Classically and alternatively activated macrophages contribute to tissue remodelling after myocardial infarction

Abstract

An important goal in cardiology is to minimize myocardial necrosis and to support a discrete but resilient scar formation after myocardial infarction (MI). Macrophages are a type of cells that influence cardiac remodelling during MI. Therefore, the goal of the present study was to investigate their transcriptional profile and to identify the type of activation during scar tissue formation. Ligature of the left anterior descending coronary artery was performed in mice. Macrophages were isolated from infarcted tissue using magnetic cell sorting after 5 days. The total RNA of macrophages was subjected to microarray analysis and compared with RNA from MI and LV-control. mRNA abundance of relevant targets was validated by quantitative real-time PCR 2, 5 and 10 days after MI (qRT-PCR). Immunohistochemistry was performed to localize activation type-specific proteins. The genome scan revealed 68 targets predominantly expressed by macrophages after MI. Among these targets, an increased mRNA abundance of genes, involved in both the classically (tumour necrosis factor alpha, interleukin 6, interleukin 1beta) and the alternatively (arginase 1 and 2, mannose receptor C type 1, chitinase 3-like 3) activated phenotype of macrophages, was found 5 days after MI. This observation was confirmed by qRT-PCR. Using immunohistochemistry, we confirmed that tumour necrosis factor alpha, representing the classical activation, is strongly transcribed early after ligature (2 days). It was decreased after 5 and 10 days. Five days after MI, we found a fundamental change towards alternative activation of macrophages with up-regulation of arginase 1. Our results demonstrate that macrophages are differentially activated during different phases of scar tissue formation after MI. During the early inflammatory phase, macrophages are predominantly classically activated, whereas their phenotype changes during the important transition from inflammation to scar tissue formation into an alternatively activated type.

Figure 1

Isolation of macrophages from infarcted area in mice. (A) Macrophages were isolated from infarcted area (I) after ligature of the left anterior descending artery. mRNA abundance of all investigated targets was also compared with the infarct border zone (BZ). (B) After isolation of macrophages, the cell population was spotted on slides using cytospin technique and immunostained using F4/80 antibodies (red). Note that the majority (>90%) of isolated cells are macrophages. (Nuclei: DAPI, blue). FACS analyses showed that when using CD11b, monocytes were successfully separated from the isolated cell population by using magnetic cell sorting (MACS). (C) Representative HE-staining of infarcted tissue, which was used for immunohistochemistry. The infarcted tissue was used to investigate the time course of activation type-specific genes. It was isolated 2, 5 and 10 days after myocardial infarction and compared with sham-operated mice. (D) qRT-PCR of genes known to be expressed by macrophages after MI. Fold changes (FC) are expressed relative to mRNA abundance in myocardial infarction (MI), which was set to 1. Both targets, toll-like receptor 2 (TLR2) and colony stimulating factor 2 (CSF2, also designated GM-CSF), are strongly expressed in macrophages compared with MI, BZ, MonoB and LV. This indicates that these genes are transcribed infarct-specific and derive predominantly from macrophages after myocardial infarction. (E) Matrix metalloproteinase 2 (MMP2) is not transcribed by macrophages after myocardial infarction. Even though up-regulated in myocardial infarction versus LV, the FC of macrophages versus LV is less than 1. (FC, fold change; LV, left ventricle; MonoB, monocytes isolated from blood; BZ, border zone of infarcted area; MI, entire infarcted area; MAC, macrophages isolated from infarcted area; each n= 6).

Figure 2

qRT-PCR of genes specific for the classical, the alternative activated type of macrophages and deactivating targets 5 days after myocardial infarction. All fold changes (FC) are expressed relative to mRNA abundance in myocardial infarction (MI), which was set to 1. Tumour necrosis factor α (TNFα) (A), interleukin 6 (IL6) (B) and interleukin 1β (IL1β) (C) are all strongly up-regulated in macrophages compared with myocardial infarction, BZ, MonoB and LV. This indicates a myocardial-specific regulation predominantly in macrophages. Arginase 1 (ARG1) (D), arginase 2 (ARG2) (E) and chitinase3 like 3 (CHI3L3, also designated YM1) (F) are mainly transcribed by macrophages compared with MI, BZ and LV. In connection with ARG2 as well as CHI3L3, a moderate transcription was also detected in monocytes isolated from blood compared with MI, BZ and LV, which indicates an incomplete cardiac-specific expression. Suppressor of cytokine signalling 3 (SOCS3) (G), interleukin 1 receptor antagonist (IL1rn) (H) and interleukin 10 (IL10) (I), which are all known to suppress the inflammatory response, are predominantly transcribed by macrophages. Note that especially IL10 shows a very strong up-regulation in macrophages compared with controls. (FC, fold change; LV, left ventricle; MonoB, monocytes isolated from blood; BZ, border zone of infarcted area; MI, entire infarcted area; MAC, macrophages isolated from infarcted area; each n= 6).

Figure 3

Immunostaining of TNFα in the infarcted area at different time points after ligature of the left descending anterior coronary artery. (A) Confocal images of infarcted tissue 2, 5 and 10 days after MI, which were stained with specific antibody F4/80 (red) and TNFα (green), representing the classical activation type of macrophages. The last panel shows co-localization in merged images (merged images, yellow; nuclei: DAPI, blue). (B) TNFα-positive macrophages were quantified at different time points (2, 5 and 10 days after myocardial infarction). For each image, the proportion (in percentage) of F4/80-positive cells which, at the same time, express TNFα was evaluated. (C) Abundance of TNFα mRNA in macrophages isolated at different time points (2, 5 and 10 days, n= 5) after myocardial infarction. The relative amount of TNFα mRNA was investigated, using quantitative real-time PCR, and normalized to RNA isolated from entire infarcted tissue (MI), which was set to 1. (LV healthy left ventricle; *P < 0.01 versus MI).

Figure 4

Time course of alternative activated macrophages at different time points after myocardial infarction using immunostaining with ARG1. (A) Confocal laser microscopy images of infarcted tissue 2, 5 and 10 days after MI, which were stained with specific antibody F4/80 (red) and ARG1 (green), representing the alternative activation type of macrophages. The last panel shows co-localization in merged images (merged images, yellow; nuclei: DAPI, blue). (B) ARG1-positive macrophages were quantified at different time points (2, 5 and 10 days after myocardial infarction). For each image, the proportion (in percentage) of F4/80-positive cells which, at the same time, express ARG1 was evaluated. (C) Abundance of ARG1 mRNA in macrophages isolated at different time points (2, 5 and 10 days, n= 5) after myocardial infarction. The relative amount of ARG1 mRNA was investigated, using quantitative real-time PCR, and normalized to RNA isolated from entire infarcted tissue (MI), which was set to 1. (LV healthy left ventricle; *P < 0.01 versus MI).

Figure 5

After LAD ligature IL10 expression increases, demonstrating that deactivation of macrophages increases over a longer period of time. (A) Confocal laser microscopy images of infarcted tissue 2, 5 and 10 days after MI, which were stained with specific antibody F4/80 (red) and IL10 (green), representing the alternative activation type of macrophages. The last panel shows co-localization in merged images (merged images, yellow; nuclei: DAPI, blue). (B) IL10-positive macrophages were quantified at different time points (2, 5 and 10 days after myocardial infarction). For each image, the proportion (in percentage) of F4/80-positive cells which, at the same time, express IL10 was evaluated. (C) Abundance of IL10 mRNA in macrophages isolated at different time points (2, 5 and 10 days, n= 5) after myocardial infarction. The relative amount of IL10 mRNA was investigated, using quantitative real-time PCR, and normalized to RNA isolated from entire infarcted tissue (MI), which was set to 1. (LV healthy left ventricle; *P < 0.01 versus MI).

Figure 6

Macrophages show a time-dependent occurrence of activation types after myocardial infarction. (A) Classically activated macrophages dominate the inflammatory phase after myocardial infarction. Alternatively activated and deactivated macrophages increase during the proliferative and maturation phase. (B) ARG1, representing the alternatively activated phenotype of macrophages, and TNFα, a target expressed in classically activated macrophages, were co-localized (arrows) using immunohistochemistry. (Scale bar 100 μm).