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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

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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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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.


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


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.


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

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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).

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, 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

.


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.


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.


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.


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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