H1N1 influenza virus infection induces dramatic and permanent alveolar remodeling mediated by p63+ progenitor cell expansion in both mice and some patients with acute respiratory distress syndrome. This persistent lung epithelial dysplasia is accompanied by chronic inflammation, but the driver(s) of this pathology are unknown. This work identified de novo appearance of solitary chemosensory cells (SCCs), as defined by the tuft cell marker doublecortin-like kinase 1, in post-influenza lungs, arising in close proximity with the dysplastic epithelium, whereas uninjured lungs are devoid of SCCs. Interestingly, fate mapping demonstrated that these cells are derived from p63-expressing lineage-negative progenitors, the same cell of origin as the dysplastic epithelium. Direct activation of SCCs with denatonium + succinate increased plasma extravasation specifically in post-influenza virus-injured lungs. Thus we demonstrate the previously unrecognized development and activity of SCCs in the lung following influenza virus infection, implicating SCCs as a central feature of dysplastic remodeling.

Alveolar type-2 (AT2) cells are necessary for the lung’s regenerative response to epithelial insults such as influenza. However, current methods to expand these cells rely on mesenchymal co-culture, complicating the possibility of transplantation following acute injury. Here we developed several mesenchyme-free culture conditions that promote growth of murine AT2 organoids. Transplanting dissociated AT2 organoids into influenza-infected mice demonstrated that organoids engraft and either proliferate as AT2 cells or unexpectedly adopt a basal cell-like fate associated with maladaptive regeneration. Alternatively, transplanted primary AT2 cells also robustly engraft, maintaining their AT2 lineage while replenishing the alveolar type-1 (AT1) cell population in the epithelium. Importantly, pulse oximetry revealed significant increase in blood-oxygen saturation in primary AT2 recipients, indicating that transplanted cells also confer increased pulmonary function after influenza. We further demonstrated that both acid installation and bleomycin injury models are also amenable to AT2 transplantation. These studies provide additional methods to study AT2 progenitor potential, while serving as proof-of-principle for adoptive transfer of alveolar progenitors in potential therapeutic applications.

Objective: R-spondin 2 (RSPO2) is required for lung morphogenesis, activates Wnt signaling, and is upregulated in idiopathic lung fibrosis. Our objective was to investigate whether RSPO2 is similarly important in homeostasis of the adult lung. While investigating the characteristics of bronchoalveolar lavage in RSPO2-deficient (RSPO2 −/−) mice, we observed unexpected changes in neutrophil homeostasis and vascular permeability when compared to control (RSPO2 +/+) mice at baseline. Here we quantify these observations to explore how tonic RSPO2 expression impacts lung homeostasis. Results: Quantitative PCR (qPCR) analysis demonstrated significantly elevated myeloperoxidase (MPO) expression in bronchoalveolar lavage fluid (BALF) cells from RSPO2 −/− mice. Likewise, immunocytochemical (ICC) analysis demonstrated significantly more MPO+ cells in BALF from RSPO2 −/− mice compared to controls, confirming the increase of infiltrated neutrophils. We then assessed lung permeability/barrier disruption via Fluorescein Isothiocyanate (FITC)dextran instillation and found a significantly higher dextran concentration in the plasma of RSPO2 −/− mice compared to identically treated RSPO2 +/+ mice. These data demonstrate that RSPO2 may be crucial for blood-gas barrier integrity and can limit neutrophil migration from circulation into alveolar spaces associated with increased lung permeability and/or barrier disruption. This study indicates that additional research is needed to evaluate RSPO2 in scenarios characterized by pulmonary edema or neutrophilia.