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The damaging and protective features of eosinophils in healthy individuals andpatients with chronic inflammatory respiratory diseases
Sabogal Piñeros, Y.S.
Publication date2019Document VersionOther versionLicenseOther
Link to publication
Citation for published version (APA):Sabogal Piñeros, Y. S. (2019). The damaging and protective features of eosinophils inhealthy individuals and patients with chronic inflammatory respiratory diseases.
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Download date:29 Apr 2021
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Eleanor Roosevelt
Chapter 3Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage,B-Cell, and Neutrophil Responses (MATERIAL)
1 Department of Respiratory Medicine, 2 Department of Experimental Immunology (Amsterdam Infection & Immunity Institute), and3 Department of Clinical Epidemiology, Bioinformatics, and Biostatistics, University of Amsterdam,
Amsterdam UMC, Amsterdam, the Netherlands
Yanaika S. Sabogal Piñeros1,2, Suzanne M. Bal1,2, Marianne A. van de Pol2,
Barbara S. Dierdorp2, Tamara Dekker2, Annemiek Dijkhuis2, Paul Brinkman1,
Koen F. van der Sluijs2, Aeilko H. Zwinderman3, Christof J. Majoor1, Peter I. Bonta1,
Lara Ravanetti1,2, Peter J. Sterk1, and Rene Lutter1,2
Chapter 3
52
ABSTRACT
RationaleEosinophils drive pathophysiology in stable and exacerbating eosinophilic asthma, and
therefore treatment is focused on the reduction of eosinophil numbers. Mepolizumab,
a humanized monoclonal antibody that neutralizes IL-5 and efficiently attenuates
eosinophils, proved clinically effective in severe eosinophilic asthma but not in mild
asthma.
ObjectivesTo study the effect of mepolizumab on virus-induced immune responses in mild asthma.
Methods Patients with mild asthma, steroid-naive and randomized for eosinophil numbers,
received 750 mg mepolizumab intravenously in a placebo-controlled double-blind
trial, 2 weeks after which patients were challenged with rhinovirus (RV) 16. FEV1, FVC,
fractional exhaled nitric oxide, symptom scores (asthma control score), viral load (PCR),
eosinophil numbers, humoral (luminex, ELISA), and cellular (flow cytometry) immune
parameters in blood, BAL fluid, and sputum, before and after mepolizumab and RV16,
were assessed.
Measurements and Main ResultsMepolizumab attenuated baseline blood eosinophils and their activation, attenuated
trendwise sputum eosinophils, and enhanced circulating natural killer cells. Mepolizumab
did not affect FEV1, FVC, and fractional exhaled nitric oxide, neither at baseline nor
after RV16. On RV16 challenge mepolizumab did not prevent eosinophil activation
but did enhance local B lymphocytes and macrophages and reduce neutrophils and
their activation. Mepolizumab also enhanced secretory IgA and reduced tryptase
in BAL fluid. Finally, mepolizumab affected particularly RV16-induced macrophage
inflammatory protein-3a, vascular endothelial growth factor-A, and IL-1RA production
in BAL fluid.
Conclusions Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-
induced immune responses in mild asthma. Although these latter effects likely are
caused by attenuated eosinophil numbers, we cannot exclude a role for basophils.
Clinical trial registered with www.clinicaltrials.gov (NCT 01520051).
Keywords: loss of asthma control; exacerbation; mepolizumab; rhinovirus 16 challenge
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
53
3
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject: Anti–IL-5–mediated attenuation of eosinophils
is an effective intervention in severe asthma, reducing corticosteroid dependency
and exacerbations. The lack of effect of anti–IL-5 in mild asthma questions the role of
eosinophils.
What This Study Adds to the Field: Anti–IL-5 in mild asthma reduced eosinophil
activation and numbers but did not prevent activation of the remaining eosinophils in
response to a rhinovirus 16 challenge. In addition, anti–IL-5 attenuated the neutrophil
and mast cell responses to the viral challenge but enhanced that of B lymphocytes and
macrophages. The lack of effect of anti–IL-5 in mild asthma may relate to the remaining
activated eosinophils and the altered humoral and cellular immune response to a viral
challenge.
Asthma is a heterogeneous chronic inflammatory disease of the respiratory tract reflected
by differences in severity, time of disease onset, and responsiveness to treatment (1).
Also, the inflammatory profile in asthma is diverse, with eosinophilic versus neutrophilic
airway inflammation as key determinants. Two major groups of patients with asthma
have eosinophilic inflammation: patients with early-onset, atopic mild asthma and a
smaller group with nonatopic, severe asthma (2). The latter is difficult to control with
standard treatment (i.e., anti inflammatory corticosteroids), especially during periodic
acute worsening of their asthma (exacerbations).
The recruitment and activation of eosinophils is considered to contribute to asthma
pathology, likely by the release of cytotoxic compounds from granules and production
of reactive oxygen species (3). In line herewith, treatment with corticosteroids in mild to
moderate asthma can be successfully directed by relative sputum eosinophil counts (4).
T-helper cell type 2–like mediators and predominantly IL-5 are considered key players
in eosinophilic inflammation (1). IL-5 is involved in the development and release of
eosinophils from the bone marrow.
Furthermore, IL-5 prolongs eosinophil proliferation, survival, and enhances eosinophil
activation and differentiation (1, 5). Treatment with anti–IL-5 in severe eosinophilic
asthma led to reductions in eosinophil numbers and, in parallel, exacerbations and
corticosteroid dependency (1, 6). This contrasts with earlier trials with anti–IL-5 of mild
to moderate asthma that did not attenuate symptoms even though eosinophils were
reduced markedly (7).
Chapter 3
54
Besides its damaging properties, both in vitro and murine studies indicate that
eosinophils are also important for innate and adaptive immune responses (3).
Eosinophils were found to direct dendritic cell and macrophage responses and underlie
IgA production, but confirmation in human studies is lacking (8). The recruitment of
eosinophils during viral infections suggested a possible antiviral role of eosinophils.
Handzel and colleagues (9) demonstrated that eosinophils with bound rhinovirus
(RV)-16 activated RV-specific T cells, suggestive of an important role in the initiation
of adaptive antiviral responses. Pioneering studies by Rosenberg and coworkers
revealed that activated eosinophils and granular products like EDN (eosinophil-derived
neurotoxin) exert antiviral effects (10, 11). The latter has been proposed to interact with
viral capsid proteins and exert RNAse activity (12). Besides these antiviral properties,
eosinophils can also exert other antiviral mechanisms, such as promoting CD8 T-cell
responses (13) or attenuating viral infectivity by nitric oxide release (14). Recently, we
found that eosinophils from patients with mild to moderate asthma, and more markedly
in those from patients with severe asthma, displayed a reduced capacity to capture
respiratory syncytial virus in vitro (Y. S. Sabogal Piñeros and colleagues, unpublished
data). In line with these findings, anti–IL-5–mediated depletion of eosinophils resulted
in enhanced influenza X31 viral loads in house dust mite–sensitized mice, whereas the
enhanced morbidity was reduced (15).
This led us to explore the contribution of eosinophils to immune responses during
viral airway infections in mild asthma, which account for most asthma exacerbations
and loss of asthma control (16), reflected by increased eosinophilic and neutrophilic
inflammation (17). We hypothesized therefore that reduced eosinophil numbers by
anti–IL-5 attenuate eosinophil-driven immune and inflammatory responses to an RV16
challenge. Because we expected that anti–IL-5 would have a more pronounced effect
in patients with high eosinophil numbers we also analyzed data stratified for eosinophil
numbers. Some of the results of this study have been previously reported in the form of
an abstract (18).
METHODS
Study DesignThe MATERIAL study was a double-blind placebo-controlled, parallel-group design,
single center study with a 1:1 randomization in mepolizumab versus placebo. Figure 1
shows the study design.
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
55
3
PatientsAll patients with asthma were steroid-naive, did not use asthma medication other
than short-acting b2-agonists, had a positive skin prick test for at least 1 out of 12
aeroallergens, a provocative concentration of methacholine bromide causing a 20%
fall in FEV1 less than or equal to 9.8 mg/ml, and baseline FEV1 greater than or equal to
80%. Furthermore, patients were between 18 and 50 years of age, nonsmoking, and
clinically stable (i.e., no exacerbation for at least 6 wk). Patients with a RV16 antibody
titer above 1:4 were excluded.
Randomization and BlindingAt the first study visit, sputum was collected and relative eosinophil counts were
determined. Patients with eosinophilia (>3% sputum eosinophils or, when no adequate
sputum sample was obtained, >300 eosinophils/ml blood) and patients without
eosinophilia (<3% sputum eosinophils or without adequate sputum sample <300
eosinophils/ml blood) were double-blind allocated to either mepolizumab or placebo
treatment and received a subject identification code. The pharmacy providing the study
medication kept the allocation key until deblinding.
Intervention and RV16 Challenge Following randomization, patients received a single
intravenous dose of either 750 mg mepolizumab, which is known to attenuate blood
eosinophils for at least 4 weeks (6, 7), or placebo containing 0.9% saline only. After 2
weeks, patients were challenged intranasally with RV16 (10TCID50) in 750 ml, using an
atomizer (DeVilbiss Model 286) to spray the virus into a single nostril. The procedure was
repeated until the complete inoculum was instilled. Before RV16 challenge a respiratory
virus PCR for adenovirus, influenza A and B virus, enterovirus, human metapneumovirus,
respiratory syncytial virus, RV, coronavirus, parechovirus, parainfluenza 1, 2, 3, and 4,
and human bocavirus was performed on a nasal swab to exclude concomitant viral
infections.
F I G U R E 1 . Flow chart showing all visits, interventions, measurements, and samplings. Bronchoscopy involved standardized BAL. FENO = fractional exhaled nitric oxide; RV16 = exposure to rhinovirus 16. d = day; v = visit.
Chapter 3
56
Procedures and OutcomesThe prebronchodilator FEV1 before and after RV16 challenge served as primary
outcomes (see sample size calculation for explanation in the online supplement). The
secondary endpoints were symptom scores (Wisconsin Upper Respiratory Symptom
Survey [WURSS] and asthma control score [ACQ]), viral loads by quantitative PCR
in nasal swabs, and immunologic parameters (inflammatory mediators, eosinophil
numbers and markers of activation and degranulation, cell differentials in blood, BAL
fluid [BALF] and sputum, and cellular immune responses toward RV16).
Statistical MethodsData concerning cell populations, activation markers, and asthma-related parameters
were expressed as mean 6 SEM and were analyzed using GraphPad Prism 5.0 software.
Luminex data were analyzed using PC analysis in R and baseline characteristics are
expressed as mean 6 SD. Some data were stratified for eosinophils, in which eosinophilic
asthma was considered greater than or equal to 3% sputum eosinophils or, when no
adequate sputum was available, greater than or equal to 300 eosinophils/ml blood and
noneosinophilic asthma less than 3% or less than 300, respectively. Blood eosinophils
correlated well with % sputum eosinophils (see Figure E1 in the online supplement)
showing that these parameters were interchangeable. P less than 0.05 was considered
significant.
Study ApprovalThe study protocol was reviewed and approved by the internal ethical review committee
and in accordance with the declaration of Helsinki. All participants provided written
informed consent.
Additional details on the methods are provided in the online supplement.
RESULTS
After informed consent, 78 subjects were screened, 41 of whom were excluded
because of preexisting antibodies against RV16, negative skin prick test, or not fulfilling
mild asthma criteria. In total 37 subjects with allergic asthma were randomized between
January 2012 and March 2015, a total of 19 in the placebo and 18 in the mepolizumab
arm. Nine subjects were excluded from the study after the first bronchoscopy, two in the
placebo group and seven in the mepolizumab-treated group. Post hoc analysis showed
that this exclusion did not significantly affect the placebo- and mepolizumab-treated
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
57
3
groups. All exposed participants were deemed infected based on either seroconversion
and/or a positive RV PCR (n = 27), or a positive WURSS21 score (n = 28). Figure 2
shows the flowchart of inclusion and exclusion of the participating subjects. No adverse
events were reported.
Table 1 shows similar baseline (visit 1/Day 14) characteristics for both groups, which
were not significantly different from the characteristics before exclusion of the nine
subjects. Mepolizumab treatment attenuated eosinophil numbers in blood and
trendwise (P = 0.053) in sputum (Figure 3). Because IL-5 affects multiple activation
markers on eosinophils (19), not surprisingly mepolizumab attenuated the activation
status of blood eosinophils as reflected by a reduced CD11b expression and by more
cells expressing CD62L, although the latter occurred only in patients with relatively low
(<3%) sputum eosinophils (see Table E1). The CD69-expressing eosinophils were also
reduced, but both in the placebo- and mepolizumab- treated groups. Mepolizumab
F I G U R E 2 . Flowchart of inclusion and exclusion of subjects with mild asthma. IC = informed consent; PC20 = airway responsiveness to methacholine bromide (the provocative concentration resulting in a 20% drop of FEV1); RV16 1 = antibody titer against rhinovirus type 16 above 1:4.
Chapter 3
58
enhanced systemic natural killer (NK) (CD32CD192CD561) cells (see Table E1), whereas
CD41 and CD81 T cells and CD191 B cells were not affected (data not shown).
RV16 challenge resulted in a modest but significant reduction of FEV1% predicted and
increase of ACQ, which was not affected by mepolizumab (Figures 4A and 4E). RV16
challenge induced a drop in FVC, only when compared with baseline, which was not
seen in the mepolizumab treatment arm (Figure 4C). Fractional exhaled nitric oxide
(FENO) was not affected by mepolizumab treatment or by the RV16 challenge (Figure 4G).
Because a reduction in eosinophils in patients with high baseline eosinophil numbers
may have a larger impact than compared with that in patients with low eosinophil
numbers, we stratified for eosinophil counts. This stratification revealed no differences
between patients with less than 3% and greater than or equal to 3% eosinophils for
FEV1, ACQ, FVC, and FENO, nor was there a differential effect of mepolizumab on these
parameters (Figures 4B, 4D, 4F, and 4H).
The main purpose of this study was to determine whether mepolizumab affected immune
and inflammatory responses to an RV16 challenge. RV16 challenge did not change
BAL and sputum eosinophil numbers or the release of eosinophil cationic protein (ECP),
although all were lower in the mepolizumab-treated patients (Table 2).
TA B L E 1 . Baseline Characteristics of Study Subjects
Baseline Parameters Placebo (n = 17 ) Mepolizumab (n = 11) P Value
Age, yr 23.00 ± 4.54 24.27 ± 5.82 0.52
M/F 6/11 1/10 0.10
ACQ 0.93 ± 0.60 0.96 ± 0.55 0.90
IgE, kU/L 372.31 ± 569.84 196.2 ± 280.5 0.35
Blood eosinophils, 109/L 0.43 ± 0.40 0.22 ± 0.12 0.11
Sputum eosinophils
% 1.87 ± 2.62 1.63 ± 3.107 0.84
< 3%/>3% 9/8 6/5 1
PC20, log2 0.40 ± 1.49 0.41 ± 1.30 0.98
FEV1 pred, L 3.78 ± 0.71 3.40 ± 0.53 0.14
FEV1% pred 104.20 ± 8.52 103.08 ± 10.84 0.75
FVC pred, L 4.41 ± 0.91 3.92 ± 0.66 0.14
FVC % pred 106.90 ± 8.53 102.5 ± 9.25 0.20
FENO, ppb 69.62 ± 47.89 54.45 ± 33.97 0.39
Definition of abbreviations: ACQ = asthma control questionnaire average score where >1.5 indicates uncontrolled asthma; FENO = fractional exhaled nitric oxide; PC20 = airway responsiveness to metacholine bromide (the provocative concentration resulting in a 20% drop of FEV1); % pred = percent of predicted value.Data are mean 6 SD unless otherwise indicated. Unpaired Student’s t test or Fisher exact test is used.
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
59
3
Eosinophils in BALF from patients in the placebo arm were activated on RV16 challenge
as shown by a significant increase in CD69 expression. The failure to increase CD69
expression on RV16 challenge in the mepolizumab arm compared with the placebo
arm indicates that CD69 expression on eosinophils is triggered by RV16- induced IL-5
release. Reduced CD62L expression too is indicative of eosinophil activation. In the
placebo arm there was a small but nonsignificant reduced CD62L expression, but in
the mepolizumab arm there was a profoundly reduced CD62L expression. ECP was not
significantly increased in either treatment arm, indicative of no or limited degranulation
of local eosinophils.
In the placebo arm, RV16 challenge reduced percentage B lymphocytes (BALF) and
that of macrophages (sputum), whereas percentage of neutrophils (sputum) and
release of their activation product myeloperoxidase increased (Table 2). None of these
were affected in patients treated with mepolizumab. Blood B lymphocytes were also
reduced on RV16 challenge, but most in mepolizumab-treated patients, which could
reflect migration into the lungs and explain why BALF B lymphocytes do not reduce in
mepolizumab-treated patients. Stratification for eosinophils showed that the effects on
B lymphocytes were predominantly observed in patients with high baseline eosinophil
numbers. Secretory IgA (sIgA) is a potent activator of eosinophils and provides essential
antimicrobial defense (20). After mepolizumab treatment, sIgA levels appeared higher
in patients with low (<3%) eosinophils as compared with placebo, and this difference
F I G U R E 3 . Effect of mepolizumab on percentage of eosinophil in blood and in sputum. Eosinophil percentages were determined at baseline at (B) v1/d214 in sputum and (A) v2/d0 in blood and after treatment at (B) v3/d11 in sputum and (A) v4/d14 in blood. d = day; v = visit. In blood, placebo n = 17 and mepolizumab n = 11; in sputum samples, placebo n = 16 and mepolizumab n = 11. Data are expressed as mean 6 SEM. Paired or unpaired Student’s t tests: *P , 0.05, **P , 0.01, and ***P , 0.001.
Chapter 3
60
FIG
UR
E 4
. Lu
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n p
aram
eter
s, a
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fter m
epol
izum
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r pla
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eatm
ent,
and
bef
ore
and
afte
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cha
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ultip
le m
easu
rem
ents
wer
e ta
ken
into
acc
ount
to
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uce
dai
ly v
aria
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(Day
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and
14
= a
fter
trea
tmen
t an
d b
efor
e R
V16
ch
alle
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Day
s 19
and
21
= a
fter
trea
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RV
16 c
halle
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. D
ata
in A
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for
all
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ticip
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ata
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>3%
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sino
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ted
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t: D
ays
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(G
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EN
O: P
n =
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, ≥ 3
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M n
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CQ
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. Dat
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M. P
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AC
Q =
ast
hma
cont
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uest
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aire
; FE
NO =
frac
tiona
l exh
aled
nitr
ic o
xid
e; M
= m
epol
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ab; P
= p
lace
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RV
16 =
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oviru
s 16
.
6080100
120
FEV1 [% pred.]
6080100
120
FVC [% pred.]
0.0
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1.0
1.5
ACQ [% pred.]
050100
FeNO [ppb]
Plac
ebo
Mep
olizu
mab
6080100
120
FEV1 [% pred.]
6080100
120
FVC [% pred.]
0.0
0.5
1.0
1.5
ACQ [mean]
020406080 FeNO [ppb]
<3
>3
<3
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>3
*
**
*
***
***
** **
**
**
**
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P n
=13
, M n
=7,
<3%
P n
=6,
>3%
P n
=7,
<3%
M n
=2,
>3%
M n
=5;
P n
=13
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=7,
<3%
P n
=6,
>3%
P n
=7,
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=2,
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M n
=5;
P n
=12
, M n
=7,
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P n
=5,
>3%
P n
=7,
<3%
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=3,
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M n
=4
P n
=10
, M n
=7,
<3%
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=4,
>3%
P n
=6,
<3%
M n
=4,
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M n
=3;
ba
selin
e t
rea
tme
nt
R
V1
6
AC
EG
BD
FG
ba
selin
e t
rea
tme
nt
R
V1
6b
ase
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tre
atm
en
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RV
16
ba
selin
e t
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nt
R
V1
6
<3
>3
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tme
nt
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ase
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RV
16
ba
selin
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rea
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R
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6b
ase
line
tre
atm
en
t
RV
16
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
61
3
remained on RV16 challenge (Figure 5A). There was no significant increase in sIgA on
RV16 challenge.
Mast cells are critical in allergic asthma (21). Tryptase in BALF is a marker of mast
cell activation, and significantly higher levels were found in patients with high
(>3%) eosinophils, which were slightly although not significantly attenuated in the
mepolizumab-treated group (Figure 5B).
RV16 challenges in subjects with mild asthma triggered inflammatory mediator
responses both in sputum and BALF, many of which were reduced by mepolizumab,
albeit without reaching statistical significance (data not shown). To clarify whether
mediators reacted in a concerted manner we performed multivariate analyses in the
subgroup of patients with high eosinophils. In Figure 6A and Figure E2, we show the
response of nine mediators in BALF clustered roughly into four groups: 1) IL-6, MIP-1b
(macrophage inflammatory protein-1b)/CCL4 (chemokine ligand 4), IL-8/CXCL8 (C-X-C
motif chemokine ligand 8); 2) IP-10 (inducible protein- 10)/CXCL10, MIP-3A/CCL20,
VEGF (vascular endothelial growth factor-A), IL-1RA; 3) GRO-a (growth-regulated
oncogene-a)/CXCL1; and 4) MMP-9 (matrix metallopeptidase-9). When looking more in
depth into the distribution of the mediators in BALF between the two treatment groups
we observed that MIP-3A/ CCL20, IL-1RA, VEGF-A, GRO-a/CXCL1, and MMP-9 show
the strongest change in the mepolizumab group, whereas IP-10/CXCL10, MIP-1b/
CCL4, IL-6, and IL-8/CXCL8 display the highest median delta in the placebo group
(Figures 6B and 6C). Calculation of P values and receiver operating characteristic
curves and areas under the curve resulted in significant outcome for component 2 (P =
0.018; receiver operating characteristic curves and areas under the curve, 0.914) and
a nonsignificant outcome for component 1 (P = 0.088; receiver operating characteristic
curves and areas under the curve, 0.800).
Finally, to determine whether reduced eosinophil numbers attenuated the antiviral
response we assessed RV16 loads in nasal swabs at Day 7 postinfection and compared
that with the number of BALF eosinophils after the RV16 challenge at Day 7 postinfection
(see Figure E3). Patients receiving mepolizumab as compared with those receiving
placebo had a significant enhanced viral load (P = 0.0404).
FIG
UR
E 4
. Lu
ng fu
nctio
n p
aram
eter
s, a
sthm
a sy
mp
tom
sco
res,
and
FE
NO b
efor
e an
d a
fter m
epol
izum
ab o
r pla
ceb
o tr
eatm
ent,
and
bef
ore
and
afte
r R
V16
cha
lleng
e. M
ultip
le m
easu
rem
ents
wer
e ta
ken
into
acc
ount
to
red
uce
dai
ly v
aria
tion
(Day
s 11
and
14
= a
fter
trea
tmen
t an
d b
efor
e R
V16
ch
alle
nge;
Day
s 19
and
21
= a
fter
trea
tmen
t an
d a
fter
RV
16 c
halle
nge)
. D
ata
in A
, C
, E
, an
d G
are
for
all
par
ticip
ants
; w
here
as in
B,
D,
F, a
nd H
, d
ata
wer
e st
ratif
ied
for
<3%
and
>3%
bas
elin
e eo
sino
phi
ls. (
A a
nd B
) FE
V1%
pre
dic
ted
det
erm
ined
at b
asel
ine:
Day
214
; at t
reat
men
t: D
ays
11 a
nd
14;
and
afte
r R
V16
cha
lleng
e: D
ays
19 a
nd 2
1. (
C a
nd D
) A
s in
A a
nd B
but
for
FV
C %
pre
dic
ted
. (E
and
F)
As
in A
and
B b
ut f
or a
sthm
a co
ntro
l sc
ore.
(G
and
H)
As
in A
and
B b
ut fo
r F E
NO. N
umb
er o
f sub
ject
s p
er p
aram
eter
and
trea
tmen
t gro
up: F
EN
O: P
n =
10,
M n
= 7
, <3%
P n
= 4
, ≥ 3
% P
n
= 6
, <3%
M n
= 4
, >3%
M n
= 3
; FE
V1:
P n
= 1
3, M
n =
7, ,
3% P
n =
6, >
3% P
n =
7, ,
3% M
n =
2, >
3% M
n =
5; A
CQ
: P n
= 1
2, M
n =
7, ,
3% P
n =
5,
>3%
P n
= 7
, ,3%
M n
= 3
, >3%
M n
= 4
. Dat
a ar
e ex
pre
ssed
as
mea
n 6
SE
M. P
aire
d o
r un
pai
red
Stu
den
t’s t
test
s: *
P ,
0.05
, **P
, 0.
01, a
nd *
**P
, 0.
001.
AC
Q =
ast
hma
cont
rol q
uest
ionn
aire
; FE
NO =
frac
tiona
l exh
aled
nitr
ic o
xid
e; M
= m
epol
izum
ab; P
= p
lace
bo;
RV
16 =
exp
osur
e to
rhin
oviru
s 16
.
Chapter 3
62
TA
BL
E 2
. E
ffect
of R
hino
viru
s 16
Cha
lleng
e on
Cel
l Per
cent
ages
and
Eos
inop
hil A
ctiv
atio
n in
Blo
od, B
ALF
, and
Sp
utum
as
a Fu
nctio
n of
Mep
oliz
umab
Tr
eatm
ent v
ersu
s P
lace
bo
V3/
d11
and
V4/
d14
V6/
d19
and
V7/
d21
P V
alue
Mep
oliz
umab
Pla
cebo
Mep
oliz
umab
Pla
cebo
(n =
11)
M v
s. M
P v
s. P
Blo
odV
4/d
14 (
n =
8)
V4/
d14
(n
= 1
1)V
7/d
21 (
n =
8)
V7/
d21
(n
= 8
)V
4 vs
. V7
V4
vs. V
7
% C
D19
5.05
(0.
89)
7.16
(2.
21)
3.64
(0.
71)
5.32
(0.
95)
0.04
0.29
≥ 3%
4.58
(0.
56)
9.09
(3.
98)
2.90
(0.
42)
6.49
(1.
76)
0.04
0.40
BA
LFV
4/d
14 (
n =
8)
V4/
d14
(n
= 1
1)V
7/d
21 (
n =
8)
V7/
d21
(n
= 8
)V
4 vs
. V7
V4
vs. V
7
% C
D8
16.7
0 (3
.81)
34.0
5 (5
.19)
16.7
9 (4
.51)
36.6
1 (3
.80)
0.97
0.40
≥ 3%
14.6
6 (5
.34)
35.8
1 (6
.87)
13.3
0 (5
.78)
39.7
3 (4
.65)
0.49
0.31
% C
D19
0.96
(0.
33)
2.81
(0.
52)
1.24
(0.
28)
0.94
(0.
19)
0.54
0.00
4
≥ 3%
1.29
(0.
63)
2.85
(0.
70)
1.14
(0.
30)
0.77
(0.
29)
0.59
0.04
% C
D69
/Eos
47.3
9 (1
0.09
)53
.07
(7.5
5)57
.40
(10.
90)
70.2
7 (5
.17)
0.24
0.01
% C
D62
L/E
os32
.50
(11.
20)
23.1
6 (5
.45)
4.39
(2.
57)
13.8
6 (6
.35)
0.02
0.12
% E
osin
ophi
ls0.
49 (
0.15
)1.
29 (
0.34
)0.
47 (
0.19
)1.
44 (
0.46
)0.
790.
53
EC
P, p
g/m
l24
1.60
(60
.09)
1042
.00
(450
.20)
436.
20 (
153.
10)
1,22
5.00
(29
4.20
)0.
250.
60
% N
eutro
phi
ls34
.54
(8.5
8)20
.53
(4.8
2)38
.69
(10.
71)
29.4
4 (5
.69)
0.52
0.14
MP
O, n
g/m
l4.
06 (
0.78
)4.
11 (
0.41
)7.
30 (
2.84
)9.
22 (
2.06
)0.
280.
03
Sp
utum
V3/
d11
(n
= 1
1)V
3/d
11 (
n =
17)
V6/
d19
(n
= 1
1)V
6/d
19 (
n =
17)
V3
vs. V
6V
3 vs
. V6
% E
osin
ophi
ls0.
36 (
0.15
)3.
23 (
0.82
)0.
36 (
0.24
)1.
72 (
0.80
)0.
840.
26
EC
P, p
g/m
l63
.41
(18.
31)
115.
30 (
27.9
4)50
.45
(16.
53)
209.
70 (
57.1
2)0.
590.
12
% M
acro
pha
ges
48.5
5 (8
.13)
53.0
9 (6
.88)
36.5
0 (1
1.07
)28
.60
(5.8
2)0.
500.
02
% N
eutro
phi
ls47
.32
(8.3
1)40
.90
(6.9
2)60
.10
(11.
58)
66.0
0 (6
.25)
0.74
0.04
MP
O, n
g/m
l69
7.80
(17
7.50
)98
9.40
(28
6.30
)1,
888.
00 (
532.
60)
803.
70 (
258.
70)
0.15
0.24
Def
initi
on o
f ab
brev
iatio
ns:
<3%
/≥3%
= s
trat
ified
for
<3%
/≥3%
bas
elin
e eo
sino
phils
; B
ALF
= B
AL
fluid
; d
= d
ay;
ECP
= e
osin
ophi
l ca
tioni
c p
rote
in;
Eos
= e
osin
ophi
ls;
M =
mep
oliz
umab
; MP
O =
mye
lop
erox
idas
e; P
= p
lace
bo;
V3/
d11
= v
isit
3 at
Day
11;
V4/
d14
= v
isit
4 at
Day
14;
V6/
d19
= v
isit
6 at
Day
19;
V7/
d21
= v
isit
7 at
Day
21.
Blo
od a
nd B
ALF
sam
ple
s P
n =
11
and
M n
= 9
/8, s
put
um s
amp
les
P n
= 1
7 an
d M
n =
11;
the
num
ber
s ar
e lo
wer
in b
lood
and
BA
LF b
ecau
se o
f inc
omp
lete
pan
el fo
r flo
w
cyto
met
ry. D
ata
are
exp
ress
ed a
s m
ean
(SE
M).
Pai
red
or
unp
aire
d S
tud
ent’s
t te
st w
as u
sed
.
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
63
3
DISCUSSION
Eosinophils in asthma are considered pathogenic, which is why treatment of
eosinophilic asthma focuses on reducing eosinophil numbers. Anti–IL-5 treatment (e.g.,
mepolizumab) effectively reduces eosinophil numbers, systemically and locally, but
mepolizumab proved therapeutically effective only in reducing the exacerbation risk
and corticosteroid-dependency in severe eosinophilic asthma and not in mild asthma.
This questioned the pathogenic role of eosinophils in patients with mild asthma and
therefore we set out to analyze the role of eosinophils in immune responses in more
detail. This led us to investigate the effects of mepolizumab on RV16-induced immune
responses in mild asthma.
We confirmed that mepolizumab reduced systemic and local eosinophils and did not
attenuate FEV1, ACQ, and FENO, and we showed that mepolizumab had no effect either
after worsening asthma by the RV16 challenge. Even though the study was powered
to reveal clinical differences it is important to note that the study was not set up to
determine a clinical effect of mepolizumab on an RV16 challenge in mild asthma. In
that case it would have been preferred to treat patients over a longer period before
the RV16 challenge, and not only once as was done in the current study. With a single
dose we expected to attenuate eosinophils without affecting asthma pathophysiology,
which allowed us to conclude that the observed effects were related to a direct effect
of mepolizumab and not indirectly by modifying asthma pathophysiology. Mepolizumab
did affect immune responses, which was most evident on the RV16 challenge.
Compared with placebo, at baseline mepolizumab enhanced circulating NK cells. On
RV16 challenge mepolizumab prevented a decrease in airway lumen B lymphocytes
and macrophages and an increase of neutrophils and their activation. Furthermore,
mepolizumab enhanced sIgA and reduced tryptase in BALF. Finally, mepolizumab
markedly influenced RV16-induced MIP-3A/CCL20, IL-1RA, and VEGF-A. Together
these findings indicate that mepolizumab, and therefore likely eosinophils, exert
profound effects on both cellular and humoral immune responses to RV16 challenge.
The increase of blood NK cells caused by mepolizumab was an unexpected finding, not
reported earlier. NK cells cross talk to eosinophils, either activating them or killing them.
Possibly the mepolizumab-induced increase of NK cell numbers is a compensatory
mechanism to sustain eosinophil functions during IL-5 depletion (22, 23). Further
investigations are needed to explore the role of IL-5 on NK cells.
Chapter 3
64
Mepolizumab reduced blood eosinophil numbers and that of activation markers, but on
RV16 challenge eosinophils were activated in both treatment groups. This activation,
however, differed between the placebo and mepolizumab treatment groups: CD69
expression increased in the placebo treatment group, whereas CD62L expression was
markedly reduced in the mepolizumab treatment group. IL-5 increases CD69 expression
and reduces that of CD62L and so the slight nonsignificant increase of CD69, but not
the decrease of CD62L, is in line with IL-5 neutralization by mepolizumab, indicating
that another pathway may activate the eosinophils in patients on mepolizumab (19).
BALF ECP levels seemed to increase on RV16 challenge in both treatment groups,
although not significant. In sputum, RV16 exposure resulted in higher, although still
nonsignificant, ECP levels in the placebo treatment group, which was collected 2 days
earlier (Day 19) after RV16 challenge than BALF (Day 21). Together, these findings
indicate that the remaining eosinophils in mepolizumab-treated patients are still activated
in response to RV16, but this may involve a later and different activation cascade than
in the placebo-treated group. In a recent study (Y. S. Sabogal Piñeros and colleagues,
unpublished data), we found that viruses directly interact and activate eosinophils,
which could explain why eosinophils after RV16 challenge are activated despite the use
0
2
4
6
sIgA
[µg/
ml]
0.0
0.5
1.0
1.5
tryp
tase
[µg/
L]MepolizumabPlacebo
** * *
*
*
< 3 % ≥3 %T o ta l
d 1 4
< 3 % ≥3 %T o ta l
d 2 1 d 1 4 d 2 1 d 1 4 d 2 1 d 1 4 d 2 1 d 1 4 d 2 1 d 1 4 d 2 1
A B
F I G U R E 5 . Effect of mepolizumab and rhinovirus 16 challenge on sIgA and mast cell activation. (A) sIgA determined at treatment (v4/d14) and after rhinovirus 16 challenge (v7/d21); data are shown for the total and divided into groups ,3% and >3% eosinophils. (B) Tryptase release determined at treatment (v4/d14) and after RV16 challenge (v7/d21); data are shown for the total and divided into groups ,3% and >3% eosinophils. sIgA: P n = 17, M n = 11, ,3% P n = 8, >3% P n = 9, ,3% Mn = 6, >3% M n = 5; tryptase: P n = 17, M n = 9, ,3% P n = 8, >3% P n = 9, ,3% M n = 4, >3% Mn = 5. Data are expressed as mean 6 SEM. Paired or unpaired Student’s t test *P , 0.05. **P , 0.01. d = day; M = mepolizumab; P = placebo; sIgA = secretory IgA; v = visit.
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
65
3
of mepolizumab. In another recent study, where patients with allergic mild asthma were
treated with mepolizumab, a similar eosinophil activation was reported after allergen
challenge (24), which may underlie the failure to reduce asthma symptoms in patients
with mild allergic asthma on mepolizumab (7).
Mepolizumab blocked the RV16-induced increase of sputum neutrophils and in
BALF the marker of neutrophil activation (myeloperoxidase). That these neutrophilic
markers are affected in both matrices strengthens the validity of these findings.
Because mepolizumab markedly attenuates eosinophils, this suggests that eosinophils
drive neutrophil activation, which could provide an explanation toward neutrophilic
inflammation during exacerbations of asthma. Whether eosinophils influence neutrophils
(e.g., directly by releasing IL-8 or indirectly) remains to be determined. In addition, both
innate and adaptive cellular immune responses were also affected by mepolizumab,
some of which were already linked to eosinophils in murine studies (8). Intriguingly,
mepolizumab seemed to increase extravasation of B lymphocytes, concomitant with
maintenance of B-cell numbers in the airway lumen, whereas in placebo-treated
individuals both decreased significantly. Furthermore, mepolizumab enhanced sIgA
levels, together indicating that mepolizumab, likely by attenuating eosinophil responses,
enhances production of antibodies at mucosal surfaces, particularly that of IgA. Like
for the B lymphocytes, mepolizumab also maintained macrophage numbers, which
may indicate that eosinophil responses also suppress those of macrophages. Because
macrophages and the antibody production at the respiratory mucosal surface primarily
drive antimicrobial responses, an excessive local eosinophil response may attenuate
antimicrobial responses.
The multivariate PLS-DA analysis led us to conclude that mepolizumab affects
multiple inflammatory mediators, such as VEGF-A, MIP-3a, and IL-1RA. VEGF-A is
linked to enhanced mucosal leakage and angiogenesis (25), MIP-3a to recruitment of
lymphocytes (26) and neutrophils, whereas IL-1RA inhibits IL-1–mediated responses
(27). These mediators, most prominently VEGF-A, have been implicated in asthma
pathophysiology (28). With this supervised classification algorithm the best possible
discrimination between the groups of interest is searched, which is subject to bias.
Therefore, these results need to be validated in an independent study. Because
current findings indicate that not all patients are responsive to mepolizumab treatment,
early recognition of nonresponders would be an important step forward. Our findings
suggest that MIP-3a, VEGF-A, and IL-1RA may be potential biomarkers for recognizing
nonresponders at an early stage.
Chapter 3
66
F I G U R E 6 . Graphical presentation of the differential effect of mepolizumab on rhinovirus 16–induced mediator responses. (A) A graphical display of the variable distribution between the two calculated PLS-DA components. Associated variables are projected in the same direction from the origin. (B and C) Horizontal bar plots providing visualization of the highest median value of the features used during PLS-DA analysis, with color code corresponding to the outcome of interest. The most important variables are ranked from the bottom of the graph. Negative and positive signs indicate the correlation structure between variables. Data are from 12 patients (placebo [red] >3% eosinophils n = 7; mepolizumab [blue] >3% eosinophils n = 5) from whom BAL fluid was collected at both visit 4 and visit 7 and analyzed. CCL = chemokine ligand; CXCL = C-X-C motif chemokine; GRO-a = growth-regulated oncogene-a; IP = inducible protein; MIP = macrophage inflammatory protein; MMP = matrix metallopeptidase; VEGF = vascular endothelial growth factor.
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
67
3
Analyses of data on stratification for patients with or without eosinophilia, as per study
protocol, showed that the effect of mepolizumab differed between both patient groups.
For example, low numbers of eosinophils may be required to halt sIgA production and
because mepolizumab may be less effective in reducing local eosinophils in patients
with eosinophilia, it does not affect sIgA levels in those patients. Along similar lines,
high eosinophil numbers may promote mast cell activation (29) and thus mepolizumab
may attenuate levels of tryptase in patients with eosinophilia, whereas there is no effect
in patients without eosinophilia. So, we consider it possible that certain thresholds for
eosinophils are required to exert a biologic response. This needs to be confirmed in a
more extensive study, particularly because numbers were low after stratification.
In contrast to the effects of mepolizumab on immune responses, there was no effect
on FENO. This was of interest because FENO is considered by some to reflect eosinophilic
inflammation (30, 31). Although there was an apparent reduction in FENO in the
mepolizumab-treated patients, the decrease in eosinophils was far bigger than the
effect on FENO and therefore we confirm the earlier findings that eosinophil responses
are not directly related to FENO (32).
Although to the best of our knowledge this study in humans with a combination of
anti–IL-5 treatment and an RV16 challenge is unique and the detailed analyses in
well-characterized patients contribute to the strength of this study, there are several
limitations to this study. The reported findings with mepolizumab here have contributed
to the attenuation of eosinophil responses. In humans, besides eosinophils also
basophils express the IL-5 receptor (33) and so we cannot exclude that mepolizumab
may also have affected basophil responses. The mepolizumab-induced decrease in
tryptase, which is expressed predominantly by mast cells and to a minor extent also by
basophils, does suggest that a contribution by basophils cannot fully be excluded (34–
36). Nevertheless, because RV16 induces profound eosinophil responses and because
eosinophils are more abundant than basophils, we consider it likely that most findings
can be attributed to eosinophil responses.
The findings in this study were obtained after one infusion of mepolizumab and in
response to 10TCID50 RV16, which is a low dose of a virus. Although this low dose
of virus likely reflects a more natural infection as compared with earlier used doses
as high as 10,000 TCID50, peak symptoms were 2–3 days delayed compared with
such authors as Message and colleagues (37). In all, the low dose of RV16 may have
influenced the kinetics of the immune responses, which could explain why we did not
see such a marked eosinophilic inflammation in response to RV16 as shown earlier (37).
Chapter 3
68
Furthermore, it remains to be determined what the effects of mepolizumab would be
ona prolonged treatment and with a more virulent virus.
Our findings are also based on a relatively low number of patients and variable sample
numbers of sputum and BAL, which may have biased results. Having mentioned that,
most results were confirmed in sputum and BAL that were taken at different time points,
which strengthens these findings. Also, the findings apply to mild asthma and do not
necessarily extend to the role of eosinophils in severe asthma, where mepolizumab
was able to attenuate the risk of exacerbation and improve lung function and symptom
scores (38). It would be relevant to study the role of eosinophils in severe asthma to
better understand the mechanism of action of anti–IL-5 treatment in these patients.
Lastly, it is important to indicate here that viral loads in nasal swabs, which could be
measured reliably, were significantly enhanced in the mepolizumab-treated group (see
Figure E3). We cannot exclude that any of the current findings have contributed to the
increased viral loads, nor that the enhanced viral loads have contributed to different
immune responses. The latter, however, we consider unlikely because previous studies
with higher RV16 loads have displayed similar responses as in our placebo group, be
it with different kinetics.
In conclusion, we have shown that mepolizumab in mild asthma, likely via attenuation of
eosinophils, modulates both innate and adaptive immune responses. Most importantly,
in response to RV16 mepolizumab attenuated neutrophil activation, enhanced sIgA
production, and prevented attenuation of B-cell and macrophage numbers. Although
mepolizumab in mild asthma attenuates eosinophil numbers, the remaining eosinophils
were still activated on RV6 challenge. The increased macrophage and B-cell numbers
under mepolizumab may explain why mepolizumab had no clinical effect after RV
challenge in mild asthma.
Author disclosures are available with the text of this article at www.atsjournals.org.
Anti–IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses
69
3
ACKNOWLEDGMENT
The authors are very grateful to all patients for their participation in the present study;
without their commitment and suggestions this study would not have been possible.
The authors acknowledge H. W. van Eijk, K. C. Wolters, Ph.D., and R. Molenkamp,
Ph.D., for performing and supervising the virus analysis; E. J. M. Weersink, M.D., Ph.D.,
for independent medical supervision; and S. Versteeg for performing IgE and tryptase
assays.
Chapter 3
70
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Anti-IL5 in Mild Asthma Alters Rhinovirus-Induced Macrophage, B Cell and Neutrophil Responses (MATERIAL): A Placebo-Controlled,
Double-Blind Study
Yanaika S. Sabogal Piñeros, Suzanne M. Bal, Marianne A. van de Pol, Barbara S.
Dierdorp, Tamara Dekker, Annemiek Dijkhuis, Paul Brinkman,
Koen F. van der Sluijs, Aeilko H. Zwinderman, Christof J. Majoor, Peter I. Bonta, Lara
Ravanetti, Peter J. Sterk, and René Lutter
ONLINE DATA SUPPLEMENT
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SUPPLEMENTARY METHODS
Sample collectionVenous blood was collected in EDTA tubes. Nasal swabs were collected in preservation
fluid. Bronchoalveolar lavage (BAL) was obtained during a standardized bronchoscopy
procedure under lidocaine anesthetics. Eight 20mL aliquots of sterile saline solution at
room temperature were instilled and immediately retrieved into separate sterile tubes.
BAL fluid from fraction 2 to 7 aspirated after each instillation was pooled together into
a single specimen and processed for flow cytometry and ELISA. BAL recovery was
71.1 ± 4.9 % (mean ± SD), which allows direct comparisons. Lung function tests were
performed using a daily-calibrated spirometer, according to European Respiratory
Society (ERS) recommendations. Measurement is based on a dynamic lung function
examination via an ‘open system’ with the JAEGER pneumotach. Broncho provocation
test was performed using MeBr according to the standardized tidal volume method.
Serial doubling concentrations were performed until reaching PC20 or 19.6 mg/ml of
MeBr. FeNO was measured with the NIOX® Flex (Aerocrine AB, Sweden) and performed
according the American Thoracic Society (ATS) recommendations. Skin prick test was
performed based on the position paper by European Academy of Allergology and
Clinical Immunology (EAACI). 12 droplets containing common aeroallergens were
placed on the skin followed by small needle pricks using SPT-lancets. Positive tests
were development of a red “wheal” from at least 3mm after 15 minutes. Sputum was
induced with hypertonic saline solution according to a severe asthma protocol using
increasing concentration of saline (3, 4 and 5%). Sputum was liquefied using DTT and
cells were processed for cell differentiation on cytospins and supernatant for ELISA/
luminex. After Diff-Quick staining differential cell counts were scored as percentage
of cells and sputum containing >80% squamous epithelial cells were excluded from
examination.
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Typically, 500 cells were scored but when required (e.g. low eosinophil numbers) we
counted up to 1000 cells.
FLOW CYTOMETRY ANALYSIS
Single cell suspensions were stained for 30 min on ice. Granulocytes were distinguished
from lymphocytes based on forward and side scatter. Eosinophils were identified as
CD45+, Siglec8+ (7C9; Biolegend), CCR3-positive (61828; R&D) and CD16-negative
(3G8; Bio Legend) cells. Neutrophils were identified as CD45+, CD16-positive (3G8;
Bio Legend) cells. Lymphocyte populations were distinguished by CD45 (HI30; Bio
Legend), CD3 (SK-7; BD), CD19 (HIB19; eBioscience), CD4 (MEM241; Immunotools),
CD8 (SK-1; BD) and CD3-CD19-CD56+ (NCAM16; BD Bioscience). Cellular activation
was assessed using mAbs against the following molecules: CD66b (G10F5; Bio
Legend), CD69 (FN50; BD Pharmingen), CD11b (ICRF44; BD Pharmingen) and CD62L
(DREG-56; Bio legend). Viability was assessed using viability dye (FVD eFluor® 780;
eBioscience). Data acquisition was done on FACSCanto II (BD Biosciences) and data
was analysed using Flowjo (Treestar).
ASSAYS
Human ECP/MPO ELISA.ECP (Eosinophil Cationic Protein) was measured using ECP monoclonal capture antibody
(clone 614, Diagnostics Development, Uppsala Sweden), ECP standard (ImmunoCAP
ECP Calibrator, Nieuwegein, the Netherlands) and biotinylated polyclonal detection
antibody (Diagnostics Development, Uppsala Sweden). MPO (Myeloperoxidase) was
measured using duoset reagents DY3174 (R&D).
Luminex immunoassaysThe following cyto- and chemokines were measured using eBioscience reagents: IL-
17A, IP-10, MIP-1b, VEGF-A, IFN-γ, TNF-α, IL-1RA, fractalkine, GM-CSF, IL-2, IL-21,
IL-8, IL-5, IL-1β, IL-4, IL-6, IL-10, IL-13, G-CSF, GRO-α, IL-1α, IL-12p70 and MIP-3a,
according to the manufacturer’s instructions. The plates were read on a Bioplex 200
(BioRad).
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Multivariate analysesSince RV16 challenge was expected to trigger the release/production of more than one
mediator, changes of mediators in BALF were also evaluated in a multivariate analysis
through Partial Least Squares Discriminant Analysis (PLS-DA). This supervised and
explorative classification algorithm can be applied to obtain maximum separation
between groups of observation and with that help revealing the most predictive or
discriminative features [11]. The input for the PLS-DA were delta’s (visit 7 – visit 4) for
IP-10 (CXCL-10), MIP-1b (CCL4), VEGF-A, IL-1RA, IL-8 (CXCL8), IL-6, GRO-α (CXCL1),
MMP-9 and MIP-3a (CCL20). Furthermore, the`number of components to be modeled
was set to the default of two. Finally Wilcoxon test p-values and receiver operating
characteristic-area under the curve (ROC-AUC) and were calculated based on the
obtained multivariate components. Multivariate analysis was performed in R studio
(v.1.0.136) engine by R (v.3.3.3). R package: MixOmics 6.1.1.
In a separate analysis of the data in table 2, we performed multivariate analysis of
variance (MANOVA) and used Hotelling’s T-square to compare to zero the average
change of the marker values as a result of the exposure to RV16 in both treatment
groups separately. In the placebo group the p-value of Hotelling’s T-square was 0.007,
suggesting that at least 1 marker was significantly changed as a result of the exposure
to Mepolizumab. In effect many markers changed significantly (see table 2). In the
Mepolizumab group the p-value of Hotelling’s T-square was 0.27, suggesting that the
null hypothesis of no change could be rejected for any of the markers.
We also used Hotelling’s T-square test to compare the average changes as a result of
exposure to RV16 of the various blood, BALF and sputum markers between patients
treated with Mepolizumab or placebo. P-value of Hotelling’s T-square was 0.0001
indicating that there was at least 1 marker with a significant different average change
between placebo and Mepolizumab patients. When comparing the different markers it
was clear that it were mainly sputum eosinophils, BALF neutrophils and BALF CD19 (B
cells) that showed a significant change between placebo and Mepolizumab patients.
SAMPLE SIZE CALCULATION
We did not have a priori data to perform an appropriate power calculation for a study
into the contribution of Mepolizumab to RV16-induced immune responses. Based on an
earlier study [12] we expected no clinical effect of Mepolizumab in RV16-exposed mild
asthma patients, which was also what we aimed for, as differences in clinical symptoms
may trigger other immune/inflammatory mechanisms that could bias the eosinophil-
driven responses. An interim analysis of the aforementioned RV16 challenge study [13]
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showed a RV16-induced fall in FEV1 of 0.34L (± 0.13L) from baseline in allergic asthma
patients. When considering an improvement of 0.15L upon Mepolizumab treatment to
be a clinically relevant change in lung function we calculated that we needed at least 14
patients per group with a test significance level of 0.05 and a power of 90%. So we used
the pre-bronchodilator FEV1 before and after RV16 challenge as primary outcomes,
expecting no effect. For an earlier RV16 challenge study into the immune modulatory
enzyme indoleamine 2,3-dioxygenase we found that inclusion of 11-13 patients per arm
sufficed to get significant differences [18], in line with the aforementioned. As volunteers
were not allowed to use inhaled corticosteroids during loss of asthma control and the
need for two bronchoscopies, we expected that not everybody would complete the
study and thus we included 38 instead of 28 patients.
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SUPPLEMENTARY FIGURES AND TABLE
F I G U R E E 1 . Correlation between sputum eosinophils and blood eosinophils in asthma patients at d-14. Sputum and blood was collected and relative eosinophil counts were determined. Threshold was determined by either </≥ 3% sputum eosinophils or when unavailable with </≥ 300 eosinophils per µl blood, since both are correlated; Spearman r 0.43, p=0.043.
Parameter
Mean ± SEM
n=8
Mepolizumab
Mean ± SEM
n=11
Placebo
Mean ± SEM
n=8
Mepolizumab
Mean ± SEM
n=11
Placebo
p-value
M vs M Pvs P
Blood V2/d0 V2/d0 V4/d14 V4/d14 V2vs V4 V2vs V4
%CD69/Eo 39.00 (4.90) 20.70 (4.17) 18.30 (6.50) 11.60 (2.12) 0.03 0.04
%CD62L/Eo 54.54 (7.25) 65.73 (7.26) 81.34 (6.84) 63.47 (8.19) 0.20 0.38
%CD62L/Eo; <3% 34.23 (2.92) 53.58 (11.38) 77.37 (4.90) 65.03 (13.07) 0.002 0.54
MFI CD11b/Eo 4552.71 (339.47) 4759.00 (438.10) 3304.29 (179.14) 3947.00 (786.60) 0.02 0.14
%CD56 30.84 (7.12) 33.86 (3.02) 46.96 (2.93) 38.77 (4.37) 0.03 0.43
TA B L E E 1 . Effect of Mepolizumab treatment on eosinophil activation and relative NK cell count in blood. %CD69 and %CD62L are percentage of CD69/CD62L-expressing eosinophils. As 100% of eosinophils expressed CD11b, the mean fluorescence intensity (MFI) of CD11b is given. </≥ 3 %: treatment group stratified by </≥3% sputum eosinophils; M: Mepolizumab; P: Placebo. P n=11 and M n=9/8 Data is expressed as mean and SEM. Paired or un-paired t-test.
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F I G U R E E 2 . Cluster analysis depicted three groups. As shown in figure 6a, associated variables are projected in the same direction from the origin. The graphical display of the variable distribution among the two calculated PLS-DA components showed nine mediators in BALF responses which cluster into four groups: 1) IL-1RA, VEGF-A, MIP-3A/CCL20, IP-10/CXCL10; 2) IL-8/CXCL8, IL-6, MIP-1b/CXCL4; 3) GRO-α/CXCL1 and 4) MMP-9.
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F I G U R E E 3 . Viral titer in nasal swab in the two treatment groups. RV16 viral load in placebo- (black; 11 patients) and Mepolizumab-treated (grey; 8 patients) groups; p=0.0404; Mann-Whitney test
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