BMP9 and BMP10 coordinate liver cellular crosstalk to maintain liver health

Reviewer #1 (Public Review):

Summary:

The aim of the present work is to evaluate the role of BMP9 and BMP10 in liver by depleting Bmp9 and Bmp10 from the main liver cell types (endothelial cells (EC), hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes (H)) using cell-specific cre recombinases. They show that HSCs are the main source of BMP9 and BMP10 in the liver. Using transgenic ALK1 reporter mice, they show that ALK1, the high affinity type 1 receptor for BMP9 and BMP10, is expressed on KC and EC. They have also performed bulk RNAseq analyses on whole liver, and cell-sorted EC and KC, and showed that loss of Bmp9 and Bmp10 decreased KC signature and that KC are replaced by monocyte-derived macrophages. EC derived from these Bmp9fl/flBmp10fl/flLratCre mice also lost their identity and transdifferentiated into continuous ECs. Liver iron metabolism and metabolic zonation were also affected in these mice. In conclusion, this work supports that BMP9 and BMP10 produced by HSC play a central role in mediating liver cell-cell crosstalk and liver homeostasis.

Strengths:

This work further supports the role of BMP9 and BMP10 in liver homeostasis. Using a specific HSC-Cre recombinase, the authors show for the first time that it is the BMP9 and BMP10 produced by HSC that play a central role in mediating liver cell-cell crosstalk to maintain a healthy liver. Although the overall message of the key role of BMP9 in liver homeostasis has been described by several groups, the role of hepatic BMP10 has not been studied before. Thus, one of the novelties of this work is to have used liver cell specific Cre recombinase to delete hepatic Bmp9 and Bmp10. The second novelty is the demonstration of the role of BMP9 and BMP10 in KC Differentiation/homeostasis which has already been slightly addressed by this group by knocking out ALK1, the high affinity receptor of BMP9 and BMP10 (Zhao et al. JCI, 2022).

Weaknesses:

This work remains rather descriptive and the molecular mechanisms are barely touched upon and could have been more explored.
Some references should be added; In particular, a work that has already demonstrated, using a different approach (in situ hybridization RNAscope), that in the liver BMP9 and BMP10 are expressed by HSC (Tillet et al., J Biol Chem 2018). Another publication (Bouvard et al., Cardiovasc Res, 2021) has previously showed that deletion of Bmp9 and Bmp10 leads to liver fibrosis and could have thus been cited. There is also a reference that is not correctly cited. Ref 26 (Herrera et al., 2014) does not say that "BMP10 is mostly expressed in the heart, followed by the liver" or that "BMP9 and BMP10 also bind to ALK2" as cited in the manuscript.
The gating strategies for cell sorting which is used for bulk RNAseq and FACS analyses should be better described in order to better follow the manuscript. This point is particularly important for KC gating as the authors show that Tim4 is very strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2c), yet, it seems that this marker is used for gating macrophages (Suppl fig4). Same question with F4/80 which is strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2d) and also used for gating. It is important to show the gating strategy for both Control and Bmp9fl/flBmp10fl/flLratCre mice.
The authors should explain how they selected the genes shown on each heatmaps and add references that can justify the choice of the genes.
Quantifications of Immunostaining and FACS data should be added as well as statistical analyses.

Reviewer #2 (Public Review):

Summary:

The authors characterized the contribution of BMP9/BMp10 expression/secretion from all different hepatic cell types and analysed their impact on the other cell types. They are able to show that HSC derived BMP9/BMP10 controls Kupffer cell and EC differentiation and functions.

Strengths:

This is the first study to my knowledge to comprehensively analyze the contribution of BMP9/BMP10 expression in such systematic fashion in vivo. This study therefore is a significant contribution to the field and further supports previous studies that have already implied BMP9 and BMP10 in Kupffer cell and EC functions but did not unravel the intercellular cross talk in such detailed fashion.

Weaknesses:

Several findings such as the impact of BMP9/10 on Kupffer cells and EC were already known. So these findings are not innovative, however I still believe that the elucidation of the cellular crosstalk makes this publication highly interesting to a broad scientific community.

Overall the authors achieved their aims and the results are well supporting the conclusions and discussion.

Author response:

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The aim of the present work is to evaluate the role of BMP9 and BMP10 in liver by depleting Bmp9 and Bmp10 from the main liver cell types (endothelial cells (EC), hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes (H)) using cell-specific cre recombinases. They show that HSCs are the main source of BMP9 and BMP10 in the liver. Using transgenic ALK1 reporter mice, they show that ALK1, the high affinity type 1 receptor for BMP9 and BMP10, is expressed on KC and EC. They have also performed bulk RNAseq analyses on whole liver, and cell-sorted EC and KC, and showed that loss of Bmp9 and Bmp10 decreased KC signature and that KC are replaced by monocyte-derived macrophages. EC derived from these Bmp9fl/flBmp10fl/flLratCre mice also lost their identity and transdifferentiated into continuous ECs. Liver iron metabolism and metabolic zonation were also affected in these mice. In conclusion, this work supports that BMP9 and BMP10 produced by HSC play a central role in mediating liver cell-cell crosstalk and liver homeostasis.

We appreciate the comprehensive summary of reviewer 1.

Strengths:

This work further supports the role of BMP9 and BMP10 in liver homeostasis. Using a specific HSC-Cre recombinase, the authors show for the first time that it is the BMP9 and BMP10 produced by HSC that play a central role in mediating liver cell-cell crosstalk to maintain a healthy liver. Although the overall message of the key role of BMP9 in liver homeostasis has been described by several groups, the role of hepatic BMP10 has not been studied before. Thus, one of the novelties of this work is to have used liver cell specific Cre recombinase to delete hepatic Bmp9 and Bmp10. The second novelty is the demonstration of the role of BMP9 and BMP10 in KC Differentiation/homeostasis which has already been slightly addressed by this group by knocking out ALK1, the high affinity receptor of BMP9 and BMP10 (Zhao et al. JCI, 2022).

We appreciate the positive comment of reviewer 1.

Weaknesses:

This work remains rather descriptive and the molecular mechanisms are barely touched upon and could have been more explored. Some references should be added; In particular, a work that has already demonstrated, using a different approach (in situ hybridization RNAscope), that in the liver BMP9 and BMP10 are expressed by HSC (Tillet et al., J Biol Chem 2018). Another publication (Bouvard et al., Cardiovasc Res, 2021) has previously showed that deletion of Bmp9 and Bmp10 leads to liver fibrosis and could have thus been cited. There is also a reference that is not correctly cited. Ref 26 (Herrera et al., 2014) does not say that "BMP10 is mostly expressed in the heart, followed by the liver" or that "BMP9 and BMP10 also bind to ALK2" as cited in the manuscript.

We agree with the comment of reviewer 1 that the molecular mechanisms were barely investigated in our work. Indeed, it has been reported that BMP9/10 induce the expression of ID1/3 in KCs and GATA4 and Maf in liver ECs in vitro culture system. These master regulators play an important role in the differentiation of the two cell types. Thus, we think that the reduced expression of these master regulators can explain the phenotype in KCs and ECs observed in Bmp9fl/flBmp10fl/flLratCre mice. In addition, according to the reviewer’s suggestion, these references will be added or corrected in our revised manuscript.

The gating strategies for cell sorting which is used for bulk RNAseq and FACS analyses should be better described in order to better follow the manuscript. This point is particularly important for KC gating as the authors show that Tim4 is very strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2c), yet, it seems that this marker is used for gating macrophages (Suppl fig4). Same question with F4/80 which is strongly decreased in Bmp9fl/flBmp10fl/flLratCre (Fig 2d) and also used for gating. It is important to show the gating strategy for both Control and Bmp9fl/flBmp10fl/flLratCre mice.

The authors should explain how they selected the genes shown on each heatmaps and add references that can justify the choice of the genes.

Thank you for your suggestion. In our study, we used CD45+ Ly6C- F4/80+ CD64+ cells to define liver macrophages. We will delete Tim4 FACS plot from Suppl fig4 to avoid the misunderstanding. Although F4/80 positive cells were reduced in the livers of Bmp9fl/flBmp10fl/flLratCre mice, double staining by anti-F4/80 and anti-CD64 fluorescence antibodies can still clearly distinguish liver macrophages based on above gating strategy. Gating strategy for both control and Bmp9fl/flBmp10fl/flLratCre mice will be presented in our revised manuscript.

Quantifications of Immunostaining and FACS data should be added as well as statistical analyses.

Quantitative data will be added in our revised manuscript.

Reviewer #2 (Public Review):

Summary:

The authors characterized the contribution of BMP9/BMP10 expression/secretion from all different hepatic cell types and analysed their impact on the other cell types. They are able to show that HSC derived BMP9/BMP10 controls Kupffer cell and EC differentiation and functions.

We appreciate the comprehensive summary of reviewer 2.

Strengths:

This is the first study to my knowledge to comprehensively analyze the contribution of BMP9/BMP10 expression in such systematic fashion in vivo. This study therefore is a significant contribution to the field and further supports previous studies that have already implied BMP9 and BMP10 in Kupffer cell and EC functions but did not unravel the intercellular cross talk in such detailed fashion.

We appreciate the positive comment of reviewer 2.

Weaknesses:

Several findings such as the impact of BMP9/10 on Kupffer cells and EC were already known. So these findings are not innovative, however I still believe that the elucidation of the cellular crosstalk makes this publication highly interesting to a broad scientific community.

Overall the authors achieved their aims and the results are well supporting the conclusions and discussion.

We appreciate the positive comment of reviewer 2. We agree with the comment of reviewer 2 that although some findings in our paper are somehow expected, the detailed investigation of the crosstalk between different liver cell types is still needed and beneficial to this field.