Platelet RIG-I Promotes Flu-Induced Severe Lung Injury in Sickle Cell Disease

Background: Sickle cell disease (SCD) affects over ~8 million people world-wide. Acute chest syndrome, a type of acute lung injury (ALI) is a major cause of mortality among SCD patients. Flu typically causes a self-resolving upper airway inflammation, however, it may progress for unknown reasons into a highly morbid ALI. Epidemiological evidence suggests that flu results in severe outcomes in SCD patients and associates with a 50-fold higher risk of hospitalization among SCD compared to non-SCD humans. However, the mechanisms underlying the flu severity and subsequent severe lung injury in SCD remains largely unknown.

Methods: We have developed a new model of A/PR/8/34 (H1N1) influenza A virus (IAV) induced respiratory infection in knock-in, humanized Townes SCD (SS) mice. SCD or control (Townes AS) mice were inoculated intranasally with a mild dose of IAV, and severity of lung injury was evaluated 14 days post infection using histological scoring, oxygen saturation was measured every other day and body weight was assessed every day. Real-time intravital (in vivo) multi-photon-excitation fluorescence microscopy (MPE) was used to assess thrombo-inflammation and vascular leakage in the lung of mice at 10 days post IAV-infection. Platelets were isolated for western blotting and co-immunoprecipitation to assess the activation of anti-viral pathways in platelets.

Results: SCD+Flu mice manifested significant drop in oxygen saturation (<90%), body weight (³ 20%) and developed severe lung injury indicated by the presence of hemorrhage, vascular congestion and edema in the lung histopathological analysis. Severe ALI in SCD+Flu mice at day 10 post infection was associated with pulmonary thrombo-inflammation leading to microvasculature occlusion by platelet-rich neutrophil-platelets aggregates (NPAs), resulting in pulmonary ischemia and loss of blood-air barrier. In contrast to SCD mice, control mice inoculated with mild dose of flu did not develop ALI and neutrophil-platelet aggregates were absent in the lung microcirculation of Control+Flu mice at day 10^th post infection. Co-immunoprecipitation showed assembly of viral-RNA sensing RIG-I-MAVS complex in SCD+Flu but not Control+Flu mice platelets, suggestive of the activation of anti-viral response in SCD+Flu but not Control+Flu mice platelets 10-days post infection.

Conclusions: These findings suggest for the first time a potential role for platelet-dependent anti-viral RIG-I signaling in promoting thrombo-inflammation and severe lung injury following flu infection in SCD. Currently, studies are in progress to identify the innate immune signaling downstream to platelet RIG-I-MAVS complex formation, and how it can be therapeutically manipulated to reduce the flu morbidity in SCD.