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.

Author Response

The following is the authors’ response to the original reviews.

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.

Response from the authors: We thank the reviewer for the thorough review and the positive feedback, and we agree that the Csf1r-FIRE mice represent an interesting model for studying the role of resident embryo-derived macrophages in different tissues and pathologies.

Recent work of the Cochain lab demonstrated by combined CITE-seq analysis and CCR2 antibody treatment that monocyte depletion does not affect levels of resident tissue macrophages after myocardial infarction (REF Rizzo et al PMID: 35950218), supporting the concept to specifically investigate the role of resident and recruited macrophages. While previous work has addressed the effects of broad CCR2-mediated monocyte depletion, information on differential macrophage subsets derived from blood monocytes has been lacking. We agree with the reviewer that targeting subsets of monocyte-derived macrophages, such as for example Ly6Chi monocytes, MHCII+Il1b+ macrophages, and Isg15hi populations (REF Rizzo et al PMID: 35950218), or interference with their recruitment at different time-points after myocardial infarction would be of interest and could help to decipher their functions in the different stages of cardiac healing. However, these studies would go beyond the scope of the current analysis and will be addressed in a separate project.

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.

1. The topic of this work is important given the emerging role of tissue resident macrophages in cardiac homeostasis and disease.

Response from the authors: We thank the reviewer for pointing out the strengths of our study, and putting the findings in context of the current view of the role of resident macrophages.

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, 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.

Response from the authors: We thank the reviewer for these comments. The reviewer addresses the specificity of the ∆FIRE mouse model for ablating resident macrophages and its potential effects on bone marrow-derived macrophages. Our single-cell sequencing data support the specificity of the ∆FIRE model regarding embryo-derived resident macrophages in two ways. First, the ∆FIRE mice are characterized by the specific reduction of embryo-derived macrophage clusters (e.g. homeostatic macrophages as well as antigen-presenting macrophages) in baseline conditions, while the abundance of recruited macrophages (e.g. Ccr2hiLy6chi macrophages, Cx3Cr1hi macrophages) is not altered (Fig. 2B-D). Second, transcriptomic analysis of bone marrow-derived macrophage clusters (e.g. Ccr2hiLy6chi macrophages, Cx3Cr1hi macrophages) and of monocytes revealed no differences in ∆FIRE compared to control mice. On the other hand, we found substantial transcriptome differences in clusters that were mainly of embryonic origins (e.g. homeostatic macrophages as well as antigenpresenting macrophages) (Fig.2 and Fig S.4). These findings indicate that the ∆FIRE model mainly induces changes in embryo-derived macrophages.

We agree with this reviewer that crossbreeding of ∆FIRE mice with Flt3CreRosa26eYFP mice would be of interest, and we have been working hard to establish this line. However, our breeding efforts have thus far been in vain, which is probably due to the necessity to keep a CBA/Ca background for the FIRE model (as reported by JAX: https://www.jax.org/strain/032783) and requires further backcrossing of Flt3CreRosa26eYFP mice with the respective CBA strain. In future work, we plan to carry out this experiment and also to specifically target monocyte-derived macrophages.

The reviewer further asks about the modality to quantify cardiac macrophages, and suggests flow cytometry to quantify their number and not only use immunohistology. The quantification of cardiac immune cells shown in Fig. 1D (formerly 1C) was in fact performed by flow cytometry. We apologize for the lack of clarity. We rearranged the figure and added this information to the figure legend. We also added quantification by immunohistology, which is now shown in Fig. 1G.

1. 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.

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.

Response from the authors: The reviewer raises the question why the ablation of resident macrophages trends towards a beneficial effect at earlier time points after I/R injury. Further, the reviewer questions the relevance of a <5% reduction in ejection fraction/stroke volume over time in the light of an otherwise modestly reduced ejection fraction.

In this study we used the experimental mouse model of ischemia-reperfusion injury with transient (1h) coronary artery occlusion. The potential disadvantage of this model is the smaller infarct size and smaller effects on cardiac function. However, it better represents the clinical picture and pathology of myocardial infarction in human patients with timely reperfusion by percutaneous coronary intervention. Infarct size after I/R was approx. 25% in control animals indicating relevant cardiac injury. Further, infarct size was reduced to approx. 16% in ∆FIRE mice 6 days after infarction, however, the difference did reach statistical significance. In line with this, the ejection fraction was numerically reduced on d6 after infarction in the control group, however with no statistical significance. In the chronic phase after infarction, the ejection fraction improved over time in the control group by approx. 5% and decreased in ∆FIRE mice by 4%, which resulted in a difference (delta) of 9% change of ejection fraction. This indicated adverse remodeling in ∆FIRE mice.

We agree that the different impact of the absence of resident cardiac macrophages during the course of myocardial healing after injury is of great interest to the field. We discuss potential mechanisms of the differential effects of resident macrophage ablation in lines 290-314 in the revised manuscript. However, to decipher the influence of embryo-derived macrophages at different time points after infarction, an inducible model for specific depletion of this macrophage population would be necessary, which to our knowledge does not exist.

In the revised manuscript, we now discuss the effects on cardiac healing in ∆FIRE and also the limitations more thoroughly.

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).

Response from the authors: We chose a time point in the “late early phase” of myocardial infarction (= day 2 post I/R) as we were also interested in the effect of resident macrophage depletion on other immune cell subsets (e.g. neutrophils) which could only be captured in this time period.

We aimed to analyse 10000 cells per condition. The applied sample size allowed us to analyse 13452 CD45+cells from ∆FIRE mice and 9152 cells from control mice in infarct condition.

Lines 299-324 "Ablation of resident macrophages altered macrophage crosstalk to non-macrophage immune cells, especially lymphocytes and neutrophils. This was characterized by a proinflammatory gene signature, such as neutrophil expression of inflammasome-related genes and a reduction in anti-inflammatory genes like Chil3 and Lcn2. Interestingly, inflammatory polarization of neutrophils have also been associated with poor outcome after ischemic brain injury (Cuartero et al, 2013). Clinical trials in myocardial infarction patients showed a correlation of inflammatory markers with the extent of myocardial damage {Sanchez, 2006 #2763} and with short- and long-term mortality {Mueller, 2002 #2780}.

Our study provides evidence that the absence of resident macrophages negatively influences cardiac remodeling in the late postinfarction phase in ∆FIRE mice indicating their biological role in myocardial healing. In the early phase after I/R injury, absence of resident macrophages had no significant effect on infarct size or LV function. These observations potentially indicate a protective role in the chronic phase after myocardial infarction by modulating the inflammatory response, including adjacent immune cells like neutrophils or lymphocytes.

Deciphering in detail the specific functions of resident macrophages is of considerable interest but requires both cell-specific and temporally-controlled depletion of respective immune cells in injury, which to our knowledge is not available at present. These experiments could be important to tailor immune-targeted treatments of myocardial inflammation and postinfarct remodelling."

Reviewer #1 (Recommendations For The Authors):

1. Fetal-derived macrophages are often involved in organ development and function during steady-state. The authors should show heart morphology/function before I/R injury to make sure that the cause for a worsened outcome in FIRE mice is not due to a developmental/functional defect.

Response from the author: We conducted a gross analysis of cardiac morphology by histology, and did not determine differences to littermate controls. However, we have not conducted a detailed investigation of cardiac development since this was not the scope of this study. Further, our study mainly shows differences in cardiac healing between d6 and d30, which is unlikely influenced by developmental defects.

1. Line 164: The authors state that they have analysed macrophages via flow cytometry, but Figure 4a only shows IF. Quantification of different macrophage subsets via flow cytometry should be included in this model.

Response from the author: The sentence “To gain a deeper understanding of the inflammatory processes taking place in the infarcted heart, we quantified macrophage distribution by immunofluorescence and flow cytometry analysis of ischemic and remote areas after I/R.” beginning line 164 describes the entire figure 4 and not only 4a. Here we show IF as well as flow cytometry to describe numbers but also different subpopulations of macrophages (BM-derived vs. resident).

1. Lines 254-255 (now starting 267): it is not entirely true that the heart does not harbor BM-derived macrophages under steady state. Of course, there are many more after I/R injury, but the authors should take also their own data into account (Figure 1c, e showing a clear reduction but not complete absence of macrophages) and not claim a "scarce" population. See also Dick et al (PMID: 30538339), where both, the Ccr2-Tim4- and Ccr2+ populations are (slowly) replaced by BM monocytes.

Response from the author: We thank the reviewer for this comment. We changed “scarce population” to “small population”.

1. Lines 269-273 (now starting line 283): The point that DT-mediated depletion of cells causes inflammation that may have an impact on macrophages is compelling. However, the approach of combining and correlating data from PLX diet and FIRE mice is not proof that the significant increase in infarct size and deterioration of left ventricular function after I/R injury is driven by monocyte-derived macrophages. The authors could use Ccr2KO mice or injection of Ly6C antibody to show the specific functions of recruited macrophages.

Response from the author: In this study we combine a specific genetic depletion of resident macrophages (FIRE) with an pharmaceutical depletion of all macrophage populations (Csf1r-inhibiton with PLX5622). We did not aim to specifically deplete monocyte-derived macrophages, which has been addressed previously by Bajpai et al. (PMID: 30582448) using the CCR2-DTR mouse line. To address the functions of recruited macrophages would go beyond the scope of the manuscript.

Along these lines: the authors discuss that neutrophils may have been targeted in the Ccr2-DTR model. However, the egress of neutrophils in the CCR2 KO model is not affected and should be a good model to look at the impact of monocyte-derived macrophages after I/R injury in the heart.

Response from the author: We agree with the reviewer that CCR2 under steady state conditions might not be important for the egress of neutrophils. However, after ischemic injury CCR2-inhibition has been shown to impair neutrophil egress as well as neutrophil recruitment to ischemic tissue in an ischemia-reperfusion injury model (PMID: 28670376).

1. Line 299 (now line 332): Reference is missing for Ccr2-DTR mice study

Response from the author: We added the respective reference.

1. Can the authors take also the timing of treatment/cell depletion into account in their discussion incoming monocytes may be required in the first days after injury to promote the regeneration process so that targeting them before the onset of the injury may be detrimental while targeting them during the chronic phase may be beneficial.

Response from the author: We thank the reviewer for this comment. We added the following sentence to the manuscript (Lines 343-346):

“An explanation of this controversy might be the timing and duration of macrophage depletion. Bajpai et al. depleted recruited macrophages only in the initial phase of myocardial infarction which improved cardiac healing (Bajpai et al., 2019), while depletion of macrophages over a longer period of time, as shown in our study, is detrimental for cardiac repair.”

1. Figure 6E, F: Why are the outgoing signals pooled? The data has the strength of distinguishing between distinct populations. This data should be used and exploited to work out distinct pathways of distinct macrophage populations in more detail. From the representation, it remains unclear which pathways are active and distinct between Ctrl and FIRE mice besides the few chosen once (inflammasome). Also, legends are missing (what is red/blue?)

Response from the author: We thank the reviewer for this comment. The aim of this analysis was to evaluate the effect of the FIRE ko on communication of immune cells in infarct conditions. To address changes in all populations which are affected by the FIRE ko we pooled the respective clusters (e.g. homeostatic, antigen-presenting and Ccr2loLy6clo Mø clusters). We provided the detailed analysis of the individual clusters in the new Supplemental Figure 9. Further, we added the respective legend to the Figure.

1. The methods part mentioned CD169-DTR mice, however, there are no experiments shown in the manuscript. Further, how did the authors breed the FIRE mice? It is known in the field that they have big developmental issues and behavioural deficits if kept on a B6 background, which was likely the case in the study, at least for the fate-mapping approach.

Response from the author: We removed the CD169-DTR reference from the methods part.
FIRE mice were kept on a CBA/Ca background. As mentioned by the reviewer this was not the case for the experiment where reporter mice were bred with FIRE mice (Csf1rΔFIRE/+RankCreRosa26eYFP) as these mice are on a C57Bl6 background. All experiments evaluating cardiac function and outcome after infarction in FIRE mice were performed on mice kept with a CBA/Ca background.

Reviewer #2 (Recommendations For The Authors):

• Please provide the sample size for Fig. 5.

We described the sample size in the methods part (lines 448-450: “Cell sorting was performed on a MoFlo Astrios (Beckman Coulter) to obtain cardiac macrophages from CD45.2; Mx1CreMybflox/flox after BM-transplantation of CD45.1 BM (n=3 for 2 days after I/R injury) for bulk sequencing,..“). We added the sample size also to the figure legend.

• Please state in the methods how the normality of data was tested.

We added the respective normality test to the methods part. “The Shapiro-Wilk test was used to test normality. “

• How did the authors ensure a standardized infarct size?

The authors ensured a standardized infarct size in mice following myocardial infarction through a carefully controlled experimental protocol. We employed the well-established I/R procedure for inducing myocardial infarction in mice by ligation of the LAD for 1h to mimic the transient blockage of blood flow to the anterior wall of the heart. Success of the ligation of the LAD and the induction of ischemia was confirmed by the pale color of the myocardium after ligation and the success of reperfusion by the return of color after removing the suture. The surgical technique was consistently performed by the same highly trained veterinarian in a blinded fashion to minimize variability.