View Abstract

A1333 - Corticosteroid Insensitivity of Monocyte-Derived Macrophages from Severe Neutrophilic Asthmatics Is Not Associated with Differentiation Bias
Author Block: N. Dauletbaev1, A. Dintakurti2, K. G. Tantisira3, P. Castaldi4, C. Fugere5, R. Olivenstein5, L. C. Lands6; 1Research Institute, McGill University Health Centre, Montreal, QC, Canada, 2McGill University, Montreal, QC, Canada, 3Brigham and Womens Hosp, Boston, MA, United States, 4Channing Division of Network Medicine, Boston, MA, United States, 5Montreal Chest Institute, Montreal, QC, Canada, 6Montreal Childrens Hospital-McGill University Health Centre, Montreal, QC, Canada.
Rationale: Severe corticosteroid-insensitive asthma is a significant burden to both patients and health care system. For some patient cohorts, such as severe neutrophilic asthma, no effective therapies exist due to deficient mechanistic understanding. Macrophages are key immune cells that may define corticosteroid insensitivity in these patients. It is often speculated that macrophage corticosteroid sensitivity may be linked to the differentiation status of these cells. Here we hypothesized that in Monocyte-Derived Macrophages (MDM) from patients with severe neutrophilic asthma, diminished corticosteroid sensitivity would be associated with altered macrophage differentiation. Methods: MDM (two healthy controls, two severe neutrophilic asthmatics) were obtained by Ficoll-Paque isolation, negative monocyte purification (EasySep Monocyte Purification kit; StemCell Technologies), and a 14-day culture in RPMI1640 (10% fetal bovine serum, 40 ng/ml Monocyte Colony-Stimulation Factor, M-CSF, PeproTech) on 48-well plates. This culture condition constituted the basal MDM phenotype, M(M-CSF). Parallel M(M-CSF) were differentiated for 18 hours. Specifically, M(M-CSF) exposure to 20 ng/ml Interferon (IFN)-ү (PeproTech) + 100 ng/ml ultrapure Lipopolysaccharide (LPS; Invivogen) yielded a classically activated phenotype (M(IFN-ү/LPS)), whereas exposure to 20 ng/ml Interleukin (IL)-4 yielded an alternatively activated phenotype (M(IL-4)). Supernatants of M(M-CSF) and two differentiated phenotypes were collected for multiplex ELISA, while cells were lysed with Lysis/Binding Buffer (MagMax 96 RNA isolation kit; Thermo Fisher). One-hundred and fifty ng of RNA were subjected to RNAseq for differential gene expression or to droplet digital PCR for confirmatory studies. To verify corticosteroid sensitivity, MDM were treated for 2 hours with increasing doses of budesonide. Corticosteroid sensitivity was judged by the Half Maximum Effective Dose (EC50) of budesonide, based on expression of the corticosteroid sensitivity biomarker FKBP5. Results: In both healthy and asthmatic MDM, differentiation followed an expected pattern. Specifically, differentiation to M(IFN-ү/LPS) led to >2 times higher number of modulated genes than the alternative differentiation to M(IL-4). Furthermore, patterns of both classical and alternative differentiation were comparable between healthy controls and severe asthmatics (Principal Component Analysis of RNAseq and droplet digital PCR results, and multiplex ELISA). Yet all three phenotypes of severe asthmatic MDM required higher corticosteroid doses to upregulate the corticosteroid sensitivity biomarker FKBP5 (e.g., EC50 of 19.3 * 10-9 M in severe asthmatic M(IFN-ү/LPS) vs. 0.88 * 10-9 M in healthy M(IFN-ү/LPS)). Conclusion: Our preliminary data indicate that MDM from severe neutrophilic asthmatics exhibit a corticosteroid-insensitive phenotype without an obvious differentiation bias. A study with higher MDM numbers is underway. ND and AD: co-first authors