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

CX₃CR1 deficient cells lack in vitro tunnelling capacity in solid matrix and form incomplete 3D tube-like structures compared to CX₃CR1 competent cells.

<p>CX₃CR1 cells isolated from bone marrow of CX₃CR1^+/gfp or CX₃CR1^gfp/gfp mice were sandwiched between two DQ-red fortified (20 µg/ml; substrate for proteases) Matrigel layers with or without 10 ng/ml CX₃CL1 gradient and were cultured for 5 days. A & B, CX₃CR1 cells remodelled into tube-like structures (tubulation) especially under CX₃CL1 gradient (Scale bar: 10 µm). However these tubular structures were incompletely formed by CX₃CR1 deficient cells. Furthermore, the CX₃CR1 deficient cells lacked tunneling capacity (dotted lines-protease activation red) in the Matrigel (C & D) and produced significantly less laminin following CX₃CL1 stimulation (E & F). Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<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

Pharmacologic inhibition of CX₃CR1 results in formation of leaky microvessels within experimental plaque.

<p>A, Drug (F1) study protocol: Carotid artery was ligated in the C57BL6J mice (wt mice) and animals were treated with a selective CX₃CR1 inhibitor (F1) (from second week post carotid ligation for another two weeks). B, Representative cross sectional images of carotid artery from C57BL6J mice treated with saline or a selective CX₃CR1 inhibitor (F1) and stained for laminin (B; Basement membrane; Red), or CD42b (D; Platelets; Red), CX₃CR1 (GFP; Green) and DAPI (Nucleus; Blue). B & C, Significantly greater number of CX₃CR1 positive microvessels were covered by basement membrane laminin in saline treated C57BL6J mice compared to F1 treated mice. D & E, Increased platelet CD42b staining was observed in the neointimal interstitial space in the F1 treated mice compared to saline treated mice (Scale bar: 50 µm). In addition the leaky microvessel phenotype in mice treated with F1 was confirmed by presence of intravenously administered (tail vein) 2–2.5 µm diameter microspheres (red spheres) in the neointimal lesion (F & G). IgG control staining for isotype-matched antibodies shown. Data is represented as mean ± SEM of 15 plaque sections/mice (n = 4 independently performed experiments); * denotes p<0.01.</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

CX₃CR1 positive cells contribute to formation of experimental plaque angiogenesis.

<p>Plaque angiogenesis was created by ligation of carotid artery at its bifurcation for 4 weeks in CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice. Perfusion fixed carotid arteries were isolated and OCT embedded. 5 µm cross sections were stained with smooth muscle marker (Calponin; Red) and confocal images were acquired. A, Representative bright field cross section image of carotid artery from CX₃CR1^+/gfp mice stained with DAPI (Nucleus; Blue). Red blood cells (RBCs) (arrow heads) were observed in the microvessels indicating these microvessels were functional. B, Mice competent for CX₃CR1 function had a higher proportion of MV containing RBCs C, Representative cross section image of carotid artery from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice were stained with calponin (Red) and DAPI (nucleus; blue). CX₃CR1 positive cells (GFP positive; Green) integrated into microvascular wall and were also present in perivascular region and co-expressed smooth muscle marker (Calponin; Red) (Scale bar: 10 µm). In the CX₃CR1 functionally deficient (CX₃CR1^gfp/gfp) mice the number of vascular and perivascular cells (GFP positive; Green) (D) and co-expressing smooth muscle marker (E) were significantly reduced. Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 4 independently performed experiments). F, Microvessels in the plaque stained with CX₃CR1 (Green) and DAPI (nucleus; blue) were 3D reconstructed using IMARIS software to depict signet ring structures and their tubular architecture. G, The number of signet-ring cells was significantly reduced in CX₃CR1^gfp/gfp mice. H, Schematic representing the three major phenotypes of CX₃CR1 cells associated with microvascular structures in the plaque. * denotes p<0.05.</p

Functional deficiency of CX₃CR1 results in leaky microvessel phenotype in experimental plaque.

<p>Representative cross sectional images of carotid artery plaques from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice stained with CD42b (Platelets; Red) (A) or laminin (Basement membrane; Red) (C) and DAPI (Nucleus; Blue). B, Significantly increased staining for platelet CD42b was observed in the neointimal interstitial space in CX₃CR1^gfp/gfp mice compared to competent CX₃CR1^+/gfp mice (Scale bar: 50 µm). D, A significantly greater number of CX₃CR1 positive microvessels were covered by basement membrane laminin in CX₃CR1^+/gfp mice compared to CX₃CR1^gfp/gfp mice (insets in panel C show GFP and laminin co-staining present in CX₃CR1^+/gfp but not CX₃CR1^gfp/gfp mice). Data is represented as mean ± SEM of 15 plaque sections/mice (n = 4 independently performed experiments); * denotes p<0.05.</p

Rho activation is essential for tubulation of CX₃CL1 stimulated CX₃CR1 cells.

<p>CX₃CR1 cells isolated from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice were treated with 100 nM CX₃CL1 and 30 minutes post probed for expression of activated RhoA. A, Representative immuno blot showing active RhoA protein expression. B, Active RhoA expression within 30 mins post CX₃CL1 stimulation was significantly higher in CX₃CR1 cells isolated from CX₃CR1^+/gfp compared to CX₃CR1^gfp/gfp mice. C, CX₃CR1 cells isolated from bone marrow of CX₃CR1^+/gfp mice were sandwiched between two Matrigel layers with 10 ng/ml CX₃CL1 gradient and were cultured for 5 days in absence or presence of Rho inhibitor (Y27632; 10 µM, Scale bar: 10 µm). C & D, Inhibition of Rho using Y27632 (10 µM) prevented the formation of tube-like structures by CX₃CR1 cells isolated from CX₃CR1^+/gfp mice. Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<0.01.</p