Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA. Department of Veterans Affairs Medical Center, Nashville, Tennessee, USA. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Address correspondence to: Timothy S. Blackwell, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, 1611 21st Avenue South, T-1218 Medical Center North, Nashville, Tennessee 37232, USA. Phone: 615.322.3412; Email: email@example.com.
First published August 13, 2018 - More info
Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell–restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc (SP-CI73T). Tamoxifen-treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7–14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2–4 weeks), commensurate with a rise in TGF-β1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, α–smooth muscle actin–positive (α-SMA–positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis.
Shin-Ichi Nureki, Yaniv Tomer, Alessandro Venosa, Jeremy Katzen, Scott J. Russo, Sarita Jamil, Matthew Barrett, Vivian Nguyen, Meghan Kopp, Surafel Mulugeta, Michael F. Beers
Although mutant forms of the gene encoding surfactant protein C (SFTPC) have been linked to interstitial lung disease, the mechanisms by which the most common of these mutations, SFTPCI73T, results in lung fibrosis are uncertain. In this issue of the JCI, Nureki et al. developed a knockin mouse model and showed that SFTPCI73T is expressed by alveolar type II (AT2) epithelial cells in the lungs. These mice developed an age-related fibrotic phenotype when the mutant allele was expressed at low levels and acute lung inflammation/injury followed by lung fibrosis when mutant SFTPCI73T expression was enhanced. This work provides important information regarding the impact of AT2 cell dysfunction on fibrotic remodeling in the lungs.
Although understanding of the genetic underpinnings of interstitial lung disease (ILD) is far from complete, identification of rare genetic variants that associate with disease in some families has provided valuable information regarding key cells and pathways that regulate fibrotic remodeling in the lungs. Outside of defined genetic disorders, such as Hermansky-Pudlak syndrome, surfactant protein C (SFTPC) was the first gene associated with ILD. Nogee and colleagues identified a heterozygous rare variant in this gene in an affected mother and child in 2001 (1). In 2002, a rare missense variant, SFTPCL188Q, was shown to segregate with disease in a large family with ILD, including idiopathic pulmonary fibrosis (IPF) in affected adults (2). Subsequently, ILD-associated mutations in another surfactant protein, SFTPA2, were identified (3). Together, these studies provide convincing evidence that alveolar type II (AT2) cells, which produce surfactant proteins in the lungs, are integral to disease pathogenesis. Rare variants that involve the C-terminal portion (or BRICHOS domain) of proSP-C result in protein misfolding, aggregation, and endoplasmic reticulum (ER) stress (4–6); however, the mechanisms linking AT2 dysfunction and disease in individuals with the most common SFTPC mutation, SFTPCI73T, in humans with ILD have remained unclear. In this issue, Nureki et al. (7) use a clever strategy to develop a knockin mouse model to investigate how this toxic gain-of-function mutation causes AT2 dysfunction and leads to progressive lung fibrosis.
Prior attempts to express mutant forms of SFTPC in AT2 cells in vivo have been hampered by perinatal mortality in response to constitutive expression of mutant SFTPC during lung development and lack of a spontaneous phenotype following induction of low levels of SFTPCL188Q in adult mice (5, 8, 9). The strategy employed by Nureki et al. involves using a construct to insert an HA-tagged SFTPCI73T mutant containing a PGK-Neo cassette in intron 1, which functions to limit expression of the mutant allele, into the endogenous Sftpc locus (7). Despite evidence of altered protein processing and trafficking, the resulting phenotype was relatively mild, with alveolar septal thickening and increased collagen content. Removal of the inhibitory PGK-Neo cassette via tamoxifen-induced expression of Flp-O recombinase enhanced SFTPCI73T expression, resulting in embryonic lethality during the saccular stage of lung development, similar to what has been observed in previous models (8, 9). However, when the PGK-Neo cassette was removed after lung development, increased SFTPCI73T expression in homozygous mice resulted in acute lung injury, with substantial mortality followed by a fibrotic ILD-like phenotype. Heterozygous SFTPCI73T/+ mice developed an intermediate phenotype. Intriguingly, while Nureki et al. found evidence of increased macroautophagy, they found no evidence of AT2 cell apoptosis in this model (7). These findings are consistent with prior studies in which SFTPCI73T was overexpressed in vitro (10) but contrast with results from a variety of animal model studies that have linked AT2 cell injury and apoptosis to subsequent development of lung fibrosis (5, 6). Instead, AT2 cells expressing SFTPCI73T had increased levels of TGF-β1, which can induce production of collagen and other matrix components by mesenchymal cells. Also, SFTPCI73T-expressing AT2 cells produced increased levels of CCL2, an important chemokine for recruitment of monocyte-derived macrophages, which were increased in SFTPCI73T-expressing mice and have been shown to participate in fibrotic remodeling (11, 12). In addition to a persistent increase in macrophages in the lungs, an early increase in neutrophils followed by a transient eosinophil influx was identified after deletion of the PGK-Neo cassette (7). This inflammatory cell influx was accompanied by only a modest increase in cytokine levels in bronchoalveolar lavage and an absence of the classic Th2 cytokine response that has been linked to fibrosis in some models (13).
The prevailing paradigm to explain IPF pathogenesis suggests that recurrent micro-injuries in the alveolar compartment culminate in lung fibrosis. While alveolar injury has been thought to result from environmental stimuli, this study by Nureki et al. raises the possibility that focal alveolar injury might be driven by endogenous AT2 cell activation. Interestingly, lung injury was worse in male SFTPCI73T-knockin mice compared with females, potentially shedding light on the preponderance of male IPF patients. Further studies will be needed to better characterize the AT2 cell phenotype that drives alveolar capillary barrier dysfunction and inflammatory cell influx. Moreover, it will be important to determine whether inflammatory cells, particularly macrophages, are essential for development of fibrosis in this model.
Although substantial apoptosis of AT2 cells was not observed in conjunction with lung injury in SFTPCI73T-knockin mice, cleaved caspase-3 was only measured at 2 and 4 weeks after tamoxifen treatment to remove the PGK-Neo cassette. Therefore, an early increase in AT2 cell apoptosis cannot be excluded as a possibility. Regardless, the total number of HA-tagged AT2 cells was increased by 7 days after tamoxifen treatment and persisted for up to 4 weeks. This finding is potentially relevant to human disease, as areas of AT2 cell hyperplasia are common in the IPF lung. The observed increase in AT2 cells in the alveolar compartment raises the question of whether there could be a defect in differentiation of AT2 cells to alveolar type I (AT1) epithelium in this model, which has been proposed in human IPF. Future studies to determine whether AT1 cell injury or death occurs in this model could be enlightening.
The SFTPCI73T-knockin mouse is the first model derived from an IPF-associated gene that develops spontaneous lung fibrosis. Other models with mutations in genes associated with familial IPF require a fibrotic stimulus, such as bleomycin, and manifest as increased fibrosis. Therefore, this model provides a translationally relevant platform to further explore disease pathogenesis and test potential therapeutic approaches. This work highlights an important strategy to operationalize genetic data by developing models wherein specific disease-associated mutations are expressed in appropriate cell types. This approach is particularly applicable in situations where the genetic variant (like SFTPCI73T) encodes a toxic gain-of-function protein.
TSB is supported by the NIH/National Heart, Lung, and Blood Institute (NIH-NHLBI; P01HL092870), the US Department of Defense, and the Department of Veterans Affairs.
Conflict of interest: TSB declares research support from Boehringer Ingelheim and Celgene.
Reference information: J Clin Invest. 2018;128(9):3745–3746. https://doi.org/10.1172/JCI122727.
See the related article at Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis.