PB0829 - Gasdermin-D promotes pulmonary thrombosis in mice exposed to cigarette smoke and Flu.

Background: Flu, primarily caused by the influenza A virus (IAV), usually results in self-resolving upper airway inflammation but can progress to severe acute lung injury (ALI) for unknown reasons. Epidemiological evidence links cigarette smoking (CS) to increased morbidity and mortality in flu patients. However, the mechanisms through which CS promotes flu severity are poorly understood. Emerging evidence indicates that the upregulation of innate immune pathways in neutrophils and platelets correlates with flu severity.

Aims: To identify innate immune pathways in platelets and neutrophils promoting severe flu in mice pre-exposed to CS.

Methods: We have established a two-hit mouse model of 4-week CS exposure and subsequent mild-dose of intranasal IAV-infection. Severity of lung injury was evaluated using scoring of histology and drop in blood-oxygen saturation. Multi-photon-excitation enabled quantitative fluorescence intravital lung microscopy (qFILM) was used to assess thrombo-inflammation in the lungs of live mice. Neutrophils and platelets were isolated from blood of mice at day 4th post infection and used in western blotting or qPCR. Plasma was collected for image flow cytometry and ELISA.

Results: Lung injury and weight-loss was more severe in mice exposed to CS than room air (RA) prior to IAV-infection. Remarkably, qFILM revealed higher accumulation of large neutrophil-platelet-aggregates (NPAs) in lung microcirculation, leading to larger areas with ischemia, neutrophil extracellular traps (NETs) and vascular leakage, accompanied by more severe hypoxemia within 4-days of IAV infection in mice pre-exposed to CS than RA. NPAs in the lung of mice receiving CS-exposure prior to flu were uniquely more abundant in platelets (platelet-rich NPAs) and associated with NETs, which was secondary to caspase-11-mediated cleavage (activation) of pore forming protein gasdermin-D-(GSDMD) in neutrophils.

Conclusion(s): Our results indicate that CS-exposure exacerbates flu-induced lung injury by promoting lung microvascular occlusion by platelet-rich NPAs, leading to GSDMD-dependent NETs generation by neutrophils in the lungs.