Diminished airway macrophage expression of the Axl receptor tyrosine kinase is associated with defective efferocytosis in asthma
Authors: Aleksander M. Grabiec, Nicholas Denny, John A. Doherty, Kaisa E. Happonen, Jenny Hankinson, Emma Connolly, Mark E. Fife, Toshifumi Fujimori, Naoya Fujino, Anu Goenka, Susan Holden, Gaël Tavernier, Rajesh Shah, Peter C. Cook, Andrew S. MacDonald, Robert M. Niven, Björn Dahlbäck, Stephen J. Fowler, Angela Simpson, Tracy Hussell
Journal: Journal of Allergy and Clinical Immunology (2017)

Up to 15% of the 300 million people affected by asthma worldwide suffer from disease refractory to available medications and associated with significant morbidity, particularly due to frequent exacerbations secondary to respiratory viral infections. Airway macrophages from patients with severe asthma are known to be defective in apoptotic cell clearance.1Fernandez-Boyanapalli R. Goleva E. Kolakowski C. Min E. Day B. Leung D.Y. et al.Obesity impairs apoptotic cell clearance in asthma.J Allergy Clin Immunol. 2013; 131 (1047.e1-3): 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 2Huynh M.L. Malcolm K.C. Kotaru C. Tilstra J.A. Westcott J.Y. Fadok V.A. et al.Defective apoptotic cell phagocytosis attenuates prostaglandin E2 and 15-hydroxyeicosatetraenoic acid in severe asthma alveolar macrophages.Am J Respir Crit Care Med. 2005; 172: 972-979Crossref PubMed Scopus (122) Google Scholar, 3Simpson J.L. Gibson P.G. Yang I.A. Upham J. James A. Reynolds P.N. et al.Impaired macrophage phagocytosis in non-eosinophilic asthma.Clin Exp Allergy. 2013; 43: 29-35Crossref PubMed Scopus (86) Google Scholar Because phagocytosis of apoptotic cells, called efferocytosis, prevents secondary necrosis and the release of proinflammatory necrotic cell debris, defects in apoptotic cell uptake contribute to perpetuation of lung inflammation.4Grabiec A.M. Hussell T. The role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation.Semin Immunopathol. 2016; 38: 409-423Crossref PubMed Scopus (101) Google Scholar Although several processes have been implicated in impaired efferocytosis in asthma, the underlying mechanisms and the links between apoptotic cell clearance, respiratory infections, and disease exacerbations remain unknown. TAM (Tyro3, Axl, MerTK) receptor tyrosine kinases mediate apoptotic cell uptake by phagocytes through the bridging molecules Protein S or growth arrest specific 6 (Gas6), and are critical for lung immune homeostasis.4Grabiec A.M. Hussell T. The role of airway macrophages in apoptotic cell clearance following acute and chronic lung inflammation.Semin Immunopathol. 2016; 38: 409-423Crossref PubMed Scopus (101) Google Scholar We and others have shown that Axl is specifically expressed on mouse airway macrophages, is constitutively bound to its ligand Gas6, and is critical for efficient efferocytosis.5Fujimori T. Grabiec A.M. Kaur M. Bell T.J. Fujino N. Cook P.C. et al.The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.Mucosal Immunol. 2015; 8: 1021-1030Crossref PubMed Scopus (73) Google Scholar, 6Schmid E.T. Pang I.K. Carrera Silva E.A. Bosurgi L. Miner J.J. Diamond M.S. et al.AXL receptor tyrosine kinase is required for T cell priming and antiviral immunity.Elife. 2016; 5: e12414Crossref PubMed Google Scholar Axl-deficient mice develop exaggerated lung inflammation upon influenza infection associated with accumulation of apoptotic cells in the airways,5Fujimori T. Grabiec A.M. Kaur M. Bell T.J. Fujino N. Cook P.C. et al.The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.Mucosal Immunol. 2015; 8: 1021-1030Crossref PubMed Scopus (73) Google Scholar indicating a critical role for Axl in apoptotic cell clearance from the inflamed lung. Here, we sought to define the TAM receptor expression profile in human airway macrophages, characterize the local factors driving their expression, and identify potential defects in TAM receptors contributing to defective efferocytosis in asthma. We first performed mRNA expression profiling of TAM receptors in peripheral blood monocytes, monocyte-derived macrophages (MDMs), and airway macrophages isolated from sputum samples obtained from 12 healthy donors (HDs) (for details of experimental procedures and study cohort characteristics, see this article's Online Repository at www.jacionline.org). TAM receptors displayed a strong cell type–specific expression pattern: MerTK was preferentially expressed in MDMs, but not monocytes or airway macrophages, whereas Axl expression was restricted to airway macrophages (Fig 1, A). Using macrophages from the healthy margins of lung resections (which contain both airway and alveolar macrophages), we confirmed that only airway/alveolar macrophages constitutively expressed high levels of Axl protein, while MDMs were strongly MerTK-dominant (Fig 1, B and C). Apoptotic cell recognition by Axl requires the bridging molecule Gas6 and, similar to mouse models,5Fujimori T. Grabiec A.M. Kaur M. Bell T.J. Fujino N. Cook P.C. et al.The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.Mucosal Immunol. 2015; 8: 1021-1030Crossref PubMed Scopus (73) Google Scholar we detected Gas6 on most airway/alveolar macrophages (see Fig E1 in this article's Online Repository at www.jacionline.org). These data reveal selective Axl expression on human airway/alveolar macrophages and constitutive binding of Gas6 rendering these cells primed for apoptotic cell recognition through Axl. We next investigated why Axl is exclusively expressed on airway/alveolar macrophages. Axl mRNA and protein expression was significantly reduced during ex vivo culture of HD airway/alveolar macrophages from lung resections (Fig 1, D and E), suggesting that these cells may require the airway microenvironment to maintain their unique TAM receptor expression profile. To identify factors that drive Axl expression, we stimulated airway/alveolar macrophages with factors known to affect their activation during infection and chronic inflammation. Strikingly, only IFNs significantly restored Axl mRNA expression during ex vivo culture (Fig 1, F). Restoration of Axl expression by IFNs, in particular IFN-α and IFN-λ, was also evident at the protein level (Fig 1, G). These findings indicate that homeostatic levels of IFNs or their sustained production by bronchial epithelial cells following previous viral infections drive selective Axl expression and regulate the efferocytic function of human airway/alveolar macrophages. Previous studies have shown clear defects in efferocytosis in severe asthma.1Fernandez-Boyanapalli R. Goleva E. Kolakowski C. Min E. Day B. Leung D.Y. et al.Obesity impairs apoptotic cell clearance in asthma.J Allergy Clin Immunol. 2013; 131 (1047.e1-3): 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 2Huynh M.L. Malcolm K.C. Kotaru C. Tilstra J.A. Westcott J.Y. Fadok V.A. et al.Defective apoptotic cell phagocytosis attenuates prostaglandin E2 and 15-hydroxyeicosatetraenoic acid in severe asthma alveolar macrophages.Am J Respir Crit Care Med. 2005; 172: 972-979Crossref PubMed Scopus (122) Google Scholar, 3Simpson J.L. Gibson P.G. Yang I.A. Upham J. James A. Reynolds P.N. et al.Impaired macrophage phagocytosis in non-eosinophilic asthma.Clin Exp Allergy. 2013; 43: 29-35Crossref PubMed Scopus (86) Google Scholar On the basis of our observations, we hypothesized that alterations in Axl may explain these defects. The extracellular domain of Axl is cleaved upon engagement, and alterations in soluble Axl (sAxl) levels have been reported in inflammatory conditions.7Ko C.P. Yu Y.L. Hsiao P.C. Yang S.F. Yeh C.B. Plasma levels of soluble Axl correlate with severity of community-acquired pneumonia.Mol Med Rep. 2014; 9: 1400-1404Crossref PubMed Scopus (11) Google Scholar We compared the levels of sAxl in serum and sputum from HDs and patients with moderate-to-severe asthma (British Thoracic Society steps 3-5). Although no difference in sAxl was detected in serum samples between the 2 groups (Fig 2, A), the levels of sAxl in sputum samples were significantly elevated in patients with asthma compared with controls (Fig 2, B), suggesting increased Axl shedding at the site of inflammation. Next, we compared Axl and MerTK mRNA expression in peripheral blood monocytes, MDMs, and airway macrophages in sputum from patients with moderate-to-severe asthma and HDs. Similar levels of MerTK were expressed in both groups in all analyzed cell types (Fig 2, C), whereas Axl expression was significantly reduced in airway macrophages from patients with asthma (P < .001). The observed difference was restricted to the site of inflammation as monocytes and MDMs from asthma patients and HDs expressed comparable, albeit low, levels of Axl (Fig 2, D). To test the functional significance of reduced Axl expression in asthma, we determined the amounts of DNA-histone complexes (nucleosomes), a hallmark of necrosis and secondary necrosis of apoptotic cells,5Fujimori T. Grabiec A.M. Kaur M. Bell T.J. Fujino N. Cook P.C. et al.The Axl receptor tyrosine kinase is a discriminator of macrophage function in the inflamed lung.Mucosal Immunol. 2015; 8: 1021-1030Crossref PubMed Scopus (73) Google Scholar in the airways of the same patients. Extracellular nucleosome levels were elevated in sputum from patients with moderate-to-severe asthma compared with HDs (Fig 2, E), and negatively correlated with airway macrophage Axl expression (Fig 2, F; R = −0.71; P < .0001). These results indicate that Axl is the main apoptotic cell recognition receptor expressed on human airway macrophages and reduced Axl expression and/or increased shedding might perpetuate chronic inflammation in asthma. In conclusion, we report for the first time that (1) the apoptotic cell recognition receptor Axl is exclusively expressed by human airway/alveolar macrophages, (2) Axl expression depends on the presence of type I/III IFNs, (3) increased shedding of sAxl occurs in sputum from patients with moderate-to-severe asthma, and (4) Axl mRNA expression in airway macrophages isolated from sputum is reduced in patients with moderate-to-severe asthma. These findings have important implications for those patients at the most difficult-to-treat end of the asthma disease spectrum, particularly in light of recent reports demonstrating the role of altered IFN responses during respiratory infections in asthma.8Contoli M. Message S.D. Laza-Stanca V. Edwards M.R. Wark P.A. Bartlett N.W. et al.Role of deficient type III interferon-lambda production in asthma exacerbations.Nat Med. 2006; 12: 1023-1026Crossref PubMed Scopus (859) Google Scholar Our results suggest that strategies aimed at restoring or activating Axl could be beneficial in patients with asthma by facilitating apoptotic cell clearance. This idea is supported by a recent clinical trial where inhaled type I IFN prevented viral infection–induced exacerbation of disease symptoms only in patients with severe asthma.9Djukanovic R. Harrison T. Johnston S.L. Gabbay F. Wark P. Thomson N.C. et al.The effect of inhaled IFN-beta on worsening of asthma symptoms caused by viral infections: a randomized trial.Am J Respir Crit Care Med. 2014; 190: 145-154Crossref PubMed Scopus (202) Google Scholar In light of our observation that IFNs restore Axl expression on airway macrophages, the beneficial effects of type I IFN might be at least partly attributed to improved efferocytic function and prevention of secondary necrosis. Collectively, data from this study provide evidence that Axl is an important regulator of lung immune homeostasis and identify a novel pathway that could be targeted to improve defective efferocytosis in asthma. We acknowledge the Manchester Allergy, Respiratory and Thoracic Surgery Biobank and the North West Lung Centre Charity for supporting this project. In addition, we thank the study participants for their contribution. Paired peripheral blood and sputum samples (spontaneous or induced by using hypertonic saline) were collected from 15 healthy individuals and 46 patients with moderate-to-severe asthma fulfilling the British Thoracic Society criteria (disease steps 3-5).E1British Guideline on the Management of Asthma.Thorax. 2008; 63: iv1-iv121Crossref PubMed Scopus (456) Google Scholar Clinical characteristics of patients are presented in Table E1. For some experiments, marginal noncancerous lung tissue was collected from patients undergoing surgery for suspected or confirmed cancer (n = 15). Patients included in this study did not have a history of any chronic inflammatory lung disease and had normal lung function (median [interquartile range] FEV1/forced vital capacity ratio, 0.74 [0.70-0.79]). All samples were collected at the University Hospital of South Manchester/North West Lung Centre under the framework of the Manchester Allergy, Respiratory and Thoracic Surgery Biobank (study nos. M2014-17 and M2015-32) with ethical approval of the National Research Ethics Service (REC reference 10/H1010/7). All patients provided written informed consent before participation in the study. Peripheral blood from healthy individuals (leukocyte cones) was also obtained from the National Blood Transfusion Service (Manchester, UK) with ethical approval by the University of Manchester ethics committee. Lung tissue was perfused with PBS. The retrieved cell suspension was centrifuged at 400g for 10 minutes, and the cell pellet was resuspended in PBS and subjected to Ficoll-Paque (GE Healthcare, Buckinghamshire, United Kingdom) density gradient centrifugation to remove granulocytes and erythrocytes. The interface of mononuclear cells, containing predominantly airway/alveolar macrophages, was collected and washed in PBS, and viable macrophages were counted using trypan blue exclusion and plated at 1 × 106 cells/mL in RPMI 1640 supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine (complete medium) for 1 hour at 37°C for adherence. Nonadherent cells were removed by extensive washing and airway/alveolar macrophages were either lysed immediately or stimulated with M-CSF (50 ng/mL), GM-CSF (50 ng/mL), IL-4 (20 ng/mL), IL-10 (20 ng/mL), IFN-γ (50 ng/mL), IFN-λ (100 ng/mL) (all from Peprotech, Rocky Hill, NJ), IFN-α (1000 U/mL) (R&D Systems, Abingdon, United Kingdom), or dexamethasone (Dex, 1 μM) (Sigma-Aldrich, Dorset, United Kingdom) for 24 hours. A 90% or more purity of airway macrophages was routinely achieved with this isolation method.E2Kent L.M. Fox S.M. Farrow S.N. Singh D. The effects of dexamethasone on cigarette smoke induced gene expression changes in COPD macrophages.Int Immunopharmacol. 2010; 10: 57-64Crossref PubMed Scopus (19) Google Scholar To purify airway macrophages from sputum, samples were processed as previously described.E3Pizzichini E. Pizzichini M.M. Efthimiadis A. Hargreave F.E. Dolovich J. Measurement of inflammatory indices in induced sputum: effects of selection of sputum to minimize salivary contamination.Eur Respir J. 1996; 9: 1174-1180Crossref PubMed Scopus (243) Google Scholar Supernatants were stored at −80°C and cells were incubated with 50 μg/mL DNase (Roche Diagnostics, Mannheim, Germany) for 30 minutes at 37°C, washed with PBS, and plated in 24-well plates in complete medium. After 1-h adherence, cells were extensively washed to remove nonadherent cells and airway macrophages were lysed in RLT buffer (Qiagen, Manchester, United Kingdom) before RNA extraction. PBMCs were isolated by Ficoll-Paque density gradient centrifugation. Monocytes were then purified by magnetic-activated cell sorting using human CD14 MicroBeads (Miltenyi Biotec, Surrey, United Kingdom), plated at 5 × 105 cells/mL in complete medium containing 50 ng/mL M-CSF, and cultured for 6 days before further processing. Total RNA was isolated using RNeasy micro kit (Qiagen), quantified with a Nanodrop spectrophotometer (Thermo Scientific, Waltham, Mass) and equivalent amounts of RNA were reverse transcribed using High-Capacity RNA-to-cDNA Kit (Life Technologies, Paisley, United Kingdom). Quantitative PCR reactions were performed using TaqMan expression assays and TaqMan Fast Universal PCR Master Mix on a QuantStudio 12K Flex PCR system and the obtained data were analyzed using QuantStudio 12K Flex Software (all from Life Technologies). Average expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and RPLP0 was used to calculate relative mRNA expression of Axl and MerTK. Protein lysates were prepared in lysis buffer containing 1% Igepal CA-630, 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, and protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Scientific) and equivalent amounts of protein were resolved by electrophoresis on 4% to 15% mini-Protean TGX precast gels (BIO-RAD, Hercules, Calif). Proteins were then transferred to nitrocellulose membranes (BIO-RAD) using a semi-dry Trans-Blot Turbo transfer system (BIO-RAD). Membranes were washed in PBS containing 0.05% Tween-20 (Sigma-Aldrich) (PBS/T), blocked in 2% milk (BIO-RAD) in PBS/T, and incubated o/n at 4°C with antibodies recognizing MerTK (D21F11, Cell Signaling Technology, Beverly, Mass), Axl (M20), or actin (I19-R) (both from Santa Cruz Biotechnology, Santa Cruz, Calif) diluted in PBS/T containing 1% BSA. After washing in PBS/T, membranes were incubated with horseradish peroxidase–conjugated antirabbit, antimouse, or antigoat immunoglobulin antibodies (Dako, Cambridge, United Kingdom), developed using a Clarity Western ECL Substrate (BIO-RAD) and visualized with a ChemiDoc MP Imaging System (BIO-RAD). Alternatively, Axl and MerTK protein levels were analyzed in cell lysates using total Axl and MerTK ELISA kits (R&D Systems) as per the manufacturer's instructions and the data were presented as ng/mg total protein in the lysate. Cells were stained with Zombie UV Flexible Viability dye (BioLegend, San Diego, Calif) for 20 minutes followed by incubation with Human TruStain FcX Fc receptor blocking solution (BioLegend) for 10 minutes. Cells were then washed with FACS buffer (PBS containing 1% FCS and 2 mM EDTA) and stained with PE/Cy7-conjugated anti-CD64 (clone 10.1, BioLegend) and biotinylated anti-Gas6 (BAF885) or control biotinylated goat IgG (BAF108) antibodies (all from R&D Systems) for 20 minutes at 4°C. After washing with FACS buffer, cells were incubated with BV421-conjugated streptavidin (BioLegend) for 20 minutes at 4°C, washed, and fixed by 10-min incubation with 2% paraformaldehyde in PBS. Cells were acquired on a BD LSRFortessa (BD Biosciences, San Jose, Calif) and data were analyzed using FlowJo software (TreeStar, Ashland, Ore). Airway/alveolar macrophages were defined as FSChiSSChiCD64+ and highly autofluorescent. The strategy using autofluorescence to define airway/alveolar macrophages was described before.E4Baharom F. Thomas S. Rankin G. Lepzien R. Pourazar J. Behndig A.F. et al.Dendritic cells and monocytes with distinct inflammatory responses reside in lung mucosa of healthy humans.J Immunol. 2016; 196: 4498-4509Crossref PubMed Scopus (48) Google Scholar, E5Desch A.N. Gibbings S.L. Goyal R. Kolde R. Bednarek J. Bruno T. et al.Flow cytometric analysis of mononuclear phagocytes in nondiseased human lung and lung-draining lymph nodes.Am J Respir Crit Care Med. 2016; 193: 614-626Crossref PubMed Scopus (117) Google Scholar Levels of nucleosomes in sputum samples were determined using the Cell Death Detection ELISA Plus (Roche) according to the manufacturer's instructions. To measure sAxl in serum samples, Maxisorp plates (Nunc, Roskilde, Denmark) were coated o/n with 500 ng/mL goat anti-Axl antibody (AF154, R&D Systems) in PBS. Plates were washed with wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and blocked in 3% fish gelatin (Norland Products Inc, Cranbury, NJ) diluted in wash buffer for 2 hours at room temperature (RT). Serum was diluted 1:10 in blocking buffer and incubated on the plates for 2 hours at 37°C. After washing, plates were incubated with polyclonal rabbit anti-Axl antibody (in-house, Ab042) diluted in blocking buffer, followed by incubation with goat antirabbit horseradish peroxidase conjugate (P0448, Dako) for 1 hour at RT. Plates were developed using an OPD-substrate (Dako, S2045) and the absorbance was measured using an Infinite 200 microplate reader (Tecan, Männedorf, Switzerland). To detect sAxl in sputum, samples from patients and controls (600 μL) or an equal volume of standard solution containing 1, 5, or 10 ng/mL sAxl was mixed with 1 mL PBS. sAxl standard was expressed and purified as described previously.E6Ekman C. Stenhoff J. Dahlback B. Gas6 is complexed to the soluble tyrosine kinase receptor Axl in human blood.J Thromb Haemost. 2010; 8: 838-844Crossref PubMed Scopus (77) Google Scholar The samples were precleared with 30 μL Protein G sepharose (Life Technologies) for 30 minutes at 4°C after which the beads were removed by centrifugation at 2000g for 5 minutes. One microgram anti-Axl antibody (AF154, R&D Systems) was added together with 30 μL Protein G sepharose to the supernatant and samples were incubated o/n at 4°C under rotation. The beads were then washed twice with PBS and incubated for 5 minutes in reducing Laemmli buffer at 95°C. Supernatants were separated on a 4% to 15% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, and probed against Axl (in-house, Ab042). Blots were analyzed using the ImageLab software and the intensities of the bands corresponding to sAxl were quantified. Normalization was performed on each blot by setting the signal of the 5 ng/mL sAxl standard solution IP reaction to 1. In all cases, samples from patients and controls were run in parallel on each blot to minimize interblot variation. Data are presented as mean ± SEM (Fig 1, B, F, and G), as boxplots with the line within the box denoting the median, boxes representing 25th and 75th percentiles, and the lines outside the box marking the minimum and maximum values (Fig 2, A and B), or as symbols representing data from individual donors. Nonparametric tests were used for comparisons between patient groups or cell types and for in vitro studies of airway macrophages: Kruskal-Wallis test followed by Dunn's multiple comparison test (Fig 1, A and B), Wilcoxon signed rank test (Fig 1, D), Friedman test followed by Dunn's multiple comparison test (Fig 1, F), or Mann-Whitney U test (Fig 2, A-E). The Spearman correlation coefficient was used for correlation analyses. P values of less than .05 were considered statistically significant.Table E1Clinical features of patients with severe asthma and healthy subjects included in the studyFeatureModerate-to-severe asthma (n = 46)Healthy control (n = 15)Sex: male/female19/274/11Age (y)55 (45-63)37.2 (30-46)Smoking history Never2111 Ex184 Current70Asthma severity BTS step 38 BTS step 412 BTS step 526FEV1 (L)2.14 (1.63-2.73)NAFEV1 % predicted77 (64-94)NAFVC (L)3.10 (2.63-3.69)NAFVC % predicted93 (83-109)NAFEV1/FVC ratio0.7 (0.59-0.79)NAThe values are expressed as counts or median (interquartile range).BTS, British Thoracic Society; FVC, forced vital capacity; NA, not applicable/available. Open table in a new tab The values are expressed as counts or median (interquartile range). BTS, British Thoracic Society; FVC, forced vital capacity; NA, not applicable/available.