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Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit (RNA-Seq)

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: GSE261307 | 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:Background: Excess fibrotic remodeling leads to cardiac dysfunction in ischemic heart disease and is driven by MAP kinase-dependent transforming growth factor-ß1 (TGF-ß1) activation by coagulation signaling of myeloid cells. How coagulation-inflammatory circuits can be specifically targeted to achieve beneficial macrophage reprogramming after myocardial infarction (MI) is incompletely understood. Methods: Mice with permanent ligation of the proximal left anterior descending artery (LAD) were used to model ischemic heart disease and analyzed by single cell RNA sequencing, protein expression changes, confocal microscopy, and longitudinal monitoring of recovery. We probed the role of the tissue factor (TF)-factor 7 (F7)-integrin ß1-protease activated receptor (PAR) 2 signaling complex by utilizing genetic mouse models and pharmacological intervention. Results: Cleavage-insensitive PAR2R38E and myeloid cell integrin ß1-deficient mice had improved cardiac function after MI compared to controls. Proximity ligation assays of monocytic cells in the infarcted myocardium demonstrated that colocalization of F7 with integrin ß1 was diminished in monocyte/macrophage F7-deficient mice. F7fl/fl CX3CR1Cre relative to littermate control mice showed reduced TGF-ß1 and MAP kinase activation, as well as cardiac dysfunction after MI, despite unaltered overall recruitment of myeloid cells into the infarct zone. Single cell mRNA sequencing of CD45+ cells 3 and 7 days after MI uncovered a trajectory from ischemic myocardium-recruited monocytes to inflammatory TF+/F7+/TREM1+ macrophages. As early as 7 days after MI, macrophage F7-deletion led to an expansion of Olfml3+ macrophages with a reparative phenotype and, conversely, to a reduction of TF+/F7+/TREM1+ macrophages, which were also attenuated in activation-resistant PAR2R38E mice. To demonstrate the therapeutic potential of inhibiting the TF-F7-PAR2 signaling complex,, we injected a specific monoclonal anti-TF antibody that lacks anticoagulant activity, but interferes with macrophage migration in vitro. Short-term treatment from day 1-5 after non-reperfused MI improved cardiac dysfunction, decreased excess fibrosis, attenuated vascular endothelial dysfunction, and increased survival 28 days after MI. Conclusions: Extravascular TF-F7-PAR2 complex signaling drives inflammatory macrophage polarization in ischemic heart disease. Targeting this signaling complex for specific therapeutic macrophage reprogramming following MI attenuates cardiac fibrosis and improves cardiovascular function.

Project description:Background: Sustained sleep insufficiency impairs immune system and increases the adverse outcomes of cardiovascular diseases. However, the role and mechanisms prolonged sleep fragmentation (SF) on the outcomes of myocardial infarction (MI) remains elusive. Methods: Among 497 MI patients with complete sleep data participating in the National Health and Nutrition Examination Survey, the association between different types of sleep disorders and post-MI survivability was evaluated. We then developed a mouse model with 10 weeks of SF followed by MI surgery. Emergency hematopoiesis and neutrophil expansion were analyzed. Potential mechanisms were explored using bone marrow (BM) transplantation, anti-SiglecF neutralizing antibodies and Interferon alpha and beta receptor subunit 1 knockout (Ifnar1-/-) in BM. Results: Frequent nocturnal awakening independently predicted reduced survival rate of MI patients, with a hazard ratio (HR) of 2.136 (95% CI=1.142-3.995). Increased infarct size, deteriorated cardiac function and lower survival rate were also observed in MI mice pretreated with 10-week SF. SF facilitated post-MI neutrophil expansion and infiltration into infarct area, differentiating into SiglecFhi subset. Inhibition of SiglecFhi neutrophils allowed for the alleviations of post-MI cardiac remodeling. Furthermore, BM progenitors were skewed toward neutrophil lineage concomitant with the clonal expansion of granulocyte-monocyte progenitors (GMPs) in MI mice pretreated with 10-week SF. Transcriptome analysis and functional experiments revealed that activation of type I interferon (IFN) signaling was involved in this process, while depletion of Ifnar1 in BM and administration of IFNAR1-neutralizing antibodies significantly alleviated post-MI cardiac remodeling and neutrophil expansion. Conclusions: SF aggravates post-MI cardiac remodeling and dysfunction through promoting GMP differentiation and neutrophil expansion. Blockage of type I IFN could alleviate this detrimental influence.

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Macrophages suppress cardiac reprogramming of fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit (RNA-Seq)

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