Macrophages have been implicated in both the resolution and progression of influenza, but the drivers of these outcomes are poorly understood. We probed mouse lung transcriptomic datasets using tissue deconvolution algorithms to predict immune cell subsets that correlated with mild or severe influenza A virus (IAV) infection outcomes. This allowed us to identify a novel lung macrophage population that was transcriptionally similar to small serosal cavity macrophages and was only present in mice that survived IAV infection. Large serosal macrophages have been implicated as a source of macrophages for visceral organs in inflammatory diseases. However, until now, the study of serosal macrophage translocation in the context of infections, and the study of small serosal macrophage translocation in general has been neglected. We used flow cytometry and microscopy to show that fluorescently-labeled pleural macrophages (PMs) migrate from the pleural cavity into the lung after infection with pH1N1 A/California/04/2009 influenza virus. The numbers of translocated PMs peak at day 9, which is during the resolution phase of the disease and after virus has been cleared. Our data show that PM recruitment to the lung parenchyma occurs across the mesothelial layer and not through the vasculature. Translocated PMs phenotypically resemble a MHCIIhi subpopulation of interstitial macrophages (IMs) that rapidly expands in number during influenza virus infection. Depletion of PMs prior to IAV infection led to increased morbidity when compared to the control group. Lack of PMs also resulted in increased pleural cavity inflammation and an influx of neutrophils within the lung. Our results show that both small and large PMs are recruited to the lung during IAV infection and contribute to recovery from influenza. This study expands our knowledge of PM plasticity and provides a new source of IMs independent of monocyte recruitment and local proliferation.

Learning Objectives:

1. Discuss the impact of pleural macrophages on influenza outcomes.
2. Describe how to identify mouse pleural and lung macrophages using flow cytometry.
3. Describe how to track pleural macrophage translocation using in vivo labeling.