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Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit (CUT&Tag)

ABSTRACT: Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-pSTAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-pSTAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

ORGANISM(S): Mus musculus

PROVIDER: GSE261308 | GEO | 2024/03/15

REPOSITORIES: GEO

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Project description:Direct conversion of cardiac fibroblasts (CFs) to cardiomyocytes (CMs) in vivo to regenerate heart tissue is an attractive approach. After myocardial infarction (MI), heart repair proceeds with an inflammation stage initiated by monocytes infiltration of the infarct zone establishing an immune microenvironment. However, whether and how the MI microenvironment influences the reprogramming of CFs remains unclear. Here, we found that in comparison with cardiac fibroblasts (CFs) cultured in vitro, CFs that transplanted into infarct region of MI mouse models resisted to cardiac reprogramming. RNA-seq analysis revealed upregulation of interferon (IFN) response genes in transplanted CFs, and subsequent inhibition of the IFN receptors increased reprogramming efficiency in vivo. Macrophage-secreted IFN-β was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-β displayed reduced reprogramming efficiency, while macrophage depletion or blocking the IFN signaling pathway after MI increased reprogramming efficiency in vivo. Co-IP, BiFC and Cut-tag assays showed that phosphorylated STAT1 downstream of IFN signaling in CFs could interact with the reprogramming factor GATA4 and inhibit the GATA4 chromatin occupancy in cardiac genes. Furthermore, upregulation of IFN-IFNAR-pSTAT1 signaling could stimulate CFs secretion of CCL2/7/12 chemokines, subsequently recruiting IFN-β-secreting macrophages. Together, these immune cells further activate STAT1 phosphorylation, enhancing CCL2/7/12 secretion and immune cell recruitment, ultimately forming a self-reinforcing positive feedback loop between CFs and macrophages via IFN-IFNAR-pSTAT1 that inhibits cardiac reprogramming in vivo. Cumulatively, our findings uncover an intercellular self-stimulating inflammatory circuit as a microenvironmental molecular barrier of in situ cardiac reprogramming that needs to be overcome for regenerative medicine applications.

Project description:Inflammatory response plays a bidirectional regulatory role in myocardial infarction (MI). TLR signaling pathway plays an important role in the development of inflammation. Additionally, we discovered that CCRR is an anti-arrhythmic lncRNA in a prior work. Here we show, it is decreased in mice with 3-day MI followed by a sharp increase of pro-inflammatory cytokines and activation of the TLR pathway. Overexpression of CCRR could effectively inhibit the inflammatory response, reduce the infarct area, and improve cardiac function. And also reduced hypoxia-induced inflammation in cardiomyocytes and macrophages induced by IFN and LPS. It produced a similar protective effect in cardiomyocytes co-cultured with macrophages. Through microarray analysis, RNA-binding protein immunoprecipitation analysis, and other related experiments verification, TLR2 and TLR4 may be as potential targets of CCRR after MI. Our findings provide the evidence that lncRNA CCRR plays a key cardioprotective role against MI injury by targeting the TLR signaling pathway. nflammatory response plays a bidirectional regulatory role in myocardial infarction (MI). TLR signaling pathway plays an important role in the development of inflammation. Additionally, we discovered that CCRR is an anti-arrhythmic lncRNA in a prior work. Here we show, it is decreased in mice with 3-day MI followed by a sharp increase of pro-inflammatory cytokines and activation of the TLR pathway. Overexpression of CCRR could effectively inhibit the inflammatory response, reduce the infarct area, and improve cardiac function. And also reduced hypoxia-induced inflammation in cardiomyocytes and macrophages induced by IFN and LPS. It produced a similar protective effect in cardiomyocytes co-cultured with macrophages. Through microarray analysis, RNA-binding protein immunoprecipitation analysis, and other related experiments verification, TLR2 and TLR4 may be as potential targets of CCRR after MI. Our findings provide the evidence that lncRNA CCRR plays a key cardioprotective role against MI injury by targeting the TLR signaling pathway.

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Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit (CUT&Tag)

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