PB1429 - NETs promote gasdermin-D-dependent pulmonary thrombosis in mice exposed to cigarette smoke and flu.

Background: Flu is an acute respiratory disease primarily caused by the influenza-A virus (IAV). Flu typically causes self-resolving upper-airway inflammation, however, it may progress for unknown reasons into highly morbid acute lung injury (ALI). Epidemiological evidence suggests that cigarette smoking(CS) is major risk for mortality among flu patients, however, mechanisms by which CS enhances flu severity remains poorly understood. Emerging evidence suggests that upregulation of immune pathways in neutrophils and platelets correlates with flu severity, but how prior CS-exposure primes innate-immunity to promote flu remains unknown.

Aims: To determine whether Gasdermin-D-(GSDMD)-dependent generation of neutrophil-extracellular-traps (NETs) promotes pulmonary thrombosis and severe ALI in mice exposed to CS and flu.

Methods: We established two-hit mouse model of 4-week CS-exposure and subsequent mild-dose of intranasal IAV-infection. Severity of lung injury was evaluated using histology scoring and oxygen saturation measurements. Real-time intravital (in vivo) multi-photon-excitation(MPE) fluorescence microscopy of the lung was used to assess the role of thrombo-inflammation in promoting ALI in two-hit mice. Neutrophils were isolated at day 4th post-IAV infection and western blots/qPCR were performed to assess the activation of GSDMD-pathway.

Results: Mild dose of IAV infection led to severe lung injury in mice pre-exposed to CS but not room air. Intravital lung microscopy revealed that severe lung injury in CS+Flu than Flu-only mice was associated with significantly higher accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) and NETs in lung microcirculation, leading to larger areas with pulmonary thrombosis and vascular leakage in lung of CS+Flu than Flu-only mice. Remarkably, western blot analysis revealed the activation of GSDMD-pathway in circulating neutrophils of CS+Flu but not Flu-only mice.

Conclusion(s): Our findings suggest that prior exposure to CS augments flu severity in mice by triggering sequestration of platelet-rich NPAs in the lung microcirculation followed by GSDMD-dependent NETs generation, leading to the development of pulmonary thrombosis in large areas of the lung.