Novel function of macrophage migration inhibitory factor in regulating post-infarct inflammation and the therapeutic significance

Fig. 1. The systemic inflammatory responses in wild type (WT) and global MIF deficient (MIFKO) mice after 3, 24 and 72 h myocardial infarction (MI). A-D, Counts of white blood cells (WBC), monocytes, lymphocytes and neutrophils in peripheral blood. n = 5–10 per group. E, Representative scatter plots of Ly-6Chigh monocytes from peripheral blood of WT and MIFKO mice at 72 h post-MI. F, Quantification of Ly-6Chigh monocytes from peripheral blood at 3, 24 and 72 h post-MI. n = 7–13 per group. G-J, mRNA levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), IL-1β and matrix metalloproteinase-9 (MMP9) in peripheral blood mononuclear cells (PBMCs). n = 3–5 per group. *P < 0.05 vs. sham in the same genotype. **P < 0.01 vs. sham in the same genotype. ***P < 0.001 vs. sham in the same genotype.

Fig. 2. The regional inflammatory responses in the infarct myocardium in wild type (WT) and global MIF deficient (MIFKO) mice after myocardial infarction (MI). A-D, mRNA levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), IL-1β and matrix metalloproteinase-9 (MMP9) in the infarct tissue from WT and MIFKO mice at 24 and 72 h post-MI. n = 3–5 per group. E, Quantification of leukocytes (CD45 positive cells) in the infarct region from WT and MIFKO mice at 72 h post-MI. n = 4–7 per group. F, Representative images of CD45 positive stained cells in sham heart and the infarct region from WT and MIFKO mice at 72 h post-MI. The pink color (white arrow) indicates overlap of CD45 staining (red) with DAPI (blue) staining for nuclei. The amplified image (inset) shows a typical overlap staining. G, Representative scatter plots of Ly-6Chigh monocytes in the infarct tissue from WT and MIFKO mice at 72 h post-MI. H, Quantification of Ly-6Chigh monocytes in the infarct tissue of WT and MIFKO mice at 3, 24 and 72 h post-MI. n = 4–7 per group. *P < 0.05 vs. sham in the same genotype. **P < 0.01 vs. sham in the same genotype. ***P < 0.001 vs. sham in the same genotype. (For…

Fig. 3. Mobilization of splenic inflammatory cells in wild type (WT) and global MIF deficient (MIFKO) mice after myocardial infarction (MI). A, Spleen weight to tibia length ratio in WT and MIFKO mice at 3 and 24 h post-MI. n = 6–13 per group. B, Quantification of splenic white pulp numbers in WT and MIFKO mice at 24 h post-MI. n = 5–8 per group. C-D, Quantification of splenic white pulp size and marginal zone size in WT and MIFKO mice at 24 h post-MI. n = 5–8 per group. E, Representative images of hematoxylin-eosin (HE) stained spleen cross-sections. In HE stained spleen, there are white pulp (arrow), marginal zone (arrow) and subcapsular red pulp (black-dotted line). Amplified images (inset) of HE-stained white pulp in the spleen reveal differential nuclear densities across MI groups. F, Quantification of mean integrated optical density (IOD) of leukocytes (CD45 positive cells) in WT and MIFKO spleens at 24 h post-MI. n = 3–5 per group. G, Quantification of Ly-6Chigh monocytes from spleens of WT and MIFKO mice at 3, 24 and 72 h post-MI. n = 7–12 per group. *P < 0.05 vs. sham in the same genotype. **P < 0.01 vs. sham in the same genotype. ***P < 0.001 vs. sham in the same genotype. (For…

Fig. 4. Macrophage migration inhibitory factor (MIF) promotes inflammatory cell migration and infiltration through C–C motif chemokine receptor 2 (CCR2) and C-X-C motif chemokine receptor (CXCR) 4 and promotes splenic monocyte mobilization via interaction with angiotensin Ⅱ type 1 receptor (AT-1R) after myocardial infarction (MI). A, peripheral blood mononuclear cells (PBMCs) chemotaxis in response to homogenized sham or infarct heart tissue from wild type (WT) mice at day 3 post-MI, or MI homogenate together with inhibition of C–C motif chemokine receptor 2 (CCR2), C-X-C motif chemokine receptor (CXCR) 2 and CXCR4, respectively. Cells/HPF, cells/high power field. PBMCs harvested from WT mice at 3 days post-MI. CCR2i, CCR2 inhibition. CXCR2i, CXCR2 inhibition. CXCR4i, CXCR4 inhibition. n = 4–8 per group. ***P < 0.001 vs. sham.B, PBMCs chemotaxis in response to recombinant human MIF (rMIF) or rMIF with inhibition of CCR2, CXCR2 and CXCR4, respectively. PBMCs harvested from WT mice at 3 days post-MI. n = 4–8 per group. ***P < 0.001 vs. control.C, Representative immunoblotting images for monocyte chemoattractant protein-1 (MCP-1), CXCR4 and internal reference protein (heat shock protein 60,…

Fig. 5. Acute inflammatory responses in chimeric mice with somatic cell-derived MIF (WTKO) and MIF derived from bone marrow cell (KOWT) after myocardial infarction (MI). A, Representative scatter plots of Ly-6Chigh monocytes from peripheral blood of WTKO and KOWT mice at 72 h post-MI. B, Quantification of Ly-6Chigh monocytes from peripheral blood of WTKO and KOWT mice at 3, 24 and 72 h post-MI. n = 5–8 per group. C, Counts of white blood cells (WBC) in peripheral blood from WTKO and KOWT mice at 3, 24 and 72 h post-MI. n = 6–12 per group. D, Representative scatter plots of Ly-6Chigh monocytes from the infarct tissue of WTKO and KOWT mice at 72 h post-MI. E, Quantification of Ly-6Chigh monocytes from the infarct tissue of WTKO and KOWT mice at 3, 24 and 72 h post-MI. n = 9–14 per group. F, Spleen weight to tibia length ratios in WTKO and KOWT mice at 3, 24 and 72 h post-MI. n = 4–8 per group. G, Quantification of splenic white pulp size in WTKO and KOWT mice at 24 h post-MI. n = 4–6 per group. H, Quantification of splenic white pulp numbers in WTKO and KOWT mice at 24 h post-MI. n = 4–6 per group. I, Representative images of hematoxylin-eosin (HE) stained spleen cross-sections. In HE…

Fig. 6. Somatic cell-derived MIF (WTKO) promoted type-2 macrophages (M2) but attenuated type-1 macrophages (M1) accumulation during the proliferative phase of myocardial infarction (MI). A, Representative scatter plots of Ly-6Clow monocytes from the infarct tissue of WTKO and KOWT mice at 7 days post-MI. B, Quantification of Ly-6Clow monocytes from the infarct tissue of WTKO and KOWT mice. n = 5–8 per group. **P < 0.01 vs. sham in the same genotype. ***P < 0.001 vs. sham in the same genotype. C, Representative images of M2 macrophages (CD68 and CD206 double-immunofluorescent staining) in the infarct region. The yellow or orange color (white arrows) indicates overlap of CD68 (red) and CD206 (green) positive staining. The amplified image (inset) shows a typical overlap staining. D, Quantification of M2 macrophages in the infarct region from WTKO and KOWT mice at 7 days post-MI. n = 4 per group. *P < 0.05 vs. sham in the same genotype. ***P < 0.001 vs. sham in the same genotype. E-I, mRNA expression of M1 macrophage markers including CD86, CD80, inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and interferon γ (IFN-γ) in peripheral blood mononuclear cells (PBMCs) from normal and…

Fig. 7. Fibroblast function in chimeric mice with specific somatic cell-derived MIF (WTKO) and bone marrow cell-derived MIF (KOWT) after myocardial infarction (MI). A-D, Changes in mRNA level of collagen 1 (Col-1), Col-3, α-smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β) in the infarct tissue of WTKO and KOWT mice at 6 days post-MI. n = 4–5 per group. E, Representative images of α-SMA positive stained cells in the infarct region. The cyan color (white arrow) indicates overlap of α-SMA staining (green) with DAPI (blue) staining for nuclei. The amplified image (inset) shows a typical overlap staining. F, Quantification of α-SMA positive cells in the infarct region from WTKO and KOWT mice at 6 days post-MI. n = 3–4 per group. G, Proliferation of primary mouse fibroblasts isolated from sham, 2-day MI and 6-day MI hearts. n = 5–6 per group *P < 0.05 vs. sham in the same genotype. **P < 0.01 vs. sham in the same genotype. ***P < 0.001 vs. sham in the same genotype. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Anti-macrophage migration inhibitory factor (MIF) treatment attenuated splenic monocyte mobilization, systemic and reginal inflammatory responses post-myocardial infarction (MI). A-B, Changes of MIF level in plasma after anti-MIF treatments in MI mice. n = 8–12 per group. C-E, Counts of white blood cells (WBC), monocytes and neutrophils in peripheral blood after anti-MIF treatment in MI mice. n = 5–8 per group. F, Spleen weight to tibia length ratios. n = 8–12 per group. G, Quantification of splenic white pulp size. n = 6–9 per group. H, Representative scatter plots of Ly-6Chigh monocytes from the spleen after anti-MIF treatment in MI mice. I, Representative scatter plots of Ly-6Chigh monocytes from peripheral blood after anti-MIF treatment in MI mice. J, Quantification of Ly-6Chigh monocytes from the spleen at 24h post-MI. n = 6–9 per group. K, Quantification of Ly-6Chigh monocytes from peripheral blood at 72h post-MI. n = 6–9 per group. L-O, Quantification of the number of leukocytes (CD45 positive cells) and macrophages (CD68 positive cells) at 3 and 6 days post-MI. n = 3–4 per group. NS-IgG, non-specific IgG was administered according to MIFAb-1 or MIFAb-2 regimen,…

Fig. 9. Anti-macrophage migration inhibitory factor (MIF) treatment did not affect the healing, but improved prognosis post-myocardial infarction (MI). A, Quantification of necrotic area. n = 4–6 per group. *P < 0.05 vs. 7d MI in the same treatment group. B, Quantification of infarct wall thickness. n = 4–6 per group. C, Quantification of collagen content (fibrosis). n = 4–6 per group. ***P < 0.001 vs. 7d MI in the same treatment group. D, Cumulative incidence of cardiac rupture in non-specific IgG (NS-IgG) and anti-MIF treatment groups within 7 days after surgery. E, Kaplan–Meier curve of the survival in NS-IgG and anti-MIF groups within 28 days after surgery. F, Representative B-mode views of long-axis (upper panel) and M-mode views of short-axis (lower panel) at the left ventricular (LV) end-diastole from different groups at baseline and 4 weeks after MI. G-J, Changes in LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), ejection fraction (EF) and stroke volume (SV) at baseline (0), 1 and 4 weeks in NS-IgG and anti-MIF treatment groups after MI. n = 6–15 per group. *P < 0.05 vs. baseline. †P < 0.05 vs. NS-Ig groups at the same time point. NS-IgG, was administered…

Fig. 10. Anti-macrophage migration inhibitory factor (MIF) treatment modulated the activity of matrix metalloproteinases (MMPs) and the expression tissue inhibitor of metalloproteinases (TIMPs). A, Representative images of gelatine zymography demonstrating changes of active and latent MMP-9 in non-specific IgG (NS-IgG) and MIFAb-1 treatment groups at 3 days post-MI. B, Quantitative analyses of MMP-9 activity in NS-IgG and MIFAb-1 treatment groups at 3 days post-MI. n = 3–4 per group. C, Representative images of gelatine zymography demonstrating changes of active and latent MMP-2 in NS-IgG and MIFAb-2 groups at 7 days post-MI. D, Quantitative analyses of MMP-2 activity in NS-IgG and MIFAb-2 groups at 7 days post-MI. n = 3–4 per group. E, Representative immunoblotting images for TIMP-2, TIMP-4 and internal reference protein (heat shock protein 60, HSP-60) in the heart from mice with sham-operation or 3-day MI received NS-IgG and MIFAb-1 treatment. F-G, Quantitative analyses of TIMP-2 and TIMP-4 expression in NS-IgG and MIFAb-1 treated mice at 3 days post-MI. n = 6 per group. H, Representative immunoblotting images for TIMP-2, TIMP-4 and internal reference protein (HSP-60) in the heart from…