2. Methods
2.1. Description of the Cohort
Our analyses were conducted using data from the PreDicta study(Post-infectious immune reprogramming and its association with
persistence and chronicity of respiratory allergic diseases), a 2-year
multi-center prospective cohort study [14]. Blood
and nasopharyngeal samples were collected from 233 preschool children
(4-6 years) with asthma (167 subjects) and matched healthy controls (66)
across 5 major European climate regions, i.e. Greece (Athens), Poland
(Lodz), Finland (Turku), Germany (Erlangen) and Belgium (Ghent)[14]. To conduct this study, cytokine measurements
and metagenomics data were used from 51 subjects from the whole cohort
(32 asthmatics, 19 healthy controls), based on the availability of the
samples, in 3 out of the 5 centres. Our population was generally
representative of the whole cohort [12](Supplementary Table 1). Written informed consent was obtained from the
parents or by the legal guardians and the study was approved by the
Regional Ethics Committee of Karolinska Institutet, Stockholm, Sweden.
Cases needed to be diagnosed with asthma within the previous 2 years and
have a minimum of 3 wheezing episodes within the last 12 months prior to
study inclusion [12]. Exclusion criteria included
severe asthma, >6 courses of oral steroids during the last
12 months, immunotherapy, chronic medication use, or the history of
chronic respiratory disease other than asthma and/or allergic rhinitis.
Control subjects had to demonstrate no history of wheezing/asthma[14]. Additionally, subjects needed to be away
from an asthma exacerbation and/or upper respiratory tract infection for
at least 4 weeks before sample collection. Subjects were balanced for
sex (50.9% males, 49.1% females), of 4.95 +/-0.65 years old (range
3,26 to 6,29). Comorbidities and other characteristics are shown in
Supplementary Table 1.
2.2. Treatment of blood samples and cytokine measurements
Blood samples were collected in tubes with lithium heparin (Vacutainer)
and diluted with an equal volume of warm PBS (Gibco, Invitrogen,
Massachusetts). Peripheral blood mononuclear cells (PBMCs) were isolated
by centrifuging at 800g for 20 min at 18-20ºC on Biocoll separating
solution (Biochrom AG, Germany). After three washes, PBMCs were
resuspended in complete medium (CM) [RPMI-1640 with HEPES 25 mM and
L-Glutamine (Gibco, Life Technologies Ltd, UK), supplemented with 10ml/L
Penicillin-Streptomycin USA, 50μl/L 1M β-mercaptoethanol, 20ml/L
L-Glutamine plus MEM Vitamin, 20ml/L Non-essential Amino Acid, Sodium
Pyruvate and 10% heat-inactivated FBS (all from Sigma-Aldrich,
Germany)] at concentration 106 cells/ml.
RV1b stock was propagated in HeLa cells and purified by centrifugation
at 2500rpm for 10 minutes (4ºC). Suspension from HeLa lysates was used
as a control. The same batch of RV1b and HeLa suspension was used
throughout the study. 1ml of cells suspension (106PBMCs) were exposed to RV1b (6.7 titration) or HeLa suspension as
follows: After centrifugation at 300g, for 15min, at room temperature
(RT), the supernatant was carefully removed by aspiration, and cells
were exposed to 0.5ml of RV or HeLa suspension for 1 hour, under
rotation at RT. Subsequently, cells were washed twice with CM at 300g
for 15 min, RT, and resuspended in 1ml of CM.
The cell suspension was seeded in a flat-bottom 48-well tissue plate
(Corning Incorporated, Costar, New York), with 5 × 105viable cells per well (500μL). PBMCs were cultured in duplicates either
with complete medium alone (unstimulated control, RV1b-exposed cells,
and HeLa-exposed cells) or with one of the following stimulants: 4μg/ml
Resiquimod (R848), 5μg/ml Endotoxin-free bacterial DNA (InvivoGen,
France), 20μg/ml
Polyinosinic–polycytidylic acid
potassium salt (Poly I:C), 1μg/ml Lipopolysaccharides from E.Coli
0111:B4 (LPS), (Sigma-Aldrich, Germany), at 37ºC, 5%
CO2.
Cultures were harvested after 48 hours and, after centrifugation at 600g
for 5 min, supernatants were stored at -80ºC until analysis. Cytokine
expression levels in the culture supernatants were quantified by
multiplex bead-based fluorometric immunoassay (Milliplex, Millipore)
using Luminex xMAP (Luminex 200, Bio-Rad) at the Swiss Institute for
Allergy and Asthma Research (SIAF) in Davos, Switzerland. The panel used
contained IFNα2, IFNγ, IFNλ-2, IL-1β, IL-5, IL-6, IL-7, IL-9, IL-10,
IL-12p70, IL-13, IL-17A, IL-23A, IL-25, IL-27, IL-33, CCL3, CCL4, CCL5,
CXCL8, CXCL10, TNF-α.
2.3. Characterisation of the nasopharyngeal virome
The presence of prokaryotic and eukaryotic viruses in the upper
respiratory tract (nasopharynx and anterior nares) was previously
investigated using metagenomic sequencing in samples obtained from 19
healthy individuals and 32 patients with asthma[12]. Briefly, based on the predominant viral
families of these individuals, three virome profile groups (VPGs) were
assigned: Prokaryotic VPG; (P-VPG, n=29), contained samples with high
prevalence of prokaryotic viruses and low/intermediate of eukaryotic
viruses and Anelloviridae, Eukaryotic VPG (E-VPG, n=11) included samples
with high eukaryotic viruses’ predominance and low/intermediate of
Anelloviridae and Anelloviridae VPG (A-VPG, n=11) contained samples with
high Anelloviridae predominance. The virome characteristics of the 51
individuals are described in the Supplementary Data 1.
2.4. Statistical analysis
The data used for analysis consisted of a set of 22 cytokine
concentrations in control medium and their inductions from different
stimuli. Inductions, representing the ratio between the stimulated
values over the baseline levels were used in the downstream analysis.
Pre-processing, necessary for subsequent clustering, included the
following steps. First, a few missing values were imputed with the use
of the random forest algorithm for imputation[15].
Second, outlier detection and correction were performed: low outliers
(stimulation values lower than 1) were converted to 1, in sake of
biological validity, while any high outliers were substituted with the
minimum outlier value, according to the default boxplot definition.
Then, all values were converted to z-scores; thus, they all possessed a
mean value equal to 0 and a standard deviation equal to 1. All such
variables were found to be non-parametric, with the use of the
Shapiro-Wilk procedure for composite normality.
Unsupervised cluster analysis was applied to group subjects according to
subsets of spontaneously released or stimulated cytokines. Stimuli were
grouped according to their nature; therefore, two major conditions were
generated: the antiviral (R848, Poly-IC & RV1b) and the antibacterial
(Endotoxin-free bacterial DNA & LPS) responses, alongside the baseline.
After pre-processing and prior to clustering, optimal number of cluster
identification took place with the use of a set of 27 appropriate
criteria [16]. Then, the hierarchical
agglomerative algorithm for clustering was used in order to group
subjects regarding their similarity, to the pre-identified number of
groups. The linkage method used was Ward’s linkage. Visualization of the
clustering outcome was performed with the use of principal component
analysis (PCA) in the dimensions of the first two dominant principal
components.
Groups identified by clustering were analysed to characterize different
types of response towards a stimulus or homeostasis. Comparisons between
groups regarding the presence of major viral families (Siphoviridae,
Picornaviridae, Anelloviridae) were performed by Pearson’s chi-squared
test of independence (Supplementary table 2). Furthermore, the studied
categorical variables (Geography, Sex, VPGs, Rhinitis, Siphoviridae,
Picornaviridae, Anelloviridae and Asthma) and age of the donors were
included in a multivariate regression. To avoid multicollinearity issues
in the analysis, we ran beforehand bivariate crosstab tests between the
predictors (immune clusters) and all target variables, namely Pearson’s
chi-squared tests of independence, to eliminate those variables that did
not provide significance to the model and only retaining those that
yielded a significant p-value to one of the predictors. In each
regression, predictors’ cluster 1 and target variables’
1st level were used as benchmark.
Additionally, stimulated cytokines values were compared between subjects
with or without the presence of pre-specified viral families
(Picornaviridae, Siphoviridae, Anelloviridae), using Wilcoxon’s rank-sum
test. Since all statistical tests were non-parametric, the descriptive
statistics provided were non-parametric as well (i.e., in the form of
median (Q1 - Q3)). All of the statistical tests were two-sided and
statistical significance was taken when p was less than 0.05. The
statistical analysis was implemented with the usage of the R language
and the RStudio interface.