Emerging yet complex role of fetal lung macrophages in patients with congenital diaphragmatic hernia

To the Editor Congenital diaphragmatic hernia (CDH) is a malformation characterized by in utero herniation of abdominal organs into the chest, resulting in pulmonary hypoplasia.1 Despite decades of research, the pathogenesis of pulmonary hypoplasia remains elusive.1 Emerging evidence suggests inflammation as a key driver in animal and human fetuses with CDH. We read with interest the letter by Riedel et al.on this topic.2 The authors first performed proteomics analysis on human lung tissue collected between 30 and 36 weeks of gestation from CDH and control fetuses. Enrichment analysis on 10 differentially expressed proteins revealed an involvement of the innate immune system. The authors then confirmed findings of upregulated CD68 + macrophages in human fetal CDH lungs that our group reported in 2024.3 Immunostaining experiments for CD206 + cells and Macrophage Migration Inhibitory Factor confirmed macrophage enrichment in fetal CDH lungs.2 Finally, the authors reported that tracheal aspirates derived from mechanically ventilated newborns with CDH had higher expression of macrophage/monocyte markers at flowcytometry compared to controls.2 We agree with the authors about the prominent role of ­macrophages in the lung immune system and their potential contribution to pulmonary hypoplasia in CDH. Nonetheless, it is important to acknowledge the complex biology of macrophages, the diversity of macrophage subtypes and their distinct roles in health and disease, including lung development, as well as the limitations of our current knowledge, which is largely based on rodent studies. Experimental evidence suggests that alveolar macrophages populate the alveolar niche during the first week of life, whereas a broad spectrum of interstitial macrophages resides in the mesenchymal or vascular niche with distinct roles during fetal development.4,5 Therefore, the contribution of alveolar macrophages to CDH pulmonary hypoplasia warrants cautious interpretation. In our study, using single-nucleus RNA sequencing in the CDH rat model, we discovered a multilineage inflammatory signature with an upregulation of macrophage genes.3 Some of the identified fetal lung macrophages in CDH rat lungs were likely derived from monocytes (CD68+/CD43high) and from the bone marrow (CD68+/ADGRE-1high). To overcome the intrinsic limitations of single-nucleus RNA sequencing, we used an algorithm trained on the only available adult lung dataset and predicted an enrichment of alveolar macrophages in CDH fetal lungs. However, given the abundance of alveolar macrophages during adult life, the prediction in our fetal model might have over-represented this cell subtype.3 Similarly, in the work by Riedel et al. the easy accessibility of alveolar macrophages through tracheal aspiration may have contributed to an overinterpretation of their role. The limited macrophage panel (CD11b, CD14, CD86, CD163, CD206) and the use of single-staining immunofluorescence did not allow for further macrophage classification, which would have provided additional insights. It is important to remember that interstitial macrophages residing in the perivascular and mesenchymal regions are key not only in defending against microbes but also in regulating tissue homeostasis, remodeling, and repair.5 To support their interpretation of a prominent role for alveolar macrophages in pulmonary hypoplasia, Riedel et al.cite a recent study reporting increased alveolar cell populations co-expressing type I and II markers in human fetal CDH lungs, a phenotype proposed to result from inflammatory signals originating from an expanded alveolar macrophage population.2,6 However, these transitional cells may also be influenced by inflammatory cues from interstitial macrophages or other immune cells within the fetal lung.7 We agree with the authors that fetal lung macrophages and inflammation represent an exciting opportunity to identify novel therapeutic targets for CDH. Yet, before moving in that direction, it will be crucial to conduct additional studies that examine all macrophage subtypes as well as other immune cells to define their dysregulation and clarify their potential contribution to the development of pulmonary hypoplasia in CDH. Supplementary material is available at American Journal of ­Respiratory and Critical Care Medicine online. Please see the ICMJE disclosure forms, which have been provided as supplementary material. Funding support for this article was provided by the Washington University School of Medicine, Department of Surgery Start-Up funds.