Impaired Myofibroblast Proliferation is a Central Feature of Pathologic Post-Natal Alveolar Simplification

Reviewer #1 (Public Review):

Summary:

In this study, the authors used both the commonly used neonatal hyperoxia model as well as cell-type-specific genetic inactivation of Tgfbr2 models to study the basis of BPD. The bulk of the analyses focus on the mesenchymal cells. Results indicate impaired myofibroblast proliferation, resulting in decreased cell number. InactzXivation of Etc2 in Pdgfra-lineaged cells, preventing cytokinesis of myofibroblasts, led to alveolar simplification. Together, the findings demonstrate that disrupted myofibroblast proliferation is a key contributor to BPD pathogenesis.

Strengths:

Overall, this comprehensive study of BPD models advances our understanding of the disease. The data are of high quality.

Weaknesses:

The critiques are mostly minor and can be addressed without extensive experimentation.

Reviewer #2 (Public Review):

Summary:

In this study, the authors systematically explore the mechanism(s) of impaired postnatal lung development with relevance to BPD (bronchopulmonary dysplasia) in two murine models of 'alveolar simplification', namely hyperoxia and epithelial loss of TGFb signaling. The work presented here is of great importance, given the limited treatment options for a clinical entity frequently encountered in newborns with high morbidity and mortality that is still poorly understood, and the unclear role of TGFb signaling, its signaling levels, and its cellular effects during secondary alveolar septum formation, a lung structure generating event heavily impacted by BPD. The authors show that hyperoxia and epithelial TGFb signaling loss have similar detrimental effects on lung structure and mechanical properties (emphysema-like phenotype) and are associated with significantly decreased numbers of PDGFRa-expressing cells, the major cell pool responsible for generation of postnatal myofibroblasts. They then use a single-cell transcriptomic approach combined with pathway enrichment analysis for both models to elucidate common factors that affect alveologenesis. Using cell communication analysis (NicheNet) between epithelial and myofibroblasts they confirm increased projected TGFb-TGFbR interactions and decreased projected interactions for PDGFA-PDGFRA, and other key pathways, such as SHH and WNT. Based on these results they go on to uncover in a sequela of experiments that surprisingly, increased TGFb appears reactive to postnatal lung injury and rather protective/homeostatic in nature, and the authors establish the requirement for alpha V integrins, but not the subtype alphaVbeta6, a known activator of TGFb signaling and implied in adult lung fibrosis. The authors then go beyond the TGFb axis evaluation to show that mere inhibition of proliferation by conditional KO of Ect2 in Pdgfra lineage results in alveolar simplification, pointing out the pivotal role of PDGFRa-expressing myofibroblasts for normal postnatal lung development.

Strengths:

(1) The approach including both pharmacologic and mechanistically-relevant transgenic interventions both of which produced consistent results provides robustness of the results presented here.

(2) Further adding to this robustness is the use of moderate levels of hyperoxia at 75% FiO2, which is less extreme than 100% FiO2 frequently used by others in the field, and therefore favors the null hypothesis.

(3) The prudent use of advanced single-cell analysis tools, such as NicheNet to establish cell interactions through the pathways they tested and the validation of their scRNA-seq results by analysis of two external datasets. Delineation of the complexity of signals between different cell types during normal and perturbed lung development, such as attempted successfully in this study, will yield further insights into the underlying mechanism(s).

(4) The combined readout of lung morphometric (MLI) and lung physiologic parameters generates a clinically meaningful readout of lung structure and function.

(5) The systematic evaluation of TGFb signaling better determines the role in normal and postnatally-injured lungs.

Weaknesses:

(1) While the study convincingly establishes the effect of lung injury on the proliferation of PDGFRa-expressing cells, differentiation is equally important. Characterization of PDGFRa expressing cells and tracking the changes in the injury models in the scRNA analysis, a key feature of this study, would benefit from expansion in this regard. PDGFRa lineage gives rise to several key fibroblast populations, including myofibroblasts, lipofibroblasts, and matrix-type fibroblasts (Collagen13a1, Collagen14a1). Lipofibroblasts constitute a significant fraction of PDGFRa+ cells, and expand in response to hyperoxic injury, as shown by others. Collagen13a1-expressing fibroblasts expand significantly under both conditions (Figure 3), and appear to contain a significant number of PDGFRa-expressing cells (Suppl Fig.1). Effects of the applied injuries on known differentiation markers for these populations should be documented. Another important aspect would be to evaluate whether the protective/homeostatic effect of TGFb signaling is supporting the differentiation of myofibroblasts. Postnatal Gli1 lineage gains expression of PDGFRa and differentiation markers, such as Acta2 (SMA) and Eln (Tropoelastin). Loss of PDGFRa expression was shown to alter Elastin and TGFb pathway-related genes. TGFb signaling is tightly linked to the ECM via LTBPs, Fibrillins, and Fibulins. An additional analysis in the aforementioned regard has great potential to more specifically identify the cell type(s) affected by the loss of TGFb signaling and allow analysis of their specific transcriptomic changes in response and underlying mechanism(s) to postnatal injury.

(2) Of the three major lung abnormalities encountered in BPD, the authors focus on alveolarization impairment in great detail, to a very limited extent on inflammation, and not on vascularization impairment. However, this would be important not only to better capture the established pathohistologic abnormalities of BPD, but also it is needed since the authors alter TGFb signaling, and inflammatory and vascular phenotypes with developmental loss of TGFb signaling and its activators have been described. Since the authors make the point about the absence of inflammation in their BPD model, it will be important to show the evidence.

(3) Conceptually it would be important that in the discussion the authors reconcile their findings in the experimental BPD models in light of human BPD and the potential implications it might have on new ways to target key pathways and cell types for treatment. This allows the scientific community to formulate the next set of questions in a disease-relevant manner.

Reviewer #3 (Public Review):

Summary:

This paper seeks to understand the role of alveolar myofibroblasts in abnormal lung development after saccular stage injury.

Strengths:

Multiple models of neonatal injury are used, including hyperoxia and transgenic models that target alveolar myofibroblasts.

Weaknesses:

There are several weaknesses that leave the conclusions significantly undersupported by the data as presented:

(1) There is no validation of the decreased number of myofibroblasts suggested by flow cytometry/scRNAseq at the level of the tissue. Given that multiple groups have reported increased myofibroblasts (aSMA+ fibroblasts) in humans with BPD and in mouse models, demonstrating a departure from prior findings with tissue validation in the mouse models is essential. There are many reasons for decreased numbers of a subpopulation by flow cytometry, most notably that injured cells may be less likely to survive the cell sorting process.

(2) The hallmark genes used to define the subpopulations are not given in single-cell data. As the definition of fibroblast subtypes remains an area of unsettled discussion in the field, it is possible that the decreased number by classification and not a true difference. Tissue validation and more transparency in the methods used for single-cell sequencing would be critical here.

(3) There is an oversimplification of neonatal hyperoxia as a "BPD model" used here without a reference to detailed prior work demonstrating that the degree and duration of hyperoxia dramatically change the phenotype. For example, Morty et al have shown that hyperoxia of 85% or more x 14 days is required to demonstrate the septal thickening observed in severe human BPD. Other than one metric of lung morphometry (MLI), which is missing units on the y-axis and flexivent data, the authors have not fully characterized this model. Prior work comparing 75% O2 exposure for 5, 8, or 14 days shows that in the 8-day exposed group (similar to the model used here), much of the injury was reversible. What evidence do the authors have that hyperoxia alone is an accurate model of the permanent structural injury seen in human BPD?

(4) Thibeault et al published a single-cell analysis of neoantal hyperoxia in 2021, with seemingly contrasting findings. How does this dataset compare in context?