Systemic and Cardiac Depletion of M2 Macrophage through CSF-1R Signaling Inhibition Alters Cardiac Function Post Myocardial Infarction

<div><p>The heart hosts tissue resident macrophages which are capable of modulating cardiac inflammation and function by multiple mechanisms. At present, the consequences of phenotypic diversity in macrophages in the heart are incompletely understood. The contribution of cardiac M2-polarized macrophages to the resolution of inflammation and repair response following myocardial infarction remains to be fully defined. In this study, the role of M2 macrophages was investigated utilising a specific CSF-1 receptor signalling inhibition strategy to achieve their depletion. In mice, oral administration of GW2580, a CSF-1R kinase inhibitor, induced significant decreases in Gr1^lo and F4/80^hi monocyte populations in the circulation and the spleen. GW2580 administration also induced a significant depletion of M2 macrophages in the heart after 1 week treatment as well as a reduction of cardiac arginase1 and CD206 gene expression indicative of M2 macrophage activity. In a murine myocardial infarction model, reduced M2 macrophage content was associated with increased M1-related gene expression (IL-6 and IL-1β), and decreased M2-related gene expression (Arginase1 and CD206) in the heart of GW2580-treated animals versus vehicle-treated controls. M2 depletion was also associated with a loss in left ventricular contractile function, infarct enlargement, decreased collagen staining and increased inflammatory cell infiltration into the infarct zone, specifically neutrophils and M1 macrophages. Taken together, these data indicate that CSF-1R signalling is critical for maintaining cardiac tissue resident M2-polarized macrophage population, which is required for the resolution of inflammation post myocardial infarction and, in turn, for preservation of ventricular function.</p></div

CD206+ M2 macrophage depletion reduces LV ejection fraction but not infarct size 2 weeks post MI in MAFIA mice.

<p>A. mRNA expression relative to GAPDH of M1 markers IL6, IL1B, and (b) M2 markers Arg1 and CD206 respectively within the heart 2 weeks post MI (n = 4–6 animals per group). C. Determination of ejection fraction based on pressure-volume loop measurements vehicle or GW2580-treated MAFIA mice 2 weeks post MI. (* <i>p</i><0.05. n = 11–13 animals per group). D. Upper panels: representative MAFIA heart sections after TTC staining. Middle panels: representative MAFIA heart section under fluorescent microscope allowing the detection of coloured microspheres distributed in the perfused area. Lower panels: Infarct areas are represented as white, non-perfused area-at risk (AAR) of infarction (dark red) and perfused areas (blue) are highlighted. The area at risk corresponds to the non-perfused area. E. AAR/LV ratio quantification in MAFIA mice 2 weeks post MI using coloured microspheres. F. IZ/LV ratio quantification in MAFIA mice 2weeks post MI using TTC staining and coloured microspheres (* <i>p</i><0.05. n = 11–13 animals per group).</p

Effect of CD206+ M2 macrophage depletion on collagen deposition and cell infiltration within the infarct 2 weeks post MI in MAFIA mice.

<p>A. Representative Sirius Red staining on histological sections from MAFIA mice 2 weeks post MI. B. Quantification of collagen staining as a percentage of LV from MAFIA mice 2 weeks post MI (Scale bar: 1 mm, n = 6 animals per group, ≥8 images per animal). C. Representative hematoxilin and eosin staining on 20x infarct or remote histological section from MAFIA mice 2 weeks post MI showing an increase in inflammatory infiltrates in animals treated with GW2580. D. Quantification of nuclei number per mm² in MAFIA remote and infarct zone in MAFIA mice 2 weeks post MI. (n = 4–6 animals per group). E. Representative images of immunofluorescent staining of CD206+ M2 macrophages, Gr1+ M1 macrophages and Ly6G+ neutrophils within the infarct zone from MAFIA mice 2 weeks post MI. Quantification of Ly6G+ neutrophil, Gr1+ M1 macrophage and CD206+ M2 macrophage infiltration within the infarct zone from MAFIA mice 2 weeks post MI (n = 4 animals per group, 10 images/animal). * <i>p</i><0.05, ** <i>p</i><0.01.</p

Depletion of the circulating monocyte and Gr1^lo and F4/80^hi populations following 1 week GW2580 treatment.

<p>A. Identification of total monocyte population in mouse blood using MAFIA-GFP. B Following one week of GW2580 treatment, no difference in total circulating monocytes was observed. FACS quantification of Gr1^hi (M1) and Gr1^lo (M2; C,D) and F4/80^hi (E,F) in total monocytes following 1 week GW2580 treatment (n = 6 and n = 4 animals per group, respectively).</p

Depletion of the M2 cardiac macrophage populations following 1 week GW2580 treatment.

<p>A. Representative profiles of digested hearts from vehicle and GW2580-treated animals. B. Quantification of cardiac tissue-resident macrophages 1 week post-GW2580 treatment (n = 6 animals per group). C. Quantification of CD206⁺ M2 macrophages within the heart, 1 week post-GW2580 treatment (n = 6 animals per group, nd:not detected). D. mRNA expression relative to GAPDH of M2 markers Arg1 and CD206 and E. M1 markers IL6, IL1β, respectively, within the heart following 1 week GW2580 treatment (n = 4 animals per group). * <i>p</i><0.05, ** <i>p</i><0.01.</p