American Journal of Respiratory Cell and Molecular Biology

Monocytes/macrophages are major effectors of lung inflammation associated with various forms of pulmonary hypertension (PH). Interactions between the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine systems that guide phagocyte infiltration are incompletely understood. Our objective was to explore the individual and combined actions of CCL2/CCR2 and CX3CL1/CX3CR1 in hypoxia-induced PH in mice; particularly their roles in monocyte trafficking, macrophage polarization, and pulmonary vascular remodeling. The development of hypoxia-induced PH was associated with marked increases in lung levels of CX3CR1, CCR2, and their respective ligands, CX3CL1 and CCL2. Flow cytometry revealed that both inflammatory Ly6Chi and resident Ly6Clo monocyte subsets exhibited sustained increases in blood and a transient peak in lung tissue, and that lung perivascular and alveolar macrophage counts showed sustained elevations. CX3CR1−/− mice were protected against hypoxic PH compared with wild-type mice, whereas CCL2−/− mice and double CX3CR1−/−/CCL2−/− mice exhibited similar PH severity, as did wild-type mice. The protective effects of CX3CR1 deficiency occurred concomitantly with increases in lung monocyte and macrophage counts and with a change from M2 to M1 macrophage polarization that markedly diminished the ability of conditioned media to induce pulmonary artery smooth muscle cell (PA-SMC) proliferation, which was partly dependent on CX3CL1 secretion. Results in mice given the CX3CR1 inhibitor F1 were similar to those in CX3CR1−/− mice. In conclusion, CX3CR1 deficiency protects against hypoxia-induced PH by modulating monocyte recruitment, macrophage polarization, and PA-SMC cell proliferation. Targeting CX3CR1 may hold promise for treating PH.

Monocytes/macrophages are major effectors of inflammation associated with pulmonary hypertension (PH). We show here that genetic or pharmacological inactivation of CX3CR1 protects against hypoxia-induced PH by modulating monocyte recruitment, macrophage phenotype, and pulmonary artery smooth muscle cell proliferation. Targeting CX3CR1 may hold promise for treating PH.

Inflammation in the lungs and pulmonary vessels is now recognized as a hallmark of various forms of pulmonary hypertension (PH), including human pulmonary arterial hypertension (PAH) and experimentally induced PH in animal models (13). The main effectors of inflammation are the myeloid leukocytes, most notably monocytes and macrophages. Among the factors that control monocyte infiltration are the two chemokines, CCL2 and CX3CL1, and their cognate receptors, CCR2 and CX3CR1. Monocytes fall into two main subsets: inflammatory monocytes, which express high levels of CCR2 but low levels of CX3CR1 (Ly6Chi CCR2hi/CX3CR1lo), and resident monocytes, which express high levels of CX3CR1 but low levels of CCR2 (Ly6Clo CCR2lo/CX3CR1hi) (4, 5). Although targeting monocyte mobilization and function is a promising approach to the treatment of PH, the molecular underpinnings of the inflammatory process and their relationships with the development and progression of PH remain ill-defined.

Previous studies of the roles for the CX3CL1/CX3CR1 and CCL2/CCR2 systems in PH produced contradictory findings. In patients with idiopathic PAH, we previously found CCR2 overexpression in pulmonary artery smooth muscle cells (PA-SMCs) from remodeled pulmonary vessels, with a stronger proliferation-inducing effect of CCL2 on PA-SMCs from patients with PAH compared with those from control patients (6). Recent studies in mice exposed to chronic hypoxia, however, showed that CCR2 deficiency aggravated PH development (7). We also found that CX3CR1 activation induced rat PA-SMC migration and proliferation (8), an effect consistent with recent reports that CX3CL1 contributes to hypoxic PH (9). Thus, it is unclear whether CCR2 activation acts in combination with CX3CR1 to alter PH development, whether the CX3CL1/ CX3CR1 and CCL2/CCR2 systems play additive or antagonistic roles, and whether they exert prominent effects on monocyte trafficking or PA-SMC functions.

In addition, both CCL2 and CX3CL1 may alter macrophage polarization into M1- or M2-like macrophages (1013). M1-like macrophages display a cytotoxic and proinflammatory phenotype characterized by strong pathogen and tumor cell clearance capabilities (1113). In contrast, M2-like macrophages suppress immune and inflammatory responses, but participate in tissue remodeling and tumor progression (12, 13). In mice with hypoxia-induced PH, lung macrophages, particularly those in the alveoli, have been shown to preferentially acquire the M2 phenotype, and subsequently to promote PA-SMC proliferation by releasing mitogenic factors (14, 15). The roles played by the CX3CL1/CX3CR1 and CCL2/CCR2 systems in this process, however, remain unknown.

Here, we explored the specific and combined actions of the CX3CL1/ CX3CR1 and CCL2/CCR2 systems in hypoxia-induced PH in mice, with special emphasis on their roles in monocyte trafficking, macrophage polarization, and pulmonary vascular remodeling. Some of the results of these studies have been previously reported in the form of an abstract (16).

Mouse Models

CX3CR1-deficient (CX3CR1−/−) mice, CCL2-deficient (CCL2 −/−) mice, and double CX3CR1- and CCL2-deficient (CX3CR1−/−/CCL2−/−) mice were produced and bred at the Centre d’Immunologie et des Maladies Infectieuses, as previously described (17). Wild-type (WT) mice (C57Bl/6j) were used according to institutional guidelines that complied with national and international regulations, including the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experiments were performed under agreement 94-28245 at a level 2 animal platform at the INSERM-Unit 955, Créteil, France.

Exposure to Chronic Hypoxia

Male mice aged 3 months were exposed to chronic hypoxia (9% O2) in a ventilated chamber (Biospherix, New York, NY). The hypoxic environment was a mixture of air and nitrogen. The chamber was opened twice a week for cleaning and replenishing the food and water supplies. In addition, when indicated by the protocol, the chamber was opened to give the mice the CX3CR1 antagonist F1 by intraperitoneal injection (50 μg in phosphate-buffered saline) throughout hypoxia exposure, as previously described (18, 19).

Hemodynamics and Ventricular Weight Measurements

At the specified points after the initiation of hypoxia exposure, mice were anesthetized and hemodynamic and ventricular weight measurements were performed as previously described (3).

Antibodies, Recombinant Proteins, and Reagents

Antibodies for immunohistochemistry, Western blot, fluorescence-activated cell sorting, and recombinant proteins (CX3CL1, CCL2) are described in the online supplement.

Mouse Lung Tissue Analysis

Lung tissue sections for immunostaining, TUNEL assay, and assessment of pulmonary vascular remodeling were prepared as described in the online supplement. Total RNA and protein were extracted from the right lung of each animal. Total RNA was used for real-time polymerase chain reaction and total protein for Western blot and ELISA. The immunostaining, Western blot, ELISA, and real-time polymerase chain reaction methods are described in the online supplement.

Flow Cytometry

Bone marrow, blood, lung, and bronchoalveolar lavage (BAL) cells were prepared and fluorescence-activated cell sorting performed as previously described (17, 20). The methods are detailed in the online supplement.

Cell Studies

Mouse PA-SMC isolation and cell proliferation assays were performed as previously described (3). Briefly, PA-SMCs were isolated by enzymatic digestion with a collagenase and elastase mixture. PA-SMC proliferation was then assessed by MTT assay. The method for preparing M1- and M2-like macrophages is described in the online supplement.

Statistical Analysis

Quantitative variables from human and animal studies are reported as mean and individual values. Data from quantitative polymerase chain reactions, Western blotting, and ELISA are reported as mean ± SEM. These variables were compared using the Mann–Whitney post hoc test with the Bonferroni correction for multiple comparisons. Cell-study results are reported as mean ± SEM and compared using standard one-way ANOVA with Sidak’s multiple comparison test (Prism 6.0; GraphPad Software, San Diego, CA). P values <0.05 were considered significant.

Increased Lung Expression of CX3CR1, CCR2, and Their Respective Ligands CX3CL1 and CCL2 During Hypoxia-Induced PH

Exposure to chronic hypoxia led to progressive increases in right ventricular systolic pressure (RVSP) and Fulton’s index of right ventricular hypertrophy (right ventricular weight/left ventricular plus septum weight, RV/LV+S), which became significant on Day 3 compared with control normoxic mice, and then continued until Day 18 (see Figure E1 in the online supplement). Lung mRNA and protein levels of CX3CR1 and CCR2 were increased on Day 18 of hypoxia exposure, whereas CX3CR1 mRNA levels increased earlier, starting on Day 8 (Figure 1A). Changes in the CX3CR1 ligand CX3CL1 were noted only after 18 days of hypoxia. In contrast, CCL2 mRNA levels showed an early peak on Day 1, followed by a return to basal levels and a second increase on Days 8 and 18 (Figure 1B). CCL2 protein levels were increased on Day 3, but not on Day 18 (Figure 1B). Immunofluorescence staining revealed that CX3CR1 was widely distributed in pulmonary vessels and pulmonary alveolar cells, whereas CCR2 was chiefly found in pulmonary vessels (Figure 1C).

Effects of Deficiencies in CX3CR1, CCL2, or Both on Hypoxia-Induced Pulmonary Hypertension in Mice

Under normoxia, body weight, RV weight/body weight, RVSP, and heart rate were similar in WT, CX3CR1−/−, CCL2−/−, and CX3CR1−/−/CCL2−/− mice (Figure E2). After 18 days of hypoxia, RVSP was lower and RV hypertrophy less severe in CX3CR1−/− mice than in WT mice (Figure 2A). Furthermore, distal pulmonary artery muscularization, which increased with the duration of hypoxia, was less marked in the CX3CR1−/− mice than in WT controls (Figure 2A). In contrast, compared with WT mice, CCL2−/− and CX3CR1−/−/CCL2−/− mice exposed to the same conditions of hypoxia developed PH of similar severity, with a similar degree of pulmonary vascular remodeling. Interestingly, a reduction in PA-SMC proliferation (as assessed by the percentage of dividing Ki67-stained cells) and an increase in PA-SMC apoptosis (as assessed by the percentage of TUNEL-positive cells) were noted in CX3CR1−/− mice, but not in CCL2−/− or CX3CR1−/−/CCL2−/− mice, compared with WT mice (Figure 2B).

Effects of Genetic Deletion of CX3CR1, CCL2, or Both on Lung Monocytes and Macrophages During Chronic Hypoxia

We first assessed the effect of hypoxia on lung myeloid cells by performing flow cytometry analyses of bone marrow, blood, and lung cells from WT mice exposed to hypoxia for 18 days. Hypoxia exposure was followed by significant reductions in bone marrow total cells and neutrophils, contrasting with a rapid and early increase in bone marrow monocytes followed by a return to basal levels, and then by a second increase after 18 days of hypoxia (Figure 3A). These changes in bone marrow cells were associated with marked increases in blood counts of total leukocytes, neutrophils, and monocytes, which started on Day 3. Of note, the increase in blood counts of inflammatory (Ly6Chi) monocytes was larger than that of resident monocytes (Ly6Clo), which became significant only on Day 8. In lungs studied after flushing of the pulmonary vessels with phosphate-buffered saline (to eliminate inflammatory blood cells not attached to the lungs), neutrophil counts decreased gradually with increasing time under hypoxia, whereas counts of both resident and inflammatory monocytes increased sharply on Day 3 and then diminished, with resident monocytes showing a significant reduction on Day 18 (Figure 3A). In contrast, the number of bronchoalveolar macrophages (measured in BAL fluid) increased rapidly during hypoxia exposure and remained elevated until Day 18, whereas perivascular macrophage counts (determined by F4/80 staining) increased linearly throughout hypoxia exposure, in parallel with pulmonary vascular remodeling (Figure 3B).

We then examined whether these changes in lung inflammatory cell populations were altered in CX3CR1−/−, CCL2−/−, and CX3CR1−/−/CCL2−/− mice after 18 days of hypoxia exposure. Flow cytometry analysis revealed that neither total lung cell nor lung neutrophil counts were affected by deletion of CX3CR1, CCL2, or both on Day 18 (Figure 4A). In contrast, the total lung monocyte count was markedly increased in CX3CR1−/− mice compared with in WT mice, whereas no difference was observed in CCL2−/− and CX3CR1−/−/CCL2−/− mice (Figure 4A). Of note, this increase in lung monocytes was chiefly ascribable to resident Ly6Clo monocytes. Inflammatory Ly6Chi monocytes remained unchanged in CX3CR1−/−/mice and, as expected, were reduced in lungs from both CCL2−/− and CX3CR1−/−/CCL2−/− mice. Interestingly, the increase in total lung monocytes in CX3CR1−/− mice was associated with significant increases in total lung macrophage counts (measured using flow cytometry) (Figure E3) and in BAL macrophages and perivascular macrophages (quantified by immunohistochemistry) (Figure 4B). No changes in lung macrophage counts were noted in CCL2−/− or CX3CR1−/−/CCL2−/− mice (Figure 4B and Figure E3).

Changes in Lung Macrophage Phenotypes in CX3CR1−/−, CCL2−/−, and CX3CR1−/−/CCL2−/− Mice During Chronic Hypoxia Exposure

To further explore whether CX3CR1 deficiency, which led to increases in lung monocyte and macrophage counts, altered the functional phenotype of lung macrophages, we characterized the macrophage phenotypes in WT and transgenic mice exposed to chronic hypoxia. As previously reported, and shown in Figure 5, hypoxia exposure was associated with marked elevations in lung mRNA levels for the M2-macrophage markers Fizz1, Mannose RC, Arg-1, and Ym-1, and to a lesser extent in lung mRNA levels for the M1-macrophage markers inducible nitric oxide synthase, TNF, and CCL2 levels, but not CD80. Interestingly, these changes in M1 and M2 macrophages associated with hypoxia were dramatically altered by CX3CR1 deficiency: in CX3CR1−/− mice, the M1 phenotype was more pronounced compared with in WT mice, whereas the M2 phenotype was less marked, with lower levels of at least two M2 markers, Fizz1 and Mannose RC. No consistent changes in the M1/M2 macrophage balance were observed in the other transgenic mice.

Functional Consequences of Lung Macrophage Phenotypes on PA-SMC Proliferation: Role for CX3CR1 Expressed by PA-SMCs

We assessed the effects of conditioned medium from bone marrow-derived cultured macrophages on PA-SMC growth. As shown in Figure 6A, conditioned media from M2 macrophages stimulated the growth of PA-SMCs from WT mice to a much greater extent than did conditioned media from M1 macrophages, which induced only a mild effect. This proliferating effect of M2 macrophages was markedly reduced with PA-SMCs from CX3CR1−/− mice or PA-SMCs from WT mice in the presence of the CX3CR1 inhibitor F1 (Figure 6A). Consistent with this finding, CX3CL1 levels were higher in conditioned medium from M2 compared with M1 macrophages (Figure 6A). Moreover, CX3CL1 markedly increased the proliferation of cultured PA-SMCs, and this effect was CX3CR1-dependent (Figure 6B and Figure E4A). In addition, CX3CL1 levels were lower in alveolar macrophages from CX3CR1−/− mice. In keeping with this finding, the growth-promoting effect of conditioned media of alveolar macrophages was weaker with cells from CX3CR1−/− mice than from control mice (Figure 6C). The CX3CR1 inhibitor F1 suppressed the growth-stimulating effect of CX3CL1, but not of platelet-derived growth factor (Figure E4B). This proliferating effect of exogenous CX3CL1 on cultured PA-SMCs contrasted with a weaker effect of CCL2 and other CCR2 ligands such as CCL8 (Figure 6B).

Pharmacological Blockade of CX3CR1: Effects on PH Development and on Lung Macrophages

To investigate whether pharmacological CX3CR1 blockade mimicked CX3CR1 gene deletion in chronically hypoxic mice, we treated WT mice with the CX3CR1 antagonist F1 (50 µg three times a week) during chronic hypoxia exposure. Mice given F1 had less severe PH, with lower values of RVSP and RV/LV+S; less vessel muscularization; and lower counts of Ki67-positive pulmonary vascular cells (Figure 7A). Importantly, this protective effect occurred concomitantly with a significant increase in perivascular macrophage counts (Figure 7B).

The main finding from the present study is that genetic or pharmacological inactivation of CX3CR1 protects against hypoxia-induced PH by modulating monocyte recruitment, macrophage phenotype, and PA-SMC proliferation. Of major interest, the protective effects of CX3CR1 deficiency on hypoxia-induced PH severity occurred concomitantly with increases in lung monocyte and macrophage counts. We suggest that the protective effect of CX3CR1 inactivation may be related to a change in the balance of M1/M2 macrophage phenotypes, which directly affects PA-SMC proliferation. That pharmacological CX3CR1 inhibition was similar to genetic CX3CR1 inactivation regarding protection against PH and increased perivascular macrophage counts further supports the possibility that CX3CR1 axis modulation may constitute a therapeutic target in PH.

These findings emphasize the complexity of the role inflammatory cells may play in the process of pulmonary vascular remodeling. Although several studies examined the role for specific chemokines and chemokine receptors in PH development (1, 3, 6, 21), whether chemokine systems interact in an additive or antagonistic manner is unknown. Of particular interest are potential interactions between the CX3CL1/CX3CR1 and CCL2/CCR2 systems, both of which are involved in monocyte/macrophage lineage recruitment and affect PA-SMC functions (5, 6, 8, 9). We therefore undertook the present studies to elucidate the individual and combined actions of these systems in mice with PH induced by chronic hypoxia exposure.

After chronic hypoxia exposure, the mice exhibited increased counts of both resident and inflammatory monocytes in blood and lungs, together with increased macrophage counts in BAL fluid and around pulmonary vessels. The classical view is that monocyte production by the bone marrow leads to an increase in blood monocyte counts with subsequent monocyte recruitment to damaged organs (22). In the present study, the increases in blood and lungs after hypoxia exposure were greatest for resident monocytes, whose counts subsequently increased also in BAL fluid and perivascular lung tissue. Thus, mobilization of resident monocytes emerges as a prominent feature preceding lung macrophage recruitment during chronic hypoxia. Our results are therefore consistent with those of previous studies showing an early increase in lung macrophages in mice exposed to chronic hypoxia (14).

Our initial hypothesis was that the CCL2/CCR2 and CX3CL1/CX3CR1 systems played complementary roles to promote lung macrophage activation during hypoxia exposure and the subsequent development of hypoxic PH. We therefore studied mice with inactivation of each or both systems. We found no additive effects: CX3CR1 deficiency protected against PH, whereas CCL2 deficiency had no effect. Moreover, the two systems seemed to exert antagonistic effects, as mice with combined CX3CR1 and CCL2 deletion showed no protection against PH.

To further investigate the roles played by the CX3CR1/CX3CL1 and CCR2/CCL2 systems on pulmonary vascular remodeling, we examined the effects of CX3CL1 and CCL2 on mouse PA-SMC proliferation. CX3CL1 produced a marked PA-SMC growth response compared with CCL2, whose effect was mild. Of note, PA-SMCs from CX3CR1−/− or CX3CR1−/−/CCL2−/− mice did not respond to treatment with CX3CL1. These in vitro results seem at variance with the dissimilar PH response of CX3CR1−/− mice and CX3CR1−/−/CCL2−/− mice to hypoxia. The roles for these two chemokine/receptor systems in PH, therefore, cannot be restricted to a direct effect on PA-SMC growth and must involve more complex mechanisms.

An unexpected finding was that protection against PH in CX3CR1-deficient mice occurred together with increased counts of total and resident lung monocytes. By contrast, CCL2−/− mice and CX3CR1−/−/CCL2−/− mice were not protected against hypoxic PH, and they had lower lung counts of inflammatory monocytes. Furthermore, CX3CR1−/− mice consistently exhibited increases in both perivascular and BAL fluid macrophages, a profile not shared by CCL2−/− mice or CX3CR1−/−/CCL2−/− mice. These apparently contradictory findings prompted us to conduct a more detailed analysis of lung macrophage phenotypes under our study conditions. Indeed, in various models, different subpopulations of monocytes are recruited to damaged organs and may acquire M1-like and/or M2-like activation modes that specifically affect repair or remodeling processes (22). In chronically hypoxic mice, alveolar macrophages were shown to acquire an activated M2 phenotype in response to hypoxia, and to play an active role in pulmonary vascular remodeling by releasing a variety of factors that stimulated PA-SMC growth (14). Thus, an important issue is the acquired specialized functional phenotype of accumulated lung macrophages in PH, which may depend on environmental signals. We therefore looked at macrophage subtype markers in whole lungs from normoxic and hypoxic mice. We found that compared with normoxia, hypoxia was associated with higher levels of both M1 and M2 macrophage markers, but with a predominance of M2 markers. These results are therefore consistent with those previously described by Vergadi et al (14) and support a preferential shift to the M2 macrophage phenotype, which may contribute to PH.

In our study, clear effects of CX3CR1 deletion during chronic hypoxia were to increase lung biomarkers for M1 macrophages and, simultaneously, to down-regulate biomarkers for M2 macrophages. These changes in macrophage polarization were seen in lungs from CX3CR1−/− mice, but not in those from CCL2−/− or CX3CR1−/−/CCL2−/− mice. Thus, a major consequence of CX3CR1 deficiency was to alter the polarized activation of lung macrophages during chronic hypoxia, shifting the balance toward the M1 phenotype at the expense of the M2 phenotype. This change may be of great importance, as we found that conditioned medium from M2 macrophages strongly stimulated PA-SMC growth compared with conditioned medium from M1 macrophages. Thus, CX3CR1 deficiency led to major changes in macrophage polarization and simultaneously affected PH development. In contrast, combined CX3CR1 and CCL2 deficiency affected neither macrophage polarization nor the development of PH. Differentiation into the M1 or M2 macrophage phenotype therefore appeared linked to the pulmonary vascular remodeling process. An additional effect of CX3CR1 was to mediate part of the proliferation-inducing effect of M2 macrophages, which was markedly reduced in PA-SMCs from CX3CR1−/− or CX3CR1−/−/CCL2−/− mice. Moreover, the amount of CX3CL1 and the PA-SMC growth-stimulating effect of conditioned media from alveolar macrophages derived from CX3CR1−/− mice was markedly reduced. CX3CR1 inactivation may therefore alter pulmonary vascular remodeling through various mechanisms, including lack of a PA-SMC growth response to CX3CL1, change in macrophage phenotype from M2 to M1, and reduction in CX3CL1 release by alveolar macrophages.

The finding that CX3CR1 is an important signaling pathway involved in differentiation into M1 or M2 macrophages is consistent with results from previous studies. Indeed, CX3CR1 ablation shifted tumor-associated macrophages toward M1 polarization while suppressing tumor growth (10). Moreover, in recent studies, CX3CR1 affected muscle repair by altering macrophage function, but not macrophage infiltration (23). CX3CR1 may therefore affect macrophage function through multiple mechanisms that may alter pulmonary vascular remodeling. Thus, targeting CX3CR1 might constitute a powerful tool to combat the development and progression of PH. To investigate whether pharmacological CX3CR1 inactivation had similar effects to those of gene deletion in mice, we treated hypoxia-exposed WT mice with a CX3CR1 inhibitor (18). We found that this compound selective for CX3CR1 suppressed PA-SMC growth induced by CX3CL1, but not platelet-derived growth factor, in vitro. Moreover, in WT mice exposed to chronic hypoxia, chronic F1 treatment led to a significant reduction in PH severity and, more important, was associated with increased counts of perivascular macrophages. Therefore, genetic and pharmacological CX3CR1 inactivation led to similar protective effects on PH via similar mechanisms. Taken together, these results strongly suggest that targeting the CX3CR1 chemokine axis might be a useful therapeutic approach for controlling the complex effects of monocytes-macrophages on the development and progression of PH. New trials are now underway to evaluate the efficacy of immunomodulatory treatments in various types of human PAH (24). Targeting CX3CR1 holds promise in this setting.

The authors are grateful to R. Souktani from the Plateforme Exploration Fonctionnelle du Petit Animal, as well as X. Decrouy and C. Micheli from the imaging platform facility; Institut Mondor de Recherche Biomédicale Inserm, Créteil, France.

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Correspondence and requests for reprints should be addressed to Serge Adnot, M.D., Ph.D., Hôpital Henri Mondor, Service de Physiologie-Explorations Fonctionnelles, 94010, Créteil, France. E-mail:

This study was supported by grants from the INSERM, Délégation à la Recherche Clinique de l’Assistance Publique–Hôpitaux de Paris, Fondation pour la Recherche Médicale, Chancellerie des Universités de Paris (Legs Poix), and Coeur et Poumon foundation. C.C. receives research grants from CNRS and Université Pierre et Marie Curie Emergence.

Author Contributions: Conception or design of the study: V.A., C.C., and S. Adnot; acquisition, analysis, or interpretation of data: V.A., S. Abid, L.P., A.P., M.R., G.G.-B., M.L., and L.L.; drafting the manuscript or critical revision for important intellectual content; V.A., J.-L.D.-R., C.C., and S. Adnot; and final approval of the version submitted for publication: all authors.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2016-0201OC on January 26, 2017

Author disclosures are available with the text of this article at www.atsjournals.org.

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