H6N6 Avian Influenza Virus Infection Induced Pyroptosis of M1 Macrophages by Activating Caspase-1

The H6N6 avian influenza virus has expanded its host range from birds to mammals. Some strains can now bind to human-like receptors, raising concerns about human infection. Although H6N6 is a low-pathogenic avian influenza virus (LPAIV), it is unclear whether it triggers pyroptosis in human lungs, a process linked to cytokine storms in infections like H7N9. Here, we found that the chicken-origin H6N6 LPAIV can effectively replicate in and infect human alveolar macrophages and their M1 macrophages. Viral infection of M1 macrophages upregulated the mRNA levels of NLRP3, caspase-1, and Gasdermin D (GSDMD). Subsequently, caspase-1 was activated and cleaved GSDMD protein into its N-terminal fragment (GSDMD-N), which formed pores in the cell membrane and triggered the release of IL-1β and IL-18. Further analysis demonstrated that inhibition of the NLRP3/Caspase-1/GSDMD pathway by specific inhibitors attenuated pyroptosis in infected M1 macrophages. In summary, our study revealed that H6N6 virus infection induces M1 macrophage pyroptosis via the NLRP3/caspase-1/GSDMD pathway. Notably, M1 macrophages inherently produce pro-inflammatory cytokines; their pyroptosis, accompanied by the release of IL-1β and IL-18, can amplify inflammation and potentially trigger a cytokine storm in the lungs. These findings reveal novel pathogenic mechanisms and potential therapeutic targets for avian influenza viruses.