Contributed equally
The aim of the present study was to characterize the phenotypic shift, quantity and role changes in different subgroups of retinal macrophages in a mouse model of oxygen-induced retinopathy (OIR). The mRNA expression levels of macrophage M1 and M2 subgroup marker genes and polarization-associated genes were analyzed by RT-qPCR. The number of M1 and M2 macrophages in our mouse model of OIR was analyzed by flow cytometry at different time points during the progression of OIR. Immunofluorescence whole mount staining of the retinas of mice with OIR was performed at different time points to examine the influx of macrophages, as well as the morphological characteristics and roles of M1 and M2 macrophages. An increased number of macrophages was recruited during the progression of angiogenesis in the retinas of mice with OIR due to the pro-inflammatory microenvironment containing high levels of cell adhesion and leukocyte transendothelial migration molecules. RT-qPCR and flow cytometric analysis at different time points revealed a decline in the number of M1 cells from a significantly high level at post-natal day (P)13 to a relatively normal level at P21, as well as an increase in the number of M2 cells from P13 to P21 in the mice with OIR, implicating a shift of macrophage polarization towards the M2 subtype. Immunofluorescence staining suggested that the M1 cells interacted with endothelial tip cells at the vascular front, while M2 cells embraced the emerging vessels and bridged the neighboring vessel sprouts. Thus, our data indicate that macrophages play an active role in OIR by contributing to the different steps of neovascularization. Our findings indicate that tissue macrophages may be considered as a potential target for the anti-angiogenic therapy of ocular neovascularization disease.
Angiogenesis is an essential process for embryonic development and tissue repair, whereas abnormal angiogenesis is a fundamental characteristic in the pathophysiology of ocular diseases, such as retinopathy of prematurity (ROP), diabetic retinopathy and choroidal neovascularization (CNV), usually associated with age-related macular degeneration. Retinochoroidal neovascularization diseases can lead to blindness in developed countries (
Increasing evidence has suggested that macrophages play a significant role in both physiological and pathological angiogenesis (
Although CD11c was a marker traditionally associated with dendritic cells (DCs), a recent study found it to be expressed by some macrophages (
All mice used in this study were pathogen-free (SPF) C57BL/6 mice and kept under the conditions in compliance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval (SYXK-2012–0026) of the Scientific Investigation Board of Shanghai Jiaotong University School of Medicine, Shanghai, China. All efforts were made to minimize animal suffering.
OIR was induced by exposure to high concentrations of oxygen, followed by the return to normal room air; this leads to ischemia. C57BL/6 pups (n=320) were exposed to 75% oxygen at post-natal day (P)7 with their nursing mother and returned to room air at P12, as previously described (
Platelet endothelial cell adhesion molecule (PECAM)-1, CD11c and CD206 were selectively stained by
C57BL/6 mice with or without oxygen-induced ischemic retinopathy were euthanized at P13 and P18, and the eyes were rapidly removed and frozen in optimum cutting temperature embedding compound (Miles Diagnostics, Elkhart, IN, USA). The frozen sections (10-
RNA was isolated from the retinas of mice with OIR and age-matched controls using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions, and as previously described (
The retinas of normal mice and those with OIR were carefully dissected out and digested in pre-warmed 16.5 U/ml papain solution (Worthington, Freehold, NJ, USA) for 30 min with gentle pipetting, and the cell digestion suspension was then transferred and passed through cell strainers (BD Falcon, Franklin Lakes, NJ, USA) to obtain single cell suspension. The cells were spinned down at 900 rpm. After gently removing the supernatant, the cell pellet was suspended with 90
CD11b+ cells from the retinas of the mice with OIR or the normal mice were resuspended in MACS buffer (BD Biosciences, San Jose, CA, USA) and incubated with PE-conjugated anti-mouse CD11c (12-0114-82; eBioscience), PE-conjugated anti-mouse F4/80 (12-4801-82; eBioscience) and Alexa Fluor 647-conjugated CD206 (MCA2235A647; AbD Serotec) and the matching control isotype IgG (MCA421; AbD Serotec) for 30 min at 4°C. The cells were then washed and rinsed again and incubated with secondary antibodies for 30 min at 4°C. The cells were then washed and re-suspended in FACS buffer (BD Biosciences, San Jose, CA, USA) and analyzed by flow cytometry (BD FACSCalibur flow cytometer; BD Biosciences, Heidelberg, Germany). M1 macrophages were identified as F4/80-positive/CD11c-positive/CD206-negative and M2 macrophages were identified as F4/80-positive/CD11c-negative/CD206-positive. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA) (6 mice were enrolled in both the OIR and normal control groups for each time point).
The high-throughput screening of differential mRNA expression between the mice with OIR and the normal mice were obtained using Affymetrix GeneChip Mouse Genome 430 2.0 arrays. Genes were identified as differentially expressed if they exhibited a fold change of at least 1.5 and a P-value <0.05. Two retinas from one mouse were considered as one sample (6 mice were enrolled in both the OIR and normal control groups).
Quantitative data are presented as the mean values ± standard deviation (SD). Statistical significance was determined by the two tailed Student's t-test and one-way ANOVA with Student-Newman-Keuls method for multiple comparisons. Differences were considered to be statistically significant at P-values of 0.05, 0.01 and 0.001. Statistical analysis was performed using SAS 9.0 software.
The expression changes associated with leukocyte transendothelial migration, cell adhesion and cell communication molecules were assessed at mRNA level by RT-qPCR. Specifically, we measured the expression ratio in the retinas of mice with OIR to that of the normal controls at P15. The significant upregulation of most leukocyte transendothelial migration and cell adhesion molecules was observed in the mice with OIR compared to the normal controls (
The retinas from the mice with OIR and the age-matched controls were dissected, fixed and immunofluorescence stained with FITC-isolectin B4 and PE-labeled F4/80 antibody (
The retinas from mice with OIR were pooled for RNA extraction from P13 to P24 with the age-matched normal retinas as controls. RNA was then reverse transcribed into cDNA and specific M1 and M2 macrophage polarization-associated genes were evaluated by qPCR (
Retina flat mounts from the mice with OIR and the controls were immunofluorescence stained with PE-labeled F4/80 anti-body and FITC-isolectin B4. In the mice with oxygen-induced ischemic retinopathy, the macrophages were located in close proximity to the area affected by RNV, as shown by immunofluorescence staining of the retinas (
The numbers of macrophages in the retinas of mice with OIR and normal mice were analyzed by flow cytometry at different time points. F4/80-positive cells were gated out from the live cells and we used CD11c and CD206 as markers to identify M1 or M2 macrophages. F4/80+, CD11c+ and CD206− cells are marked as M1-positive cells, and F4/80+, CD11c− and CD206+ cell are marked as M2-positive cells. The cell distribution patterns in the retinas from the normal mice, and the mice with OIR during the early stage (P13) and later stage (P21) are shown in
Arginase 1 and iNOS were stained to indicate M2 and M1 polarized macrophages as previously reported (
To explore the phenotypic and functional differences of M1 and M2 macrophages, the retinas of mice with OIR at P13 and P18 were analyzed. Retinal flat mounts were immunofluorescence stained with isolectin B4 and CD11c or CD206 (
It has been well established that blood vessels grow into networks through a process involving sprouting, anastomosis and maturation (
The present study demonstrated that the retinas of mice with OIR expressed high levels of mRNAs associated with leukocyte transendothelial migration and cell adhesion, indicating an active interplay between inflammation and angiogenesis under conditions of OIR in this animal model. Sato
In order to investigate the phenotype of the increased inflammatory cells and their correlation with RNV, we performed qPCR for inflammation-associated genes and immunofluorescence staining for retinal vessels together with macrophages from P12 to P24. The results of qPCR suggested a significant increase in the expression of the macrophage marker, F4/80, accompanied with upregulated expression levels of M1-associated genes. The staining results indicated a close association of macrophages with protruding bulbous networks of neovascularization in ischemic retinas from the mice. Further assays to classify the infiltrated macrophages demonstrated enhanced M1 phenotype polarization at P13 and enhanced M2 phenotype polarization at P15–P18, indicating a shift in the macrophage polarization towards the M2 subtype. In addition, Spiller
The distinct features of M1 and M2 macrophages phenotypically and functionally in RNV has attracted increasing interest. Marchetti
This study demonstrated the specific contributing roles of M1, M2 macrophages in different steps of RNV following OIR. During this angiogenic process, M1 macrophages dominated the first 2–3 days following OIR, while M2 macrophages represented the overwhelming macrophage subset thereafter, which is in accordance with the studies of post-myocardial infarction and wound healing process (
The present study provides an outlook of macrophage polarization during OIR, in an aim to shed light on the therapeutic potential target of macrophages in the treatment of neovascular eye diseases in addition to anti-VEGF therapy. However, there are still disadvantages in this study. First, there are different subgroups within M2 macrophages. In addition to traditional M2 macrophages, called M2a, macrophages stimulated with IL-10 are classified as M2c. Yet the different function of these two subsets in RNV remains unclear. Further studies are necessary to investigate their distinctions in this process. Second, clearly it is the combined effect of cytokine profiles that drives the angiogenic phenotype of macrophages. For instance, TNF-α has been shown to be pro-angiogenic in cancers, but it is also secreted at functionally significant levels by the anti-angiogenic M1 macrophages (
We are now at a circumstance where identifying additional VEGF-independent pathways that trigger abnormal angiogenesis in the eye is critical. Ma
In conclusion, the findings of this study demonstrate that M1 and M2 macrophages play an active role in OIR by contributing to different steps of RNV. Thus, tissue macrophages may be considered as a potential target for the anti-angiogenic therapy of ocular neovascularization diseases.
The study was supported by grants from the National Nature Science Foundation of China, 81470639 and 81570853; the Shanghai Nature Science Foundation Grant 14411968400; the Shanghai Charity Cancer Research Center Program 2013, and 2015 Doctoral Innovation Fund Projects BXJ201414 from Shanghai Jiaotong University School of Medicine, China. The authors would like to thank Professor Honglin Wang for assisting with the flow cytometry experiment.
neovascularization
retinal/choroidal neovascularization
macrophage
retinopathy of prematurity
post-natal day n
The mRNA expression levels of focal adhesion (red bars)-, cell communication (black bars)-, cell adhesion molecule (CAM) (white bars)- and leukocyte transendothelial migration-related genes (green bars) were mostly upregulated as shown in the graph. The expresion of MMP-9, PDGFa, FGF-1, FGF-10 (genes associated with the regulation of the actin cytoskeleton, yellow bars) slightly decreased, whereas that of other genes shown in this graph increased (n=6 mice/group). OIR, oxygen-induced retinopathy; P, post-natal day. *P<0.05 and **P<0.01.
Immunofluorescence staining of retinal wholemounts for vasculature and macrophages in retinas of mice with OIR from P13 to P24 [lectin, green (A–D); F4/80, red (E–H)] and age-matched controls [F4/80, red (I–L)]. Retinas from mice with OIR were stained with FITC-isolectin B4 and PE-labled anti-mouse F4/80 antibody for 45 min at room temperature. We observed an increasing area of retinal neovascularization (RNV) from P13 to P24, accompanied by the ascending influx of macrophages. Both RNV and macrophages on the surface of the retinas from mice with OIR were estimated at P13 (A and E), P18 (B and F), P21 (C and G) and P24 (D and H). Macrophages influx of the age-matched controls was also examined at P13 (I), P18 (J), P21 (K) and P24 (L). RNV significantly increased from P18, peaking at P21, and then decreased [(M) P<0.05], accompanied by a significant macrophage influx ascending at P18 and P21 compared to the controls and then decreasing (N) (n=6 mice/group). OIR, oxygen-induced retinopathy; NOR, normal; P, post-natal day. *P<0.05.
The mRNA expression of macrophage polarization-associated genes in mice with OIR and age-matched normal controls. The mRNA levels of (A) iNOS, (B) CD16, (C) CD11c, (D) tumor necrosis factor-α (TNF-α), (E) CD163 and (F) CD206 were compared between the mice with OIR and the normal controls from P13 to P24 (*P<0.05) (n=10 mice/group). OIR, oxygen-induced retinopathy; NOR, normal; P, post-natal day. *P<0.05.
Immunofluorescence staining for retinal vasculature (lectin, green) and macrophages (F4/80, red) of retinas from mice with OIR and age-matched normal controls at different time points. The eyes of mice with OIR and age-matched control mice were fixed and retinas were dissected, incubated in FITC-isolectin B4 and PE-labeled anti-mouse F4/80 for 45 min at room temperature. Retinas were then flat-mounted and examined by fluorescence microscopy. Ischemic retinas from mice with OIR exhibited small tufts and bulbous networks of neovascularization from the retinal capillary bed (A–F) from P12 to P24, and a close association with macrophages was observed by counterstaining with F4/80 [(H–M) and (A′–F′) merged images]. Clear vasculature of normal mice was examined (N–S) counterstained with F4/80 [(T–Y) and (N′–S′) merged images] (n=4 mice/group). OIR, oxygen-induced retinopathy; NOR, normal; P, post-natal day.
Flow cytometric analysis of mice with OIR at different time points. (A) The selection of M1- or M2-positive cells. In brief, we first gated out the live cells, and then gated out the F4/80-positive cells. In this subgroup, we used CD11c and CD206 as markers to identify M1 or M2 macrophages. F4/80+, CD11c+, CD206− cells are markered as M1 positive cells, F4/80+, CD11c−, CD206+ cells are marked as M2-positive cells. (B) The cell distribution pattern in normal mice and mice with OIR in the early stage (P13) and later stage (P21). (C–E) The percentage of total F4/80+ macrophages, the M1 macrophages, and the M2 macro-phage in mice with OIR and the age-matched controls. M2 macrophage number (E) in the mice with OIR at P12 and P13 was lower than that of the normal group, but then increased sharply after the mice with OIR were returned to normal air, and remained at a relatively high level at P15–P21, returning to normal levels at P24. The M1 macrophage number (D) increased at P13, and rapidly decreased at P18, and at P24 it returned to relatively normal levels. The total number of F4/80+ macrophages (C) in mice with OIR at P12 and P13 was lower than that of the age-matched control mice, but it rapidly increased at P15 and onwards; we observed a sharp decrease in the M1/M2 cells; the macrophages in the mice with OIR were polarized towards the M2 subtype macrophage. At P24, the M1, M2, M1/M2 distribution pattern returned to normal (n=6 mice/group). OIR, oxygen-induced retinopathy; NOR, normal; P, post-natal day.
Immunofluorescence staining of iNOS/arginase 1 (red) and isolectin B4 (green) in the eyes of normal mcie and mice with OIR at P13 and P18. C57BL/6 mice with or without ischemic retinopathy were euthanized at P13 and P18, and their eyes were rapidly removed and frozen sections were created. After staining with primary anti-mouse iNOS (A, D, G and J) and arginase 1 (M, P, S and V) antibody, followed by Alexa-555 labeled secondary antibody, and FITC-isolectin B4 (B, E, H and K; N, Q, T and W) the sections were examined under fluorescent microscopy. Merged images are shown in panels C, F, I and L for iNOS and lectin B4 and in panels O, R, U and X for arginase 1 and lectin B4. Nuclei were stained with DAPI (blue) (n=3 mice/group). OIR, oxygen-induced retinopathy; NOR, normal; P, post-natal day.
M1, M2 macrophages contribute to the different steps of retinal neovascularizaion (RNV). RNV at P13 in the retinas of mice with OIR. Retinas were stained with isolectin B4 and CD11c, and at P18 retinas were stained with isolectin B4 and CD206. Flatmounts were examined by laser scanning confocal microscopy. Isolectin B4-positive endothelial tip cells and macrophages are shown in (A–C). Yellow indicates the co-localization of endothelial cells and CD11c-positive cells [M1 macrophages (D–F)] or CD206-positive cells [M2 macrophages (G–I)]. Costaining of CD11c with F4/80 at P13 is shown in (J–L) and CD206 with F4/80 at P18 in (M–O). CD11c-positive cells interact with endothelial tip cells at the vascular front, while CD206-positive cells embrace the emerging vessels and bridge the neighboring vessel sprouts, suggesting a promotive function for tip cell fusion (n=4 mice/group). OIR, oxygen-induced retinopathy; NOR, normal; P, post-natal day.