Abstract Introduction Precision-cut lung slices (PCLS) are a valuable tool in translational respiratory research. PCLS preserve the native extracellular structure and cellular diversity, enabling observation of cellular responses to various disease-modifying conditions. Emphysema is a key component of chronic obstructive pulmonary disease (COPD), primarily induced by cigarette smoke exposure. During emphysema progression, recruited neutrophils release collagenase in the lung, compromising mechanics of alveolar walls. 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. In this study, we developed a novel mouse PCLS model (mPCLS) in which mechanically collapsed and stretched regions were created within the same PCLS and studied neutrophil migration in the presence or absence of cigarette smoke extract (CSE). Methods Mice received lipopolysaccharide (LPS) intraperitoneally 6h 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 thick PCLS. PCLS were placed in a 12-well plate with custom 3D-printed needles that secured the PCLS to the bottom of the plate (Fig. 1d, top row). The medium was supplemented with: (i) CSE (0.01 cig/ml) or (ii) TRPV4 inhibitor GSK219 (4.6uM). Slices were subsequently incubated at 37 °C to allow the gelatin within the PCLS to melt, thereby creating spatially collapsed and stretched, outside and inside the needle-supported regions, respectively within the same PCLS, which were then used for live imaging of neutrophil migration (Fig. 1d). Results and Conclusions Migration in CSE treated PCLS is faster compared to the control (Fig. 1a and 1b). In both control and CSE-treated groups, neutrophils migrated faster in collapsed regions after 4 hours of alveolar collapse (p = 0.012 for control; p = 0.001 for CSE-treated). Interestingly, this difference between speeds on collapsed and stretched tissue was nearly eliminated by TRPV4 inhibition (Fig. 1c). 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. Figure 1 Neutrophil migration speed in (a) Control, (b) CSE treated, and (c) TRPV4 inhibitor treated PCLS. (d) Gelatin-inflated PCLS setup with 3-D printed needles. Collapsed and Stretched region were generated from the same PCLS after 35min incubation under 37 °C. This abstract is funded by: DP2HL168562, Beckman Young Investigator Award, NSF CAREER Award
Deng et al. (Fri,) studied this question.