Administration of DT to mice expressing a lung specific DTR transgene caused scarring of the lung as assessed by lung collagen content and histological appearance. This induction of fibrosis was associated with a decrease in animal survival in a DT dose-dependent fashion and a decrease in the expression of type II cell gene products (i.e., SPC and DTR). However, the fibrosis and the changes in alveolar epithelial functions occurred in the absence of a detected decrease in type II cell number. Taken together, our results demonstrate that injury of the type II epithelium can directly induce pulmonary fibrosis.
The DT-mediated cell ablation strategy has been successfully used to model a variety of disorders, including glomerulosclerosis with the depletion of podocytes, osteoporosis with the depletion of osteocytes, acute hepatitis with the depletion of hepatocytes, and cardiomyopathy with the depletion of cardiomyocytes (16
). This model has also been used to deplete inflammatory cell populations including dendritic cells and monocyte/macrophages. In our investigation, 2 weeks of daily DT exposure reproducibly induced pulmonary fibrosis. The 10-μg/kg dose of DT is in the range of what others have used in studies to ablate podocytes (20
) and to specifically target neurons (23
), hepatocytes (15
), osteocytes (23
), and monocytes/macrophages (21
). A significantly higher dose (5,000 μg/kg) was used to ablate cardiomyocytes (24
). Our dosing approach differs from many of these prior studies (e.g., hepatocytes, cardiomyocytes, podocytes, and osteocytes), in that more than one administration of DT was used to achieve the fibrotic phenotype. A similar prolonged administration was used to assess the role of monocytes/macrophages in atherosclerotic plaque formation (21
). There are several possible explanations for why repeated doses of DT were required for a phenotype in our model. One potential explanation is that alveolar progenitor cells can rapidly divide in response to cell injury, thus minimizing the consequences from a single dose of DT. Our data showing increased type II cell proliferation at Day 7 of DT exposure are consistent with this possibility. The most likely progenitors are residual uninjured type II cells that are not exposed to or are resistant to DT, although it is possible that the injured cells can also proliferate. A similar capacity for cell replacement has been seen in DTR-expressing monocyte/macrophages and splenic dendritic cells that recover to pretreatment numbers within 6 days after DT-induced depletion (25
). Because of this ability to regenerate depleted cells, a more prolonged dosing may be required to achieve persistent alteration of function. In contrast, the kidney has a limited capacity to regenerate podocytes, and therefore one dose of DT in rats that express podocyte-specific DTR causes glomerulosclerosis (22
). Another potential mechanism for why multiple DT doses were required to generate pulmonary fibrosis is that the level of DTR expression may be relatively low in our transgenic mice. In the model of acute hepatitis, the amount of DTR expressed in the different founder lines was inversely correlated with the dose of toxin required to cause hepatic injury and death (16
). We must also consider the possibility that type II cells become resistant to DT after multiple administrations. This scenario was also postulated to explain why prolonged DT dosing produced only a 50% decrease in peripheral monocytes that were engineered to express DTR (21
). Cleavage of the receptor from the surface of expressing cells by proteases may provide a mechanism of resistance (26
). Finally, a shorter course of DT treatment may be sufficient to cause significant type II cell depletion and resultant fibrosis in our model. Thus far, we have compared only single doses of DT with the 14-day administration protocol. Our observations that DTR expression is significantly decreased after 3 days of toxin exposure and SPC expression is decreased after 7 days of treatment suggest that a shorter course of treatment may be adequate to induce scarring.
DT mediates cell toxicity by catalyzing the ADP-ribosylation of elongation factor 2, thereby interfering with protein synthesis (16
). This disruption of protein synthesis is classically reported to induce apoptotic cell death. However, the DT model has also been shown to induce autophagy in cardiomyocytes (24
). Furthermore, when this approach was used to target osteocytes, electron microscopic examination revealed that the DT-induced injury could lead to necrosis as well as apoptosis (23
). Cell-specific targeting with DT can also induce cellular dysfunction without causing death, as was demonstrated for monocyte/macrophage injury in an atherosclerosis model (21
). Targeting the monocyte/macrophage population with DT in this report not only reduced the number of cells but also impaired LDL uptake by 54% in the remaining viable cells. In our studies, we demonstrate an alteration of type II cell function after DT treatment of the transgenic mice. DT may also induce type II cell death based on several observations. First, we found that type II cell proliferation increases in the SPC-DTR animals after DT exposure while the total number of type II cells remains constant. The stable number of total cells in the setting of proliferation suggests that the dividing cells are replacing a transiently depleted subset of the type II epithelium. Second, our in vitro
results demonstrated a trend toward increased cell death in isolated transgenic type II cells after 24 hours of DT treatment. Perhaps a longer in vitro
exposure to the toxin would enhance this effect.
One other prior report has examined the effect of DT-mediated lung cell ablation (27
). In this model, the DT receptor was expressed by the lysozyme M promoter with the intent of depleting macrophages. The authors found DT administration at doses of 10 and 40 μg/kg to cause acute lung injury and death in the transgenic mice within 4 to 6 days. Evaluation of histological sections revealed a loss of type II cells and macrophages. The authors then performed a WT bone marrow transplant into the transgenic group to replace the DT-sensitive alveolar macrophages with DT-resistant cells. Treatment of these mice with DT caused acute lung injury with loss of type II cells even though the alveolar macrophages were now resistant to the toxin. This led the authors to conclude that the DT-induced acute lung injury was the result of an insult to the type II epithelium. Several explanations may account for why the ablation of type II pneumocytes in this model caused a different phenotype as compared with our model. First, expressing DTR off of the lysozyme M promoter may not be specific for type II cells as is the SPC promoter (28
). As a result, other cells comprising the alveolar wall in addition to macrophages and type II cells may be damaged. Second, the more severe phenotype observed in the lysozyme M model may be the result of this promoter inducing higher levels of DTR expression in the type II cells or an increased number of DTR-expressing type II cells. One might predict that the more efficient targeting of a substantial proportion of the type II cells or a more diffuse injury to the alveolar wall would cause acute lung injury and death. Regardless of the mechanism of acute lung injury in their model, no data regarding the development of fibrosis in response to the insult were presented.
The mechanism by which impaired type II function leads to fibrosis requires further study. One possibility is that injury impairs the homeostatic ability of the lung to replace type I cells lost during normal attrition. Damage of the type II epithelium may also lead to the loss of important signals that suppress lung fibroblasts proliferation and collagen production. For example, alveolar epithelial cells are an important source of prostaglandin E2
, which has been shown to inhibit multiple aspects of the fibroproliferative response, including fibroblast chemotaxis, fibroblast proliferation, and collagen synthesis (29
). A loss of type II alveolar epithelial cells could diminish intraalveolar levels of this antifibrotic mediator. Finally, the residual injured type II epithelium may be a source of profibrotic factors such as TGF-β that directly activate local fibroblasts.
Establishing a causal link between type II cell loss and lung scarring provides new insights into the pathogenesis of familial and sporadic cases of IPF. First, our findings support the conlusions of Thomas and colleagues that the SPC mutation they identified in familial IPF, which reduces type II cell function and viability, predisposes to fibrosis (14
). Our findings also provide support for a casual relationship between the type II cell apoptosis seen in IPF biopsies and the development of alveolar scarring (32
). The link between telomerase mutations and pulmonary fibrosis identified in multiple kindreds implies that some key cell in the lungs of patients with IPF has decreased proliferative capacity (34
). From our data, we speculate that the type II alveolar epithelial cell may be this critical cell. Aberrant type II cell reparative capacity could also explain why IPF is diagnosed in older adults (36
) in whom senescence of these alveolar progenitor cells may prevent adequate replacement when epithelium is damaged during normal daily life.
In summary, we have generated a new mouse model of pulmonary fibrosis caused by the targeted depletion of type II alveolar epithelial cells. Our findings provide direct evidence specifically linking the targeting of type II cells for injury to the development of lung fibrosis. Based on our findings and mounting clinical evidence, we speculate that alveolar epithelial cell damage may play a central role in the pathogenesis of IPF. Our model should be useful for elucidating the downstream mechanisms by which targeting of type II cells leads to fibroblast proliferation, collagen deposition, and alveolar disruption.