Virus-triggered exacerbation in allergic asthmatic children: neutrophilic airway inflammation and alteration of virus sensors characterize a subgroup of patients
Deschildre et al. Respiratory Research
Virus-triggered exacerbation in allergic asthmatic children: neutrophilic airway inflammation and alteration of virus sensors characterize a subgroup of patients
Antoine Deschildre 0 1 2 6 7 8 11
Muriel Pichavant 0 1 2 7 8
Ilka Engelmann 5 10
Carole Langlois 9
Elodie Drumez 3 9
Guillaume Pouessel 12
Sophie Boileau 6
David Romero-Cubero 6
Irina Decleyre-Badiu 6
Anny Dewilde 5 10
Didier Hober 5 10
Véronique Néve 3 13
Caroline Thumerelle 0 1 2 6 7 8
Stéphanie Lejeune 0 1 2 6 7 8
0 University Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille , F-59000 Lille , France
1 Inserm, U1019 , F-59000 Lille , France
2 CNRS, UMR 8204 , F-59000 Lille , France
3 University Lille, EA 2694 - Santé publique: épidémiologie et qualité des soins, Département d
4 Biostatistique , F-59000 Lille , France
5 CHU Lille, Service de Virologie , F-59000 Lille , France
6 CHU Lille, Unité de Pneumologie et Allergologie Pédiatrique, Hopital Jeanne de Flandre , F-59000 Lille , France
7 Institut Pasteur d
8 CHU Lille , F-59000 Lille , France
9 CHU Lille, Departement de Biostatistiques , F-59000 Lille , France
10 University Lille, EA 3610 - Pathogenèse virale du diabète de type 1 , F-59000 Lille , France
11 INSERM U1019-CNRS UMR8204, CIIL, “Lung infection and innate immunity” research group, Institut Pasteur de Lille, 1 Rue du Professeur Calmette , F-59019 Lille cedex , France
12 CH Roubaix, Service de Pédiatrie, Hôpital Victor Provo , F-59100 Roubaix , France
13 CHU Lille, Service d'Exploration Fonctionnelle Respiratoire , F-5900 Lille , France
Background: Viruses are important triggers of asthma exacerbations. They are also detected outside of exacerbation. Alteration of anti-viral response in asthmatic patients has been shown although the mechanisms responsible for this defect remain unclear. The objective of this study was to compare in virus-infected and not-infected allergic asthmatic children, aged 6 to 16 years, admitted to hospital for a severe exacerbation, the innate immune response and especially the expression of pattern recognition receptor (PRR) and their function. Methods: Virus identification was performed both during the exacerbation and at steady state (eight weeks later). Data assessed at both periods included clinical features, anti-viral response and inflammation (in sputum and plasma), and PRR expression/function in blood mononuclear cells. Results: Viruses were identified in 46 out of 72 children (median age 8.9 years) during exacerbation, and among them, in 17 at steady state. IFN-β, IFN-γ and IL-29 levels in sputum and plasma were similar between infected and not infected patients at both times, as well as the expression of TLR3, RIG-I and MDA5 in blood monocytes and dendritic cells. Airway inflammation in infected patients was characterized by significantly higher IL-5 concentration and eosinophil count. Compared to patients only infected at exacerbation, the re-infected children significantly exhibited lower levels of IFN-γ in plasma and sputum at exacerbation associated with modifications in PRR expression and function in blood mononuclear cells. These re-infected patients also presented an airway neutrophilic inflammation at steady state. Conclusion: Our results reports in asthmatic children that impaired anti-viral response during virus-induced exacerbation is more pronounced in a subgroup of patients prone to re-infection by virus. This subgroup is characterized by altered PRR function and a different pattern of airway inflammation. Trial registration: This multicenter prospective study was approved by the regional investigational review board (ref: 08/07).
Allergic asthma; Exacerbation; Viral infection; Pattern recognition receptor; Interferon
Respiratory viruses, mainly human rhinoviruses (hRV)
are major triggers of exacerbation in asthmatic children
]. Viruses first target airway epithelial cells (AEC)
and then antigen-presenting cells (APC), including
conventional and plasmacytoid dendritic cells (cDC and
pDC, respectively), via the mobilization of pattern
recognition receptors (PRR), such as toll-like receptors (TLR)
and RNA helicases (RIG-I, MDA5) [
]. hRV induce
innate interferon (IFN) production in AEC via RIG-I,
MDA5 and TLR3 [
] whereas influenza virus requires
TLR7 . Impairment in anti-viral response has been
reported in asthmatic patients infected with hRV, as shown
by altered production of type I IFNs (IFN-α/β) and/or
type III IFNs [interleukin (IL)-28 and IL-29] [
However, if the deficient IFNs response has been
reported in severe asthma, associated with a defect in TLR
activation, it was not observed in well controlled
To our knowledge, no data are available regarding the
expression and function of PRR during exacerbation. We
hypothesized that alteration of the expression and/or
function of virus sensors is associated with impaired
innate immune response during virus-induced asthma
exacerbation. Moreover, these alterations might impact on
clinical and inflammatory profiles. To test this
hypothesis, the anti-viral response and the expression and
function of the virus sensors in blood mononuclear cells
were explored in a cohort of allergic asthmatic
children admitted to hospital with a diagnosis of severe
exacerbation. Evaluation also included clinical features
and airway and blood inflammation and was done at
exacerbation and repeated at steady state, 8 weeks
later. First, we compared virus infected to not-infected
patients at exacerbation. Secondly, as our results showed
that an infection with a different virus is frequently
detected in asthmatic patients at steady state [
focused on the virus-infected patients at exacerbation in
order to compare patients infected at both times to those
only infected at the exacerbation.
Our data demonstrated that impairment of IFN
production and virus sensor function was mainly observed
in the subgroup of asthmatic children re-infected at
Study design and patients
This multicenter prospective study, approved by the
regional investigational review board (Comité de
protection des personnes Nord Ouest, ref.: 08/07) involved the
Pediatrics Departments of Lille University Hospital
(Lille, France) and Roubaix Hospital (Roubaix, France).
Parental written informed consents were obtained for
Children aged between 6 and 16 years with a diagnosis
of allergic asthma who were admitted to hospital for a
severe exacerbation were eligible for inclusion. The
severity of the exacerbation was assessed according to the
]. All the patients were treated with
systemic corticosteroids. Allergic sensitisation was defined
by at least one allergen-specific IgE ≥ 0.35 kUA/L and/or
or a positive skin prick test. Exclusion criteria were
congenital or acquired chronic illnesses other than asthma.
Study protocol and outcomes
Subjects were assessed twice: at exacerbation during
hospitalization and at steady state during a follow-up
visit scheduled 8 weeks later (±1 week).
Baseline characteristics including demographic
characteristics, personal comorbidities (allergic rhinitis, atopic
dermatitis, food allergy), history of asthma exacerbations
and passive tobacco exposure were recorded. Maintenance
treatment was documented and inhaled corticosteroid
dose was expressed in fluticasone equivalent μg per day
(μg/d). The lengths of the oxygenotherapy (days) and of
the hospitalization (days) were collected.
Viral status, local (sputum) and systemic IFN response
and inflammatory reaction (cytokines; sputum
inflammatory cell counts), and PRR expression and function were
studied at both times.
At steady state, asthma control was evaluated and
spirometry was performed. Asthma control was assessed
according to GINA criteria (well controlled, partially
controlled or uncontrolled) (www.ginasthma.com).
Spirometry and bronchodilator reversibility were measured
according to American Thoracic Society and European
Respiratory Society Recommendations [
]. Forced vital
capacity (FVC) and FEV1 were expressed in percentage
of predicted value (%VP), FEV1 / FVC in absolute value.
Exhaled nitric oxide (eNO) was also measured and
expressed in ppb [
Subjects were first grouped and compared according
to viral infection at exacerbation: infected (V+) and
notinfected (V-) patients. Following the description of
patients who were infected at both exacerbation and steady
state (V + V+ patients), we compared this subgroup to
the patients only infected at the exacerbation (V + V-
]. The low number of patients infected at
steady state among the V- patients did not allow
studying this subgroup.
Blood and sputum collection
Spontaneous or induced sputum, peripheral blood
mononuclear cells (PBMC) and plasma were collected at
exacerbation (first 2 days) and at steady state. Plasma
from blood samples was used to measure cytokine
concentrations. Blood mononuclear cells (MNC) were isolated
using a Ficoll-Paque density gradient. After washings, cells
were resuspended in RPMI 1640 supplemented with
10% heat-inactivated fetal calf serum and antibiotics
(Life technologies) or with PBS with 2% heat-inactivated
fetal calf serum for cell culture or flow cytometry,
respectively. Isolated MNC were stimulated with a ligand
for TLR3: synthetic double-stranted RNA (poly(IC))
(5 μg/ml), a ligand for RNA-helicases: liposome-polyIC
(lipoP(I:C), 2 μg/ml), a ligand for TLR7-8: Guardiquimod
(2 μg/ml), and phytohemagglutinin (PHA) as a positive
control (Invivogen, San Diego, Ca). Supernatants were
collected after 24 h of culture.
Induced sputum samples were collected after
nebulization of isotonic (at exacerbation) or hypertonic (steady
state) saline solution as previously described [
Plugs were isolated from the sputum, weighted and
processed as previously described [
]. Briefly, plugs
were diluted with sputolysin (VWR) and then, sputum
fluids and cells were separated by centrifugation. The
isolated cells were used for differential cell counts and
the fluid for cytokine measurements. Cytospins were
prepared from the cell pellets and the supernatants were
stored at −80 °C. Samples with more than 30% of
squamous cells were excluded from further analysis and
differential leukocyte cell counts were undertaken by
counting 300 non-squamous cells in sputum samples.
Nasal secretions were collected for each patient at
inclusion (exacerbation) and at steady state. Samples were
frozen (−80 °C) before RNA extraction. A commercially
available multiplex reverse transcription–polymerase
chain reaction (RT-PCR) screened 15 respiratory viral
pathogens including influenza virus A and B, respiratory
syncytial virus A and B, adenovirus, metapneumovirus,
coronavirus 229E/NL63 and OC43, parainfluenza virus
1–4, rhinovirus A/B/C, enterovirus, and bocavirus 1–4
(Seeplex RV15 ACE Detection, Seegene, Seoul, Korea).
Specimens with detection of rhinovirus were typed
by amplification and sequencing of the viral protein
(VP) 4/VP2 region using the primers described by
Wisdom et al. [
Quantitation of HRV RNA was performed according to
Tapparel et al. [
]. Briefly, one step real-time RT PCR
was performed using the QuantiTect probe RT-PCR kit
(Qiagen) and the primers and probes: AGCCTGCGT
GGCKGCC, CYlnaAGCClnaTGCGTGG, FAM-CTCCGG
ACCCAAAGTAGT. Reactions were run on a TaqMan
7500 (Applied Biosystems) thermocycler under the
following cycling conditions: 50 °C for 30 min, 95 °C
for 15 min and 45 cycles of 94 °C for 15 s and 60 °C
for 1 min.
Rhinovirus A9 was propagated on MRC5 cells and
supernatant was quantified in TCID50/mL. RNA of
culture supernatant was extracted and serial 10-fold
dilutions submitted to the quantitative RT PCR in
order to establish a standard curve for quantification.
Results of quantification are expressed as TCID50/mL
To analyze the activation and the expression of PRR
(TLR3, MDA5 and RIG-I) within blood DC and
monocytes, PBMC were incubated for 30 min on ice with
isotype-matched control antibodies for lymphocytes and
granulocytes (lin-1), DC (HLA-DR, CD11c, CD123 and
CD86) and monocytes (CD14). Monocytes were defined
as CD14+ cells. cDC and pDC subsets were respectively
defined by the Lineage1− CD14− HLA-DR+ CD123+ and
Lineage1− CD14−CD11c+ HLA-DR+ phenotypes as
illustrated in the Additional file 1. Cell activation in APC
was analyzed by measurement of the median of
fluorescence (MFI) for HLA-DR and CD86 (BD-Biosciences).
Moreover, the expression of TLR3, RIGI and MDA5
(Santa-Cruz Biotechnology) was estimated by indirect
labeling after cell permeabilization. A corresponding
isotype control was included to define the background level
and the results were expressed after subtraction of the
value obtained with the isotype control.
PBMC were stimulated with ligands for TLR3
[polyinosinic:polycytidylic acid, poly(I:C)], RNA-helicases [poly(I:C)
liposome, lipopoly(I:C)] and TLR7-8 (gardiquimod)
(InvivoGen, San Diego, CA). PBMC supernatants were
collected at baseline and 24 h after stimulation. Levels
of IL-4, IL-5 (Th2 cytokines), CXCL8, IL-17, IL-22
(R&D Systems, Abingdon, UK), IFN-γ, IL-1β, IL-6,
IL29 (IFN-λ) (eBiosciences, San Diego, CA) and IFN-β
(Elabsciences Biot., Wuhan, China) in plasma, sputum
fluids and supernatants from PBMC were measured by
ELISA. On the whole population, P(I:C) increased the
secretion of IL-4, IL-5, IL-6, IL-29, IFN-β, IFN-γ and
CXCL8 as compared to cells in medium alone, whereas
gardiquimod and lipoP(I:C) upregulated the levels of
IL-1β, IL-6, IFN-β, IFN-γ and CXCL8 (data not shown).
Statistical analyses were performed by SAS 9.3 software
(SAS Institute Inc., Cary, NC 25513). Qualitative
variables are reported as the number or the percentage and
compared via the chi-square test or Fisher’s exact test.
Continuous variables, reported as median [interquartile
range (IQR)], were compared via the Mann-Whitney
test. Normality was assessed via the Shapiro-Wilk test.
A p-value <0.05 was considered to be statistically
significant. To evaluate the magnitude of differences
between groups, we calculated the absolute standardized
differences; a standardized difference between 20 and
50, 50 and 80 and higher than 80% denotes low,
medium and large imbalance, respectively [
First, V+ were compared to V- patients at exacerbation
and at steady state and in a second step, V+ V+ were
compared to V + V- patients. Then, exacerbation
conditions were compared to Steady state in each group: V+,
V-, V + V- and V + V+.
Seventy-two patients (median age 8.9 years [IQR:
7·711·7]; boys: 73%) were included (Table 1) among which 32
(43%) were under a maintenance treatment at inclusion. A
virus was detected in 46 patients (62%), hRV in 37 of them
(Fig. 1). Median hRV load was 2341 TCID50/mL
equivalents [1492 – 18,689]. Clinical features of the exacerbation
were similar in V+ and V- patients (Table 1 and see
Additional file 2).
Sixty-six patients (91%) were evaluated at steady state.
None had symptoms of exacerbation. According to
GINA, asthma was well controlled in 22 patients (33%)
(Table 2). A virus was identified in 24 patients (36%),
including 7 without infection during exacerbation (Fig. 1).
Different viruses were detected at exacerbation and at
steady state in 17 V + V+ patients (24%), as previously
]. Moreover, the repartition of the
inclusions during the year was not different among V+ and
V- patients as well as for V + V- and V + V+ patients
[see Additional file 3].
Comparison of the immune responses among V+ and
Production of IFNs
Similar levels of IFN-β, IL-29 and IFN-γ were detected
in the sputum (Fig. 2a) and in the plasma [see
Additional file 2] of V+ and V- patients during
exacerbation and at steady state.
Characteristics of airway and blood inflammation
At both times, V+ patients had significantly higher
IL-5 levels in sputum (Fig. 2b) and plasma [see
Additional file 4] than in V- patients, these levels
being associated with higher percentages of sputum
eosinophils (p < 0·05) (Fig. 2c). Eosinophil percentages
were significantly correlated with the levels of IL-5 in
sputum (r = 0·66, p < 0·005) but not in plasma (r =
0·47, p = NS). In contrast, sputum neutrophil
percentages and numbers were not different between V+
and V- (Fig. 2c and Table 3). During exacerbation
only, IL-6 levels were significantly greater in sputum
(Fig. 2b) and plasma of V+ patients [see Additional
file 4]. A trend towards increased CXCL8 levels in
sputum was also observed. Plasma IL-22 levels were
significantly lower in V+ patients at steady state
whereas the levels of the other cytokines, including
IL-17 and IL-4 were not different [see Additional file 4
and data not shown].
Compared to steady state, only CXCL8 and IL-6
levels in the sputum of V+ patients were significantly
higher during exacerbation [see Additional file 4].
V+ identification of viral infection (PCR) at inclusion, V– no identification of viral infection at inclusion, V + V+ identification of viral infection at inclusion
and at the steady state, V + V– identification of a viral infection at inclusion but not at the steady state, ND not done, ASD absolute standardized
Results were expressed as numbers and medians with interquartile range between brackets or
TLR expression and function in PBMC
Expression levels of TLR3, RIG-I and MDA5 by blood
DC and monocytes were similar in V+ and V-
patients at both times [see Additional file 5]. Expression
of the costimulatory molecule CD86 was greater in
cDC and monocytes of V+ than V- patients during
exacerbation and in pDC at steady state (p < 0·05, Fig.
3a-b). In contrast, HLA-DR levels were similar.
Compared to V-, IL-1β was increased in unstimulated
PBMC of V+ (p < 0·05) [see Additional file 4]. In
response to PRR ligands, IL-29 levels were higher in
gardiquimod (TLR7-8 ligand)-stimulated PBMC from V+
-FEV1 (% of PV)Post β2 agonist [IQR] 109 [99–117] 108 [97–116] 113 [100–118] 108 [100–115] 108 [95–119] 0.22 (5.7)
-FEV1/FVC (%)Pre β2 agonist [IQR]
V+ identification of a virus (PCR) at exacerbation, V– no identification of a virus at exacerbation, V + V+ identification of a virus at exacerbation and steady state,
V + V– identification of a viral infection at exacerbation but not at steady state. Results were expressed as numbers or medians with interquartile range between
brackets. ASD absolute standardized difference (%)
patients at both time points whereas IFN-β and IFN-γ
levels remained similar whatever the stimulus [see
Additional file 4]. During exacerbation only, IL-1β and IL-5
levels were greater in gardiquimod-stimulated PBMC
among V+ patients (p < 0.05), whereas IL-22 levels
were lower (p < 0·05) (Fig. 3c). At steady state, IL-1β
and IL-6 levels were higher in poly(I:C) (TLR3
ligand)-stimulated PBMC among V+ patients (p < 0·05) [see
Additional file 4].
Compared to the steady state, the levels of IFN-γ,
IL-1β and IL-6 at exacerbation were lower in
unstimulated and poly(I:C)-stimulated PBMC among V+
and V- patients [see Additional file 4]. IL-5 production by
unstimulated PBMC from V+ patients and IL-1β
secretion in gardiquimod-stimulated PBMC were also
lower (p < 0.05). The other cytokines did not change.
Characteristic features of the immune response and the
PRR in V + V+ patients
Production of IFNs
Compared to V + V- patients, sputum IFN-γ levels
(p < 0.05) at exacerbation were lower in V + V+
patients, with no significant change for IFN-β and
IL29 (Fig. 4a). In plasma, IFN-γ levels were lower in
V + V+ whereas the IL-29 concentrations were
greater (p < 0.05) and IFN-β levels did not differ (Fig. 4b).
Patients were evaluated during the exacerbation and at steady state. Results were expressed as numbers and medians with interquartile range between brackets.
Differences were considered as statistically significant after analysis by Mann–Whitney (*: p < 0.05; **: p < 0.01 versus V–) (#: p < 0.05 versus V + V–). ASD:
absolute standardized difference (%)
At steady state, levels of IFN-β (p < 0.05) and IL-29
(p = NS) were lower in V + V+ patients in sputum
(Fig. 4a). Concentrations of blood IFNs were similar
between V + V+ and V + V- patients at steady state
[see Additional file 6].
Characteristics of airway and blood inflammation
Sputum IL-5 concentrations were lower in V + V+ than
in V + V- patients at both periods (p < 0.05) (Fig. 4c)
whereas eosinophils did not differ. In contrast, the
percentages of neutrophils were significantly higher in V +
V+ patients at steady state (p < 0.05) (Table 3). During
exacerbation and at steady state, sputum concentrations
of IL-1β (p < 0.05) were higher in V + V+ than in V +
Vpatients (Fig. 4c). Higher IL-22 levels were detected in
sputum of V + V+ patients at steady state (p < 0.05)
(Fig. 4d). Finally, plasma IL-6 concentrations were
greater in V + V+ than V + V- patients during
exacerbation (p < 0.05) whereas there were no differences
for the other cytokines both in sputum and plasma
[see Additional file 6].
The comparison between steady state and
exacerbation among V + V+ and V + V- groups revealed that
plasma and sputum cytokine levels did not differ.
those of V + V- patients (Fig. 5a-b), but not MDA5
[see Additional file 7]. At exacerbation, the RIGI
expression was higher in cDC and pDC but not in
monocytes from V + V+ patients (p < 0.05) (Fig. 5a-b).
There was no difference for CD86 and HLA-DR [see
Additional file 7].
Secretion of cytokines by unstimulated PBMC was
similar in V + V- and V + V+ patients at both times
[see Additional file 6]. The production of IL-29 in
response to gardiquimod was significantly lower in
PBMC of V + V+ patients during exacerbation (p <
0.05) [see Additional file 4]. IFN-γ secretion in
response to poly(I:C) or lipopoly(I:C) was also
significantly decreased (Fig. 5c). At steady state, the same
trend was observed for lipopoly(I:C)-induced
production of IL-29 (Fig. 5c, p < 0.05) and IFN-γ (p = NS)
[see Additional file 6]. The levels of the other
cytokines did not differ between groups.
Compared to steady state, IFN-γ levels were
significantly lower in poly(I:C)-stimulated PBMC at
exacerbation in V + V- patients but not in V + V+ patients
[see Additional file 6]. The same was observed for
IL-1β production in gardiquimod-stimulated PBMC
(p < 0.05).
TLR expression and function in PBMC
At exacerbation and steady state, V + V+ cDC and
monocytes expressed significantly more TLR3 than
This study was designed to assess the alteration of
virus sensors and its association with impaired innate
immune response during virus-induced asthma
exacerbation in children. To test our hypothesis,
patients were their own control and were analyzed
according to their viral status. We further focused
our work on a previously described subgroup prone
to viral re-infection (V + V+) [
]. Each patient was
evaluated during exacerbation and at steady state to
distinguish the inflammation induced by exacerbation
from the one due to asthma itself, which was done in
very few other studies [
Exacerbations were triggered by a virus in 62% of
the children. Among them, 80% were hRV and type C
was the most commonly identified [
]. We first
observed that the clinical characteristics of the different
groups of patients were not different at both times.
We have shown that IFN-β and IL-29 levels in V+
did not differ from those in V- patients. Low
production of type I and III interferons by AEC and alveolar
macrophages experimentally infected by hRV has been
reported in asthmatic children and adults [
more profound in severe atopic asthma . In the
present study, patients infected with hRV appeared to
display the same impairment (data not shown). In
contrast, Bergauer et al. recently reported that IFN-α
levels were markedly increased in a small cohort of
hRV-infected symptomatic asthmatic children (4.8 ±
0.64 years) as compared with infected but
asymptomatic patients whereas IL-29 synthesis remains
]. Our results suggest that IFN-β and
IFN-λ production is altered during virus-induced
exacerbations in asthmatic children. Compared to our
study, the population differs from that of Bergauer et
al. by the age and also the severity of the
]. Furthermore, their results seems to be
inconsistent on a larger cohort. At last in our V+
patients, the alteration of the anti-viral response was
not linked to a modification of the expression of PRR
of blood DC and monocytes nor with a blockade of
their function. These results might also suggest that
the alteration of the anti-viral response might
preferentially be observed in the airways compared with
The antiviral response was further analyzed among
the V+ patients. We observed that V + V+ patients,
infected at both times, produced lower levels of
IFNγ than V + V- patients during exacerbation. IL-29 and
IFN-γ production were also lower at steady state,
despite the presence of a new virus. As compared with
V + V-, TLR3 and RNA-helicases were overexpressed
on circulating APCs in V + V+ patients. During
exacerbation, their PBMC showed an altered IFN-γ and
IL-29 production after TLR3 and RIGI activation, that
might facilitate the viral re-infection according to the
IL-29 function [
]. The IL-29 production in
response to TLR7-8 ligand was also impaired,
suggesting a defective function of these receptors in PBMC
from V + V+ patients, as recently reported in alveolar
]. IFN-γ and IL-29 production
impairment may be due to an altered signaling.
Different mechanisms might be responsible for this
defective production. Transforming growth factor-β
inhibits IFN production in response to hRV [
The implication of suppressor of cytokine signaling
(SOCS)1 overexpression in airway epithelium of
severe asthmatic children have been suggested [
although this is controversial [
]. Our data suggest
that a specific alteration of the anti-viral response
related to a dysfunction of virus sensors characterize
these re-infected patients.
Viral infection during the exacerbation has also an
impact on the airway inflammation as shown by the
persistent increased sputum eosinophilia and its
correlation with the IL5 concentrations, in agreement with
Norzila et al. [
]. Th2 cytokines alter the innate
immune response to viral infection and favor the
development of a specific inflammatory reaction [
]. In V + V+,
the lack of increase in IL-5 levels suggests that
virusinfections don’t directly influence IL-5 in this subgroup.
Nevertheless, it has been reported that viral infection in
an allergic environment can induce IL-5 synthesis by
CD8+ T cells, probably due to PRR activation in DC
]. Interestingly, the production of IL-5 after TLR7-8
stimulation was primed in PBMC from V+ patients, a
result probably due to the V + V- subgroup.
Concomitantly, DC activation during exacerbation was
demonstrated by the over-expression of CD86 and contributes
to the APC propensity to induce IL-5 secretion by T
lymphocytes in infected patients. The link between
Th2 inflammation and antiviral response is also
illustrated by the restoration of IFN-α production by
plasmacytoid DC in allergic asthmatic children
treated with omalizumab [
In re-infected patients, the inflammatory reaction was
characterized by a strong secretion of IL-1β, which
might be involved in the neutrophil recruitment
observed at steady state. This feature was also
associated with the production of IL-22, which
promotes smooth muscle cells proliferation [
Interestingly, Simpson et al. reported that adult asthmatic
with a neutrophilic inflammation and a high level of
IL-1β in sputum have a reduced ability to produce
IFN-α in response to hRV [
]. We suggest a link
between this population and our group of V + V+
children. Long term follow-up is needed to define if the
specificities of inflammation are mostly related to the
repeated viral infection.
Our results support a more pronounced defect in IFN-γ
and IFN-λ secretion during virus-triggered exacerbation
in the asthmatic children prone to viral re-infection.
This defect is associated with an overexpression of virus
sensors, a defective response to the corresponding
ligands and with a specific airway inflammation. The
benefit of strategies integrating an antiviral approach in
this subgroup of patients should be further explored.
Additional file 1: Gating strategy for the analysis of conventionnal and
plasmacytoid dendritic cell (cDC and pDC, respectively) in peripheral
blood mononuclear cells (PBMC) from asthmatic children. (PDF 300 kb)
Additional file 2: Characteristics of the exacerbation at inclusion in
the overall population, and comparison according to the viral status.
(PDF 262 kb)
Additional file 3: Repartition of the exacerbations during the year
according to the viral status. a) According to the viral status at the
exacerbation. b) According to viral status at steady state in virus infected
patients at the exacerbation. (PDF 353 kb)
Additional file 4: Concentrations of cytokines in asthmatic patients by viral
status during the exacerbation and at steady state. Cytokines concentrations
were measured during exacerbation (upper part) or at steady state (lower
part) in plasma, sputum fluids and supernatants of MNC stimulated with
Poly(I:C), Gardiquimod, lipopoly(I:C) or not (Medium). Patients infected by virus
(V+) or not infected (V-) during the exacerbation were compared. Results are
expressed as pg/ml (median with interquartile range [IQR]). ND: not
detectable, NE: Not evaluated. (PDF 555 kb)
Additional file 5: Phenotype of blood antigen-presenting cells from
asthmatic children by viral status during the exacerbation and at steady
state. The upper part reported data collected during exacerbation from
infected (V+) or not infected (V-) patients during the exacerbation,
whereas the lower part showed the data obtained at steady state. The
phenotype was analyzed in conventional and plasmacytoid DC (cDC and
pDC, respectively) as well as in monocytes during the exacerbation and
at steady state, respectively. Results are expressed as median of fluorescence
intensity (MFI) with interquartile range [IQR]. ND: not detectable, NE: Not
evaluated. (PDF 309 kb)
Additional file 6: Concentrations of cytokines in asthmatic patients
prone to re-infection at steady state. Cytokines concentrations were
measured during exacerbation or at steady state in plasma, sputum
fluids and supernatants of MNC stimulated with Poly(I:C), Gardiquimod,
lipopoly(I:C) or not (Medium). Patients only infected during the exacerbation
(V + V-) were compared to those infected during both periods (V + V+).
Results are expressed as pg/ml (median with interquartile range [IQR]).
ND: not detectable, NE: Not evaluated. *:p < 0.05 significantly different from
V- patients. (PDF 435 kb)
Additional file 7: Phenotype of blood antigen presenting cells in asthmatic
patients prone to re-infection at steady state. Patients only infected during the
exacerbation (V + V-) were compared to those infected during both periods
(V + V+). The upper and the lower part showed the data collected in
conventional and plasmacytoid DC (cDC and pDC, respectively) as well
as in monocytes during the exacerbation and at steady state, respectively.
Results are expressed as median of fluorescence intensity (MFI) with
interquartile range [IQR]. (PDF 354 kb)
AEC: Airway epithelial cell; APC: Antigen-presenting cell; cDC: Conventionnal
dendritic cell; CXCL: CXC chemokine ligand; DC: Dendritic cell; IFN: Interferon;
IL: Interleukin; IQR: Interquartile range; MDA5: Melanoma
DifferentiationAssociated protein 5; PBMC: Peripheral blood mononuclear cell;
pDC: Plasmacytoid DC; PRR: Pathogen recognition receptor; RIG-I: Retinoic
Acid-Inducible Gene-I; Th2: T helper type 2; TLR: Toll like receptor;
V-: Not-infected patients at exacerbation; V + : Virus infected patients at
exacerbation; V + V-: Patients infected at exacerbation but not at steady state;
V + V + : Patients infected both at exacerbation and steady state
The present study is dedicated to the memory of Pr Isabelle Tillie-Leblond
who initiated the study concept and design.
We thank Pr A. Duhamel for the advices and the reviewing of our statistical
analysis. We thank Eva Vilain and Gwenola Kervoaze for their excellent technical
assistance, Laurent Beghin (Centre d’investigation clinique pédiatrique, Hôpital
Jeanne de Flandre, CHRU de Lille) for his contribution to data management.
We thank Dr. Laura Ravasi and Pr Pascal Chanez for the reading of the
manuscript. We also thank Hélène Bauderlique for her help for advice on flow
cytometry (BICel Cytometry Plateform, Institut Pasteur de Lille, France).
We thank all the families and the children and families who participated in
Availability of data and materials
ADes and PG are the guarantor of the content of this article, including the
data and the analysis. All the data and the material are available upon
ADes supervised the study in Lille university hospital and with PG, equally
defined the design of the study and coordinated its management, the
analysis of the results and the writing of the manuscript. MP participated in
the design of the study, in the analysis of the results and the writing of the
manuscript. IE performed the analysis of viruses and participated in the
writing of the manuscript. ADew and DH contributed to study concept and
design, virus studies, analysed and interpreted data and co-wrote the report.
CL and ED performed the statistical analysis and participated in the data
interpretation. GP, IB, CT, CM, DR contributed to patients’ selection, inclusion
and data collection. VN contributed to study design and data collection. SB,
DR and CM performed the analysis of biological parameters with the
supervision of PG. ADes, CM and PG generated the figures. All authors have
read and approved the final manuscript.
Ethics approval and consent to participate
This multicenter prospective study, approved by the regional investigational
review board (Comité de protection des personnes Nord Ouest, ref.: 08/07)
involved the Pediatrics Departments of Lille University Hospital (Lille, France)
and Roubaix Hospital (Roubaix, France). Parental written informed consents
were obtained for all children.
Consent for publication
A Des reports grants from Région Nord-Pas de Calais, grants from Société
Française d’Allergologie, grants from Comité de Maladies Respiratoires, during
the conduct of the study; personal fees from Novartis, personal fees from ALK,
personal fees from TEVA, personal fees and other from GSK, personal fees from
Stallergenes, personal fees from MSD, personal fees from MEDA, outside the
submitted work. CM reports personal fees from NOVARTIS, outside the submitted
work. MP and PG have received funding from GSK for an unrelated work.
All other authors (IE, CL, ED, GP, DR, SB, IB, ADew, CT, VN, DH) declare that
they have no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
The VIRASTHMA research program was supported by the Conseil Régional
du Nord-Pas de Calais, the Société Française d’Allergologie, and the Comité
National contre les Maladies Respiratoires. Lille University Hospital was the
regulatory trial sponsor (2007/0725).
1. Jackson DJ , Johnston SL . The role of viruses in acute exacerbations of asthma . J Allergy Clin Immunol . 2010 ; 125 : 1178 - 87 . quiz 1188- 1179
2. Johnston SL , Pattemore PK , Sanderson G , Smith S , Lampe F , Josephs L , Symington P , O'Toole S , Myint SH , Tyrrell DA , et al. Community study of role of viral infections in exacerbations of asthma in 9-11 year old children . BMJ . 1995 ; 310 : 1225 - 9 .
3. Takeuchi O , Akira S. Innate immunity to virus infection . Immunol Rev . 2009 ; 227 : 75 - 86 .
4. Yoo JK , Kim TS , Hufford MM , Braciale TJ . Viral infection of the lung: host response and sequelae . J Allergy Clin Immunol . 2013 ; 132 : 1263 - 76 .
5. Pichlmair A , Schulz O , Tan CP , Rehwinkel J , Kato H , Takeuchi O , Akira S , Way M , Schiavo G , Reis e Sousa C. Activation of MDA5 requires higher-order RNA structures generated during virus infection . J Virol . 2009 ; 83 : 10761 - 9 .
6. Slater L , Bartlett NW , Haas JJ , Zhu J , Message SD , Walton RP , Sykes A , Dahdaleh S , Clarke DL , Belvisi MG , et al. Co-ordinated role of TLR3, RIG-I and MDA5 in the innate response to rhinovirus in bronchial epithelium . PLoS Pathog . 2010 ; 6 : e1001178 .
7. Diebold SS , Kaisho T , Hemmi H , Akira S , Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA . Science . 2004 ; 303 : 1529 - 31 .
8. Contoli M , Message SD , Laza-Stanca V , Edwards MR , Wark PA , Bartlett NW , Kebadze T , Mallia P , Stanciu LA , Parker HL , et al. Role of deficient type III interferon-lambda production in asthma exacerbations . Nat Med . 2006 ; 12 : 1023 - 6 .
9. Sykes A , Edwards MR , Macintyre J , del Rosario A , Bakhsoliani E , TrujilloTorralbo MB , Kon OM , Mallia P , McHale M , Johnston SL . Rhinovirus 16- induced IFN-alpha and IFN-beta are deficient in bronchoalveolar lavage cells in asthmatic patients . J Allergy Clin Immunol . 2012 ; 129 : 1506 - 14 .
10. Wark PA , Johnston SL , Bucchieri F , Powell R , Puddicombe S , Laza-Stanca V , Holgate ST , Davies DE : Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus . J Exp Med . 2005 ; 201 : 937 - 947 .
11. Bergauer A , Sopel N , Kross B , Vuorinen T , Xepapadaki P , Weiss ST , Blau A , Sharma H , Kraus C , Springel R , et al. IFN-alpha/IFN-lambda responses to respiratory viruses in paediatric asthma . Eur Respir J . 2017 ; 49 : 1600969 .
12. Edwards MR , Regamey N , Vareille M , Kieninger E , Gupta A , Shoemark A , Saglani S , Sykes A , Macintyre J , Davies J , et al. Impaired innate interferon induction in severe therapy resistant atopic asthmatic children . Mucosal Immunol . 2013 ; 6 : 797 - 806 .
13. Sykes A , Edwards MR , Macintyre J , Del Rosario A , Gielen V , Haas J , Kon OM , McHale M , Johnston SL . TLR3, TLR4 and TLRs7-9 induced Interferons are not impaired in airway and blood cells in well controlled asthma . PLoS One . 2013 ; 8 : e65921 .
14. Rupani H , Martinez-Nunez RT , Dennison P , Lau LC , Jayasekera N , Havelock T , Francisco-Garcia AS , Grainge C , Howarth PH , Sanchez-Elsner T . Toll-like receptor 7 is reduced in severe asthma and linked to an altered MicroRNA profile . Am J Respir Crit Care Med . 2016 ; 194 : 26 - 37 .
15. Engelmann I , Mordacq C , Gosset P , Tillie-Leblond I , Dewilde A , Thumerelle C , Pouessel G , Deschildre A . Rhinovirus and asthma: reinfection, not persistence . Am J Respir Crit Care Med . 2013 ; 188 : 1165 - 7 .
16. Wood LG , Powell H , Grissell TV , Davies B , Shafren DR , Whitehead BF , Hensley MJ , Gibson PG . Persistence of rhinovirus RNA and IP-10 gene expression after acute asthma . Respirology . 2016 ; 16 : 291 - 9 .
17. Reddel HK , Taylor DR , Bateman ED , Boulet LP , Boushey HA , Busse WW , et al. An official American thoracic Society/European Respiratory Society statement : asthma control and exacerbations. Standardizing endpoints for clinical asthma trials and clinical practice . Am J Resp Crit Care Med . 2009 ; 180 : 59 - 99 .
18. ATS/ERS recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide , 2005. Am J Respir Crit Care Med . 2005 , 171 : 912 - 930 .
19. Zapletal A , Motoyama EK , Van De Woestijne KP , Hunt VR , Bouhuys A . Maximum expiratory flow-volume curves and airway conductance in children and adolescents . J Appl Physiol . 1969 ; 26 : 308 - 16 .
20. Wallaert B , Gosset P , Lamblin C , Garcia G , Perez T , Tonnel AB . Airway neutrophil inflammation in nonasthmatic patients with food allergy . Allergy . 2002 ; 57 : 405 - 10 .
21. Gershman NH , Wong HH , Liu JT , Mahlmeister MJ , Fahy JV . Comparison of two methods of collecting induced sputum in asthmatic subjects . Eur Respir J . 1996 ; 9 : 2448 - 53 .
22. Paggiaro PL , Chanez P , Holz O , Ind PW , Djukanovic R , Maestrelli P , Sterk PJ . Sputum induction . Eur Respir J Suppl . 2002 ; 37 : 3s - 8s .
23. Wisdom A , Leitch EC , Gaunt E , Harvala H , Simmonds P . Screening respiratory samples for detection of human rhinoviruses (HRVs) and enteroviruses: comprehensive VP4-VP2 typing reveals high incidence and genetic diversity of HRV species C . J Clin Microbiol . 2009 ; 47 : 3958 - 67 .
24. Tapparel C , Cordey S , Van Belle S , Turin L , Lee WM , Regamey N , Meylan P , Muhlemann K , Gobbini F , Kaiser L. New molecular detection tools adapted to emerging rhinoviruses and enteroviruses . J Clin Microbiol . 2009 ; 47 : 1742 - 9 .
25. Schibler M , Yerly S , Vieille G , Docquier M , Turin L , Kaiser L , Tapparel C . Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR . J Clin Microbiol . 2012 ; 50 : 2868 - 72 .
26. Cohen J . Statistical power analysis for the behavioral sciences . 2nd ed. Hillsdale: Lawrence Erlbaum Associates; 1988 .
27. Norzila MZ , Fakes K , Henry RL , Simpson J , Gibson PG . Interleukin-8 secretion and neutrophil recruitment accompanies induced sputum eosinophil activation in children with acute asthma . Am J Respir Crit Care Med . 2000 ; 161 : 769 - 74 .
28. Subrata LS , Bizzintino J , Mamessier E , Bosco A , McKenna KL , Wikstrom ME , Goldblatt J , Sly PD , Hales BJ , Thomas WR , et al. Interactions between innate antiviral and atopic immunoinflammatory pathways precipitate and sustain asthma exacerbations in children . J Immunol . 2009 ; 183 : 2793 - 800 .
29. Bizzintino J , Lee WM , Laing IA , Vang F , Pappas T , Zhang G , Martin AC , Khoo SK , Cox DW , Geelhoed GC , et al. Association between human rhinovirus C and severity of acute asthma in children . Eur Respir J . 2011 ; 37 : 1037 - 42 .
30. Wolk K , Witte K , Witte E , Raftery M , Kokolakis G , Philipp S , Schonrich G , Warszawska K , Kirsch S , Prosch S , et al. IL -29 is produced by T(H)17 cells and mediates the cutaneous antiviral competence in psoriasis . Sci Transl Med . 2013 ; 5 : 204ra129 .
31. Thomas BJ , Lindsay M , Dagher H , Freezer NJ , Li D , Ghildyal R , Bardin PG . Transforming growth factor-beta enhances rhinovirus infection by diminishing early innate responses . Am J Respir Cell Mol Biol . 2009 ; 41 : 339 - 47 .
32. Gielen V , Sykes A , Zhu J , Chan B , Macintyre J , Regamey N , Kieninger E , Gupta A , Shoemark A , Bossley C , et al. Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons . J Allergy Clin Immunol . 2015 ; 136 : 177 - 88 . e111
33. Doran E , Choy DF , Shikotra A , Butler CA , O'Rourke DM , Johnston JA , Kissenpfennig A , Bradding P , Arron JR , Heaney LG . Reduced epithelial suppressor of cytokine signalling 1 in severe eosinophilic asthma . Eur Respir J . 2016 ; 48 : 715 - 25 .
34. Contoli M , Ito K , Padovani A , Poletti D , Marku B , Edwards MR , Stanciu LA , Gnesini G , Pastore A , Spanevello A , et al. Th2 cytokines impair innate immune responses to rhinovirus in respiratory epithelial cells . Allergy . 2015 ; 70 : 910 - 20 .
35. Coyle AJ , Erard F , Bertrand C , Walti S , Pircher H , Le Gros G . Virus-specific CD8+ cells can switch to interleukin 5 production and induce airway eosinophilia . J Exp Med . 1995 ; 181 : 1229 - 33 .
36. Teach SJ , Gill MA , Togias A , Sorkness CA , Arbes SJ Jr, Calatroni A , Wildfire JJ , Gergen PJ , Cohen RT , Pongracic JA , et al. Preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations . J Allergy Clin Immunol . 2015 ; 136 : 1476 - 85 .
37. Chang Y , Al-Alwan L , Risse PA , Halayko AJ , Martin JG , Baglole CJ , Eidelman DH , Hamid Q. Th17-associated cytokines promote human airway smooth muscle cell proliferation . FASEB J . 2012 ; 26 : 5152 - 60 .
38. Simpson JL , Carroll M , Yang IA , Reynolds PN , Hodge S , James AL , Gibson PG , Upham JW . Reduced antiviral interferon production in poorly controlled asthma is associated with Neutrophilic inflammation and high-dose inhaled corticosteroids . Chest . 2016 ; 149 : 704 - 13 .