Macrophages suppress cardiac reprogramming of  fibroblasts in vivo via IFN-mediated intercellular self-stimulating circuit (RNA-Seq)
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-ß was identified as the dominant upstream signaling factor after MI. CFs treated with macrophage-conditioned medium containing IFN-ß 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-ß-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. Overall design: In order to investigate the in vivo suppressors of CFs reprogramming, we established a transplantation system that enables comparison of the same cells expressing a combination of reprogramming factors between in vivo and in vitro conditions, to preclude potential differences in gene delivery efficiency. Briefly, we digested in vitro cultured MICFs (cardiac fibroblasts isolated from adult mice with myocardial infarction) infected with lentiviruses tetracycline-inducible expressing MGT (polycistronic construct including Mef2c, Gata4 and Tbx5), Myo+S (separates virus driving expression of Myocd and Sall4) (Zhao et al., 2021), and eGFP, respectively, and transplanted them into hearts of mice with surgically induced MI (MI hearts hereafter). A portion of these infected MICFs were retained for concurrent in vitro culture with the addition of doxycycline (Dox). On days 7 and days 14 after transplantation and Dox administration, we used FACS to isolate eGFP+ transplanted MICFs from the hearts of MI mice and compare them with FACS-isolated eGFP+ MICFs with sustained in vitro culture