Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia

Reviewer #1 (Public Review):

Weinberger et al. use different fate-mapping models, the FIRE model and PLX-diet to follow and target different macrophage populations and combine them with single-cell data to understand their contribution to heart regeneration after I/R injury. This question has already been addressed by other groups in the field using different models. However, the major strength of this manuscript is the usage of the FIRE mouse model that, for the first time, allows specific targeting of only fetal-derived macrophages.
The data show that the absence of resident macrophages is not influencing infarct size but instead is altering the immune cell crosstalk in response to injury, which is in line with the current idea in the field that macrophages of different origins have distinct functions in tissues, especially after an injury.
To fully support the claims of the study, specific targeting of monocyte-derived macrophages or the inhibition of their influx at different stages after injury would be of high interest.
In summary, the study is well done and important for the field of cardiac injury. But it also provides a novel model (FIRE mice + RANK-Cre fate-mapping) for other tissues to study the function of fetal-derived macrophages while monocyte-derived macrophages remain intact.

Reviewer #2 (Public Review):

In this study Weinberger et al. investigated cardiac macrophage subsets after ischemia/reperfusion (I/R) injury in mice. The authors studied a ∆FIRE mouse model (deletion of a regulatory element in the Csf1r locus), in which only tissue resident macrophages might be ablated. The authors showed a reduction of resident macrophages in ∆FIRE mice and characterized its macrophages populations via scRNAseq at baseline conditions and after I/R injury. 2 days after I/R protocol ∆FIRE mice showed an enhanced pro inflammatory phenotype in the RNAseq data and differential effects on echocardiographic function 6 and 30 days after I/R injury. Via flow cytometry and histology the authors confirmed existing evidence of increased bone marrow-derived macrophage infiltration to the heart, specifically to the ischemic myocardium. Macrophage population in ∆FIRE mice after I/R injury were only changed in the remote zone. Further RNAseq data on resident or recruited macrophages showed transcriptional differences between both cell types in terms of homeostasis-related genes and inflammation. Depleting all macrophage using a Csf1r inhibitor resulted in a reduced cardiac function and increased fibrosis.

Strengths
1. The authors utilized robust methodology encompassing state of the art immunological methods, different genetic mouse models and transcriptomics.
2. The topic of this work is important given the emerging role of tissue resident macrophages in cardiac homeostasis and disease.

Weaknesses:
1. Specificity of ∆FIRE mouse model for ablating resident macrophages.
The study builds on the assumption that only resident macrophages are ablated in ∆FIRE mice, while bone marrow-derived macrophages are unaffected. While the effects of the ∆FIRE model is nicely shown for resident macrophages, the authors did not directly assess bone marrow-derived macrophages. Moreover, in the immunohistological images in Fig. 1D nearly all macrophages appear to be absent. It would be helpful to further address the question of whether recruited macrophages are influenced in ∆FIRE mice. Evaluation of YFP positive heart and blood cells in ∆FIRE mice crossed with Flt3CreRosa26eYFP mice could clarify whether bone marrow-derived cardiac macrophages are influenced in ∆FIRE mice. This would be even more relevant in the I/R model where recruitment of bone marrow-derived macrophages is increased. A more direct assessment of recruited macrophages in ∆FIRE mice could also help to discuss potential similarities or discrepancies to the study of Bajpai et al, Circ Res 2018 (https://doi.org/10.1161/CIRCRESAHA.118.314028), which showed distinct effects of resident versus recruited macrophages after myocardial infarction. Providing the quantification of flow cytometry data (fig. 1E-F) would be supportive.

2. Limited adverse cardiac remodeling in ∆FIRE mice after I/R.
The authors suggested an adverse cardiac remodeling in ∆FIRE mice. However, the relevance of a <5% reduction in ejection fraction/stroke volume within an overall normal range in ∆FIRE mice is questionable. Moreover, 6 days after I/R injury ∆FIRE mice were protected from the impairment in ejection fraction and had a smaller viability defect. Based on the data few questions may arise: Why was ablation of resident macrophages beneficial at earlier time points? Are recruited macrophages affected in ∆FIRE mice (see above)? Overall, the manuscript could benefit if the claim of an adverse remodeling in ∆FIRE mice would be discussed more carefully.

3. Underlying mechanisms.
The study did not functionally evaluated targets from transcriptomics to provide further mechanistic insights. It would be helpful if the authors discuss potential mechanisms of the differential effects of macrophages after ischemia in more detail.

Other:
- It is unclear why the authors performed RNAseq experiments 2 days after I/R (fig. 5/6), while the proposed functional phenotype occurred later.
- A sample size of 2 animals per group appears very limited for RNAseq in ∆FIRE mice (fig. 6).