ALX receptor ligands define a biochemical endotype for severe asthma
Isabell Ricklefs, Ioanna Barkas, Melody G. Duvall, Manuela Cernadas, Nicole L. Grossman, Elliot Israel, Eugene R. Bleecker, Mario Castro, Serpil C. Erzurum, John V. Fahy, Benjamin M. Gaston, Loren C. Denlinger, David T. Mauger, Sally E. Wenzel, Suzy A. Comhair, Andrea M. Coverstone, Merritt L. Fajt, Annette T. Hastie, Mats W. Johansson, Michael C. Peters, Brenda R. Phillips, Bruce D. Levy, the National Heart Lung and Blood Institute’s Severe Asthma Research Program-3 Investigators
Isabell Ricklefs, Ioanna Barkas, Melody G. Duvall, Manuela Cernadas, Nicole L. Grossman, Elliot Israel, Eugene R. Bleecker, Mario Castro, Serpil C. Erzurum, John V. Fahy, Benjamin M. Gaston, Loren C. Denlinger, David T. Mauger, Sally E. Wenzel, Suzy A. Comhair, Andrea M. Coverstone, Merritt L. Fajt, Annette T. Hastie, Mats W. Johansson, Michael C. Peters, Brenda R. Phillips, Bruce D. Levy, the National Heart Lung and Blood Institute’s Severe Asthma Research Program-3 Investigators
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Clinical Research and Public Health Inflammation Pulmonology

ALX receptor ligands define a biochemical endotype for severe asthma

Abstract

BACKGROUND. In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). METHODS. ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). RESULTS. Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. CONCLUSIONS. Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. TRIAL REGISTRATION. Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov NCT01606826) FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology.

Authors

Isabell Ricklefs, Ioanna Barkas, Melody G. Duvall, Manuela Cernadas, Nicole L. Grossman, Elliot Israel, Eugene R. Bleecker, Mario Castro, Serpil C. Erzurum, John V. Fahy, Benjamin M. Gaston, Loren C. Denlinger, David T. Mauger, Sally E. Wenzel, Suzy A. Comhair, Andrea M. Coverstone, Merritt L. Fajt, Annette T. Hastie, Mats W. Johansson, Michael C. Peters, Brenda R. Phillips, Bruce D. Levy, the National Heart Lung and Blood Institute’s Severe Asthma Research Program-3 Investigators

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Figure 6

SAA and 15-epi-LXA4 signaling via ALX receptors regulates production of the neutrophil chemoattractant IL-8.

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SAA and 15-epi-LXA4 signaling via ALX receptors regulates production of ...
A549 human epithelial cells expressing human ALX receptors (A549hALX) were exposed to BALF from HD, NSA, or SA subjects (24 hours, 37°C, 5% CO2) and IL-8 levels were measured in the cell-free supernatant by ELISA (see Methods). (A) Mean levels of LXA4, 15-epi-LXA4, and SAA in BALF from n = 15 subjects used for A549hALX cell incubations are shown in stacked bar graphs and the relative proportions are noted in pie charts. (B) IL-8 production by A549hALX cells was measured after incubation with saline control or SAA (2.5 ng/ml or 10 ng/ml) for 24 hours. (C) IL-8 production by A549hALX cells was measured after 24 hours of exposure to BALF from HD (n = 3), NSA (n = 6), or SA (n = 5) and is expressed as an increase relative to saline controls. Incubations with BALF without an increase in IL-8 production relative to saline control were assigned a value of zero. (D) A549hALX epithelial cells were exposed to BALF from subjects with endogenous levels that were LXA4hiSAAlo (n = 5) or LXA4loSAAhi (n = 6) followed by exposure to exogenous 15-epi-LXA4 (100 nM). Percentage inhibition of IL-8 production after 15-epi-LXA4 exposure was calculated. *P < 0.05, ****P < 0.001 by (B) 1-way ANOVA, (C) Kruskal-Wallis test, and (D) Mann-Whitney test. ALX, airway lipoxin A4 receptor; LXA4, lipoxin A4; 15-epi-LXA4, 15-epimer lipoxin A4; SAA, serum amyloid A; HD, healthy donors; NSA, nonsevere asthma; SA, severe asthma; IL-8, interleukin-8; BALF, bronchoalveolar lavage fluid.