Study design
This study consists out of three series of experiments: two studies in mice and some measurements performed in patients with COPD. In the mice study, the impact of vitamin D deficiency combined or not with cigarette smoking on inflammatory cells and immunoglobulin production was first addressed on non-infected animals (series 1). Then, the effects of vitamin D deficiency and smoking were examined in animals repeatedly infected with NTHi in whom lung function and histopathology, bacterial clearance, inflammatory mediators, lymph node activation and immunoglobulin (Ig) production were measured (series 2). Finally, Ig production against NTHi and DNA was measured in serum of COPD patients with different baseline 25-OHD serum levels.
Animal model
Animals: Three week-old male C57Bl/6JolaH mice were fed with a vitamin D deficient (VDD; n= 32 and n=30 mice for series 1 and series 2, respectively) (TD.87095, Envigo Teklad custom diet, Madison, Wis. USA) or vitamin D sufficient diet (VDS; n=32 and n=26 mice for series 1 and series 2, respectively) (Ssniff Bioservices, Uden, The Netherlands) and were kept in an ultraviolet light-free environment during the whole study, as described in [32]. Mice were housed in individually ventilated cages with a 12/12 h light-dark cycle.
Exposure to cigarette smoking or room air: At the age of eight weeks, mice were randomly divided into two separate groups: nose-only CS- and air-exposed groups. CS-exposed mice were first acclimatized for one week with an increasing number of cigarettes (3R4F research cigarettes with filter, Kentucky Tobacco R&D center, university of Kentucky) per session, until the limit of 6 cigarettes per session was reached while control mice were exposed to ambient air and kept in soft restraints for the same duration as established previously [32]. Afterwards, mice were exposed twice a day to CS or to ambient air, 5 days/week for 14 weeks (16 controls and 16 smokers for series 1; 13/15 controls and 13/15 smokers for series 2).
Induction of repeated infections in series 2 only: On week 6 of CS or air-exposure, mice were oropharyngeally infected with 1 × 106 colony forming units (CFU) NTHi in a total volume of 50 ml PBS (kindly provided by J.V.) and subsequently repeated on week 10 and 14 (Figure 1). Before infection, mice were anaesthetized intraperitoneally (i.p.) with a mixture of xylazine (4.25 mg/kg, RompunÒ, Bayer, Belgium) and ketamine (65 mg/kg, KetalarÒ Pfizer, Belgium). After each infection mice were kept in quarantine for 1 week during which they were not exposed to CS. CS or air exposure sessions were resumed after this one-week of quarantine.
Measurements: For series 1, inflammatory cells in broncho-alveolar lavage (BAL) and immunoglobulin production in BAL and serum were measured while for series 2, lung function and histopathology, bacterial clearance, inflammatory mediators, lymph node activation and Ig production were assessed 72h after the last infection. Levels of serum 25-OHD were determined in the two mouse studies.
Patients characteristics
Serum samples from 137 vitamin D-naïve COPD participants were included in our study. These patients were issued from a previous single-center, double-blind, randomized, placebo-controlled trail (RCT) (University hospitals Louvain, Belgium), approved by the local ethics committee of University Hospital of Louvain (S50722, EudraCT number: 2007-004755-11) and ClinicalTrails.gov (NCT00666367). In this RCT study, the patients were treated with either a high-dose of vitamin D3 (100,000 IU, D-Cure) or a placebo (arachidic oil) every 4 weeks for one year. Details of the vitamin D intervention trial have been previously published [10]. In this post-hoc analysis, we assessed total IgG and IgG against NTHi and DNA in serum of placebo or vitamin D-treated COPD patients. Patients were matched according to gender, current smokers, GOLD stage, serum 25-OHD level, Forced Expiratory Volume in 1s (FEV1), Forced Vital Capacity (FVC) and FEV1/FVC (Table S2). Participants from the RCT did not receive any antibiotics or oral steroid treatment for an acute exacerbation during the one-year study period. A subgroup analysis was also performed by dividing the participants into severe deficient (< 10ng/ml 25-OHD), deficient (< 20ng/ml 25-OHD) and insufficient (<30ng/ml) patients. Sufficient patients (> 30ng/ml 25-OHD) were excluded, because of insufficient numbers for reliable statistics (n=7, Table S2).
Animal model measurements:
Lung function (series 2)
Mice were anaesthetized i.p. with a Xylazine (10 mg/kg, Rompun®, Bayer, Belgium), Ketamine (100 mg/kg, Ketalar®, Pfizer, Belgium) and Acepromazine (3 mg/kg, Placivet®, Kela, Belgium) mixture 3 days after the last NTHi infection. Sedated mice were tracheotomized and placed in the whole-body plethysmograph (Force Pulmonary Maneuvers, Buxco®) to measure total lung capacity (TLC) and chest wall compliance (Cchord) [33].
Broncho-alveolar lavage (both series)
BAL was performed with 500 ml and 3 times 1000 ml of saline (B. Braun Medical, Diegem, Belgium). The supernatant of the first fraction was collected for cytokine analysis by centrifugation (10 min, 1000 g, 4°C) (series 2 only), while total cell count was measured on all pooled cell fractions using a Bürker hemocytometer with trypan blue. Differential cell counts were determined after centrifugation (6 min, 300 g) of the BAL cells onto microslides with cytospins (Shandon, TechGen, Zellik, Belgium) and staining with Diff-Quick® (Medical Diagnostics, Düdingen, Germany). On each slide, 100 cells were counted 3 times to determine the various differentiated cells.
For series 2, viable NTHi was assessed at sacrifice, by plating serial dilutions onto Haemophilus agar plates. The number of colonies were counted 24 and 48h after incubation (37°C) and expressed as the number of CFU per milliliter of BAL.
25-OHD serum levels (both series)
Blood collected from the vena cava was kept at room temperature for 1 hour and centrifuged at 1500 g for 15 min (4°C). Serum was collected and stored at -80°C. Serum 25-OHD levels were measured by liquid chromatography tandem-mass spectrometry (LC-MS/MS) (LC, Shimadzu, and MS, Qtrap 5500, Sciex) at the laboratory of medicine, UZLeuven. Samples were extracted with methanol mixture spiked with internal standard 25-OH Vit-D3-d6.
Histopathology of the lungs (series 2)
The heart-left lung block was fixated for 24 h under a constant hydrostatic pressure (20 - 25 cm H2O) with 6% paraformaldehyde. The left lung was isolated and cut in three transversal pieces (apical, center and basal), dehydrated and embedded in paraffin. Transversal lung slides were stained with H&E and scored for inflammation in a blinded manner by a lung pathologist (E.V.). Each compartment of the lung was scored separately e.g. broncho-vascular bundle, parenchymal and venous compartment for lymphoid aggregates and diffuse inflammation. Scoring ranged from 0 to 3: 0 = normal, 0.5 = minimal, 1 = mild, 2 = moderate and 3 = severe. Airspace enlargement was quantified by measuring the mean linear intercept (Lm) in 25 fields over the whole lung using an in-house macro in ImageJ.
Expression of proteases/antiproteases (series 2)
The right lung was removed, snap frozen in liquid nitrogen and stored at -80°C for RNA extraction. After homogenization in TRIzol® (ThermoFisher scientific, Life Technologies, Waltham, MA USA), total RNA was extracted with chloroform and purified with RNAeasy mini kit (Qiagen, Leudsen, Netherlands) according to manufacturer’s instructions. 1µg of RNA was reverse transcribed with random hexametric primers and Superscript III reverse transcriptase was used for cDNA synthesis at 42°C for 50 min, followed by 15 minutes incubation at 70°C. The quantitative Real-Time PCR amplifications (qRT-PCR) were performed with 10 ng cDNA and Platinum®SYBR® Green qPCR SuperMix-UDG (ThermoFisher scientific) using a thermal cycler (Eco Real-Time PCR system, Illumina). Threshold data was normalized using a housekeeping gene, Ribosomal Protein L27 (RPL27), and groups were compared using the comparative cycle threshold (∆∆CT) method. Primers are given in Table S1.
Inflammatory mediators and lung permeability (series 2)
Inflammatory mediators for IFN-γ, IL-1β, IL-4, IL-6, IL-10, IL-13, IL-17A, TNF-α, KC and MIP-2 were analyzed in cell-free BAL and serum with a MSD U-plex® multiplex assay (Meso Scale Discovery®, Rockville, USA). Lung permeability was assessed via measurement of total protein concentration in cell-free BAL using the Bradford method (Bio-Rad, Temse, Belgium) and surfactant protein (SP)-D (Duoset ELISA R&D Systems Inc., DY6839-05) in the serum.
Flow cytometry analysis of mediastinal lymph nodes (series 2)
Single-cell suspensions were prepared from mediastinal lymph nodes by mechanical disruption. Cells were stained with conjugated antibodies directed against CD3, CD4, CD8α, CD44, CD62L and CD69 (eBioscience, Thermo Fisher Scientific). Dead cells were excluded from analysis following staining using Zombie Aqua Fixable Viability dye (BioLegend). Samples (> 100,000 cells) were acquired on a BD Canto II (BD Biosciences) flow cytometer, and data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR).
Immunoglobulin production in serum and BAL (both mouse series and patient study)
In the mouse studies, mouse IgA, M and G in cell-free BAL and serum were measured via ELISA‘s (Ready-Set-Go, Invitrogen, Merelbeke, Belgium). In the post-hoc analysis of the patient study, human IgG was measured in serum via ELISA (Ready-Set-Go, Invitrogen, Merelbeke, Belgium). All assays were performed according to manufacturer’s instructions. Indirect ELISA against NTHi and DNA (calf thymus activated, Sigma-Aldrich), were performed by coating 50μl/well of 10μg/ml antigen in PBS. 96-well plates were incubated for 1h at 37°C. Plates were washed 3 times with PBS-Tween 0.05% and blocked with 150 μl PBS-BSA 1% for 1h at 37°C. Plates were washed 3 times and incubated with 1:4 dilution of BAL fluid or 1:32 dilution of serum samples. An arbitrary standard curve was made from pooled BAL fluid or serum samples with a dilution from 1:2 to 1:128. Samples were incubated for 2h at 37°C, washed 3 times and incubated for 1h at room temperature with anti-mouse IgA, IgM and IgG biotinylated (Sigma-Aldrich) for the mice studies or anti-human IgG biotinylated (Sigma-aldrich) for human study. Plates were washed 3 times and incubated for 30min with Streptavidin-HRP. TMB and stop solution was used for coloration and measured at 450nm.
Statistical analysis
Datasets were analyzed using GraphPad Prism 8.1.1 and are presented as median±IQR. Population distribution, for each dataset, was examined with the Shapiro-Wilk normality test. For the animal studies: if the dataset was non-parametrically distributed, it was transformed with Log10 to a parametric dataset and further analyzed as a Two-way ANOVA with a Bonferroni post-hoc test for group comparison. In the post-hoc analysis of the patient study: baseline characteristics were analyzed using an unpaired T-test or Mann-Whitney test for parametric or non-parametric distributions. For the subgroup analysis dividing the participants into severe deficient (< 10ng/ml 25-OHD), deficient (< 20ng/ml) and insufficient (<30ng/ml) patients, datasets with two variables were transformed, if non-normally distributed, with Log10 to a parametric dataset and analyzed with a Mixed-effects model for missing values and a Bonferroni post-hoc group comparison. Correlations were performed with a Spearman correlation between Danti-NTHi antibodies and serum 25-OHD levels measured after one year of supplementation. This was performed with the total population and in the insufficient subgroup. A Poisson regression analysis was performed between the exacerbation/year and the Danti-NTHi antibodies. Differences were considered significant when p-value was less than 0.05.