Administration of amniotic fluid stem cell extracellular vesicles promotes development of fetal hypoplastic lungs by immunomodulating lung macrophages

Abstract

Congenital diaphragmatic hernia (CDH) is a devastating condition characterized by incomplete closure of the diaphragm and herniation of abdominal organs into the chest. As a result, fetuses have pulmonary hypoplasia, whose severity is the main determinant of poor outcome. The pathogenesis of pulmonary hypoplasia secondary to CDH is at least in part explained by lack or dysregulation of miRNAs that are known to regulate lung developmental processes. Herein, we report that intra-amniotic administration of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) rescues lung growth and maturation in a fetal rat model of CDH. To understand which fetal lung cells and biological pathways are affected by AFSC-EVs, we conducted whole lung single nucleus RNA-sequencing. We discovered that CDH lungs have a multilineage inflammatory signature with macrophage enrichment, and confirmed these findings in autopsy samples of lungs from human fetuses with CDH. Transcriptomic analysis of CDH fetal rat lungs also showed that AFSC-EV treatment reduced macrophage density and inflammation to normal levels. Analyzing the miRNAs contained in the AFSC-EV cargo with validated mRNA targets, we found that the downregulated genes in AFSC-EV treated CDH lungs were involved in inflammatory response and immune system processes. This study reports a single cell atlas of normal and hypoplastic CDH fetal rat lungs and provides evidence that AFSC-EVs restore lung development by addressing multiple pathophysiological aspects of CDH.

Overactivated epithelial NF-κB disrupts lung development in human and nitrofen CDH

“…Here, we identified increased TNF-α abundance in the airway epithelium of nitrofen CDH lungs, which corresponds to a previous report that described increased TNF-α abundance in distal CDH lungs when compared to age-matched non-CDH controls 44 . In line, we and others previously found increased pro-inflammatory markers (e.g., MCP-1; STAT3) and enrichment of immune cells such as pulmonary macrophages in nitrofen CDH lungs 19,23,34,45 . Accordingly, different groups have reported positive effects of late gestational anti-inflammatory treatment with dexamethasone (DXM) on distal lung development in the nitrofen rat and a sheep model of CDH [30][31][32]46 .…”

“…In this context, we had previously reported an increase of pulmonary macrophages and monocyte chemoattractant protein 1 in nitrofen CDH lungs, as a potential source of pro-inflammatory signaling in CDH lungs 23,38 . A more recent and unbiased single nuclear RNA sequencing approach confirmed an influx of macrophages and other immune cells, together with an increase of inflammatory pathways in nitrofen CDH lungs at E21 34 . While these studies do not report on the polarity and molecular function of recruited macrophages in CDH lungs, they indicate that immune cells and in particular lung macrophages and their pro-inflammatory effects should be investigated in CDH pathophysiology and potential therapeutic targeting.…”

“…Lung underdevelopment remains the main cause of death in neonates with CDH. While the established nitrofen rat model and other genetic mouse models have revealed different developmentally important pathways that are altered in CDH 4,7 , more recent reports focused on inflammatory pathways as a common event between animal models and human CDH 19,20,34 .…”

“…Promising antenatal surgical interventions, such as fetoscopic endoluminal tracheal occlusion (FETO) 48 , in combination with established prenatal anti-inflammatory therapeutics such as corticosteroids; or novel experimental approaches (e.g. : extracellular vesicles 34 ) deserve further investigation.…”

Fetal lung vascular development is disrupted by mechanical compression and rescued by administration of amniotic fluid stem cell extracellular vesicles via regulation of the Hippo signaling pathway

“…Next, we sought to explore the underlying mechanisms of compression-induced vascular remodeling in fetal lungs. Using a dataset that we previously obtained by single nucleus RNA-sequencing of rat fetal lungs with CDH 25 , we investigated possible signaling networks that are known to be associated with mechanical stimuli response. We found that CDH lungs had dysregulated expression of Hippo pathway genes across the main cell populations of the fetal lung, i.e., endothelium, epithelium, mesenchyme, and immune cells ( Figure 3A-C ).…”