Atf3 defines a population of pulmonary endothelial cells essential for lung regeneration

  1. Terren K Niethamer
  2. Lillian I Levin
  3. Michael P Morley
  4. Apoorva Babu
  5. Su Zhou
  6. Edward E Morrisey

  • Curated by eLife

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    eLife Assessment

    The study has advanced our mechanistic understanding of lung regeneration. While the importance of regeneration of alveolar capillaries for long response to injury has been long recognized, the regulation of this process has not been well understood. Your study provides novel, comprehensive, and compelling evidence that the expression of the transcription factor Atf3 in alveolar capillary endothelial cells plays a critical role in the regeneration of alveolar capillaries following lung injury.

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Abstract

Following acute injury, the capillary vascular bed in the lung must be repaired to reestablish gas exchange with the external environment. Little is known about the transcriptional and signaling factors that drive pulmonary endothelial cell (EC) proliferation and subsequent regeneration of pulmonary capillaries, as well as their response to stress. Here, we show that the transcription factor Atf3 is essential for the regenerative response of the mouse pulmonary endothelium after influenza infection. Atf3 expression defines a subpopulation of capillary ECs enriched in genes involved in endothelial development, differentiation, and migration. During lung alveolar regeneration, this EC population expands and increases the expression of genes involved in angiogenesis, blood vessel development, and cellular response to stress. Importantly, endothelial cell-specific loss of Atf3 results in defective alveolar regeneration, in part through increased apoptosis and decreased proliferation in the endothelium. This leads to the general loss of alveolar endothelium and persistent morphological changes to the alveolar niche, including an emphysema-like phenotype with enlarged alveolar airspaces lined with regions that lack vascular investment. Taken together, these data implicate Atf3 as an essential component of the vascular response to acute lung injury that is required for successful lung alveolar regeneration.

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  1. Version published to 10.7554/elife.83835 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    We thank the reviewer for their assessment that our work “supports the idea that epithelial-endothelial crosstalk is important for lung regeneration and proposes a potential candidate for this process” and their helpful suggestions for strengthening and clarifying our work.

    1. The scRNA-seq analysis is performed in two separate objects ("control lung" and "H1N1 infected lung 14dpi"). For these two sets of data to be comparable, the authors should have integrated the objects and analyzed them together. This is not only important for deciding the clusters' identities and making sure that the same clusters are compared between control and infected, but also necessary to compare gene expression.

    We have integrated the control and H1N1-infected scRNA-seq datasets and reanalyzed the integrated data. We then analyzed CAP1_A and CAP1_B populations, comparing their gene expression between control and influenza conditions. Unbiased clustering of the integrated dataset reveals the same clusters we identified in the individual datasets, with cells from control and flu contributing to each cluster (with the exception of proliferating endothelial cells, which are found only in the H1N1-infected lung). We have added a supplemental figure outlining these data (Figure 1 – Figure Supplement 3).

    1. ATF3 is not only present in Cap1_B, in the infected lung there seems like Cap1_A express less ATF3. The authors should comment on this difference.

    We have added violin plots to Figure 1, which we feel will better represent the greater Atf3 expression in CAP1_Bs relative to other endothelial cell subtypes. The reviewer is correct that Atf3-expressing cells are found in large vessels, but they are also numerous in the alveolar capillary space and increase with influenza in these regions. We have added lower-magnification, higher-resolution images of Atf3CreER; ROSA26tdTomato animals, both control and influenza-infected, to illustrate this expansion in a new Figure 2 – Figure Supplement 3. This increase is also quantified in Figure 2C. We have also clarified this in the text.

    1. It is unclear how the clusters Cap1_A and Cap1_B were decided. The manuscript would benefit from clarification.

    We have added text to the Materials and Methods section to clarify this.

    1. It would be beneficial to see via immunofluorescence the morphological and spatial differences between ATF3-expressing and non-expressing endothelial cells since this transcription factor is expressed in multiple endothelial cell types.

    We have added lower-magnification, higher-resolution images of Atf3CreER; ROSA26tdTomato animals, both control and influenza-infected, to illustrate the spatial distribution of Atf3-expressing endothelial cells. This data is now shown in the new Figure 2 – Figure Supplement 3. We have also added further data to the new Figure 5 – Figure Supplement 1 to include the cytoplasmic endothelial marker Endomucin-1 (EndoM1) in an analysis of the spatial distribution of endothelial cells in wild-type and Atf3-knockout animals at 21 dpi.

    1. The authors mention ATF3 is not endothelial-specific. Expression of ATF3 in other cell types should be evaluated via immunofluorescence.

    This data is present in Figure 2 – Figure Supplement 2.

    1. The authors should have shown evidence of the deletion in their Atf3EC-KO mouse and addressed whether they had residual ATF3. If there is no antibody available, RNAscope could be used, or Western Blot or RT-PCR on sorted endothelial cells.

    We agree that this is an important quantification to make. We have performed qRT-PCR for Atf3 in both the animals used to perform the RNA sequencing experiment as well as a new cohort of animals to confirm Atf3 deletion. We have added these results to a new supplemental figure accompanying Figure 4 (Figure 4 – Figure Supplement 1).

    1. The authors only show the epithelium as evidence that the alveolar region is altered in their mutant after infection. The endothelium should have also been investigated, especially since their mutant is an endothelial-specific deletion. Within this, the different endothelial cells should have been assessed by a method other than RNAscope such as immunofluorescence, given that this method is unable to show morphology and there are antibodies available.

    This data is present in Figure 5. We have also added additional data to the new Figure 5 – Figure Supplement 1 to extend our analysis to 21 dpi and to incorporate a cytoplasmic marker of endothelial cells, Endomucin (EndoM1).

    1. Bulk RNA-seq from endothelial cells is used in the manuscript. However, because ATF3 is not specific to Cap1_B cells or even capillaries alone, the downstream gene expression analysis of bulk RNA should be placed into the context of lung endothelial heterogeneity.

    We have added qRT-PCR analysis of several downstream genes to address the comments of Reviewer #3, point #3. To place this into the context of endothelial heterogeneity, we have added dot plots to show the expression of selected genes from the RNA-seq analysis in each endothelial subtype from the H1N1 scRNA-seq dataset. These data can be found in the new Figure 4 – Figure Supplement 1. However, because of the relatively low sequencing depth of scRNA-seq compared to bulk RNA-seq, many of the transcripts examined were only present in a small percentage of endothelial cells in the scRNA-seq dataset, so the differences seen are more striking in the RNA-seq data.

    1. Although the authors mentioned that the infection with H1N1 influenza can have regional differences, they do not show how they picked regions for their analysis and quantification, and whether ATF3 upregulation was found in more severely affected regions. Furthermore, since they quantified via FACS, this heterogeneity in the infection itself could have affected their observations.

    We agree that it is essential both to define the extent of H1N1-mediated inflammation in Atf3 wild-type and knockout mice and to compare this factor between genotypes. We have therefore used a previously published method for quantifying regions of severe, damaged, and normal tissue structure (Liberti et al., Cell Reports 2021) in both Atf3 wild-type and knockout animals. Our results show that Atf3 wild-type and knockout mice have similar levels of tissue damage, and we have added a supplemental figure demonstrating these data (new Figure 3 – Figure Supplement 2). We have also clarified how regions were selected for quantification of alveolar area.

    H1N1 influenza injury in mice is heterogeneous, with regions of severe alveolar destruction marked by densely packed immune cells, adjacent regions of damaged tissue, and regions of tissue that appear to have normal tissue structure, as we and others have previously described (Zacharias, Frank et al., Nature 2018; Liberti et al., Cell Reports 2021; Niethamer et al., eLife 2020). However, it has become increasingly apparent that these regions where tissue structure appears normal are actually regions of active regeneration, and endothelial cell proliferation is increased in these regions (Niethamer et al., eLife 2020). We therefore selected 20X fields in these areas to use for quantifying alveolar area, as these are actively regenerating regions where alveolar structures are present for quantification. Because of the changes to tissue structure seen in damaged or destroyed tissue areas, we did not select these regions for quantification, although they were present at similar frequency in Atf3 wild-type and knockout animals.

  3. eLife

    eLife Assessment

    The study has advanced our mechanistic understanding of lung regeneration. While the importance of regeneration of alveolar capillaries for long response to injury has been long recognized, the regulation of this process has not been well understood. Your study provides novel, comprehensive, and compelling evidence that the expression of the transcription factor Atf3 in alveolar capillary endothelial cells plays a critical role in the regeneration of alveolar capillaries following lung injury.

  4. eLife

    Reviewer #1 (Public Review):

    Here the authors investigate the mechanisms by which pulmonary endothelial cells (EC) contribute to alveolar repair post-H1N1-mediated acute lung injury and the molecular basis for the heterogeneity of this response among different EC subpopulations. Using single-cell transcriptomic analysis they identify the CREB family factor Atf3 differentially enriched in CAP1B cells, a subpopulation of EC previously known for its proliferative behavior in response to alveolar injury. They report a crucial role for Atf3 in injury repair but not during homeostasis. Using a combination of lineage tracing and loss function approach and an influenza mouse model in vivo, they show that Atf3 inactivation in ECs results in the inability of CAP1B ECs to initiate a proliferative response to repair the vascular compartment and ultimately regenerate the lung. Notably, the decreased number of Atf3 lineage-labeled EC capillaries was shown to correlate with the alveolar regions that failed to repair the post-H1N1 injury. They conclude that Atf3 is an essential factor for repair damaged capillaries in alveolar injury.

    The study is carefully designed and the results provide novel important information about a previously undisclosed role of Atf3 in the regeneration of the lung vascular component. The work has many strengths and is supported by impressively coherent data from the analysis of mouse genetic models, single-cell transcriptomic, and phenotypic characterization.

  5. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Niethamer et al. investigate the role of the transcription factor ATF3 in lung regeneration after H1N1 influenza. They focus on endothelial ATF3 which is present in a subset of lung capillaries in the adult mouse lung. Interestingly, they found that influenza infection upregulates endothelial ATF3 and that endothelial deletion of Atf3 results in impaired regeneration, leading to enlarged airspaces after viral infection. They further show that this effect may be due to an increase in apoptosis and a decrease in proliferation, suggesting that endothelial ATF3 is necessary for pulmonary vascular regeneration, as well as recovery of the alveolar architecture.
    Given the recent publications in the field describing lung endothelial heterogeneity, as well as its possible role in injury repair, this work is relevant to the community. It also supports the idea that epithelial-endothelial crosstalk is important for lung regeneration and proposes a potential candidate for this process.

    Strengths:
    The authors identified and tested the role of endothelial Atf3 in lung regeneration using well-established techniques. They identified this transcription factor as a candidate using state-of-the-art scRNA-seq. They also carefully lineage traced ATF3 expressing cells using an inducible reporter before and after infection and then used a pan-endothelial driver Cdh5 to delete Atf3 specifically in the endothelium. Thus, the authors successfully show significant changes in the alveolar structure after infection in their mutant model.

    Weaknesses:
    Although there is evidence that the author's claims have biological relevance, this paper would benefit from strengthening and/or clarifying some things:

    • The scRNA-seq analysis is performed in two separate objects ("control lung" and "H1N1 infected lung 14dpi"). For these two sets of data to be comparable, the authors should have integrated the objects and analyzed them together. This is not only important for deciding the clusters' identities and making sure that the same clusters are compared between control and infected, but also necessary to compare gene expression.
    • ATF3 is not only present in Cap1_B, in the infected lung there seems like Cap1_A express less ATF3. The authors should comment on this difference.
    • It is unclear how the clusters Cap1_A and Cap1_B were decided. The manuscript would benefit from clarification.
    • It would be beneficial to see via immunofluorescence the morphological and spatial differences between ATF3-expressing and non-expressing endothelial cells since this transcription factor is expressed in multiple endothelial cell types.
    • The authors mention ATF3 is not endothelial-specific. Expression of ATF3 in other cell types should be evaluated via immunofluorescence.
    • The authors should have shown evidence of the deletion in their Atf3EC-KO mouse and addressed whether they had residual ATF3. If there is no antibody available, RNAscope could be used, or Western Blot or RT-PCR on sorted endothelial cells.
    • The authors only show the epithelium as evidence that the alveolar region is altered in their mutant after infection. The endothelium should have also been investigated, especially since their mutant is an endothelial-specific deletion. Within this, the different endothelial cells should have been assessed by a method other than RNAscope such as immunofluorescence, given that this method is unable to show morphology and there are antibodies available.
    • Bulk RNA-seq from endothelial cells is used in the manuscript. However, because ATF3 is not specific to Cap1_B cells or even capillaries alone, the downstream gene expression analysis of bulk RNA should be placed into the context of lung endothelial heterogeneity.
    • Although the authors mentioned that the infection with H1N1 influenza can have regional differences, they do not show how they picked regions for their analysis and quantification, and whether ATF3 upregulation was found in more severely affected regions. Furthermore, since they quantified via FACS, this heterogeneity in the infection itself could have affected their observations.

  6. eLife

    Reviewer #3 (Public Review):

    Although the response to stress has been extensively studied in pulmonary epithelium and mesenchyme, the post-injury proliferation and subsequent regeneration of pulmonary capillary endothelial cells remain poorly understood. Following their previous study on identifying mouse lung endothelial cell heterogeneity, Niethamer et al. reported a lung capillary subpopulation, CAP1_B with highly enriched Atf3. This capillary subpopulation expanded and increased the expression of genes involved in vascular regeneration in response to influenza-induced lung injury. Loss of Atf3 in lung endothelial cells led to abnormal alveoli structure and loss of endothelial cells through inhibiting cell proliferation and inducing apoptosis. This manuscript provided strong evidence to demonstrate the importance of Atf3 in mediating endothelial response to lung injury, which is novel to the field.

  7. Version published to 10.1101/2022.10.14.512212v1 on bioRxiv