Abstract: Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.

Abstract: In conclusion, our studies address two important topics in lung biology: the lineage relationships of epithelial cells in the distal gas exchange region of the lung and the cellular origins of pulmonary fibrosis. A deeper understanding of epithelial progenitors will help to identify therapies to reverse the cell loss that occurs in a number of lung diseases and not just fibrosis. For fibrosis, defining the relative contributions of each of the different stromal cell types, including pericytes, to the fibrotic process will be important in future studies. In particular, we need to understand whether contributions are direct (through synthesis of ECM) or indirect (through production of growth factors and cytokines). This knowledge has the potential to lead to novel therapeutic strategies for the treatment of pulmonary fibrosis. It has been suggested that 30–50% of the myofibroblasts in the bleomycin model originate from epithelial cells, including type 2 alveolar epithelial cells (AEC2), through a process known as epithelial to mesenchymal transition. According to this model, epithelial cells lose their polarity and assume a fibroblast phenotype, including the expression of stromal markers and secretion of ECM. We addressed this popular hypothesis by generating a knock-in allele to induce the heritable expression of a fluorescent lineage tag in AEC2 cells. In the distal lung, these cells are a source of surfactant proteins, which are critical for proper lung function, and they may also act as a population of stem cells capable of differentiating into type 1 alveolar epithelial cells (AEC1s) ( 4 ). The lineage tag also enabled us to purify AEC2s and their descendants to confirm the morphological findings by examining the expression of different marker genes. Using confocal microscopy and our panel of stromal markers, we saw no evidence that lineage-labeled AEC2 gave rise to any kind of stromal cell. Rather, they differentiate into AEC1 cells, supporting classical models for the lineage relationship of these two cell types. Significantly, the slow conversion of AEC2 to AEC1 cells seen in control lungs is greatly enhanced in response to bleomycin-induced lung injury. Currently, we do not understand the mechanism underlying this change in behavior, but additional analysis of gene expression in AEC2 cells will address this question. We also used our Secretoglobin1a1-CreER knock-in allele to follow the response of epithelial cells of the airways (bronchioles) and a subset of alveolar epithelial cells to bleomycin. Unlike our previous findings with other injury models ( 5 ), we found striking changes in the proliferation and differentiation of these cells after bleomycin injury. However, again, we found no evidence that this labeled population generates fibroblasts. With this foundation in place, we used genetic lineage tracing in the mouse to determine the origin of the fibroblasts within the bleomycin-induced lesions. First, we tested the hypothesis that pericytes are a source of myofibroblasts. We used two different mouse strains carrying transgenes, Ng2-CreER and FoxJ1-CreER, to induce the expression of a fluorescent protein that is heritable and specifically expressed in pericyte-like cells within the alveolar wall. This florescent marker allowed us to follow the fate of this cell type in response to bleomycin. We found that these cells proliferated and expanded within fibroblast foci. Surprisingly, most of the lineage-labeled cells did not express high levels of aSMA. We began by staining tissue sections with antibodies against a panel of commonly used markers for stromal cells and viewing them by confocal microscopy to characterize these cell populations before bleomycin treatment and at different times after it. The markers included α-smooth muscle actin (aSMA and Acta2), which is generally considered a hallmark of myofibroblasts. We also examined other markers including S100a4 (Fsp1), desmin, vimentin, Pdgfra, Pdgfrb, and the surface glycoprotein Ng2 (Cspg4). Some of these markers mark pericytes, a population of cells implicated as a source of myofibroblasts in other fibrotic processes ( 2 , 3 ). These studies revealed a surprising diversity of cells in the fibrotic lesions, with aSMA-positive cells showing the greatest abundance relatively early after the administration of bleomycin. A survey of fresh biopsy samples from patients with IPF suggested that a similar diversity exists in human fibrotic lesions. One factor complicating the study of IPF is that surprisingly little is known about the identities, behaviors, and lineage relationships of the different cell types that make up the gas exchange region of the lung where the disease occurs. This region has a complex 3D structure in which a vast network of capillaries and nonepithelial stromal cells surrounds the millions of thin-walled, air-filled epithelial sacs known as alveoli ( Fig. P1 ; ref. 1 ). To fully understand the process of fibrosis, we focused on high-resolution microscopy of intact normal and fibrotic lung tissue rather than study of cells grown in culture. Idiopathic pulmonary fibrosis (IPF) is a debilitating disease in which the delicate gas exchange region of the lung is gradually replaced by accumulations of ECM and myofibroblasts. These cells are commonly thought to produce components of the ECM, including collagen. Conflicting ideas about the cellular origin of the fibrotic lesions may slow progress to the development of effective therapies for IPF. To address this problem, we combined the power of cell lineage tracing in mice with a model of pulmonary fibrosis induced by bleomycin, an anticancer agent that often results in pulmonary fibrosis as a side effect. We provide evidence that the behaviors of pericytes, a cell type associated with blood vessels, and epithelial cells are modulated in response to bleomycin-induced lung injury. However, neither cell type is a major source of myofibroblasts. These findings should help to focus future efforts at developing therapies for the treatment of IPF.