Endocardially-derived macrophages are essential for valvular remodeling

The heart tube is a de novo source of macrophages

Defining the origin of hematopoietic cells is complicated by their mobile nature. Once effective heartbeat is initiated at around 8 somite stages (~E8.5), blood cells may migrate to the heart. To examine whether the heart tube generates macrophages in situ, two ex vivo colony formation assays were performed. First, the heart explants from Ncx1-mutant embryos were examined for their colony-forming activity. Ncx1 is a sodium-calcium exchanger specifically expressed in the heart. Ncx1-mutant embryos die at around E10.5 due to the lack of systemic circulation (Koushik et al., 2001), which makes them a useful model for examining local hematopoietic emergence. Consistent with a previous report (Nakano et al., 2013), hematopoietic colonies were grown from Ncx1-mutant hearts (Fig. 1A). Interestingly, significant number of macrophage colonies was generated even in the absence of circulation (Fig. 1A). Second, the colony-forming activity of the heart tube was examined using the heart explants at pre-circulation stages (1–7 somite stages or ~E8.0), when the heart tube is not contaminated by hematopoietic cells from circulation (Fig. 1B). The heart tubes were pre-cultured on an OP9 feeder layer, which supports erythro-myeloid differentiation, followed by methylcellulose culture supplemented with cytokines/growth factors (Kodama et al., 1994). Consistent with the results from Ncx1-mutants (Fig. 1A), macrophage colonies with typical morphology were observed from the heart tubes and other established hemogenic tissues (Fig. 1C, D). They were manually picked up, prepared by Cytospin and validated by CD68 and May-Giemsa stainings (Fig. 1C). Interestingly, the heart explant generated more macrophage colonies (Fig. 1E, left) and macrophages (Fig. 1E, right) than other tissues. The results from these two complementary studies suggest that the heart is a de novo source of macrophages.

Endocardial cells contribute to the macrophages in vivo

Next, the in vivo fate of endocardial cells was traced using Nfatc1 as a marker. Nfatc1 is expressed specifically in the endocardium from E9.5 to E13.5 and Nfatc1-Cre knockin line labels the endocardium in highly specific and sensitive manner (Wu et al., 2012). To further confirm the specificity, the Nfatc1lineage contribution was quantified by flow cytometry using Nfatc1^Cre/+;R26^{Tomato reporter/+} embryos at E9.5. As CD31⁺ CD41⁺ hematopoietic progenitors are found in circulation at this stages, pure endocardial cells were gated as CD31⁺ CD41⁻ CD45⁻ in this experiment. As shown in Fig. 2A, B, Nfatc1-derived Tomato⁺ cells were found in the purified endocardial/endothelial cells in the heart but only marginally in two other established de novo site for hematopoiesis, the yolk sac and caudal half (containing aorta-gonad-mesonephros (AGM) region) at E9.5and E18.5 (Fig. 2A, B). Consistent with previous observation, The progenies of Nfatc1-positive endocardial cells were found in the endothelium of the fetal liver, which is not a de novo hematopoietic source (Fig. 2C) (Zhang et al., 2016). Therefore, Nfatc1-Cre knockin line is useful to trace the hematopoietic fate of the endocardial cells.

Immunostaining of Nfatc1^Cre/+; R26^{YFP reporter/+} hearts revealed that YFP-labeled CD45⁺ cells were enriched in the endocardial cushion. These cells showed mesenchymal morphology (Fig. 3A, B) and coexpressed pan-macrophage markers (CD68, F4/80, Csf1r) and an M2 macrophage marker (CD206), suggesting their macrophage identity (Fig. 3C-I, Fig. S1A). These Nfatc1-lineage-labeled macrophages were identified from E10.5 and often found immediately under endocardial layer in the heart tube (Fig. S1A). CD68, Csf1r, and CD206 were expressed from E10.5 and F4/80 (a marker for mature macrophages) was found after E13.5 (Fig. S1B-E). Semi-quantitative cell counting suggests that, at E13.5, 58% of F4/80⁺ macrophages in the cushion were YFP-positive (Fig. S1F). At the postnatal stages, YFP⁺ macrophages were enriched in the aortic and mitral valves and atrial myocardia (Fig. S1G-J). Although the hemogenic activity of the endocardium is transient (Nakano et al., 2013), lineage-labeled macrophages were identified in the postnatal valve mesenchyme derived from cushion mesenchyme (Fig. 3J-Y). Flow cytometry analyses revealed that endocardially-derived cardiac tissue macrophages (defined by CD45⁺ CD11b⁺ F4/80⁺ Nfatc1-lineage⁺ in the Nfatc1^Cre/+; R26^{Tomato reporter/+} heart; hereafter EcTMs) contributed to 2.6–17.4% of CD45⁺ CD11b⁺ F4/80⁺ macrophages in the whole heart during embryonic stages (Fig. 4A, bar graph). While the number of EcTMs did not change significantly during postnatal stages, non-Nfatc1-derived macrophages (Nfatc1-lineage⁻ fraction of CD45⁺ CD11b⁺ F4/80⁺ cells; hereafter non-EcTMs) decreased after postnatal day 8, resulting in a gradual increase in the proportion of EcTMs to 37.9% by adult stages (Fig. 4A, black line).

By flow cytometry, Nfatc1-derived macrophages (EcTMs) were identified at E9.5 in the heart and at E10.5 in circulation (Fig. 4B). However, in any organs examined, Nfatc1-derived cells were hardly identified in monocytes (CD45⁺ CD11b^high F4/80^low Ly6C^high Csf1r⁺), granulocytes (CD45⁺ CD11b^high F4/80⁻ Ly6G⁺ Csf1r⁻), erythro-myeloid progenitors (EMPs; CD45⁺ CD41⁺ c-kit⁺), or hematopoietic stem cells (HSCs; Lineage⁻ c-kit⁺ Sca1⁺ CD150⁺ CD48⁻) (Fig. 4C-F, S2A), suggesting that endocardially-derived macrophages are not generated through monocytes or multipotent stem/progenitors. Thus, (1) macrophage colonies arise from the heart tube ex vivo even in the absence of circulation (Fig. 1), (2) a fraction of cardiac tissue macrophages are labeled by Nfatc1-Cre knockin line that is highly specific and sensitive to the endocardium (Fig. 2), and (3) Nfatc1-labeled macrophages appear in the heart tube prior to other organs (Fig. 4B). These data support that Nfatc1-labeled macrophages originate from the endocardium.

To further examine whether EcTMs are derived from multipotent stem/progenitors, Runx1 was conditionally deleted in endocardial specific manner. Runx1 is a key regulator of definitive hematopoiesis. Runx1 mutant embryos die at around E12.5 due to the absence of entire definitive hematopoietic cells (North et al., 1999; North et al., 2002). Nfatc1^cre/+; Runx1 ^fl/fl mutants survived until adult stages with no detectable phenotype. Analysis of Nfatc1^cre/+; Runx1 ^fl/fl mutant hearts revealed no significant decrease in the number of macrophages in the mutant hearts compared to the control littermates at E10.5 and 11.5 (Fig. S2B). Thus, EcTMs are not dependent on Runx1, suggesting that they are not definitive in nature.

Endocardially-derived macrophages are more phagocytic than macrophages of other origins in the heart

Macrophages are activated in response to the local tissue environment and differentiate into specific subsets including pro-inflammatory M1 macrophages (Ly6C^high CD206⁻) and alternatively-activated M2 macrophages (Ly6C^low CD206⁺) that play phagocytic and pro-tissue reparatory roles (Pinto et al., 2012). Surface marker characterization revealed that M1 macrophages were not present in EcTMs during embryonic stages whereas 8.6–11.2% of non-EcTMs were M1 (Fig. 5A, B, P < 0.05). On the other hand, the percentage of M2 macrophages was significantly higher in EcTMs compared to non-EcTMs at E15.5 (Fig. 5C). These data suggest that EcTMs are more M2 type during embryonic stages. In both EcTMs and non-EcTMs, the percentage of M2 macrophages progressively increased during embryonic and postnatal development (Fig. 5C). The surface characteristics of EcTMs and non-EcTMs gradually became less distinct towards adult stages (Fig. 5B, C, Fig. S2C). Expression of MHCII (a marker for antigen-presenting macrophages) and CCR2 (a marker for monocyte-derived macrophages) was examined to further characterize the postnatal maturation of EcTMs. In both EcTMs and non-EcTMs, phagocytic-type macrophages (Ly6C^low MHCII^low)(Epelman et al., 2014) were dominant from P1 to 28, followed by adoption of an antigen presenting role (Ly6C^low MHCII^high) at adult stages (Fig. 5D). However, EcTMs and non-EcTMs were clearly distinct in that CCR2⁺ macrophages (monocyte-derived) were rarely found among EcTMs. Although possibly oversimplified, surface marker categorization supports that EcTMs play more phagocytic role than macrophages of other origins during heart development.

To further examine the role of EcTMs, we performed RNA-Seq on these cells at postnatal day 28, when the total number of cardiac tissue macrophages is at peak. 40 genes were differentially expressed with a P_adj < 0.05 between the two groups: 36 were higher and 4 genes were lower in EcTMs compared to non-EcTMs (Fig. 6A). Gene Ontology analysis revealed that EcTMs were enriched for genes related to antigen presentation, lysosomes, and phagosomes (H2-Ab1, H2-Aa, H2-Eb1, CD74, Laptm5; Fig. 6B, C). To examine their phagocytic function, EcTMs and non-EcTMs were sorted from Nfatc1^Cre/+; Rosa26^{Tomato reporter/+} hearts at P8 and incubated with FITC-labeled latex beads. Consistent with the gene expression data, EcTMs showed 44.4% higher phagocytic activity than non-EcTMs (Fig. 6D, P < 0.05). Moreover, electron microscopy analysis revealed large phagocytic cells with phagosomes engulfing apoptotic cell debris in the cushion mesenchyme at E13.5 (de Lange et al., 2004), although filopodia were less developed compared to typical tissue macrophages in adult (Fig. 6E-J). Together, these data suggest that EcTMs predominantly play a phagocytic role in the endocardial cushion and valvular mesenchyme.

Endocardially-derived macrophages are required for the valve formation

Previous reports suggest that tissue macrophages of different origins compensate for each other in diseased tissues (Bain et al., 2016; Epelman et al., 2014). To examine whether EcTMs are dispensable for the valve formation, we generated mutant mice in which EcTMs were ablated by deleting Csf1r (also known as MCSF receptor and CD115) in the Nfatc1⁺ endocardial lineage. Csf1 and Csf1r constitute an autocrine system essential for macrophage development, and Csf1r is specifically expressed in the macrophage lineage. Germ-line knockout mice of Csf1 and Csf1r show embryonic lethality with nearly identical phenotypes due to complete ablation of the macrophages (Chitu and Stanley, 2006; Ginhoux et al., 2010; Sauter et al., 2014). Nfatc1^cre/+; Csf1r^fl/fl mutant mice were underrepresented at weaning (Fig. S3A, P < 0.05,) and had enlarged hearts (Fig. S3B, C). Histological analysis of the survivors revealed a spectrum of valvular anomalies including ectopic elastic tissue with hypercellularity in aortic valves and typical myxomatous changes with the accumulation of collagen and mucin in mitral valve leaflets (Fig. 7A, B). Less severe phenotypes include thickening of the valves with an increase in both cellular and matrix components although the matrix pattern was not significantly affected until adulthood (Fig. S4A, B). Typical bicuspid aortic valve, calcification or right heart valve deformity was not identified in our analyses (Fig. S4C, D). The valvular thinckening and anomalies were obvious at neonatal stages by histological analyses and 3D imaging by light-sheet microscopy (Fig. 7D, S5A). However, expression of αSMA and Vimentin, markers for activated valve interstitial cells, were higher in the mutants at E15.5 and P1, indicating that a pathological activation of interstitial cells starts prior to the morphological deformity (Fig. 7C, D, Fig. S5B). TUNEL staining revealed that apoptotic cells were not cleared efficiently in valvular mesenchyme in Nfatc1^cre/+; Csf1r^fl/fl hearts at P1, suggesting an inefficient clearance of apoptotic cells (Fig. 7E). Cell proliferation assay revealed no evidence of increase in pH3⁺ cells in mutant aortic or mitral valves in surviving P8 mutants (Fig. S5C). Inefficient clearance of apoptotic cells likely contributed to the activation of mesenchymal cells in the valves in our mutants. No sign of cardiac contractile dysfunction was noticed in surviving adult mutants by μMRI or echo (Fig. S6A, B). Interestingly, significant prolongation of PR interval was observed on EKG (Fig. S6C). These results suggest that EcTMs are required for the formation of the valves through their phagocytic role. Notably, no significant morphological abnormality was identified in any other organs (Fig. S3E, F), indicating that these locally-derived tissue macrophages are specifically required for local tissue remodeling.

Monocyte-derived macrophages compensate the total number of macrophages in the absence of endocardially-derived macrophages

To examine how the ablation of EcTMs impacts the subpopulations of macrophages in the heart, we profiled surface marker expression in Nfatc1^cre/+; Csf1r^fl/fl mutant hearts. While the numbers of total, Ly6C^high and Ly6C^low macrophages were not significantly different, monocyte-derived CCR2⁺ macrophages were significantly increased in Nfatc1^cre/+; Csf1r^fl/fl hearts (638±43 vs 1424±218 per 100,000 live cells, P < 0.05, Fig. 7F). The increase in CCR2⁺ macrophages was mainly due to the MHCII^low macrophages. Thus, the total number of macrophages was compensated by monocyte recruitment in the absence of EcTMs; however, the monocyte-derived macrophages failed to rescue the remodeling of valves as the phagocytic function was not fully restored. These data suggest that EcTMs play a unique role during valvular formation.

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Atsushi Nakano ([email protected]).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

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QUANTIFICATION AND STATISTICAL ANALYSIS