Montelukast reverses airway remodeling in actively sensitized young mice
E. Le remodelage bronchique précoce est-il modulable in vitro?
E. Le remodelage bronchique précoce est-il modulable in vitro?
Nous n’avons constaté aucun effet significatif de la Dexaméthasone, du Montelukast, des anti cholinergiques de synthèse type Tiotroprium, Omalizumab sur la prolifération des CML d’enfants atteints d’asthme sévère préscolaire, lorsque l’on diluait les traitements dans le milieu de culture classique des CML pendant 5 jours. Nous avons donc testé des traitements non conventionnels tels que les macrolides Azithromycine et inhibiteurs calciques Methoxy-vérapamil.
L’Azithromycine diminuait de manière significative la prolifération des CML à J5 de traitement en comptage cellulaire manuel comme en cytométrie en flux avec le marquage par le CFSE mais uniquement dans l’endotype GM ou High BSM. (Figure 24)
Pour le Gallopail (Methoxy-vérapamil), il y avait une diminution significative de la prolifération des CML à J5 de traitement en cytométrie en flux pour l’ensemble des CML préscolaires (n = 15, p = 0,04 Wilcoxon pairé) mais cette diminution n’était pas significative en ANOVA, ni en analyse par endotype (Figure 23).
Les mécanismes physiopathologiques de modulation de la prolifération cellulaire par le Gallopail avaient été précédemment décrits et publiés (88).
Concernant les mécanismes physiopathologiques de l’Azithromycine, ils sont décrits dans l’article 5
«Azithromycin decreases bronchial smooth muscle cell proliferation in severe asthmatic preschool children».
71
Figure 23. Effet de l’Azithromycine et des inhibiteurs calciques sur la prolifération des CML en fonction de l’endotype
Dans le GM ou High BSM (A), l’Azithromycine diminue la prolifération à J5 de traitement, n=9, *p=0,028. Il n’y avait pas d’effet significatif de l’Azithromycine et inhibiteur calcique dans le PM ou Low BSM (B) n=10.
L’inhibiteur calcique Methoxy‐vérapamil diminue de manière non significatif la prolifération des CML dans les 2 groupes p=0,16
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Azithromycin decreases bronchial smooth muscle cell proliferation in severe
1
preschool wheezers
2
F Beaufils1,3,*, V Siao-Him Fa1,2,3,*, T Trian1,3, G Simon2, E Maurat1,3, H Begueret1,2,3, S 3
Debelleix2,3, P Berger1,2,3, M Fayon 1,2,3 4
*FB and VS contributed equally to this article 5
6
1 Université de Bordeaux, Centre de Recherche Cardio-thoracique de Bordeaux, INSERM 7
U1045, F-33000 Bordeaux, France 8
2 CHU de Bordeaux, Service d’Exploration Fonctionnelle Respiratoire, Département de 9
Pédiatrie, INSERM CIC1401, Service d’Anatomopathologie, F-33076 Bordeaux, France 10
3 INSERM, Centre d’Investigation Clinique (CIC1401), Centre de Recherche Cardio-11
thoracique de Bordeaux (U1045), F-33000 Bordeaux, France 12
13
Short version title: Azithromycine in preschool BSM
14 15
This study has received a research grant by the Société Pédiatrique de Pneumologie et 16
d’Allergologie, with the contribution of Astra-Zeneca 17
18
Manuscript word count: ==== words 19 20 Corresponding author: 21 Pr Michael Fayon, MD PhD 22
Département de Pédiatrie, Hôpital Pellegrin-Enfants 23
Place Amélie Raba-Léon 24
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33076 Bordeaux Cedex, FRANCE 25 Tel: +33 5 56 79 56 43 26 Fax +33 5 56 79 61 13 27 e-mail: michael.fayon@chu-bordeaux.fr 28 29 At a glance summary : 30
Azithromycin in preschool BSM, page 3
Version du 11/06/2018 ABSTRACT:
31
Background: Severe wheezing in childhood is associated with decreased lung function in 32
adulthood. This is linked to airway remodeling and increased BSM mass. Preschool 33
children with more BSM mass are at a greater risk of developing asthma at school age. 34
Adult asthmatic BSM cells have an increased mitochondrial content leading to greater 35
BSM cell proliferation. Hypothesis: Mitochondria may represent a new target to treat 36
BSM proliferation in severe childhood wheezing. Objectives: To investigate in vitro BSM 37
cell proliferation in severe preschool wheezers. Moreover, we sought to determine the 38
effect of conventional and non-conventional anti-asthma treatments on BSM proliferation 39
and verify whether the underlying mechanisms involve mitochondria. Methods: BSM cells 40
were cultured from bronchial biopsies obtained from severe preschool wheezing children 41
(1 to 5 years old). Cells were attributed to 2 groups according to muscle mass on 42
histochemical analysis: high and low BSM content. BSM cell proliferation was assessed 43
by manual counting and flow cytometry (CFSE, CellTrace proliferation kit) after 5 days in 44
culture medium containing 10% fetal calf serum (10% FCS). Cells were then treated with 45
Dexamethasone, Montelukast, Tiotropium Bromide, Omalizumab, Methoxy-Vérapamil or 46
Azithromycin (10-9 to 10-6M) in the presence of 10% FCS. Cell viability was assessed 47
using flow cytometry after diamidino-phenylindol (DAPI) staining. Cellular cycle and 48
apoptosis were assessed after DAPI and Annexin-Propidium iodide (Annexin-PI) staining, 49
respectively. Mitochondrial mass, biogenesis and autophagia were assessed by Western 50
Blot and Flow Cytometry, using anti-Porin, anti-mitochondrial transcription factor A and 51
anti-LC3A/B antibody, respectively. Results: The mean±SEM number of cells in the high 52
BSM group increased significantly after 5 days of culture vs. the low BSM group: 186 700 53
± 35 800 vs. 61 560 ± 11 900 BSM cells, respectively, p < 0.001). Only Azithromycin (10
-54
7M) significantly decreased BSM cell proliferation by 40% without increasing the number 55
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of dead or apoptotic cells or blocking the cell cycle. Azithromycin decreased 56
mitochondrial mass by 1/4 in particular in HighBSM cells, by increasing autophagia but 57
had no influence on mitochondrial biogenesis. Conclusion: BSM cells from severe 58
preschool wheezers show varying degrees of proliferation. Increased BSM mass is an 59
indicator of increased mitochondrial content in BSM cells. Azithromycin decreased 60
mitochondrial mass by increasing autophagia, and decreasing BSM cell proliferation, in 61
particular in the HighBSM group. 62
63
Abstract word count: 372/500 words 64
65
KEY WORDS: asthma; child,preschool; azithromycin; cells, smooth muscle; humans;
66 airway remodeling 67 68 ABBREVIATIONS: 69
BSM(C) Bronchial smooth muscle (cells) 70 DAPI Diaminodophenylindol 71 DMSO Dimethylsulfoxyde 72 PI Propidium iodide 73 74
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INTRODUCTION
75 76
Asthma is a chronic airway disease characterized by recurrent attacks of breathlessness and 77
wheezing. It represents a significant public health problem in both developed and 78
developing countries. Asthma is characterized by airway inflammation, obstruction and 79
hyperresponsiveness leading to structural alterations of bronchi, also known as bronchial 80
remodeling. Increased bronchial smooth muscle (BSM) mass appeared to be the most 81
important aspect of this remodeling, and a key feature of severe asthma in adults (1). 82
Indeed, increased BSM mass is correlated with decreased lung function (2, 3) and worse 83
clinical outcome (4). In asthmatic adults, this increased BSM mass is related to various 84
complex mechanisms (5), in particular increased proliferation of BSM cells (BSMC) which 85
has been demonstrated both in vivo (6) and in vitro (7, 8). Moreover, this increased cell 86
proliferation, which is unresponsive to current anti-asthmatic treatments such as 87
corticosteroids (9), has been attributed to gallopamil-dependent mitochondrial biogenesis in 88
severe asthma (8). A recent clinical trial performed in our institution has shown that this 89
calcium inhibitor decreases BSM thickness of severe adult asthmatics in vivo (10). 90
Bronchial remodeling has also been reported in severely wheezing preschool children (11, 91
12) and is associated with increased BSM mass and worse lung function in adulthood (2). 92
To the best of our knowledge, BSMC physiology from preschool wheezers has not 93
evaluated so far. Very recently, we have demonstrated that preschool children with severe 94
wheezing and a high BSM content experience higher exacerbation rates and have higher 95
BSM mitochondrial mass compared to those with low BSM mass (Siao submitted 2018). 96
However, little is known regarding the mechanisms underlying BSM remodeling in 97
children. Understanding the implicit mechanisms is of capital importance to limit bronchial 98
remodeling at a very early stage. Mitochondrial autophagy, an unspecific intracellular 99
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mechanism of mitochondrial degradation, represents an interesting pathway. Its mechanism 100
involves the “light chain” 3A and 3B (LC3A and LC3B) proteins necessary for 101
autophagosome formation. Agents such as rapamycin, which targets mTOR (mammalian 102
target of rapamycin) (13-16), increase autophagosome formation and mitochondrial 103
destruction in various cell types, excluding BSMC. Rapamycin a macrolide with 104
immunosupressor properties, is commonly prescribed in cancer or severe immunological 105
diseases, but not in asthma (15). Recent publications indicate that azithromycin, another 106
macrolide, may curb preschool wheezing (17, 18). 107
108
Moreover, long-term azithromycin is prescribed in other airway diseases such as cystic 109
fibrosis, due to its immunomodulatory and anti-inflammatory effect (19) leading to 110
favorable clinical outcomes (20). 111
In the present study, we hypothesized that azithromycin may decrease BSMC proliferation 112
in severe preschool wheezing children with high BSM, by increasing autophagy and 113
decreasing mitochondrial mass. Consequently, the aims of the present study were to assess 114
(i) the proliferation of BSMC obtained from severe wheezing preschool children according 115
to BSM mass, (ii) the effect of current asthma treatments on BSMC proliferation, and (iii) 116
the effect of azithromycin on BSMC proliferation, mitochondrial mass and autophagy. 117
118 119
Azithromycin in preschool BSM, page 7 Version du 11/06/2018 METHODS 120 Study Population 121
The study was conducted in Bordeaux’s tertiary-care University Hospital. Preschool 122
children with severe wheezing (modified ATS criteria (21) were prospectively recruited in 123
the “P’tit asthme” cohort. The study was approved by the Regional Institutional Review 124
Board (Comité de Protection des Personnes du Sud-Ouest et Outre Mer III). Parents or 125
guardians provided written informed consent. 126
127
Immunochemistry: BSM remodeling assessment 128
Bronchial specimens were obtained using disposable biopsy forceps (Radial Jaw, 1.8 mm 129
OD, Boston Scientific) during fiberoptic bronchoscopy and fixed in 10% buffered 130
formalin as previously described in adults (4, 8). The total area of BSM in actin-satined 131
samples was assessed quantitatively using appropriate software as previously described 132
(4). The BSM area was normalized to the submucosal surface area of the corresponding 133
tissue (8). 134
135
Cell culture and BSM proliferation 136
Isolation and primary culture of BSM cells from bronchial specimens were carried out as 137
previously described (22). BSM cells were grown in DMEM containing 10% fetal calf 138
serum (FCS) with 1% penicillin, streptomycin and amphotericin B and with 1% 139
unessential amino acids (10% FCS medium) at 37°C in a humidified incubator under 5% 140
CO2. All experiments were performed using cells during the first five passages to preserve 141
their initial phenotype. Cells were seeded at a density of 20 000 cells.well-1 into 6-well 142
plates for 24h in 10% FCS medium. The cells were then washed twice with PBS and 143
incubated in serum-free medium for 24h prior to further treatment in order to synchronize 144
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cells. Serum starved-cells were then incubated in free FCS medium, 10% FCS medium or 145
10% FCS medium containing the therapeutic agent (on day 0, D0). BSM cells were then 146
treated for 2 or 5 days (D2 and D5, respectively). The medium was changed after 48h, 147
after two washes using phosphate-buffered saline (PBS). The therapeutic agents studied 148
were dexamethasone, montelukast, tiotropium bromide, omalizumab and azithromycin (all 149
at 10-9M to 10-6M concentrations except for azithromycin (10-9M to 10-7M)). Controls 150
were cells cultured with 10% FCS, and 10% FCS containing dimethylsulfoxyde was used 151
to dilute azithromycin. 152
Cell proliferation was assessed on D0, D2 and D5 by manual counting using a Neubauer 153
Chamber, or using carboxyfluoresceinsuccimidyl ester (CFSE) labeled cells (23) 154
according to the manufacturer’s instructions (CellTraceTM CFSEcell proliferation kit, 155
Thermofisher, Villebon sur Yvette, France). After manual counting, the doubling time 156
(DT) was calculated between D0 and D5 (24). Flow cytometry (FACS Canto, San Jose, 157
CA) was used to measure the decrease in CFSE signal and results were expressed as the 158
mean fluorescence recorded (FITC-M). 159
BSM cells viability was assessed on D5 by flow cytometry after labeling with 160
diaminidophenylindol (DAPI) (25) at a concentration of 1µg/ml. Apoptosis was assessed 161
by flow cytometry using Annexin and Propidium iodide on D5 according to the 162
manufacturer’s instructions (APC Annexin V –PI Apoptosis detection kit, BD Pharmigen, 163
Rungis, France). DAPI at a concentration of 1µg/ml was also used to analyze the cellular 164
cycle of treated cells by flow cytometry (26). 165
166
Assessment of mitochondrial activity 167
Regarding BSM mass the expression of VDAC1/Porin (voltage-dependent anion selective 168
channel protein 1), a mitochondrial outer membrane channel, was determined by flow 169
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cytometry and immunoblotting using a mouse monoclonal anti-VDAC1/Porin primary 170
antibody (ab14734, ABCAM, Paris, France) (8). Mitochondrial biogenesis and BSM cell 171
autophagy were assessed by immunoblotting using a mouse monoclonal anti-transcription 172
factor A primary antibody (ab119684, ABCAM, Paris, France) (8) and a rabbit polyclonal 173
anti LC3A/B (ab128025, ABCAM, Paris, France) (13) antibody, respectively. For flow 174
cytometry, an Alexa 488 coupled anti-mouse antibody was used as secondary antibody for 175
mitochondrial mass and mitochondrial biogenesis (8); an Alexa 488 coupled anti-rabbit 176
antibody was used as secondary antibody for autophagy. The signal was analyzed within 177
the hour and results expressed as the mean fluorescence recorded. For western blotting, 178
anti-mouse or anti-rabbit horseradish peroxidase were used as secondary antibodies and 179
analyzed as previously described (8). Respective concentrations of antibodies are 180
presented in Tables 1 and 2. 181
182
Statistical analysis 183
Analyses were performed using Graphpad Prism 5 software (La Jolla, CA 92037 USA). 184
Results are presented as the means±standard error of the mean or as medians [range] as 185
appropriate. Cell proliferation results were analyzed using MANOVA and the Bonferroni’s 186
post hoc test for subgroup analyses. In cell viability and mitochondrial activity 187
experiments, differences were analyzed using the Friedman test and the Dunn’s post-hoc 188
tests for subgroup analyses. A p-value < 0.05 was considered statistically significant. 189
190 191
Azithromycin in preschool BSM, page 10 Version du 11/06/2018 RESULTS 192 193 Study population 194
The characteristics of the 19 included children are presented in Table 1. 195
196
Immunochemistry 197
The median percentage of BSM surface normalized to total bronchial wall surface area 198
was 20.3% (Figure 1A). Consequently we divided the population into 2 groups based on 199
the percentage of the normalized BSM surface area: High bronchial smooth muscle 200
(HighBSM) (>20.3% (50th percentile), n=9) and Low bronchial smooth muscle 201
(LowBSM) (<20.3%, n=10) (Figure 1A-B). Beside BSM surface area, there were no 202
significant clinical differences between these two groups (Figure 1B). 203
204
BSM proliferation 205
From bronchial biopsy specimens, 19 different primary cells cultures were obtained and 206
used in the study. Regarding manual counting (Figure 2), after five days, the number of 207
BSMC from the HighBSM group was greater (n=6, 186 000±35 800 BSMCs) than cells 208
from the LowBSM group (n=8, 61 500±11 900 BSMCs), p = 0.0293 (figure 2A)). The D0 209
to D5 doubling time of cells from HighBSM was lower than cells from the LowBSM 210
group (52.6±19.2 h vs 89.6±19.6 h, p = 0.029) (Figure 2B). Moreover, mitochondrial mass 211
was increased in HighBSMC (n=7) vs. LowBSMC (n=7) (Western Blot porin/actin ratio: 212
HighBSM (n=8) vs. LowBSM (n=8), 0.344±0.064 vs. 0.230±0.0531, p = 0.046; Mean 213
flow cytometry Porin fluorescence (Porin FITC-M) (HighBSM (n=7) vs. LowBSM (n=7): 214
14010±1546 vs. 9605±578, p=0.011) (Figure 2C). We also demonstrated a significant 215
correlation between the percentage of BSM normalized to total bronchial wall surface area 216
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and Porin FITC-M as determined by flow cytometry (n=14, spearman correlation r=0.71, 217
p=0.005) (Figure 2D) and with porin signal measured by western blot (Spearman 218
correlation r=0.55, p=0,02 (Figure E1B). 219
220 221
Effect of treatment 222
Dexamethasone, montelukast, tiotropium bromide and omalizumab did not reduce BSMC 223
proliferation (data not shown). Only azithromycin decreased the number of BSMC in a 224
dose-dependent manner after five days of treatment (figure 3A). Indeed, no effect could be 225
observed when cells were treated with 10-9 or 10-8M azithromycin, in contrast to 10-7M, 226
which significantly decreased proliferation by 40% compared to control (number of cells 227
on D5 assessed by manual counting (n=12 cell lines): 10% FCS, 129167±25110 BSMC; 228
10% FCS+DMSO, 128958±24658; 10% FCS+Az 10-7M, 82083±14661 (p<0.0001, see 229
figure 3A for differences between groups). This result was confirmed by flow cytometry, 230
the CFSE signal being decreased similarly by 40% in BSMCs treated with azithromycin 231
(Figure 3C). Noteworthy, cell proliferation attenuation was significant in HighBSMC only 232
(figure 3D). 233
234
To understand the underlying mechanisms we assessed the effect of 10-7M azithromycin 235
on cell death using DAPI and apoptotic cell death using Annexin and Propidium iodide. 236
We also assessed the effect of the treatment on the cellular cycle. Azithromycin did not 237
modify cell death assessed by DAPI or Annexin-PI. Indeed, the percentage of live BSMCs 238
(DAPI-negative cells or Annexin and propidium ioide-negative cells) was similar in all 239
groups (Figure E2A-B). The percentage of late or early apoptotic cells was unaltered after 240
treatment (Figure E2C). We did not observe any change in the cell cycles (data not 241
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shown). Mitochondrial mass, as assessed by flow cytometry, in 10-7M azithromycin-242
treated BSMCs was decreased by 30% vs. untreated BSMCs (n=11): Porin FITC-M in 243
10% FCS, 12356±1193; 10% FCS+DMSO, 13352±1135; 10%FCS+Az 10-7M 9115±848; 244
p<0.0001) (Figure 4A-B). This was significant in the HighBSMC group (n=7, Porin FITC-245
M; 10%FCS 14012±1546 vs. 10%FCS+Az 10-7M 9591±1262; p=0.016) but not 246
LowBSMC group (Figure 4C-D). 247
248
To further explore the causes explaining the decrease in mitochondrial mass in the 249
presence of azithromycin we assessed mitochondrial biogenesis and autophagy. 250
Mitochondrial biogenesis as assessed by flow cytometry (data not shown) and western blot 251
(Figure 5A-B) was not influenced by azithromycin. However, autophagy assessed by 252
western blotting using an anti-LC3 A/B antibody was increased in azithromycin-treated vs. 253
-untreated cells: azithromycin (n=7, LC3B/LC3A ratio in 10%FCS, 0.636±0.180; 254
10%FCS+DMSO, 0.791±0.301; 10%FCS +Az10-7M, 8.299±3.875, p=0.0027) (Figure 5A-255
C). 256
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DISCUSSION
258
This study demonstrates that severe preschool wheezers’ BSMC show different 259
proliferative properties depending on whether they originate from bronchi with high or 260
low BSM content. Indeed cells from HighBSM children proliferated faster than cells from 261
LowBSM. Moreover, mitochondrial mass was increased in HighBSMC compared to 262
LowBSM. Although current anti-asthma therapeutic agents such as corticosteroids were 263
unable to decrease BSMCs proliferation, the latter was significantly decreased in the 264
presence of 10-7M azithromycin, without influencing cell cycle or death. Azithromycin 265
reduced mitochondrial mass preferentially in BSMCs from patients with the HighBSM 266
endotype. One of the mechanisms involved may be the activation of BSMC autophagy. 267
268
Compared to adults, neonatal human BSMC are in a rapid and robust proliferative phase 269
and have the capacity to respond disproportionately under abnormal environmental 270
conditions, through increased mitochondrial biogenesis and altered calcium homeostasis 271
(27). As in the present study, in asthmatic adults, a correlation between mitochondrial 272
mass and the BSM mass has been previously shown (8, 10). 273
274
Azithromycin is a macrolide with a long half-life that accumulates in lung tissue leading to 275
a lung concentration a fifty to a hundred times greater than in the serum. A tissue 276
concentration of 10-7M represents the serum concentration obtained in children treated for 277
5 days for a total dose of 50 mg/kg (). This suggests the possibility of using long-term 278
alternate-day azithromycin treatment at clinically accessible doses. In the meantime some 279
studies in pediatric asthma suggest that the incidence, severity and duration of acute 280
wheezing may be improved by the short-term administration of this drug (17, 18). Its 281
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pharmacological properties as an anti-inflammatory and immunomodulatory agent are 282
multifaceted (19, 28). 283
284
The results of the present study suggests a link between mitochondrial mass, smooth 285
muscle proliferation and the overall size of bronchial smooth muscle, similar to what has 286
been described in adults (8). On the one hand, BSMCs with greater mitochondrial mass 287
have increased proliferation and on the other hand such cells are most susceptible to 288
azithromycin for the reduction the mitochondrial mass as well as BSMC proliferation. This 289
data reinforces the predominant role of mitochondria in asthma and wheezing and the new 290
therapeutic targets they represent. In adult, the use of methoxy-verapamyl, a calcium 291
inhibitor, decreased mitochondrial biogenesis and mitochondrial mass of BSMCs leading 292
to a decrease in BSMC proliferation (10). However, this agent proved to be unsuccessful 293
in pediatric asthma, suggesting that different pathophysiologic mechanisms are involved in 294
children. We have shown that another target, i.e. BSMC mitophagy, may be of clinical 295
relevance since azithromycin was able to activate this process, thus decreasing 296
mitochondrial mass. Autophagy is a mechanism cells use to destroy or recycle proteins, 297
organelles and dysfunctional components. Mitophagy represents the same mechanism but 298
is target specifically mitochondria in cells (15, 29). Autophagy and mitophagy are 299
regulated by a large number of proteins such as mTOR and other macrolide analogues (29, 300
30). 301
This study consolidates the prospects that azithromycin may represent a new treatment in 302
severe wheezing. An in vitro study in a murine model of chronic asthma has shown that 303
long-term azithromycin improves both airway inflammation and airway remodeling, 304
particularly smooth muscle area (31, 32). As shown in rabbit, azithromycin treatment 305
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down regulates BSMC proliferation by inducing autophagy and blocking autophagy up-306
regulates BSMC proliferation (33, 34). 307
The data presented suggests that children with severe wheeze can be separated into two 308
endotypes based on BSM mass: children with HighBSM having BSMC with increased 309
mitochondrial mass, faster proliferation and an increased sensibility to azithromycin, and 310
children with LowBSM having BSMC with a lower mitochondrial mass, lower 311
proliferation with little response to azithromycin. This may be of value when weighing the 312
pro’s and con’s of performing bronchoscopy and lung biopsy in early wheezing. Targeting 313
specific endotypes as well as phenotypes are becoming of capital importance to target 314
specifically populations for clinical trials in asthma. This may explain why the various 315
clinical trials performed with macrolides have not provided adequate proof of the efficacy 316
of macrolides in asthma (35). Moreover, these studies were performed in adults with 317
different dosing regimens, mostly of short- and medium-term duration (36, 37).