The role of endogenous Smad7 in regulating macrophage phenotype following myocardial infarction [JunLi_MyS7KOinvitro]

Induction of the inhibitory Smad, Smad7 serves as a negative feedback mechanism that restrains TGF-b-mediated actions in injured tissues. In inflammatory cells, Smad7 has been suggested to exert both pro-inflammatory actions attributed to inhibition of TGF-b-induced suppression of inflammation, and anti-inflammatory effects due to disruption of the TAK-1/NF-kB system. Myocardial infarction triggers a macrophage-driven inflammatory response that plays a central role in cardiac repair, but also contributes to adverse remodeling and fibrosis. We hypothesized that Smad7 upregulation in infarct macrophages may play a modulatory role in cardiac repair, by restraining effects of TGF-b. To test the hypothesis, we investigated the response of Myeloid cell-specific Smad7 knockout mice (MyS7KO) following myocardial infarction protocols, and we examined the in vitro effects of Smad7 in isolated macrophages. Smad7 was upregulated in a subset of infarct macrophages, peaking 7 days after infarction. Myeloid cell-specific Smad7 loss did not affect baseline macrophage gene expression and had no significant effects on homeostatic functions. Although RNA-seq analysis predicted that, in the absence of Smad7, infarct macrophages may have attenuated activation of inflammatory pathways and suppressed TREM1 signaling, myeloid cell-specific Smad7 loss had no significant effects on ventricular dysfunction, adverse remodeling, scar remodeling and collagen deposition after myocardial infarction. In isolated macrophages, TGF-b attenuated pro-inflammatory cytokine and chemokine expression, modulated synthesis of matrix remodeling genes, and had profound effects on macrophage profile, inducing genes associated with activation of sphingosine-1 phosphate and integrin signaling pathways, and inhibiting cholesterol biosynthesis genes. However, RNA-seq and PCR array experiments showed that Smad7 loss has minimal effects on TGF-b-mediated macrophage responses, restraining synthesis of only a small fraction of TGF-b-induced genes, such as Itga5, Olfml3 and Fabp7. Smad7 absence did not affect the anti-inflammatory actions of TGF-b in TNF-stimulated cells. In conclusion, our findings suggest a limited role for macrophage Smad7 in regulation of post-infarction inflammation and repair, and demonstrate that the anti-inflammatory effects of TGF-b in macrophages are not restrained by endogenous Smad7 induction. In order to study the role of Smad7 in myeloid cells in vitro, we generated mice with inducible global loss of Smad7 (S7KO). We used a transgenic mouse line in which Cre recombinase is driven by CAGG promoter (Jackson Laboratory, stock No: 004682). CAGGCre-ER mice were bred with Smad7fl/fl mice (Jackson Laboratory, stock No: 017008) to generate CAGGCre-ER;Smad7fl/fl animals (S7KO) and corresponding Smad7fl/fl control littermates. For the experiment, both, control mice (Smad7 fl/fl) and global inducible S7KO mice (CAGGCre-ER;Smad7fl/fl), were injected with tamoxifen (20 mg/kg). Tamoxifen was administered intraperitoneally once for 5 consecutive days to 1 month old mice. Mice were sacrificed 2 weeks after tamoxifen injection and Bone marrow macrophages were isolated and culture in the presence or absence of 10ng/ml TGF- β1. The loss of Smad7 in S7KO macrophages has been documented by qPCR and western blot. >>> Raw data are no longer available. <<<