Acute lung injuries can be caused by avariety of factors including smoke inhalation and microbial infections like pneumonia, COVID-19, and H1N1 viral infection. These injuries reduce the respiratory capacity of the lungs by destroying resident epithelial cells and damaging alveolar architecture. A recent study published by Derek Liberti, a 5th-year DSRB candidate from Edward Morrisey’s lab, elucidates how the alveolar stem cells choose between proliferation and differentiation during alveolar restoraton following acute lung injury.
Gas exchange between the blood and atmospheric air takes place across the thin epithelial surface of pulmonary air sacs called alveoli. Two major epithelial cell types make up the lung alveolar tissue: Alveolar Type 1 (AT1) and Alveolar Type 2 (AT2) cells. AT1 cells form the single-cell thick alveolar surface across which gas exchange takes place. AT2 cells generate and secrete surfactants to reduce surface tension and prevent the alveolar sacs from collapsing. However, outside of this homeostatic state, AT2 cells have another important function. Following lung damage caused by injuries like viral infections, AT2 cells act as pulmonary stem cells, dividing and differentiating into AT1 cells to restore alveolar architecture. The balance between proliferation and differentiation of AT2 cells is precariously maintained, and signaling through the Fibroblast Growth Factor (FGF) has previously been reported to develop alveolar architecture neonatally1 maintain AT2 cell homeostasis through its receptor FGFR22. However, how AT2 cells divide or differentiate into AT1 cells, and what the role of FGFR2 is in this stem cell fate decision is not completely understood.
To address this critical knowledge gap, Liberti et al investigated the function of FGFR2 signaling during postnatal lung development and during adult lung regeneration following acute injury. First, the authors determined the role of FGFR2 during alveologenesis, a period of postnatal lung development during which alveoli form. They performed a postnatal lineage trace of control and loss-of-function Fgfr2 mutant mouse lungs using immunofluorescence and showed that loss of Fgfr2 function from AT2 cells leads to an increase in their differentiation into AT1 cells. This finding suggested that FGFR2 restricts AT2 cell fate during developmental alveologenesis. However, when Fgfr2 was inactivated in AT2 cells of the adult lung, lung architecture and morphology were maintained for 1 month, 6 months, and 12 months following the loss of FGFR2 expression. These findings demonstrated that FGFR2 is required to maintain AT2 cell identity during alveologenesis but is dispensable for adult AT2 lung homeostasis.
Following injury and in regions of acute damage, Liberti and colleagues identified a more active role for FGFR2 signaling. Lung damage caused by acute injuries is highly heterogeneous in nature and elicits varying degrees of regenerative and restorative responses depending on the degree of damage. This heterogeneity makes it difficult to study and visualize acute alveolar injury. Recognizing this caveat, Liberti et al developed a lung damage assessment program, which used computer vision on histological samples to assess the degree of lung damage and categorize injured regions as “severe”, “damaged”, or “normal”. This new and robust analytical approach helped the authors in the assessment of lung regions and their regenerative response as a factor of the degree of injury.
Combining their zonal analysis with immunohistochemistry, Liberti et al characterized these zones according to AT1 and AT2 cell behavior and density. They demonstrated that regions of severe injury following influenza infection were largely populated by Keratin 5 (KRT5)-expressing epithelial cells, which indicated a quick but temporary response to damage. These severe zones also had negligible AT1 cells but a small number of highly proliferative, non-differentiating AT2 cells. In contrast, damaged regions largely lacked KRT5+ cells, but contained both a large number of AT1 cells and rapidly dividing and differentiating AT2 cells.
After identifying these distinctive zonal characteristics, the authors next compared damaged and severe zones of control and Fgfr2-deficient AT2 cells following influenza infection. Using immunohistochemistry for Ki67, a marker of proliferative cells, the authors found that FGFR2 loss reduced the proliferation of AT2 cells in damaged zones, but not in the severe zones. This finding suggested that FGFR2 is especially important for AT2 cell proliferation for the purpose of tissue regeneration in damaged zones.
The authors next switched to an ex vivo organoid model to further define the function of FGFR2 in regulating AT2 cell proliferation. Control and Fgfr2-deficient organoids collected from the uninjured mouse lung grew comparably, however, control organoids showed a robust growth response to exogenously-supplied FGF7, an FGFR2 ligand. Conversely, Fgfr2-deficient AT2 cells did not proliferate in response to Fgf7. Given previous studies3,4 suggesting a role for inflammatory inputs in regulating lung regeneration, the group next tested whether FGFR2 is required for the AT2-cell response to three cytokines: IL-1𝞪, IL-1𝛃, and TNF-𝞪. Interestingly, the authors observed no significant difference in growth between control and Fgfr2-deficient organoids in response to these cytokines. These findings suggested that restorative AT2 cell proliferation in damaged regions can be activated in response to other stimuli besides FGFR2 signaling, for example, cytokine and inflammatory signaling.
Returning to an in vivo Fgfr2 control and mutant AT2 cell model, the authors performed a second round of lineage-tracing experiments and determined that loss of Fgfr2 function not only reduced the proliferation of AT2 cells but also increased the proportion of AT1:AT2 cells, resulting in imperfect alveolar structure of the mutant lungs with the formation of large extended air spaces in damaged zones. This result was possible either if FGFR2 indirectly suppressed differentiation of AT2 cells by promoting their proliferation, or if FGFR2 directly controlled differentiation of AT2 cells. Finally, using an Ect2 genetic deletion model (where cytokinesis and cell division are blocked) the authors showed that AT2 cells paused in a binucleated state of cell division can still differentiate into AT1 cells in damaged zones. This result demonstrated that AT2 cells do not need to divide in order to differentiate into AT1 cells when restoring a damaged alveolar zone, suggesting that cell proliferation and differentiation are decoupled in AT2 cells through FGFR2 function.
Alveolar architecture provides more than 100m2 of surface area for the exchange of gases between the blood and the atmosphere, and encounters 5-8 liters of atmospheric air per minute. This high degree of exposure renders the alveolar epithelium extremely susceptible to injury via pollutants, microbial pathogens, airborne chemicals, and particulate matter. Any damage to lung architecture calls for urgent repair and restoration to reestablish the gas exchange capacity of the lungs. Derek’s results show how diverse signaling pathways, such as FGFR2, cytokine, and inflammatory signaling, converge to restore and maintain the alveolar epithelium in injured lungs. His paper also introduces a revolutionary method of categorizing the degree of damage on heterogeneous lung tissue, and deftly shows how FGFR2 controls the cell fate of AT2 stem cells in damaged zones. Commenting on the translational potential of his study Derek states that, “I think a major challenge in identifying therapeutic targets to ameliorate lung disease lies in our limited understanding of both the essential factors that control cell fate decisions and the consequences of those decisions for lung regeneration. Without knowing the major signals regulating cell fate decisions in the lung, we have no way to enhance the regenerative process.” Combined with his previous work on neonatal lung regeneration following lung injury5, Derek Liberti’s research contextualizes how important and precariously balanced cell fate decisions are for the purposes of restoring damaged tissue and maintaining tissue homeostasis.
1. De Moerlooze, L. et al. An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 127, 483–492 (2000).
2. Yuan, T. et al. FGF10-FGFR2B Signaling Generates Basal Cells and Drives Alveolar Epithelial Regeneration by Bronchial Epithelial Stem Cells after Lung Injury. Stem Cell Reports 12, 1041–1055 (2019).
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4. Choi, J. et al. Inflammatory Signals Induce AT2 Cell-Derived Damage-Associated Transient Progenitors that Mediate Alveolar Regeneration. Cell Stem Cell vol. 27 366–382.e7 (2020).
5. Penkala, I. J. et al. Age-dependent alveolar epithelial plasticity orchestrates lung homeostasis and regeneration. Cell Stem Cell (2021) doi:10.1016/j.stem.2021.04.026.