Gene expression profile of mouse CD206+F4/80+CD11b+ M2-like macrophages
ABSTRACT: Characterisation of M2-like macrophage in terms of gene expression level. The hypothesis tested in the present study was that M2-like macrophages in myocardial infarction (MI) heart have upregulation of genes relevant to cardiac repair. The results obtained showed tha various tanti-inflammatory and reparative genes were upregulated in M2-like macrophage from MI heart compared to those from intact hearts. M2-like macrophages from the heart (either MI or intact) were distinct from those in the peritoneal cavity.
Project description:Adverse cardiac remodeling after myocardial infarction (MI) causes structural and functional changes in the heart leading to heart failure. The initial pro-inflammatory response followed by an anti-inflammatory or reparative response post-MI is essential for minimizing the myocardial damage, healing, and scar formation. Bone marrow-derived macrophages (BMDMs) are recruited to the injured myocardium and essential for cardiac repair as they can adopt both pro-inflammatory (M1) or anti-inflammatory/reparative (M2) phenotypes to modulate inflammatory and reparative response, respectively. YAP and TAZ are the key mediators of the Hippo signaling pathway and essential for cardiac regeneration and repair. However, their role in macrophage polarization and post-MI inflammation, remodeling, and healing are not well established. Here, we demonstrate that expression of YAP and TAZ is increased in macrophages undergoing M1 or M2 polarization. Genetic deletion of YAP/TAZ leads to impaired M1 polarization and enhanced M2 polarization. Consistently, YAP activation/overexpression enhanced M1 and impaired M2 polarization. We show that YAP/TAZ promote M1 polarization by increasing IL6 expression, and impede M2 polarization by decreasing Arg1 expression through interaction with the HDAC3-NCoR1 repressor complex. These changes in macrophages polarization due to YAP/TAZ deletion results in reduced fibrosis, and hypertrophy and increased angiogenesis, leading to improved cardiac function after MI. Also, YAP activation augmented MI-induced cardiac fibrosis and remodeling. In summary, we identify YAP/TAZ as important regulators of macrophage-mediated pro- and anti-inflammatory responses post-MI.
Project description:Myocardial infarction (MI) causes sterile inflammation, which is characterized by recruitment and activation of innate and adaptive immune system cells. We have delineated the temporal dynamics of immune cell accumulation following MI by flow cytometry. Macrophages were numerically the predominant cells infiltrating the infarcted myocardium, increasing in number over the first week post-MI. Macrophages are functionally heterogeneous, and can be classified into M1 and M2 macrophages, respectively, based on surface-marker expression. M1 macrophages dominated at 1-3 days post-MI, whereas M2 macrophages represented the predominant macrophage subset after 5 days. We used microarrays to examine the gene expression profiles of the macrophages sorted from the hearts at different timepoints after MI. We identified the kinetics of gene expression of cardiac macrophage after MI.
Project description:Background—Mesenchymal stem cells (MSCs) have shown therapeutic potency for treating cardiovascular diseases, but their therapeutic efficacy shows significant heterogeneity depending on their tissue of origin. We herein sought to identify the optimal source of MSCs for cardiovascular disease therapy. Methods—Heart- and bone marrow (BM)-derived GFP+ cells positive for the MSCs marker, Nestin, were flow cytometrically sorted from 7-day-postnatal Nestin-GFP transgenic mice. To study their biological characteristics in vitro, we characterized their self-renewal capacity, multi-lineage differentiation ability, and surface markers. To investigate their therapeutic potential in vivo, we intramyocardially injected Nestin+ cells (3×105) into the infarct border zone of mice subjected to acute myocardial infarction (MI), and performed echocardiography and Masson’s-Trichrome staining at 1 and 3 weeks post-MI. We characterized gene profiles with RNA-sequencing analysis, and analyzed the putative reparative mechanism, that of Periostin-mediated M2 macrophages polarization, by immunofluorescence staining, qPCR, ELISA, and flow cytometry in vivo and in vitro. Results—In vitro, the heart- and BM-derived Nestin+ cells (Nes+cMSCs and Nes+bmMSCs, respectively) were found to possess self-renewal ability, show similar tri-lineage differentiation potentials, and express some MSCs-related surface markers. Importantly, Nes+cMSCs significantly improved cardiac function, attenuated left ventricular (LV) remodeling, and decreased infarct size in the mouse MI model, compared with Nes+bmMSCs- or saline-treated MI controls. Nes+cMSC treatment notably reduced the total number of CD68+ pan-macrophages while inducing the polarization of macrophages toward an anti-inflammatory M2 phenotype in ischemic myocardium, compared with the saline-treated MI control. Periostin, which was highly expressed in Nes+cMSCs, could promote the polarization of M2-subtype macrophages in vitro and in vivo. Finally, Periostin knockdown remarkably reduced the therapeutic effects of Nes+cMSCs, inhibiting the survival and LV remodeling of MI model mice and significantly decreasing the number of M2 macrophages at lesion sites. Conclusions—Nestin+ cMSCs have greater efficacy than Nestin+ bmMSCs for cardiac repair following acute MI, using a reparative mechanism that acts at least partly through Periostin-mediated M2 macrophage polarization.
Project description:Myocardial infarction (MI) leads to cardiomyocyte death, which triggers an immune response that clears debris and restores tissue integrity. In the adult heart, the immune system facilitates scar formation, which repairs the damaged myocardium but compromises cardiac function. In neonatal mice, the heart can regenerate fully without scarring following MI; however, this regenerative capacity is lost by P7. The signals that govern neonatal heart regeneration are unknown. By comparing the immune response to MI in mice at P1 and P14, we identified differences in the magnitude and kinetics of monocyte and macrophage responses to injury. Using a cell-depletion model, we determined that heart regeneration and neoangiogenesis following MI depends on neonatal macrophages. Neonates depleted of macrophages were unable to regenerate myocardia and formed fibrotic scars, resulting in reduced cardiac function and angiogenesis. Immunophenotyping and gene expression profiling of cardiac macrophages from regenerating and nonregenerating hearts indicated that regenerative macrophages have a unique polarization phenotype and secrete numerous soluble factors that may facilitate the formation of new myocardium. Our findings suggest that macrophages provide necessary signals to drive angiogenesis and regeneration of the neonatal mouse heart. Modulating inflammation may provide a key therapeutic strategy to support heart regeneration. Total RNA was isolated from CD11b+Ly6G- cells sorted from hearts 3 days following ligation of LAD. 6 samples total: Triplicates of cells from P1 mice and from P14 mice
Project description:The objective of this study was to characterise macrophage subsets in bone marrow (BM) isolated from Csf1r-EGFP mice. A concurrent imaging flow cytometry study conducted by our team unexpectedly revealed macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Expression data from sorted BM “macrophage” populations was found to be consistent with macrophage fragments associated with non-macrophage cells. Granulocyte-specific genes were enriched within the CD11b+ “macrophage” (CD11b+F4/80+Ly6G-GFPloVCAM1+) populations, whereas CD11b- “macrophages” (CD11b-F4/80+GFP+VCAM1+) were consistent with a mixed cell population including both plasma cells and erythroblasts. This data demonstrates how fragmentation of hematopoietic tissue macrophages can result in misattribution of macrophage identity to non-macrophage populations, thereby undermining accuracy of macrophage ex vivo molecular profiles.
Project description:Measure the effect of TCRβ expression on the transcriptional profile of CD11bhighCD14+F4/80+ macrophages sorted from mouse spleen on day 6 post-infection with Plasmodium berghei ANKA malaria
Project description:Myocardial infarction (MI) leads to cardiomyocyte death, which triggers an immune response that clears debris and restores tissue integrity. In the adult heart, the immune system facilitates scar formation, which repairs the damaged myocardium but compromises cardiac function. In neonatal mice, the heart can regenerate fully without scarring following MI; however, this regenerative capacity is lost by P7. The signals that govern neonatal heart regeneration are unknown. By comparing the immune response to MI in mice at P1 and P14, we identified differences in the magnitude and kinetics of monocyte and macrophage responses to injury. Using a cell-depletion model, we determined that heart regeneration and neoangiogenesis following MI depends on neonatal macrophages. Neonates depleted of macrophages were unable to regenerate myocardia and formed fibrotic scars, resulting in reduced cardiac function and angiogenesis. Immunophenotyping and gene expression profiling of cardiac macrophages from regenerating and nonregenerating hearts indicated that regenerative macrophages have a unique polarization phenotype and secrete numerous soluble factors that may facilitate the formation of new myocardium. Our findings suggest that macrophages provide necessary signals to drive angiogenesis and regeneration of the neonatal mouse heart. Modulating inflammation may provide a key therapeutic strategy to support heart regeneration.
Project description:Macrophages infiltrate the infarcted heart and play a critical role in repair, remodeling and fibrosis. Macrophages sense changes in the extracellular matrix (ECM) environment through Integrins, thus activating signaling pathways that regulate their function. Our data show that av integrin is highly expressed in isolated bone marrow macrophages and is markedly upregulated in response to TGF-β, a growth factor known to be activated in the infarcted heart. Accordingly, we hypothesize that aV integrin induction in infarct macrophages may regulate macrophage phenotype, function and response to key macrophage-activating signals following MI.
Project description:Ly6Clow macrophages promote scar formation and prevent early infarct expansion after myocardial infarction (MI). Although CD4+ T cells influence the regulation of Ly6Clow macrophages after MI, the mechanism remains largely unknown. Here, we focused on IL-21 and uncovered its physiological relevance in post-MI hearts. CD4+ T cells harvested from the infarcted heart produce IL-21 upon stimulation, and IL-21 receptor was expressed on Ly6Clo macrophages in the infarcted heart. The survival rate after MI was significantly improved in IL-21-deficient mice compared with WT mice. Moreover, transcriptome analysis of infarcted heart tissue demonstrated that inflammation was persistent in WT mice compared with IL-21-deficient mice. The number of neutrophils was significantly decreased, whereas the number of Ly6Clow macrophages was significantly increased in IL-21-deficient mice. Consistently, IL-21 enhanced the apoptosis of Ly6Clow macrophages. Furthermore, RNA-seq analysis of Ly6Chi and Ly6Clo macrophages stimulated with or without IL-21 for 24 hours revealed that IL-21 induces inflammatory responses in both Ly6Chi and Ly6Clo macrophages. Finally, the treatment with IL-21 receptor Fc protein significantly increased the survival after MI. Thus, the deletion of IL-21 improves survival after MI by preventing Ly6Clo macrophage apoptosis.
Project description:Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative M2-like macrophage (M2) polarization, essential for cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time inactivation of EZH2 activity, linked to ectopic cytoplasmic localization of the epigenetic enzyme, during monocyte differentiation in vitro as well as in M2 macrophages in vivo during post-MI cardiac inflammation. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation at the promoter of bivalent genes, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction after MI in vivo. In conclusion, our study reveals that epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.