Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration

Abstract

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first-week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, ROS homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations (hbaa+ Mac 2 and timp4.3+ Mac 3) were enriched only in regenerative hearts and barely recovered after -1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 days (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and ECM remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration.

Defining mononuclear phagocyte distribution and behaviour in the zebrafish heart

“…Adult zebrafish are known to have populations of cTMs in the steady state and cardiac MNP populations expand following cardiac injury 24,29,62 . A recent study has highlighted the heterogeneity of resident cTMs and their involvement in promoting regeneration 42 echoing what has been described in neonatal mice 63 . However, we are yet to fully understand the spatial distribution of these cells in the heart, the roles played by distinct MNP lineages and to confirm strategies to identify these subpopulations.…”

“…Multiple studies have provided important insight into the ontogeny and maintenance of microglia 65,67,70,79 , but much of this hasn't been investigated in other tissues. Recent studies using single cell sequencing (scRNA-seq) are advancing our understanding of immune cell heterogeneity in the zebrafish heart and other organs [41][42][43]66,70 but much of this has been derived from mpeg1.1 expressing cells and is yet to be translated into new tools to define the distribution of these subpopulations. Our data indicates that using csf1ra and mpeg1.1 transgenic lines it is possible to identify four separate immune cell populations in the homeostatic heart and we have shown that these cells have distinct morphology, distribution, and behaviour in cardiac tissue.…”

“…However, much of the initial characterisation of these markers was performed in larval zebrafish and, although expression in tissue macrophages was confirmed, whether expression extends to monocytes and DCs has not been well defined. Recent technological advancements and accessibility to RNA sequencing technology has facilitated the molecular characterisation of MNPs [40][41][42][43] .…”

ptx3a+ fibroblast/epicardial cells provide a transient macrophage niche to promote heart regeneration

“…Further, we examined the spatial correlation of ptx3a + cells with macrophages in the hearts of injured mpeg:EGFP;ptx3a:mCherry fish and found many EGFP + cells associated with mCherry + signals ( Figure 3B ). As different macrophage subtypes exist in the wound, 12 , 37 , 51 we then assessed macrophage type with known markers: tnfα for pro-inflammatory macrophages 52 and mrc1b 53 and cxcr4b 54 , 55 for reparative macrophages ( Figures 3C , 3D , S1F , and S1G ). We also quantified the spatial correlation of macrophages and ptx3a + cells by the distance of these cells in three situations: (1) macrophages surrounded by ptx3a + cells, (2) macrophages within 1 μm of ptx3a + cells, excluding situation 1, and (3) macrophages more than 1 μm away from ptx3a + cells.…”

Harnessing the regenerative potential ofinterleukin11to enhance heart repair

“…This regenerative ability of cardiac muscle cells can be achieved by dedifferentiation and proliferation of pre-existing CMs [7][8][9] . The regenerative events in CMs are orchestrated by molecular signals provided by activated non-muscular cells, including epicardium, endocardium, and immune cells, which create a regenerative niche 7,[10][11][12][13][14][15] . Furthermore, similar to injured mammalian hearts, zebrafish exhibit an acute response to cardiac injury by forming fibrotic tissue at the injury site 16 .…”