High-throughput quantitation of human neutrophil recruitment and functional responses in an air-blood barrier array

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Abstract
Dysregulated neutrophil recruitment drives many pulmonary diseases, but most preclinical screening methods are unsuited to evaluate pulmonary neutrophilia, limiting progress toward therapeutics. Namely, high-throughput therapeutic assays typically exclude critical neutrophilic pathophysiology, including blood-to-lung recruitment, dysfunctional activation, and resulting impacts on the air-blood barrier. To meet the conflicting demands of physiological complexity and high throughput, we developed an assay of 96-well leukocyte recruitment in an air-blood barrier array (L-ABBA-96) that enables in vivo-like neutrophil recruitment compatible with downstream phenotyping by automated flow cytometry. We modeled acute respiratory distress syndrome (ARDS) with neutrophil recruitment to 20 ng/mL epithelial-side interleukin 8 and found a dose-dependent reduction in recruitment with physiologic doses of baricitinib, a JAK1/2 inhibitor recently Food and Drug Administration-approved for severe Coronavirus Disease 2019 ARDS. Additionally, neutrophil recruitment to patient-derived cystic fibrosis sputum supernatant induced disease-mimetic recruitment and activation of healthy donor neutrophils and upregulated endothelial e-selectin. Compared to 24-well assays, the L-ABBA-96 reduces required patient sample volumes by 25 times per well and quadruples throughput per plate. Compared to microfluidic assays, the L-ABBA-96 recruits two orders of magnitude more neutrophils per well, enabling downstream flow cytometry and other standard biochemical assays. This novel pairing of high-throughput in vitro modeling of organ-level lung function with parallel high-throughput leukocyte phenotyping substantially advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.
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Funding Information
  • National Institutes of Health (R01HL136141)
  • Center for Tobacco Products (75F40122C00146)
  • U.S. Environmental Protection Agency (84045201)
  • College of Engineering, Georgia Institute of Technology (BME COVID Seed grant)
  • College of Engineering, Georgia Institute of Technology (Georgia Tech EPICenter)
  • National Institutes of Health (5T32EB006343-08)
  • National Institutes of Health (R01HL158058)
  • Cystic Fibrosis Foundation (TIROUV22G0)
  • National Institutes of Health (K24 NR018866)
  • National Institutes of Health (5K23HL151897-04)

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