Deleterious Anti-Inflammatory Macrophage Recruitment in Early Post-Infarction Phase: Unraveling the IL-6/MCP-1/STAT3 Axis

Abstract

Using a translational approach with an ST-segment myocardial infarction (STEMI) cohort and mouse model of myocardial infarction, we highlighted the role of the secreted IL-6 and MCP-1 cytokines and the STAT3 pathway in heart macrophage recruitment and activation. Cardiac myocytes secrete IL-6 and MCP-1 in response to hypoxic stress, leading to a recruitment and/or polarization of anti-inflammatory macrophages via the STAT3 pathway. In our preclinical model of myocardial infarction, neutralization of IL-6 and MCP-1 or STAT3 pathway reduced infarct size. Together, our data demonstrate that anti-inflammatory macrophages can be deleterious in the acute phase of STEMI.

Keywords: IL-6; MCP-1; STAT3; inflammation; macrophages; myocardial infarction.

Graphical abstract

Figure 1

IL-6 and MCP-1 Levels Peak 24 Hours After Admission for a STEMI and Are Associated With Adverse Clinical Outcomes (A and B) Kinetics of IL-6 and MCP-1 concentrations in ST-segment elevation myocardial infarction (STEMI) patient sera measured by enzyme-link immunosorbent assay. (C and D) Major adverse cardiovascular events are presented according to IL-6 or MCP-1 serum levels. Data are presented as median with 95% CI. Wilcoxon rank sum test was used for statistical analysis. (E to G) Kaplan-Meier curves (log-rank test) are used to present adverse cardiac events according to the median concentration of IL-6 or MCP-1 independently or additionally at 24 hours. (H) Unadjusted HR and 95% CI for experiencing a composite endpoint during the median of 24 months of follow-up when having high IL-6 level (>median) or high MCP-1 (>median), both high IL-6 and MCP-1, elevated C-reactive protein (CRP >median), and high troponin peak (>median). The peak used is the maximum value of troponin, or CRP, measured for each patient individually. N = 239. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 2

IL-6 and MCP-1 Levels Peak at 3 Hours in the Mouse STEMI Model (A) Experimental outline: peripheral blood and heart were collected at different time points of the ischemia-reperfusion (IR) sequence. (B and C) Kinetics of IL-6 and MCP-1 cytokine levels were quantified by enzyme-link immunosorbent assay from mouse sera after ischemia-reperfusion times; n = 3 to 8 mice. (D and E) IL-6 and MCP-1 mRNA expression expressed in fragments per kilobase million (fpkm) in the area at risk normalized by the healthy area. n = 3 to 8 mice. (F) Experimental outline, isolated cardiac myocytes adult (CMA) underwent in vitro hypoxia-reoxygenation (HR) sequence. Conditioned mediums were collected at 24 hours post-reperfusion. (G and H) IL-6 and MCP-1 levels in the conditioned medium were measured by enzyme-link immunosorbent assay under normoxia or HR. n = 7 to 9 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Data were represented as median + interquartile range. Wilcoxon rank sum test for unpaired data with Dunnett's post hoc test was used for statistical analysis. STEMI = ST-segment elevation myocardial infarction.

Figure 3

CMA Release IL-6 and MCP-1 in Response to HR Stress to Recruit and Differentiate Macrophages (A) Experimental outline of recruitment experiment. After hypoxia, a cell culture insert containing PKH-labeled macrophages was added to the CMA well for 24 hours. The dynamic recruitment of macrophages was followed and quantified by confocal microscopy. (B) Proinflammatory (blue line) vs anti-inflammatory (red line) macrophage recruitment by CMA in response to HR sequence. Data are expressed as numbers of macrophages/ mm². n = 10 independent experiments. (C to E) Effects of the presence of neutralizing antibody against IL-6 (C), MCP-1 (D) or STATTIC inhibitor on the recruitment of anti-inflammatory macrophages when they are added in the medium at reoxygenation time. n = 4 independent experiments. (F) Experimental outline of differentiation experiment. Neutralizing antibodies or STATTIC inhibitor was added at the time of macrophages stimulation with the conditioned medium. (G) Characterization of macrophage phenotype following the treatments by flow cytometry. Data are presented as median + SEM. For statistical analysis, 2-way repeated measures analysis of variance with Sidak's post hoc test (B to E) and Wilcoxon rank sum test for unpaired data with Dunnett's post hoc test (G). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Abbreviations as in Figure 2.

Figure 4

The Early Recruitment of Anti-Inflammatory Macrophages Is Deleterious in Vivo During the First Hours of MI (A) Infarct size quantification using Unisperse blue to measure perfused area followed by triphenyl tetrazolium chloride (TTC) staining to measure necrosis area after treatment by STATTIC or the mix of IL-6 and MCP-1 neutralizing antibodies. (B and C) Effect of treatments on infarct size are represented by plotted necrosis area as a function of the area at risk (expressed as a percentage of left ventricular mass) or expressed by a ratio of necrosis area on area at risk in percentages. n = 8 to 10 per group. (D and E) Impact of STATTIC treatment on the phenotype of macrophages evaluated by flow cytometry; P = 0.057 (D and E). Data are presented as median + SEM. Mann-Whitney test with Sidak’s post hoc test was used for statistical analysis ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Abbreviations as in Figure 2.