Differential roles of cardiac and leukocyte derived macrophage migration inhibitory factor in inflammatory responses and cardiac remodelling post myocardial infarction

Abstract

Myocardial infarction (MI) provokes regional inflammation which facilitates the healing, whereas excessive inflammation leads to adverse cardiac remodelling. Our aim was to determine the role of macrophage migration inhibitory factor (MIF) in inflammation and cardiac remodelling following MI. Wild type (WT) or global MIF deficient (MIFKO) mice were subjected to coronary artery occlusion. Compared to WT mice, MIFKO mice had a significantly lower incidence of post-MI cardiac rupture (27% vs. 53%) and amelioration of cardiac remodelling. These were associated with suppressed myocardial leukocyte infiltration, inflammatory mediators' expression, and reduced activity of MMP-2, MMP-9, p38 and JNK MAPK. Infarct myocardium-derived or exogenous MIF mediated macrophage chemotaxis in vitro that was suppressed by inhibition of p38 MAPK or NF-κB. To further dissect the role of MIF derived from different cellular sources in post-MI cardiac remodelling, we generated chimeric mice with MIF deficiency either in bone marrow derived-cells (WT(KO)) or in somatic-cells (KO(WT)). Compared to WT and KO(WT) mice, WT(KO) mice had reduced rupture risk and ameliorated cardiac remodelling, associated with attenuated regional leukocyte infiltration and expression of inflammatory mediators. In contrast, KO(WT) mice had delayed healing and enhanced expression of M1 macrophage markers, but diminished expression of M2 markers during the early healing phase. In conclusion, global MIF deletion protects the heart from post-infarct cardiac rupture and remodelling through suppression of leukocyte infiltration and inflammation. Leukocyte-derived MIF promotes inflammatory responses after MI, whereas cardiac-derived MIF affects early but not ultimate healing process.

Keywords: Healing; Inflammation; Macrophage migration inhibitory factor; Myocardial infarction.

Figure 1. Global MIF deletion (MIFKO) reduced incidence of cardiac rupture and alleviated cardiac remodelling and dysfunction following myocardial infarction (MI)

A, Kaplan-Meier survival analysis of wild type (WT) and MIFKO mice up to 4 weeks following MI, all sham operated animals survived (not shown). n=49 for WT and 29 for MIFKO group. B, Cumulative incidence of cardiac rupture leading to mortality in WT and MIFKO mice. Numbers represent the group size. C, Quantitative analysis of infarct size at autopsy in those mice dying of rupture within 3–5 days (acute phase) and in surviving mice at 4 weeks post MI (chronic phase). Numbers in the bar represent the group size. D, Echocardiographic data showing that left ventricular end-diastolic and end-systolic dimensions (LVEDd, LVESd) were smaller and fractional shortening (FS) was greater in MIFKO than in WT mice at 4 weeks after MI, indicating less severe LV remodelling and dysfunction in MIFKO mice. *P<0.05 vs. WT, n=15–20 per time point.

Figure 2. Temporal changes in leukocyte infiltration and gene expression of cytokines, chemokine and adhesion molecules in global MIF deficient (MIFKO) and wild type (WT) mice following myocardial infarction (MI)

A, Representative images of CD45 positive immunofluroescent staining for leukocytes in the infarct region at 3 days after MI. The purple colour indicates overlap of CD45 positive staining (red) with DAPI (blue) staining for nuclei. Bar=100 μm. B, Quantification of leukocytes (CD45 positive cells) in the infarct and border regions from different time points after MI. C–G, Changes in mRNA level of monocyte chemoattractant protein-1 (MCP-1), intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), interlukin-1β (IL-1β) and IL-10 in the infarct tissue of WT and MIFKO mice at different time points after MI. H, Temporal change of IL-1β plasma level in WT and MIFKO mice with either sham-operated or MI. *P<0.05 vs. respective sham, †P<0.05 vs. WT at the same time point. n=4 per sham group, n=7–8 per MI group.

Figure 3. Global MIF deletion (MIFKO) attenuated activity and gene expression of matrix metallanoprotienase-9 (MMP-9) and MMP-2 following myocardial infarction (MI)

A, Representative images of gelatine zymography demonstrating temporal changes of MMP-9 and MMP-2 activities in WT and MIFKO mice following MI. B, Quantitative analyses of active MMP-9 and MMP-2 by zymography in WT and MIFKO mice from sham-operated and infarcted tissues. n=3 for each sham group, n=5 for each MI group. C, temporal changes in mRNA levels of MMP-9 and MMP-2 from WT and MIFKO mice following MI. n=4 per sham group, n=7–8 per MI group. *P<0.05 vs. sham, †P<0.05 vs. WT at the same time point.

Figure 4. Phosphorylation of pro-inflammatory signalling kinases was reduced in global MIF deficient (MIFKO) mice and MIF promoted macrophage infiltration in the infarct myocardium

A, Representative immunoblotting images for phospho- and total-p38 mitogen-activation protein kinase (p/t-p38) and c-Jun N-terminal kinase (p/t-JNK) in hearts of sham-operated (SH) or infarct wild type (WT) and MIFKO mice. B, Quantitative analysis of p/t-p38 MAPK and p/t-JNK in WT and MIFKO mice. *P<0.05 vs. sham, †P<0.05 vs. WT at day 3, n=3 per sham group, n=5 per MI group). C, Macrophage chemotaxis in response to sham or homogenised infarct tissue from WT or MIFKO mice at day 3. Cells/HPF, cells/high power field. *P<0.05 vs. other conditions. MCP-1 Ab, monocyte chemoattrant protein-1 neutralizing monoclonal antibody (eBioscience, 5 μg/ml). D, Chemotaxis by quantitative migration analysis of WT macrophage or pre-treated WT macrophages with inhibitors for p38 MAPK (p38i, SB203580, 10 μM) or NF-κB inhibitor (NF-κBi, Bay 11-7082, 2 μM) for 1 hr in response to recombinant MIF (rMIF, 40 ng/ml) in trans-well experiments. *P<0.05. Experiments were performed in triplicates.

Figure 5. Survival, incidence of cardiac rupture, cardiac expression of MIF and leukocytes infiltration in wild type (WT), global MIF deficient (KO) or chimeric mice with either bone marrow derived cell-MIF deficiency (WT^KO) or somatic cell-MIF deficiency (KO^WT) following myocardial infarction (MI)

A, Kaplan-Meier survival analyses for WT, KO, WT^KO and KO^WT mice after MI. B, Cumulative incidence of cardiac rupture leading to mortality in WT, KO, WT^KO and KO^WT mice. Numbers represent group size. C, Time course of CD45 positive leukocyte densities in the infarct and border zones of WT, WT^KO and KO^WT mice following MI. D, Temporal change in MIF gene expression by qPCR in WT mice and both chimeric models following MI. *P<0.05 vs. sham, ^#P<0.05. n=3 per sham group, n=5–8 per MI group. E, Temporal change in MIF protein expression in WT mice and both chimeric models following MI. *P<0.05 vs. sham, †P<0.05 vs. WT^KO at each time point. n=3 per sham group, n=5 per MI group.

Figure 6. Expression pattern of markers for M1 and M2 macrophages in wild type (WT) and chimeric mice with either bone marrow derived cell-MIF deficiency (WT^KO) or somatic cell-MIF deficiency (KO^WT) following myocardial infarction (MI)

A, mRNA expression of M1 macrophage markers, interleukin 1β (IL-1β), interferon-γ (IFN-γ), tumor necrosis factor α (TNFα) and IL-6 in WT, WT^KO and KO^WT mice. B, mRNA expression of M2 macrophage markers, transforming growth factor β1 (TGFβ1), arginase 1 (Arg-1), macrophage mannose receptor 1 (MRC-1) and CD163 in WT, WT^KO and KO^WT mice. Data expressed as fold change from WT sham expression. n=4 per sham group, n=6 per MI group. *P<0.05 vs. sham, ^#P<0.05.