3 .
Figure E3.Antiprotease levels in sputum at 4-6 weeks post-treatment.A) Western blotting of proteins in sputum supernatants from infrequent and frequent exacerbators and B) corresponding densitometry of cystatin SA and cystatin S. C) SLPI levels determined by ELISA in sputum supernatants from infrequent and frequent exacerbators.Data are presented as mean ± SEM of n=5/6.

Table E3 : Sputum bacterial responses to IV-treatment.
Table displaying intra-cohort changes in sputum inflammatory biomarkers between visits.Data are log10 transformed and are presented as mean (±SEM).