biostudies-literatureZhang Jthe Joint-innovation Program in Healthcare for Special Scientific Research Projects of Guangzhouthe International Collaboration Program of Natural Science Foundation of China and US NIHNatural Science Foundation of Jilin Provincethe National Special Research Program of China for Important Infectious Diseasesthe National Natural Science Foundation of Chinae1008334https://www.ebi.ac.uk/biostudies/studies/S-EPMC7062283biostudies-literatureUnknown16(2)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.PLoS pathogensTwo waves of pro-inflammatory factors are released during the influenza A virus (IAV)-driven pulmonary immunopathogenesis.PMC70622832018ZX10101004003001201803040002817019902018ZX10302103, 2017ZX10202102-003816017598173006081561128007Huang FLiu JMa RXi ZPan TZhang JZhang HDeng KLuo BWang JZhao MLu GZhang YZhang XLiu BLuo YYuan YfalseTwo waves of pro-inflammatory factors are released during the influenza A virus (IAV)-driven pulmonary immunopathogenesis.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.2020-01-01T00:00:00Z2020 Feb2026-04-15T23:24:18.688Z2020-05-22T13:51:03ZS-EPMC70622833210159610.1371/journal.ppat.1008334