{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,18]],"date-time":"2026-06-18T15:24:19Z","timestamp":1781796259530,"version":"3.54.5"},"reference-count":48,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:00:00Z","timestamp":1762905600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["32160032"],"award-info":[{"award-number":["32160032"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>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-1\u03b2 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-1\u03b2 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.<\/jats:p>","DOI":"10.3390\/v17111492","type":"journal-article","created":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T10:56:47Z","timestamp":1762945007000},"page":"1492","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["H6N6 Avian Influenza Virus Infection Induced Pyroptosis of M1 Macrophages by Activating Caspase-1"],"prefix":"10.3390","volume":"17","author":[{"given":"Hui","family":"Zhu","sequence":"first","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dongfang","family":"He","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sicong","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaohui","family":"Fan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5471-2682","authenticated-orcid":false,"given":"Lingxi","family":"Gao","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4006-3095","authenticated-orcid":false,"given":"Liping","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zengfeng","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Guangxi Medical University, Nanning 530021, China"},{"name":"Key Laboratory of Basic Research on Regional Diseases (Guangxi Medical University), Education Department of Guangxi Zhuang Autonomous Region, Nanning 530021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mallapaty, S. (2024). The pathogens that could spark the next pandemic. Nature, 632.","DOI":"10.1038\/d41586-024-02513-3"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/S0140-6736(97)11212-0","article-title":"Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus","volume":"351","author":"Claas","year":"1998","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1888","DOI":"10.1056\/NEJMoa1304459","article-title":"Human infection with a novel avian-origin influenza A (H7N9) virus","volume":"368","author":"Gao","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1507","DOI":"10.3201\/eid2208.160146","article-title":"Novel reassortant avian influenza A(H5N6) viruses in Humans, Guangdong, China 2015","volume":"22","author":"Shen","year":"2016","journal-title":"Emerg. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3953","DOI":"10.1128\/JVI.03292-13","article-title":"H6 influenza viruses pose a potential threat to human health","volume":"88","author":"Wang","year":"2014","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Zhu, P., He, X., Chen, Y., Yan, Z., Li, Q., Zhou, Q., Lin, W., and Chen, F. (2024). Characterization and Pathogenicity of Novel Reassortment H6N6 Avian Influenza Viruses in Southern China. Transbound. Emerg. Dis., 2024.","DOI":"10.1155\/2024\/4005909"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1038\/440435a","article-title":"Avian flu: Influenza virus receptors in the human airway","volume":"440","author":"Shinya","year":"2006","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"van Riel, D., Munster, V.J., de Wit, E., Rimmelzwaan, G.F., Fouchier, R.A., Osterhaus, A.D., and Kuiken, T. (2006). H5N1 virus attachment to lower respiratory tract. Science, 312.","DOI":"10.1126\/science.1125548"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Deng, L., Jian, Z., Xu, T., Li, F., Deng, H., Zhou, Y., Lai, S., Xu, Z., and Zhu, L. (2023). Macrophage polarization: An important candidate regulator for lung diseases. Molecules, 28.","DOI":"10.3390\/molecules28052379"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1002\/cbin.11914","article-title":"Origin, production and molecular determinants of macrophages for their therapeutic targeting","volume":"47","author":"Chowdhury","year":"2022","journal-title":"Cell Biol. Int."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Ni, R., Jiang, L., Zhang, C., Liu, M., Luo, Y., Hu, Z., Mou, X., and Zhu, Y. (2023). Biologic mechanisms of macrophage phenotypes responding to infection and the novel therapies to moderate inflammation. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24098358"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1146\/annurev-med-042921-112837","article-title":"Cytokine storm syndrome","volume":"74","author":"Cron","year":"2022","journal-title":"Annu. Rev. Med."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Malireddi, R.K.S., Kesavardhana, S., and Kanneganti, T.D. (2019). ZBP1 and TAK1: Master regulators of NLRP3 Inflammasome\/Pyroptosis, Apoptosis, and Necroptosis (PANoptosis). Front. Cell. Infect. Microbiol., 9.","DOI":"10.3389\/fcimb.2019.00406"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/j.tibs.2016.10.004","article-title":"Pyroptosis: Gasdermin-mediated programmed necrotic cell death","volume":"42","author":"Shi","year":"2017","journal-title":"Trends Biochem. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"660","DOI":"10.1038\/nature15514","article-title":"Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death","volume":"526","author":"Shi","year":"2015","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1038\/nature13683","article-title":"Inflammatory caspases are innate immune receptors for intracellular LPS","volume":"514","author":"Shi","year":"2014","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Chu, L.H., Indramohan, M., Ratsimandresy, R.A., Gangopadhyay, A., Morris, E.P., Monack, D.M., Dorfleutner, A., and Stehlik, C. (2018). The oxidized phospholipid oxPAPC protects from septic shock by targeting the non-canonical inflammasome in macrophages. Nat. Commun., 9.","DOI":"10.1038\/s41467-018-03409-3"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"666","DOI":"10.1038\/nature15541","article-title":"Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling","volume":"526","author":"Kayagaki","year":"2015","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1038\/nature22393","article-title":"Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin","volume":"547","author":"Wang","year":"2017","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1264","DOI":"10.1038\/s41556-020-0575-z","article-title":"PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis","volume":"22","author":"Hou","year":"2020","journal-title":"Nat. Cell Biol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Liu, Y., Fang, Y., Chen, X., Wang, Z., Liang, X., Zhang, T., Liu, M., Zhou, N., Lv, J., and Tang, K. (2020). Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome. Sci. Immunol., 5.","DOI":"10.1126\/sciimmunol.aax7969"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Cerato, J.A., da Silva, E.F., and Porto, B.N. (2023). Breaking bad: Inflammasome activation by respiratory viruses. Biology, 12.","DOI":"10.3390\/biology12070943"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Wan, X., Li, J., Wang, Y., Yu, X., He, X., Shi, J., Deng, G., Zeng, X., Tian, G., and Li, Y. (2021). H7N9 virus infection triggers lethal cytokine storm by activating gasdermin E-mediated pyroptosis of lung alveolar epithelial cells. Natl. Sci. Rev., 9.","DOI":"10.1093\/nsr\/nwab137"},{"key":"ref_24","first-page":"41","article-title":"ZBP1\/DAI-dependent cell death pathways in influenza A virus immunity and pathogenesis","volume":"442","author":"Thomas","year":"2023","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Ren, R., Wu, S., Cai, J., Yang, Y., Ren, X., Feng, Y., Chen, L., Qin, B., Xu, C., and Yang, H. (2017). The H7N9 influenza A virus infection results in lethal inflammation in the mammalian host via the NLRP3-caspase-1 inflammasome. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-07384-5"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2215","DOI":"10.3201\/eid2512.190261","article-title":"Avian influenza A viruses among occupationally exposed populations, China, 2014\u20132016","volume":"25","author":"Quan","year":"2019","journal-title":"Emerg. Infect. Dis."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Zhong, W., Gao, L., Wang, X., Su, S., Lin, Y., Huang, K., Zhou, S., Fan, X., and Zhang, Z. (2022). Influenza A (H6N6) viruses isolated from chickens replicate in mice and human lungs without prior adaptation. J. Virus Erad., 8.","DOI":"10.2139\/ssrn.4188553"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1007\/978-1-62703-523-1_7","article-title":"Detection of pyroptosis by measuring released lactate dehydrogenase activity","volume":"1040","author":"Rayamajhi","year":"2013","journal-title":"Methods Mol. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Yan, Z., Li, Y., Huang, S., and Wen, F. (2023). Global distribution, receptor binding, and cross-species transmission of H6 influenza viruses: Risks and implications for humans. J. Virol., 97.","DOI":"10.1128\/jvi.01370-23"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Du, Y., Xia, J., Wang, Z., Xu, J., Ji, Y., Jin, Y., Pu, L., and Xu, S. (2024). Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events. Emerg. Microbes Infect., 13.","DOI":"10.1080\/22221751.2024.2341142"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.meegid.2014.09.008","article-title":"Phylogenetic analysis of a novel H6N6 avian influenza virus isolated from a green peafowl in China and its pathogenic potential in mice","volume":"28","author":"Fan","year":"2014","journal-title":"Infect. Genet. Evol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1203","DOI":"10.1038\/nm1477","article-title":"Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia","volume":"12","author":"Simmons","year":"2006","journal-title":"Nat. Med."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Guo, J., Huang, F., Liu, J., Chen, Y., Wang, W., Cao, B., Zou, Z., Liu, S., Pan, J., and Bao, C. (2015). The serum profile of hypercytokinemia factors identified in H7N9-infected patients can predict fatal outcomes. Sci. Rep., 5.","DOI":"10.1038\/srep10942"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Gu, Y., Zuo, X., Zhang, S., Ouyang, Z., Jiang, S., Wang, F., and Wang, G. (2021). The mechanism behind influenza virus cytokine storm. Viruses, 13.","DOI":"10.3390\/v13071362"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Yamasaki, K., and van Eeden, S.F. (2018). Lung macrophage phenotypes and functional responses: Role in the pathogenesis of COPD. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19020582"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1038\/ni.3375","article-title":"Innate cell communication kick-starts pathogen-specific immunity","volume":"17","author":"Rivera","year":"2016","journal-title":"Nat. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.cellimm.2018.02.001","article-title":"The interstitial macrophage: A long-neglected piece in the puzzle of lung immunity","volume":"330","author":"Liegeois","year":"2018","journal-title":"Cell. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1189","DOI":"10.1136\/thoraxjnl-2015-207020","article-title":"Pulmonary macrophages: Key players in the innate defence of the airways","volume":"70","author":"Byrne","year":"2015","journal-title":"Thorax"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1056\/NEJMc2112416","article-title":"Human infection with an avian-origin influenza A (H10N3) virus","volume":"386","author":"Qi","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"429","DOI":"10.7326\/L22-0376","article-title":"Second identified human infection with the avian influenza virus H10N3: A case report","volume":"176","author":"Zhang","year":"2023","journal-title":"Ann. Intern. Med."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Zhao, Z., Luo, S., Gao, Y., Dai, M., Yan, J., Yang, Y., Li, H., Zhang, Y., and Mao, Z. (2024). A case report of human infection with avian influenza H10N3 with a complex respiratory disease history. BMC Infect. Dis., 24.","DOI":"10.1186\/s12879-024-09830-y"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1016\/S0140-6736(14)60111-2","article-title":"Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: A descriptive study","volume":"383","author":"Chen","year":"2014","journal-title":"Lancet"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3468","DOI":"10.4049\/jimmunol.1900045","article-title":"Gasdermin D protects from melioidosis through pyroptosis and direct killing of bacteria","volume":"202","author":"Wang","year":"2019","journal-title":"J. Immunol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3662","DOI":"10.4049\/jimmunol.1800788","article-title":"Gasdermin D promotes AIM2 inflammasome activation and is required for host protection against francisella novicida","volume":"201","author":"Zhu","year":"2018","journal-title":"J. Immunol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Xu, X., Chen, Q., Tan, M., Liu, J., Li, X., Yang, L., Shu, Y., Wang, D., and Zhu, W. (2023). Epidemiology, evolution, and biological characteristics of H6 avian influenza viruses in China. Emerg. Microbes Infect., 12.","DOI":"10.1080\/22221751.2022.2151380"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e28790","DOI":"10.1002\/jmv.28790","article-title":"Baicalin inhibits influenza A (H1N1)-induced pyroptosis of lung alveolar epithelial cells via caspase-3\/GSDME pathway","volume":"95","author":"Wei","year":"2023","journal-title":"J. Med. Virol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Zhang, M., Xu, G., Zhou, X., Luo, M., Ma, N., Wang, X., Wang, Z., Tang, H., Wang, X., and Li, Y. (2023). Mesenchymal stem cells ameliorate H9N2-induced acute lung injury by inhibiting caspase-3-GSDME-mediated pyroptosis of lung alveolar epithelial cells. Eur. J. Pharmacol., 960.","DOI":"10.1016\/j.ejphar.2023.176148"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Wang, X., Wang, X., Hao, X., Gao, R., Lu, X., Yang, W., Chen, Y., Hu, J., Gu, M., and Liu, X. (2025). The Novel H10N3 Avian Influenza Virus Triggers Lethal Cytokine Storm by Activating Multiple Forms of Programmed Cell Death in Mammalian Lungs. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26051977"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/11\/1492\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T05:23:05Z","timestamp":1763097785000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/11\/1492"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,12]]},"references-count":48,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["v17111492"],"URL":"https:\/\/doi.org\/10.3390\/v17111492","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,12]]}}}