The Roles Of Cytoskeletal Regulating Proteins On The Modulation Of Cortical Actin And The Impact On Neutrophil Deformation And Transit Through Model Pulmonary Capillary Constrictions

Neutrophils are vital for host defense, but dysregulation in the pulmonary microvessels can cause acute lung injury and lead to acute respiratory distress syndrome. Understanding neutrophil trafficking in the lung is critical for developing effective immunomodulatory therapies to treat these pathologies. Neutrophil trafficking in the lung is unique due to the diameter of the capillaries (5μM) being smaller than the neutrophils (8μM). This disparity results in the neutrophil having to deform to pass through the pulmonary capillaries. Additionally, neutrophils exposed to chemical stimuli develop cortical actin and stiffen, resulting in even greater neutrophil retention. To investigate the biochemistry of neutrophil stiffening, we use conditionally immortalized neutrophil progenitors that can be genetically manipulated and then differentiated into bona fide neutrophils. To quantify the effects of f-MLP and GM-CSF on the stiffness of nonadherent neutrophils, we use a microfluidic platform to measure the transit times through 5μm constrictions. The dimensions and force parameters were chosen to mimic those of the pulmonary capillary system. Our preliminary results show that the addition of f-MLP slows the rate of transit, resulting in a hazard ratio 0.7943 [95% CI 0.7135 to 0.8]) compared to the unstimulated control. GM-CSF has a similar impact on the rate with a hazard ratio 0.8070 [95% CI 0.7042 to 0.9248]). The most dramatic effect was with combining stimuli resulting in a hazard ratio of 0.5523 [95% CI 0.4799 to 0.6357]). Ultimately, we are interested in proteins that are known to regulate cytoskeletal organization. Early results show that f-MLP no longer induces stiffening, resulting in faster rates for vinculin-/- neutrophils; (∼ hazard ratio 1.109) and for HS1-/- (hematopoietic lineage cell-specific protein 1) neutrophils; (∼hazard ratio 1.412).