Influenza virus pneumonia causes apoptosis of alveolar epithelial cells, disruption of the epithelial barrier and edema formation that affects gas exchange dramatically, resulting in the acute respiratory distress syndrome with poor outcome. The pathology of influenza virus-induced injury is well studied, but repair mechanisms of the distal lung epithelium, which may influence the outcome are not well understood. It has been demonstrated that epithelial progenitor cells in the adult murine lung can repopulate injured tracheobronchial or alveolar regions. Therefore, this project investigated repair mechanisms of distal epithelial stem/progenitor cell (EpiSPC) after severe influenza virus pneumonia. The EpiSPC express the surface markers EpCam high alpha6 high CD24 low beta4+ Sca-1+. They highly proliferate after influenza virus injury, but show low apoptosis rates after infection as compared to other epithelial subsets. Characterization of their phenotype in ex vivo 3D cultures revealed that flow sorted EpiSPC clonally expand in presence of growth factors, including Fgf10, and upregulate markers associated with terminally differentiated bronchiolar and alveolar cells. Lung resident mesenchymal cells defined as CD45 neg CD31 neg EpCam neg Sca-1 high revealed to be the primary source of Fgf10, and supported lung-like outgrowth in the absence of further growth factors. During influenza virus infection, the Fgf10 receptor Fgfr2b was highly upregulated on non-infected EpiSPC, whereas the infected population poorly upregulated the Fgfr2b, resulting in severe limitation of their proliferative response. Interestingly, the pathogenicity of different influenza virus strains correlated with infection rates of EpiSPC in vivo, suggesting a causal relation between the extent of EpiSPC infection and their capacity to restore lung function. Targeting the Fgf10/Fgfr2b axis by induction of dominant negative soluble Fgfr2b in transgenic mice resulted in increased alveolar permeability, weight loss, and decreased proliferative capacity of EpiSPC. Application of recombinant Fgf10 (rFgf10) as a therapeutic approach in the acute phase of influenza virus infection enhanced the proliferative response of EpiSPC, decreased alveolar leakage and improved survival rates. Additionally, lung sections revealed better resolution of inflammation and restoration of lung structure after rFgf10 application. In accordance with decreased alveolar leakage, staining of lung sections revealed improved cell to cell connections in the alveolar compartment. With respect to the human lung, a population similar to EpiSPC, expressing EpCam high a6 high CD24 low-neg lgr6+ was identified, which similarly depended on growth factors, including Fgf10 and formed cystic spheres in 3D culture. As demonstrated in murine organoid cultures, co-cultures of human EpiSPC with primary human lung fibroblasts promoted outgrowth without addition of growth factors, whereas infection with pandemic influenza virus resulted in a reduced proliferative response. In conclusion, this work identifies Fgf10/Fgfr2b-dependent EpiSPC as primary drivers of lung regeneration after influenza virus-induced lung injury. Influenza virus-induced inhibition of Fgf10-mediated repair caused by influenza virus infection could be overcome by therapeutic application of Fgf10, highlighting this approach as putative therapy for patients with influenza virus-induced ARDS.
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