Nuclear Factor-κB Keeps Basal Cells Undifferentiated in Congenital Diaphragmatic Hernia

Among the congenital anomalies impacting lung development, congenital diaphragmatic hernia (CDH) is among the most common, affecting 1 in 2,500 pregnancies, with the most severe defects having mortality rates exceeding 50% (1) and survivors having substantially impaired lung function (2). There are at least three notable derangements in the CDH lung that contribute to morbidity and mortality. First, the mass effect of abdominal organs entering the chest causes hypoplasia of ipsilateral and contralateral lungs. In a multivariate analysis of magnetic resonance imaging–derived body measurements of 63 third-trimester fetuses with CDH, only percent predicted lung volume was predictive of the need for extracorporeal membrane oxygenation and survival to discharge (3). In addition, not only the lung parenchyma but also the conducting airway cross-sectional area are reduced and are prone to dynamic collapse (4). Second, the pulmonary microvasculature is severely pruned in CDH (5), and pulmonary hypertension is common (6). In a cohort of 220 infants with CDH, B-natriuretic peptide (a marker of atrial distention) and echocardiographic measures of pulmonary hypertension were significantly associated with mortality (7). Third, and perhaps less well known, the lung in general, and the epithelium, in particular, are less mature (8, 9) and have altered protein homeostasis (10). In this issue of the Journal, Wagner and colleagues (pp. 1214–1226) use basal cells from the tracheal aspirates of infants with CDH and a rat CDH model to show that nuclear factor (NF)-κB signaling plays a key role in this respiratory epithelial cell dysfunction (11).

Although the embryonic lung epithelium is derived from epithelial progenitor cells (12), the canonical stem cell of the fetal conducting airway epithelium is the same as in the adult conducting airway epithelium: the P63 + basal cell. The key finding of Wagner and colleagues is that basal cells of infants with CDH have an epigenetic predisposition to inflammasome activation that is absent in the basal cells of term and premature infants. Through a series of in vitro and in vivo experiments with correlation in CDH and non-CDH human lungs, NF-κB activity prevented the differentiation of basal cells into club and ciliated cells and preserved the immature epithelial phenotype in cell culture and in fetal rat airways. Thus, NF-κB is responsible for maintaining the CDH lung epithelium in a persistently immature state. In this study, general and specific inhibition of NF-κB with dexamethasone and JSH-23, respectively, restored basal cell differentiation.

Although the authors are to be commended for their creative approach in culturing basal cells from the tracheal aspirates of recently born infants, they did not definitively show that the behavior of basal cells that become dislodged from the basement membrane is representative of basal cells at large, except insofar as to show that fetuses with CDH have an expansion of basal cells compared with non-CDH fetuses. However, there are two reasons to think this is likely the case. First, like the basal cell culture model, expansion and impaired differentiation of basal cells were observed in the rat CDH model. Second, in human subjects with CDH, whatever process caused the observed epigenetic changes in the detached basal cells was likely present before birth and throughout the epithelium. It would be interesting to evaluate the tracheal epithelium of CDH and control fetal specimens for these same epigenetic marks and to test whether the loss of such marks occurs during maturation.

The interplay of lung development and inflammation is complex, and this paper adds to our understanding of how best to use antiinflammatory agents to optimize lung function in CDH. Chorioamnionitis accelerates lung maturation, and prenatal corticosteroids improve alveolar type 2 cell function (13), but late prenatal corticosteroids for fetuses with CDH do not improve survival or lung function (14). There is a growing understanding of how inflammatory signaling interacts with progrowth programs such as transforming growth factor-β (15) and how macrophages influence lung development (16). Mesenchymal stem cells (MSCs) and their extracellular vesicles attenuate many NF-κB signaling cascades and have been variably effective at reducing injury and improving outcomes in a host of conditions (17). MSCs and MSC extracellular vesicles improved lung development in the rat nitrofen CDH model (18, 19). However, the effects may not be purely antiinflammatory, because Zani and colleagues recently demonstrated that extracellular vesicles from amniotic fluid MSCs acted via transfer of microRNAs to improve lung development (20). It is notable that in the present study, Wagner and colleagues administered dexamethasone on three consecutive days during the pseudoglandular stage of lung development. Negative human studies administered four doses during the late saccular stage of lung development (14). Therapies that aim to improve lung structure and cell composition would need to begin sooner and to be administered more frequently than has been done in the past.

Understanding how antiinflammatory therapies impact other CDH treatments is important. There is a curious similarity in the cellular phenotype of CDH lung and the lung cell populations of experimental animal fetuses subjected to fetal tracheal occlusion. Fetal tracheal occlusion is a prenatal surgical procedure for CDH in which a fetoscopically placed balloon temporarily prevents egress of epithelial secretions with resultant growth of the lung. The procedure improves survival in fetuses with severe CDH (12). Inflammatory cytokines were increased in an ovine model of tracheal occlusion with and without surgical CDH (2), and basal cells were increased in a mouse model of tracheal occlusion without CDH (13). This finding was attributable to Yap and not NF-κB. Humans with congenital high airway obstruction also had basal cell expansion (13). Although the basal cell epigenetic landscape is not known in these cases, it could be that prenatal airway inflammation in general leads to basal cell persistence and that the phenotype is not restricted to CDH. With the emergence of newer technologies, we can better test such hypotheses and gain a better understanding of how the respiratory epithelium matures in utero and how best to target NF-κB to improve neonatal lung health.