Combination Therapy Targeting Platelet Activation and Virus Replication Protects Mice against Lethal Influenza...

Excessive neutrophils recruited during influenza pneumonia contribute to severe lung pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. We have recently shown that activation of platelets during influenza enhances pulmonary microvascular thrombosis, leading to vascular injury and hemorrhage. Emerging evidence indicates that activated platelets also interact with neutrophils, forming neutrophil-platelet aggregates (NPAs) that contribute to tissue injury. Here, we examined neutrophil-platelet interactions and evaluated the formation of NPAs during influenza pneumonia. We also evaluated the efficacy of clopidogrel (CLP), an antagonist of the ADP-P₂Y₁₂ platelet receptor, alone or in combination with an antiviral agent (oseltamivir) against influenza infection in mice. Our studies demonstrated increased platelet activation and induction of NPAs in influenza-infected lungs, and that these NPAs led to NET release both in vitro and in vivo. Furthermore, neutrophil integrin Mac-1 (macrophage-1 antigen)-mediated platelet binding was critical for NPA formation and NET release. Administration of CLP reduced platelet activation and NPA formation but did not protect the mice against lethal influenza challenge. However, administration of CLP together with oseltamivir improved survival rates in mice compared with oseltamivir alone. The combination treatment reduced lung pathology, neutrophil influx, NPAs, NET release, and inflammatory cytokine release in infected lungs. Taken together, these results provide the first evidence that NPAs formed during influenza contribute to acute lung injury. Targeting both platelet activation and virus replication could represent an effective therapeutic option for severe influenza pneumonia.