Poster Presentation: Yuqing Deng

ABSTRACT

Alveolar collapse and emphysema-associated agents accelerate neutrophil migration in mouse precision-cut lung slices

Yuqing Deng1, Byungjun Kang2, Linzheng Shi2, Kathryn Regan2, Adam Kobayter2, Gabrielle N. Grifno2, Kenneth R. Lutchen2, William Boley1, Elizabeth Bartolák-Suki2, Béla Suki2, and Hadi T. Nia2

Departments of 1Mechanical Engineering; 2Biomedical Engineering, Boston University

Introduction: Precision-cut lung slices (PCLS) preserve the native extracellular structure and cellular diversity, enabling observation of cellular responses to various disease-modifying conditions. Emphysema, a key component of chronic obstructive pulmonary disease and primarily induced by cigarette smoke, involves neutrophil recruitment and collagenase release compromising alveolar wall mechanics. Mechanical forces on the weakened tissue lead to rupture and airspace enlargement which promotes regional collapse around enlarged and stretched alveoli. These structural changes may in turn regulate how neutrophils migrate within the lung’s microenvironment, yet this relationship has not been studied. We developed a mouse PCLS model in which mechanically collapsed and stretched regions were created within the same PCLS and studied neutrophil migration in the presence or absence of collagenase and cigarette smoke extract (CSE).

Methods: Mice received lipopolysaccharide (i.p.) 6 h before euthanasia to recruit neutrophils to the lung, and fluorescent anti–Ly6G antibody was administered retro-orbitally 3.5 h before euthanasia to label neutrophils. The lung was inflated at 37 °C with 2 mL warm gelatin, then sectioned into 250 μm PCLS. PCLS were placed in a 12-well plate with custom 3D-printed needles that secured the PCLS to the bottom of the plate. The medium was supplemented with: (i) collagenase (0.5mg/ml), (ii) CSE (0.01 cig/ml), (iii) ROCK inhibitor Y-27632 (10uM), or (iv) TRPV4 inhibitor GSK219 (4.6uM). Incubation at 37°C melted the gelatin within the PCLS, creating spatially collapsed and stretched (non-collapsed needle-supported) regions within the same PCLS, which were then used for live imaging of neutrophil migration.

Results and conclusions: Collagenase and cigarette smoke extract (CSE) increased neutrophil migration speed in both collapsed and stretched regions. In control PCLS, neutrophils migrated faster in collapsed regions, and the difference between speeds on collapsed and stretched tissue was nearly eliminated by ROCK or TRPV4 inhibition. These results suggest that local mechanics and emphysema-associated agents jointly regulate neutrophil motility and have implications for emphysema progression through neutrophil migration and activation.