Influenza A Virus (IAV)-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a severe complication of IAV infection in humans with often fatal outcome due to lack of effective therapeutic options. It is characterized by severe inflammation in the alveolar compartment of the lung, associated with apoptotic injury of the alveolar epithelium, resulting in loss of barrier function, edema formation and impaired gas exchange capacity with respiratory failure. Alveolar exudate macrophages (ExMa) have been shown to be key players in both driving inflammatory injury to the alveolar epithelium, but also in promoting resolution of inflammation and driving tissue repair processes, and these different functions have been suggested to be associated with the M1 versus M2 polarization phenotype of macrophages, respectively. However, to date, methods to define these phenotypes in pneumonia models in vivo have not been established, nor have the functional properties of M1 and M2ExMa and the signaling pathways or mediators associated with these functions been elucidated, particularly in the context of IAV infection.The presented data provide evidence that ExMa reveal high functional plasticity during IAV-induced ALI/ARDS. Different polarization phenotypes, M1 and M2ExMa, can be defined and separated by a newly established FACS gating strategy, allowing analyses of their gene expression profiles and correlation to their functional properties in IAV-induced lung injury. Quantitative analyses revealed that in the early, acute phase of IAV infection (D7pi), large numbers of M1ExMa infiltrate the alveolar and, to lesser extent, the interstitial space of the lung. Later on, ExMa numbers decline and increasing proportions of M2ExMa are present. By D21pi, low numbers of ExMa are present which are completely polarized towards an M2 phenotype. Of note, bone marrow chimeric mouse models and adoptive ExMa transfer studies into ExMa recruitment-deficient CCR2-/- mice demonstrated that the functional phenotype of M2ExMa is associated with both preservation and replenishment of the rAM pool depleted upon IAV infection, and with regeneration of the alveolar epithelium and improved epithelial barrier function in IAV-induced ALI/ARDS. Transcriptomic profiling of M1 versus M2ExMa revealed highly distinct gene expression profiles, with M1ExMa expressing pro-inflammatory/pro-apoptotic and host defense-associated genes, whereas M2ExMa upregulating anti-inflammatory/anti-apoptotic genes and a high number of epithelial growth factors. The most highly regulated gene in M2 versus M1ExMa was found to be Placenta-expressed transcript 1 (Plet1), a growth factor previously associated with development of epithelial layers, epithelial cell proliferation and formation of epithelial tight junctions. In vitro infection experiments using primary murine alveolar epithelial cells (mAEC) demonstrated that recombinant Plet1 prevented AEC apoptosis and IAV replication, upregulated tight junction-associated proteins and increased tightness of the AEC monolayer. Blockade of Plet1 in M2ExMa by neutralizing antibodies abolished the epithelial-protective properties of M2ExMa in IAV infection in vivo. Orotracheal treatment of IAV infected mice with recombinant Plet1 attenuated inflammation, induced AEC repair, improved alveolar barrier function and increased survival of IAV-induced ALI/ARDS. Together, these data indicate that M1 and M2ExMa are functionally distinct phenotypes evolving during IAV infection, and that M2 programming of ExMa in vivo is protective with respect to alveolar barrier function due to expression of Plet1. Moreover, therapeutic intervention using alveolar deposition of Plet1 might be a useful strategy to improve outcome after ALI/ARDS in humans
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