Two waves of pro-inflammatory factors are released during the influenza A virus (IAV)-driven pulmonary immunopathogenesis

by Junsong Zhang, Jun Liu, Yaochang Yuan, Feng Huang, Rong Ma, Baohong Luo, Zhihui Xi, Ting Pan, Bingfeng Liu, Yiwen Zhang, Xu Zhang, Yuewen Luo, Jin Wang, Meng Zhao, Gen Lu, Kai Deng, Hui Zhang

Influenza A virus (IAV) infection is a complicated process. After IAVs spread to the lung, extensive pro-inflammatory cytokines and chemokines are released, which largely determine the outcome of infection. Using a single-cell RNA sequencing (scRNA-seq) assay, we systematically and sequentially analyzed the transcriptome of more than 16,000 immune cells in the pulmonary tissue of infected mice, and demonstrated that two waves of pro-inflammatory factors were released. A group of IAV-infected PD-L1⁺ neutrophils were the major contributor to the first wave at an earlier stage (day 1–3 post infection). Notably, at a later stage (day 7 post infection) when IAV was hardly detected in the immune cells, a group of platelet factor 4-positive (Pf4⁺)-macrophages generated another wave of pro-inflammatory factors, which were probably the precursors of alveolar macrophages (AMs). Furthermore, single-cell signaling map identified inter-lineage crosstalk between different clusters and helped better understand the signature of PD-L1⁺ neutrophils and Pf4⁺-macrophages. Our data characteristically clarified the infiltrated immune cells and their production of pro-inflammatory factors during the immunopathogenesis development, and deciphered the important mechanisms underlying IAV-driven inflammatory reactions in the lung.