1. Stem Cells and Regenerative Medicine
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Role of IL-4 in bone marrow driven dysregulated angiogenesis and age-related macular degeneration

  1. Takashi Baba  Is a corresponding author
  2. Dai Miyazaki  Is a corresponding author
  3. Kodai Inata
  4. Ryu Uotani
  5. Hitomi Miyake
  6. Shin-ichi Sasaki
  7. Yumiko Shimizu
  8. Yoshitsugu Inoue
  9. Kazuomi Nakamura
  1. Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Japan
  2. Division of Pathological Biochemistry, Department of Biomedical Sciences, Faculty of Medicine, Tottori University, Japan
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Cite this article as: eLife 2020;9:e54257 doi: 10.7554/eLife.54257

Abstract

Age-associated sterile inflammation can cause dysregulated choroidal neovascularization (CNV) as age-related macular degeneration (AMD). Intraocular fluid screening of 234 AMD patients identified high levels of IL-4. The purpose of this study was to determine the functional role of IL-4 in CNV formation using murine CNV model. Our results indicate that the IL-4/IL-4 receptors (IL4Rs) controlled tube formation and global proangiogenic responses of bone marrow cells. CCR2+ bone marrow cells were recruited to form very early CNV lesions. IL-4 rapidly induces CCL2, which enhances recruitment of CCR2+ bone marrow cells. This in vivo communication, like quorum-sensing, was followed by the induction of IL-4 by the bone marrow cells during the formation of mature CNVs. For CNV development, IL-4 in bone marrow cells are critically required, and IL-4 directly promotes CNV formation mainly by IL-4R. The IL-4/IL-4Rα axis contributes to pathological angiogenesis through communications with bone marrow cells leading to retinal degeneration.

Introduction

Age-related macular degeneration (AMD) is a neurodegenerative disorder which develops in elderly individuals and is a major cause of visual impairments in developed countries. In the early stages of AMD, lipoprotein deposits called drusen accumulate in the subretinal space between the photoreceptors and retinal pigment epithelium (RPE). Drusen are associated with the degeneration of the RPE which then leads to a dysfunction or loss of the photoreceptors. Choroidal (CNVs) develop in the subretinal space, and the CNVs lead to degeneration of the photoreceptor cells, infiltration by inflammatory cells, activation of microglia, and ganglion cell loss (Beck et al., 2016; Copland et al., 2018).

The pathology of AMD is coupled with senescence-associated para-inflammation, which is characterized by the secretion of IL-6, IL-8, CCL2, and CX3CL1 (Sasaki et al., 2010). Of these, CCL2 plays an important role in recruiting bone marrow cells, monocytes, and macrophages to the ocular neovascularizations. In this disease process, the bone marrow plays an important role by supplying new vascular endothelial cells and macrophages to the retina (Gao et al., 2016; Zhou et al., 2017). Thus, the bone marrow plays a key role in the repair of damaged tissues.

The M1 macrophages are functionally classified as pro-inflammatory, and the M2 macrophages are classified as anti-inflammatory, and both types are recruited to damaged tissues. The M2 macrophages are induced by IL-4, and it has been suggested that they have disease-regulating functions as opposed to the M1 macrophages (Zhou et al., 2017).

The concept of IL-4 as a regulatory and neuroprotective cytokine is supported by the findings in other neurodegenerative diseases including Alzheimer’s disease (Kiyota et al., 2010) and Parkinson’s disease (Chitnis and Weiner, 2017; Zhao et al., 2006). Moreover, IL-4 is known to be a potent inhibitor of angiogenesis (Haas et al., 2006; Volpert et al., 1998), and thus may prevent pathological angiogenesis in eyes with AMD.

The purpose of this study was to determine whether bone marrow cells and IL-4 protect the photoreceptors from neurodegeneration, and whether they play regulatory roles in eyes with g AMD. To accomplish this, we first determined the concentration of IL-4 and other inflammatory cytokines in the aqueous humor of the eyes of AMD patients (Sasaki et al., 2012). We then determined whether IL-4 and bone marrow cells play roles in protecting the eye from abnormal angiogenesis. This was done by functional assays and global transcriptional profiling of bone marrow cells derived from endothelial progenitor cells (EPC).

Results

Increased levels of IL-4 in aqueous humor of eyes with AMD and clinical subtypes of AMD

We first examined the levels of IL-4 and related cytokines in the aqueous humor of human eyes with AMD. To accomplish this, aqueous humor was collected from the eyes of 234 patients with clinically-diagnosed AMD and impaired central vision and 104 normal subjects undergoing routine cataract surgery. The mean age of the patients with AMD was 74.1 ± 0.6 years, and it was 74.9 ± 1.0 years for the normal subjects. The results showed that the AMD patients had significantly higher levels of IL-4 in their aqueous than in normal subjects (Table 1, Table 2). In contrast, there was no significant elevation of IL-13.

Table 1
Increase of IL-4 concentration in aqueous humor of eyes with age-related macular degeneration.
Cytokines
(pg/ml)
Control
(n = 104)
age-related macular degeneration
(n = 234)
P value
IL-40.3 ± 0.10.9 ± 0.1p=0.0000
IL-133.8 ± 0.75.2 ± 0.7NS
  1. Two-tailed t test; Mean ± standard error of the means (SEMs).

Table 2
Association of IL-4 concentration in aqueous humor with subtype of age-related macular degeneration.
Relative risk ratioRelative risk ratio
NIL-4
(quintile)
P valueIL-13
(quintile)
P value
Control104----
Typical AMD332.11 ± 0.330.0002.09 ± 0.390.000
Polypoidal choroidal vasculopathy (PCV)781.70 ± 0.170.0001.39 ± 0.150.002
Retinal angiomatous proliferation (RAP)112.46 ± 0.690.0011.48 ± 0.370.11
  1. Multinomial logistic regression analysis after age adjustment; Mean ± standard error of the means (SEMs).

We next examined whether the IL-4 levels were significantly associated with the different subtypes of AMD. The results showed that the level of IL-4 was significantly higher in the three clinical subtypes of AMD, for example, typical AMD, polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). The degree of elevation of IL-4 (quintile) had the highest relative-risk ratio of 2.5 for RAP (p=0.001, logistic regression analysis after age adjustments), followed by 2.1 for typical AMD (p=0.000) and 1.7 for PCV (p=0.000) (Table 2).

IL-4 induction in murine experimental choroidal neovascularization

The level of IL-4 expression was evaluated in a murine laser-induced CNV model to determine whether IL-4 is associated with subretinal neovascularization. First, we assessed whether the mRNA of IL-4 was induced in the CNV lesions. Our results showed that the mRNA of IL-4 was elevated and peaked at 3 days after the laser exposure and then decreased (Figure 1a). The mRNA of IL-4Rα also had similar induction kinetics. The mRNA of CCR2, a myeloid cell recruitment marker, was elevated, and the elevation preceded the mRNA of IL-4 induction by peaking at 1 day. The mRNA of CD11b gradually increased after the exposure.

Figure 1 with 2 supplements see all
Induction of Il4 and Ccl2 in laser-exposed retinas and choroids of mice.

(a) Induction kinetics of the mRNAs of IL-4, IL-4Rα, CCR2, and CD11b. The induction of Ccl2 peaked at 1 day after the exposure followed by the peak induction of Il4 and Il4ra. (n = 4–14 eyes/group). (b) Kinetics of IL-4-expressing cells by immunohistochemical analyses. IL-4 expressing cells (yellow) accumulated along the margin of laser treated area at 3 days and then move inwards. The IL-4-expressing cells (yellow) were mainly CD11b positive (green) in laser treated areas at 3 days. (c) Localization of CCL2 expression in retinal tissue by immunohistochemistry. CCL2 induction is observed at 1 day after treatment, and CCL2-positive cells (red) are colocalized with the iba1-positive microglial cells (green). The nuclei were stained by TO-PRO-3 iodide (blue). Scale 10 μm. *p<0.05, **p<0.01. ANOVA with post hoc test and linear mixed-effects regression analysis.

To examine the spatial expression of IL-4, we examined the CNV lesions by immunohistochemistry. Three day after the laser exposure, the IL-4-expressing cells were observed along the margins of the lesions and were present more centrally on day 7 (Figure 1b). The IL-4-expressing cells were largely CD11b+, and they were considered to be myeloid- or macrophage-lineage cells. The kinetics of Iba1-, CCL2-, and CD11b-positive cells after laser exposure was consistent with that of the mRNA induction (Figure 1—figure supplement 1).

We then examined which type of lineage cells can produce the CCL2 as an early recruitment signal for myeloid cells. Our results showed that the CCL2 was mainly associated with iba1-positive retinal microglial cells (Figure 1c). The microglial cells migrated to surface of the CNV (Figure 1—video 1). This indicated that they were the initial stimulators. Thus, IL-4 expressions followed by myeloid cell activation were early events acting at the inductive phase of the CNV formation.

Requirement of IL-4 in inductive phase of choroidal neovascularization

The kinetic observations suggested that IL-4 appeared in the inductive phase of CNV formation. To determine whether IL-4 had inhibitory or stimulatory effects on the pathological angiogenesis, mice were laser-treated to induce the formation of CNVs, and IL-4 was injected intravenously on day 0 and day 3 during the inductive phase. The IL-4 significantly exacerbated the CNV formation in a dose dependent way (Figure 2a).

Figure 2 with 2 supplements see all
Requirements of IL-4/IL-4Rα in the inductive phase of choroidal neovascularization (CNV).

(a) Effect of systemic administration of recombinant murine IL-4 (rIL-4) or recombinant IL-13 (rIL-13) in the inductive phase in a CNV model. (n = 7–19 eyes/group). (b) Inhibitory effect of systemic administration of anti-IL-4 antibody in the inductive phase of CNV formation. (n = 7–20 eyes/group). (c) Impaired CNV development in Il4 deficient mice. CNV development is significantly impaired in Il4-/- mice compared to wild type. (n = 8–9 eyes/group). (d) Impaired CNV development by IL-4 receptors deficiency. CNV development is significantly impaired in Il4ra-/- and Il4ra+/- mice compared to wild type. This impairment is more marked in the homozygotes. CNV development is not impaired for Il13ra1-/- mice. (n = 7–17 eyes/group) (e) Bone marrow chimeric mice reconstituted with GFP transgenic bone marrow cells that were exposed to laser to induce CNVs. The CNV lesions after 14 days were analyzed for lineage cell markers by immunohistochemistry. CNVs are formed as clusters of isolectin IB4-positive vascular endothelial cells (red). Bone marrow-derived cells (green) were co-localized with isolectin-positive vascular endothelial cells. IL-4 positive cells (yellow) are distributed at the margins of the CNVs and precisely match the location of the bone marrow-derived cells (green). IL-4Rα-positive cells (cyan) partly overlapped the bone marrow-derived cells, and precisely match the location of the vascular endothelial cells in the CNV lesion. *p<0.005, **p<0.001, ***p<0.0005. Nested ANOVA with post hoc test. Scale 10 μm.

It is known that IL-4 generally signals through IL-4Rα which is also a ligand of IL-13. Therefore, we also tested whether IL-13 had any stimulatory effect on CNV formation. Our results showed that a systemic administration of IL-13 in the inductive phase had no significant effect on CNV formation.

To examine the role of IL-4 in the inductive phase of CNV development in more detail, laser-treated mice were injected intravenously with an anti-IL-4 antibody on day 0 and day 3 to try to inhibit the expression of IL-4 (Figure 2b). Consistent with the effects of IL-4 administration, an IL-4 blockade significantly reduced the size of the CNV. In contrast, a block of IL-13 by an antibody injection had no significant effect on the CNV formation.

We next evaluated the contribution of IL-4 to the formation of CNVs using Il4-deficient mice (Figure 2c). Consistent with the outcomes of the anti-IL-4 antibody exposure, Il4-deficient mice were significantly impaired in CNV formation which supports our finding that IL-4 is involved in the inductive phase of CNV. To confirm that IL-4 contributed to the CNV formation with canonical signaling by IL-4Rα in more detail, Il4ra-deficient mice were tested for CNV formation. Il4ra-deficiency impaired the CNV formation significantly (Figure 2d).

Next, we evaluated role of the IL-13 receptors as alternative receptors of IL-4. IL-13R is composed of IL-4Rα and IL-13Rα1. When IL-13Rα1-deficient mice were tested for CNV formation, no significant impairment was observed.

Incorporation of circulating angiogenic cells and bone marrow-derived cells into CNV lesion

It has been shown that bone marrow cells are recruited into the CNV lesions during the inductive phase of CNV formation (Gao et al., 2016). Therefore, we examined the roles played by bone marrow-derived cells in CNV formation using bone marrow chimeric mice. Bone marrow chimeric mice were generated by reconstitution with a GFP transgenic mice-bone marrow, and they were evaluated for laser-induced CNV formation (Figure 2e).

Recruitment of GFP+ bone marrow-derived cells (green) peaked at 3 days after irradiation. Thus, bone marrow-derived cell recruitment also contributed to the inductive phase process. Bone marrow-derived cells in this phase were CD11b+ lineage, and they were positive for CCL2. These bone marrow cells did not express iba1 and were morphologically distinct from microglial cells. This suggested that these cells will amplify the recruitment of CCR2+ lineage cells (Figure 2—figure supplement 1). Seven days after irradiation, bone marrow-derived cells were incorporated into structures formed by CD31+ endothelial cells (Figure 2—video 1).

Two weeks after laser exposure, CNVs were formed as clusters of isolectin-positive vascular endothelial cells (red; Figure 2e). In the CNV lesion, bone marrow-derived cells (green) were localized to isolectin-positive vascular endothelial cells and CD31+ endothelial cells. The co-localization of the marrow-derived cells with CD31+ endothelial cells indicated that the bone marrow-derived cells may be able to differentiate into endothelial cells. IL-4-positive cells (yellow) were distributed at the margins of the CNVs and precisely matched the bone marrow-derived cells (green). The IL-4Rα-positive cells (cyan) in the CNV, partly overlapped the bone marrow-derived cells.

Profiles of angiogenic mRNAs of endothelial progenitor cells

These findings suggested that the IL-4 from bone marrow-derived vascular endothelial cells played disease-promoting roles in CNV formation, and they were not anti-angiogenic. To confirm this, we examined how IL-4 affected the differentiation of vascular endothelial progenitor cells (EPC) from bone marrow cells. To do this, bone marrow cells were cultured for differentiation to late EPCs for 2 weeks and exposed to IL-4. We then screened for the induction of angiogenesis-related mRNAs, including Ccl2, Vegf, VEGF receptors (Kdr, Flt4), angiopoietin-1 (Angpt1), endothelin receptor (Ednrb), thrombin receptors (F2r, F2rl1), P-selectin (Selp), and vascular endothelial cadherin (Cdh5) (Figure 3a, Figure 3—figure supplement 1, Figure 3—figure supplement 2). Of these, Ccl2 and Flt1 were significantly induced in a dose dependent manner after IL-4 exposure. Kdr and Flt4 were not induced.

Figure 3 with 2 supplements see all
Induction of Ccl2 and Flt1 in bone marrow-derived endothelial progenitor cells (EPC) and retinal vascular endothelial cells by IL-4.

(a) Induction of Ccl2 and Flt1 in bone marrow-derived endothelial progenitor cells by murine IL-4. IL-4 stimulated bone marrow-derived EPCs induced Ccl2 and Flt1 in a dose dependent manner. This induction is abolished by anti-IL-4Rα antibody. (n = 5/group). (b) Induction of Ccl2 and Flt1 in retinal vascular endothelial cells by IL-4. IL-4 stimulated vascular endothelial cells to express Ccl2 and Flt1 in a dose dependent manner. (n = 5/group). (c) Inhibition of IL-4/IL-13-mediated Ccl2 and Flt1 induction in EPCs by Il4ra deficiency (n = 6/group). IL-4 and IL-13 exposure induced Ccl2 and Flt1 in EPCs. This induction is not present in the EPCs of Il4ra-/- mice. (d) Inhibition of IL-13-mediated Ccl2 and Flt1 induction in EPCs by Il13ra1 deficiency (n = 6/group). The IL-13-induced the expression of Ccl2 and Flt1 is significantly reduced in Il13ra1-/- EPCs of mice. IL-4-induced Ccl2 and Flt1 mRNA is not affected in Il13ra1-/- EPCs of mice. (e) EPCs of Il4 -/- mice respond to induce Ccl2/Flt1 mRNA by IL-4/IL-13 exposure. (n = 6/group). *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, *****p<0.0005. ANOVA with post hoc test.

We also examined whether mature vascular endothelial cells can induce comparable transcriptional responses. When retinal microvascular cells were tested for their effect on IL-4 by real-time reverse transcription PCR (RT-PCR), IL-4 was found to stimulate the induction of Ccl2/Flt1 (Figure 3b).

An upregulation of the translation of CCL2 and VEGFR-1 in EPCs was confirmed by ELISA. IL-4-exposed EPCs had a significant increase in the secretion of CCL2 (p=0.000) and VEGFR-1 (p=0.000) after 24 hr exposure to IL-4 (Figure 3—figure supplement 2).

We next examined how IL-4Rα and IL-13Rα1 contributed to the induction of Ccl2 and Flt1 in EPCs by IL-4, IL-13, and VEGF. Both IL-4 and IL-13 significantly induced Ccl2 and Flt1 in EPCs (Figure 3). However, VEGF did not significantly induce Ccl2 and Flt1. When IL-4Rα was inhibited by an anti-IL-4Rα antibody, IL-4 failed to stimulate the EPCs from inducing the expression of Ccl2 and Flt1 (Figure 3a).

To determine the contribution of IL-4Rα to CNV formation, we examined the effect of Il4ra deficiency. EPCs from Il4ra-deficient mice did not induce Ccl2 and Flt1 in response to IL-4 or IL-13 (Figure 3c).

We also examined the contribution of IL-13Rα1 to the formation of CNVs. When Il13ra1-deficient EPC mice were stimulated by IL-4, Ccl2 and Flt1 were still induced (Figure 3d) indicating that IL-13Rα1 was not necessary for IL-4 stimulation. When Il13ra1-deficient EPC mice were stimulated with IL-13, Ccl2 and Flt1 were not induced. Collectively, these findings indicate that IL-4Rα is the major receptor for IL-4 to induce the expression of CCL2 and VEGFR-1, and the IL-13Rα1 can substitute for their induction mainly through IL-13.

We next examined whether the Ccl2 and Flt1 induction by IL-4 required intrinsic IL-4-mediated differentiation. The results indicated that the EPCs of Il4-deificient mice still induced IL-4 and the IL-13-mediated Ccl2 and Flt1 induction (Figure 3e).

Tube formation by endothelial progenitor cells and vascular endothelial cells stimulated by IL-4

To confirm a vasculogenic property of IL-4, mature vascular endothelial cells were assessed for tube formation on Matrigel-coated plates (Figure 4a). When murine retinal microvascular endothelial cells were tested for tube formation by IL-4 or VEGF, both stimulated significant tube formation (Figure 4a). Anti-IL-4 and IL-4Rα antibodies abolished the IL-4-induced tube formation.

Figure 4 with 1 supplement see all
IL-4-induced tube formation in endothelial progenitor cells (EPCs) and retinal vascular endothelial cells.

(a) IL-4-induced tube formation of retinal vascular endothelial cells. Human and murine IL-4 exposure (10 ng/ml) significantly stimulated tube formation of human and murine retinal microvascular endothelial cells in vitro, respectively. Anti-IL-4 or IL-4Rα antibodies abolished the IL-4 induced-tube formation. VEGF exposure (10 ng/ml) also stimulated tube formation. (n = 7–10/group). (b) IL-4-induced tube formation in bone marrow-derived EPCs. IL-4 exposure (10 ng/ml) significantly stimulated tube formation of EPCs. The IL-4-induced tube formation was significantly reduced in EPCs from Il4ra-/- mice but was not affected by inhibition of VEGF receptor tyrosine kinase (ZM 306416) or VEGFR-2 (ZM 323881) (n = 13–45/group). (c) Requirements of IL-4 for tube formation response of EPCs. IL-4 (10 ng/ml) and IL-13 (10 ng/ml) induced tube formation of bone marrow-derived EPCs. These actions were abolished in the EPCs from Il4-/- bone marrow cells. EPCs from Il4ra-/- mice did not respond to IL-4, however they responded to IL-13 by tube formation. EPCs from Il13ra1-/- mice did not respond to IL-13 but responded to IL-4 by tube formation. (n = 35–72/group). *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, *****p<0.0005. ANOVA with post hoc test and linear mixed-effects regression analysis.

We next confirmed the effects of IL-4 using human retinal cells (Figure 4a). IL-4 exposure stimulated tube formation by human retinal vascular endothelial cells, and anti-IL-4 and IL-4Rα antibodies blocked this effect.

Next, EPCs were examined for IL-4-mediated tube formation. Murine bone marrow cells were cultured under conditions appropriate for the differentiation of EPCs and were tested for tube formation. For the wild type bone marrow cells, IL-4 significantly stimulated tube formation by the EPCs (Figure 4bc). This IL-4-induced tube formation was blocked when the bone marrow cells were deficient of Il4ra. However, the inhibition of the VEGF receptor tyrosine kinase or VEGF receptor 2 did not significantly inhibit tube formation (Figure 4b). This indicated that this IL-4 effect was independent of canonical VEGF signaling.

The IL-4-induced tube formation was blocked when the cells were deficient of Il4 but not by Il13ra1 deficiency (Figure 4c). IL-13 also stimulated tube formation by bone marrow-derived EPCs. This response was abolished when the bone marrow cells were deficient in Il4 and Il13ra1. These findings further confirmed the roles of IL-4 as a differentiation and vasculogenic factor which signaled mainly through IL-4Rα. The results also indicated that IL-13Rα1 could serve as an alternative receptor.

IL-4Rα-dependent transcriptional networks of tube forming endothelial progenitor cells from bone marrow cells

The results suggested that IL-4 also served as a differentiation factor for cells of endothelial lineage. To characterize the vasculogenic roles of IL-4 for bone marrow cells, the mRNA of tube-forming EPCs were extracted and examined by network analysis. Analysis of the functions of IL-4-stimulated EPCs indicated significant association with the homing of the cells (Z score = 2.798, p=8.3 × 10−5), angiogenesis (Z score = 2.781, p=1.8 × 10−4), activation of macrophages (Z score = 2.731, p=1.0 × 10−5), and recruitment of myeloid cells (Z score = 2.606, p=2.8 × 10−10) (Figure 4—figure supplement 1).

Requirements of IL-4 in bone marrow-mediated choroidal neovascularization

The results suggested that the IL-4 and IL-4Rα interactions contributed to the pathological angiogenesis of bone marrow-derived EPCs. To examine how bone marrow-derived cells contributed to IL-4-stimulated CNV formation, bone marrow chimeric mice were constructed on a wild type background of Il4- or Il4ra-deficient mice (Figure 5).

Figure 5 with 1 supplement see all
Role of IL-4/IL-4Rα on bone marrow-mediated choroidal neovascularization in bone marrow chimeric mice.

(a) Requirement of IL-4 in bone marrow for choroidal neovascularization (CNV). Bone marrow chimeric mice were constructed on backgrounds of wild type, Il4-/-, or Il4ra-/- mice by transfer of wild type Il4-/-, or Il4ra-/- bone marrow cells. Il4-/- mice with Il4-/- bone marrow are most significantly impaired in CNV formation. This impairment of CNV formation is restored when bone marrow reconstituted with wild type bone marrow. Il4ra-/- mice with Il4ra-/- bone marrow cells are impaired in CNV formation. This impairment is partially restored when reconstituted with wild type bone marrow cells. (n = 4–12 eyes/group). Six of 10 IL-4-/- bone marrow chimeric mice in each group did not survive through procedures and/or were euthanized. *p<0.05, **p<0.005, ***p<0.0005. Nested ANOVA with post hoc test. (b) Immunohistochemical analysis of CNV of the bone marrow chimeric mice on Il4-/- background 14 days after laser treatment. Endothelial cells in the CNV were labeled with isolectin IB4 (red). In the Il4-/- mice reconstituted with wild type bone marrow, the CNV lesion contained IL-4 (yellow) secreting bone marrow cells (green). The IL-4Rα-positive cells (cyan) partly overlapped with bone marrow-derived cells. Scale 10 μm.

The Il4-deficient mice with Il4-/- bone marrow developed the smallest size CNVs of all the chimeric mice. This impaired CNV formation was completely restored by the transplantation of bone marrow cells from wild type mice. This indicated the crucial role played by IL-4 in bone marrow cells. In contrast, wild type mice with Il4-/- bone marrow were still impaired for CNV formation which indicated that the host resident cell-derived IL-4 is limited in this activity (Figure 5a).

Consistent with the results shown in Figure 2, Il4ra-deficient mice with Il4ra-/- bone marrow cells were impaired in the formation of CNVs. This impairment was restored by the transplantation of wild type bone marrow cells (Figure 5a).

We next examined whether IL-4 secreting cells were recruited from bone marrow or were derived from the host. To do this, we conducted immunohistochemical analyses of Il4-deficient mice reconstituted with wild type bone marrow cells. The results showed IL-4- and IL-4Rα-positive cells were present in the CNV lesion (Figure 5b; Figure 5—figure supplement 1). The IL-4-positive cells precisely matched the bone marrow-derived cells (green). In contrast, the IL-4Rα positivity partly overlapped with that of the bone marrow-derived cells. This indicated that bone marrow-derived cells are the major producer of IL-4, and bone marrow-derived cells and resident cells in the CNV via IL-4Rα recognized their signals.

To summarize, interactions of IL-4/IL-4Rα interactions with bone marrow cells are required for pathological CNV formation.

Discussion

Our results showed that IL-4 played a crucial role in the pathogenesis of CNVs by directing the migration and activating the angiogenic bone marrow cells. IL-4 is the canonical Th2 cytokine and is secreted by an array of inflammatory cells including macrophages, monocytes, and activated retinal pigment epithelial cells (Leung et al., 2009). IL-4 is also recognized as a neuroprotective cytokine, and its action is not limited to the retina (Adão-Novaes et al., 2009). In axotomized retinas, the retinal ganglion cells are severely damaged by nitric oxide synthesis by activated glial cells. IL-4 significantly increases the survival of retinal ganglion cells, and prevents neurodegeneration caused by glial cell activation (Koeberle et al., 2004). In the thapsigargin-induced rod photoreceptor cell death model, IL-4 can completely block the death of the photoreceptors (Adão-Novaes et al., 2009). During the differentiation of the retina, IL-4 modulates the proliferation of the retinal cells and promotes photoreceptor differentiation (da Silva et al., 2008). In addition, a number of studies have shown that IL-4 can inhibit the death of photoreceptors and RGCs.

IL-4 is a multifaceted cytokine and is known to have anti-angiogenic capabilities. IL-4 inhibits tumor growth by inhibiting angiogenesis (Volpert et al., 1998) and also blocks corneal neovascularization induced by basic fibroblast growth factor. Thus, IL-4 can function as an anti-inflammatory cytokine and prevent neuronal death and angiogenesis. However, such properties of IL-4 appear to be context dependent.

We found that the IL-4 level was significantly elevated in the aqueous humor of patients with AMD (Table 1, Table 2; Sasaki et al., 2012). Together with this, our analyses of the bone marrow cells and chimeric mice supports the idea that elevations of IL-4- and IL-4 receptor-bearing cells are associated with the development of abnormal vessels in the lesions of eyes with AMD.

Although the IL-4/IL-4Rα axis appears neuroprotective in the retina, retinal injury requires a recruitment or activation of cells with regenerative properties for its repair. The bone marrow cells are a major supplier of mesenchymal stem cells and hematopoietic stem cells. However, abnormally activated bone marrow cells by IL-4 in the retina promote pathological angiogenic responses.

To explain the dysregulated repair process after organ damage, the concept of cell level and organ level quorum sensing has been recently proposed (Antonioli et al., 2018). Quorum sensing was originally proposed as a phenomenon of bacterial cells, and it was described as a mechanism that senses the environment and integrity of a population of cells. Hair follicle injury sensed by a macrophage-mediated circuit via CCL2 is a well-known example of quorum sensing at the organ level (Chen et al., 2015; Feng et al., 2017). The quorum sensing circuit mediated by microglia-derived CCL2 also appears to operate in new vessel formation in the retina together with a late IL-4 modulator.

We observed the presence of CD11b in the CNV lesions at 12 hr which would indicate that monocyte/macrophage cells had arrived soon after the beginning of the CNV. In contrast, the induction of IL-4 and IL-4 receptors was delayed and peaked at 3 days (Figure 1). CCL2 recruits circulating mononuclear cells from the bone marrow, and the retinal pigment epithelial cells and microglial cells are the major sources of the CCL2 cells in the retina (Feng et al., 2017). Thus, the monocyte/macrophage recruiting signal, including CCL2, appears to be the first signal in the formation of CNVs.

It was reported that CCL2 is involved in the formation of retinal neovascularization (Sennlaub et al., 2013; Yoshida et al., 2003). However, the CCL2/CCR2 signals recruit a heterogeneous collection of monocyte/macrophage lineage cells and presumably do not determine their fate (Grochot-Przeczek et al., 2013; Pearson, 2010).

Bone marrow-derived cells and macrophages are critical contributors to retinal and choroidal neovascularization (Gao et al., 2016; Zhou et al., 2017). Higher levels of M1 than M2 type mRNAs were observed in advanced stage AMD patients (Cao et al., 2011). The M1 macrophages counteract the M2 type by the secretion of interferon-γ. Based on this, the M2 macrophages were considered to play a regulatory role in CNV formation. Consistent with this, Wu et al showed that the M2 type cytokine, IL-4, and conditioned macrophages become the regulatory phenotype to suppress the disease processes (Wu et al., 2015). However, the M1 types of IFNAR1+ macrophages have been reported to be protective in laser-induced CNV (Lückoff et al., 2016). Thus, the M1/M2 paradigm does not clearly explain how abnormal vessels are formed in AMD lesions or the CNVs of the model mice.

Wu et al also showed a contradictory role of IL-4 for CNV formation (Wu et al., 2015). This was shown using vitreous injection of IL-4 at very high concentration (600 ng/ml). This may cause toxic damage to endothelial cells or recruited cells which may not reflect physiological role of IL-4.

In the retina, vascular repair and neovascularization are performed largely by circulating EPCs because mature vascular endothelial cells for proliferation are limited (Caballero et al., 2007; Grant et al., 2002). EPCs are present in the bone marrow or are peripheral blood mononuclear cells (Rohde et al., 2006; Schatteman and Awad, 2003; Schmeisser et al., 2001), and they can differentiate into endothelial cells as late EPCs and be incorporated into the vasculature system. Alternatively, bone marrow cells will also differentiate into non-endothelial cell lineage and serve as providers of CNV-forming signals.

In the inductive phase of CNVs, a mobilization of circulating angiogenic cells and monocyte/macrophage lineage cells begins by the early recruitment of IL-4 secreting CD11b+ bone marrow cells (Figure 1). As a late phase phenomenon, the IL-4R+ bone marrow-derived cells are incorporated into the CNV presumably as late EPCs or non-endothelial lineage cells, together with the resident cell-derived endothelial cells. We propose that the bone marrow-derived cells contribute to both phases using IL-4 as the fate determinant.

We also noted that the IL-4/IL-4Rα axis is involved in pathologic angiogenesis. For example, IL-4 induces proinflammatory phenotypes and causes vascular leakage or increased turnover of endothelial cells (Kotowicz et al., 2004; Lee et al., 2010). IL-4 stimulates human umbilical vascular endothelial cells (HUVEC) cells to induce proinflammatory cytokines including CCL2, VCAM-1, and IL-6, as a signature of global transcriptional responses (Lee et al., 2004). These responses appear to be a general characteristics of vascular endothelial cells including coronary arterial endothelial cells (Skaria et al., 2016).

To identify an endothelial lineage, we used CD31 or isolectin staining because CD31 is highly expressed on endothelial cells and is commonly used as an endothelial cell marker. However, CD31 can also be expressed on other lineage cells including T cells, B cells, dendritic cells (DCs; Clement et al., 2014), neutrophils, monocytes, and macrophage (Merchand-Reyes et al., 2019).

Our data support the idea that bone marrow-derived cells may be able to differentiate into endothelial cells in the CNV lesions. However, whether the endothelial differentiation is complete or bone marrow cells serve as immature or of different lineage was not definitively determined. Importantly, bone marrow-derived cells do play pivotal roles in the CNV formation.

In conclusion, damages of the retina and choroidal tissue release signals to the bone marrow to repair the vascular damage. This signal induces a recruitment of the bone marrow-derived cells for differentiation into or establishment of new vessels. Calling and/or fate determining signals are governed by IL-4. IL-4 may serve as a therapeutic target to treat this visual disorder.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (M. musculus)C57BL/6J (wt)PMID:15729571RRID:IMSR_JAX:000664
Genetic reagent (M. musculus)C57BL/6-Tg(CAG-EGFP)PMID:9175875RRID:IMSR_JAX:003291
Genetic reagent (M. musculus)C57BL/6-Il4tm1Nnt/JPMID:8906833RRID:IMSR_JAX:002518
Genetic reagent (M. musculus)BALB/c-Il4ratm1Sz/JPMID:9380721RRID:IMSR_JAX:003514
Genetic reagent (M. musculus) Il13ra1tm1TwyPMID:18066066RRID:MGI:3772446Dr. Marc E Rothenberg, Cincinnati Children's Hospital Medical Center
University of Cincinnati College of Medicine
Cell line (M. musculus)C57BL/6 Mouse Primary Retinal Microvascular Endothelial CellsCell BiologicsC57-6065
Cell line (H. sapiens)Primary Human Retinal Microvascular Endothelial CellsCell SystemsACBRI 181
Antibodyanti IL-4 (rat monoclonal)BiolegendCat. #: 504108, RRID:AB_315322IHC(1:200)
Antibodyanti IL-4 (rabbit polyclonal)abcamCat. #: ab9622, RRID:AB_308736IHC(1:200)
Antibodyanti Phospho-Tyr497 IL-4R/CD124 (rabbit polyclonal)Assay Biotechnology CompanyCat. #: A1064, RRID:AB_10683571IHC(1:200)
anti CD124 (rat monoclonal)BD PharmingenCat. #: 552288, RRID:AB_3943560.1–10 ng/ mL TVI
Antibodyanti IL-13 receptor alpha 1 (rabbit polyclonal)abcamCat. #: ab-79277, RRID:AB_1640587IHC(1:200)
anti IL13 antibody (rabbit polyclonal)GeneTex, IncCat. #: GTX59763,0.1–10 ng/ mL TVI
Antibodyanti CD11b (rat monoclonal)eBioscienceCat. #: 14-0112-82, RRID:AB_467108IHC(1:200)
Antibodyanti CD11b (rat monoclonal) Alexa Fluor 594BiolegendCat. #: 101254, RRID:AB_2563231IHC(1:100)
AntibodyCD11b (M1/70) (rat monoclonal) FITCeBioscienceCat. #: 11-0112-41, RRID:AB_11042156IHC(1:100)
Antibodyanti CCR2 (rabbit polyclonal) DyLight 550Novus BiologicalsCat. #: NBP1-48338RIHC(1:100)
Antibodyanti MCP-1 (hamster monoclonal)BiolegendCat. #: 505906, RRID:AB_2071552IHC(1:200)
Antibodyanti Iba1 (rabbit polyclonal)FUJIFILM Wako Pure Chemical CorporationCat. # 019–19741, RRID:AB_839504IHC(1:200)
Antibodyanti CD31 (rabbit polyclomal)abcamCat. #: ab28364, RRID:AB_726362IHC(1:200)
Antibodyanti-mouse CD31(rat monoclonal) Alexa Fluor 647BiolegendCat. #: 102516, RRID:AB_2161029IHC(1:100)
Antibodyanti rat IgG (goat polyclonal) Brilliant Violet 421BiolegendCat. #: 405414, RRID:AB_10900808IHC(1:100)
Antibodyanti rabbit IgG (goat polyclonal) DyLight 488Vector LaboratoriesCat. #: DI-1488, RRID:AB_2336402IHC(1:100)
Antibodyanti rabbit IgG (donkey polyclonal) Alexa Fluor 555BiolegendCat. #: 406412, RRID:AB_2563181IHC(1:100)
Antibodyanti rabbit IgG (goat polyclonal) HiLyte Fluor 555AnaSpecCat. #: AS-61056–05 H555IHC(1:100)
Antibodyanti hamster IgG (goat polyclonal) DyLight 594BiolegendCat. #: 405504, RRID:AB_1575119
Antibodyanti rabbit IgG (goat polyclonal) PESanta Cruz BiotechnologyCat. #: sc-3739, RRID:AB_649004IHC(1:100)
Antibodyanti rabbit IgG (donkey polyclonal) Alexa Fluor 647abcamCat. #: ab150075, RRID:AB_2752244IHC(1:100)
Antibodyanti rabbit IgG (donkey polyclonal) DyLight 649BiolegendCat. #: 406406, RRID:AB_1575135IHC(1:100)
Antibodyanti mouse IgG2A (rat monoclonal)R and DCat. #: mab006, RRID:AB_3573490.1–10 ng/ mL TVI
Antibodyanti mouse Fc gamma RII/RIII (CD32/CD16)(goat polyclonal)R and DCat. #: AF1460-SP, Accession # P08101IHC
(0.2 μg/mL)
Peptide, recombinant proteinrecombinant murine IL-4R and DCat. #: 404 ML
Peptide, recombinant proteinrecombinant human IL-4PeprotecCat. #: AF-200–04
Peptide, recombinant proteinrecombinant murine IL-13PeprotecCat. #: 210–13
Chemical compound, drugZM306416 hydrochlorideabcamCat. #: ab144576
Chemical compound, drugZM323881 hydrochlorideR and DCat. #: 2475/1
Chemical compound, drugbovine serum albuminSigma-AldrichCat. #: A2153
Chemical compound, drugfetal bovine serumSigma-AldrichCat. #: 12103C
Chemical compound, drugMedetomidineChemscene LLCCat. #: 86347-14-0
Chemical compound, drugButorphanol tartrateFUJIFILM Wako Pure Chemical CorporationCat. #: 58786-99-5
Chemical compound, drugMidazolamFUJIFILM Wako Pure Chemical CorporationCat. #: 59467-70-8
Chemical compound, drugTropicamide, Phenylephrine HydrochlorideSanten Pharmaceuitical Co., Ltd.Cat. #: 1319810Q1053
Chemical compound, drugHydroxy methyl celluloseSENJU Pharmaceutical Co.,LtdCat.#: 131980AQ1038
Chemical compound, drugRNAlater solutionAmbionCat. #: AM7021
Chemical compound, drugTriton X-100Sigma-AldrichCat. #: X100 100ml
Chemical compound, drugTween 20Sigma-AldrichCat. #: P1379-100ml
Chemical compound, drugParaformadehydeElectron Microscopy ScienceCat. #: 15710
Commercial assay or kitISOLECTIN B4 FluoresceinVector LaboratoriesCat. #: FL-1201, RRID:AB_2314663IHC(1:100)
Commercial assay or kitISOLECTIN B4 DyLight 594Vector LaboratoriesCat. #: FL-1207IHC(1:100)
Commercial assay or kitDAPIRoche DiagnosticsCat. #: 10 236 276 0011 μg/ml
Commercial assay or kitTO-PRO-3 iodideMolecular Probes, IncCat. #: T-3605IHC(1:100)
Commercial assay or kitVECTASHIELDAntifade Mounting MediumVector
Laboratories
Cat. #: H-1000, RRID:AB_2336789
Commercial assay or kitFluorescence Mounting MediumDAKOCat. #: 15710
Commercial assay or kitSurePrint G3 Mouse GE 8 × 60K MicroarrayAgilent TechnologiesCat. #: AGLMO002
Commercial assay or kitQuantiTect Reverse Transcription KitQiagenCat. #: 205311
Commercial assay or kitQuantiTect SYBR Green PCR kitQiagenCat. #: 204143
Commercial assay or kitELISA kitsThermoFisher ScientificCat. #: BMS6005, EMFLT1
Commercial assay or kitPKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane LabelingSigma-AldrichCat. #: PKH26GL-1KT
Commercial assay or kitRNeasy Mini KitQiagenCat. #: 74104
Commercial assay or kitDMEM:F12ThermoFisher ScientificCat. #: 11330057
Commercial assay or kitrecombinant human GMCSFPeprotecCat. #: AF-300–03
Commercial assay or kitrecombinant murine GMCSFPeprotecCat. #: 315–03
Software, algorithmIngenuity Pathway Analysis, March 2020QiagenRRID:SCR_008653
Software, algorithmAdobe Photoshop CS5, Version 12.0.5AdobeRRID:SCR_014199
Software, algorithmSTATA 16.1StataCorp LLCRRID:SCR_012763
Software, algorithmGeneSpring SoftwareAgilent TechnologiesRRID:SCR_009196

Patient selection and measurement of aqueous humor IL-4

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The diagnosis of AMD and subtypes of AMD including PCV and RAP was made by the clinical characteristics. The presence of a CNV or retinal angiomatous proliferation (RAP) was determined by fluorescein angiography, indocyanine green angiography, and spectral domain optical coherence tomography (SD-OCT).

The inclusion criteria were the presence of active CNVs or RAP lesions determined by the angiographic images showing macular edema or subfoveal hemorrhages. Eyes with laser photocoagulation, photodynamic therapy, or intraocular surgery within the past 3 months were excluded. For the control groups, aqueous humor was collected from normal patients who were undergoing routine cataract surgery.

The levels of IL-4 and IL-13 in the aqueous humor samples were measured by commercial ELISA kits as described in detail (Chono et al., 2018; Sasaki et al., 2012).

Animals

Il4-deficient mice, C57BL/6-Il4tm1Nnt/J, Il4ra-deficient mice, and BALB/c-Il4ratm1Sz/J, were obtained from The Jackson Laboratory (Bar Harbor, ME). The Il4ra-deficient mice were backcrossed with C57BL/6 for 9 generations. The Il13ra1-deficient mice were obtained from the Regeneron Pharmaceuticals (Tarrytown, NY). Green fluorescent protein (GFP) transgenic C57BL/6 and wild type C57BL/6 mice were purchased from Japan SLC Inc (Shizuoka, Japan).

Induction of choroidal neovascularization

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Choroidal neovascularization (CNV) was induced by laser irradiation of the retina of mice, an established model for choroidal or retinal neovascular formation. This model has many characteristics of age-related macular degeneration. Mice were anesthetized and one eye was exposed to argon laser irradiation of 150 mW for 0.10 s. Three laser spots were created in each eye. The spot size was approximately 50 μm, and it was delivered with the Novus 2000 argon laser system (Coherent, Santa Clara, CA).

To analyze the CNVs, the laser-treated eyes were enucleated from euthanized mice 14 days after the photocoagulation. Choroidal sheets were isolated from the eyes and fixed in 4% paraformaldehyde at 4° C for 1 min. The choroidal sheets were stained with FITC or DyLight 594 conjugated Isolectin IB4 (Vector Laboratories, Peterborough, UK) and flat-mounted. The stained flat mounts sections were examined and photographed with a fluorescence stereo microscope (MZ-III, Leica Microsystems, Wetzlar, Germany). The isolectin IB4 reactive areas were analyzed as the CNV area by masked investigators to measure the CNV size.

Immunohistochemistry of choroidal neovascularization

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For the immunohistochemical analyses of the CNVs, isolated choroidal sheets with or without the retina were fixed in 4% paraformaldehyde and incubated with isolectin IB4 and primary antibodies including anti-IL-4 (11B11, Biolegend, San Diego, CA), anti-IL-4Rα (mIL4R-M1, BD Biosciences, Franklin Lakes, NJ), anti-Iba1 (FUJIFILM, Tokyo, Japan), anti-IL-13R (ab-79277, abcam, Cambridge, UK), anti-CD11b (M1/70, eBioscience, San Diego, CA), anti-CCL2 (2H5, Biolegend), anti-CCR2 (NBP1-48338R, Novus Biologicals, Centennial, CO), or anti-CD31 (390, Biolegend) with anti-Fc RII/RIII blocking antibody (R and D Systems, McKinley Place, NE) overnight at 4° C. Then, the choroidal sheets were rinsed and incubated with the secondary antibodies labeled with either Brilliant Violet 421, DyLight 488, Alexa Fluor 555, PE, Alexa Fluor 647, or DyLight 649, or control antibodies. DAPI and TO-PRO-3 iodide (T-3605, Molecular Probes, Eugene, OR) were used for nuclear staining. A confocal microscope (LSM730, Carl Zeiss, Oberkochen, Germany), or a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) was used for photographing the whole mounts.

Intravenous injection of IL-4/IL-13 and blockade by antibody

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After the laser irradiation, recombinant mouse IL-4 (R and D Systems, Minneapolis, MN), IL-13 (Peprotec, Rocky Hill, NJ), or vehicle was injected through a tail vein on days 0 and 3. To block the induction of IL-4 or IL-13, anti-IL4 antibody (50 μg/mouse, BioLegend), anti-IL13 antibody (50 μg/mouse, Gene Tex, Irvine, CA), or control IgG was injected through the tail vein on days 0 and 3.

Generation of bone marrow chimeric mice

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Bone marrow cells were collected from the femur and tibia as described in detail (Wu et al., 2015). Recipient mice were irradiated (600 rad × 2) with a MX-160Labo Irradiator (MediXtec, Chiba, Japan) and then injected with a bone marrow cell suspension (1 × 107 cells) through the tail vein. The transplanted mice were allowed to recover for 5 weeks to reconstitute their myeloid cells. The reconstitution was confirmed by flow cytometry and the staining of the bone marrow or blood samples.

GFP transgenic mice-derived bone marrow or PKH26 (Sigma, Saint Louis, MO) labeling was used to examine the bone marrow cells. The stability of the PKH labeling was examined in the chimeric mice transplanted with PKH-labeled GFP-transgenic bone marrow. A stable co-localization of PKH and GFP in the bone marrow cells for more than 6 weeks was confirmed (data not shown).

Real-time reverse transcription PCR (RT-PCR)

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The eyes of laser-irradiated mice were enucleated at the selected times after the photocoagulation. Total RNA was extracted from the retina and choroidal sheets with the RNeasy mini kit (Qiagen, Hilden, Germany) and transcribed using the QuantiTect Reverse Transcription Kit (Qiagen). The cDNAs were amplified with QuantiTect SYBR Green PCR kit (Qiagen) with primer pairs (Supplementary file 1), and quantified using the LightCycler (Roche, Mannheim, Germany).

Retinal microvascular endothelial cells and endothelial progenitor cells

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Primary retinal vascular endothelial cells were collected from murine (C57-6065, Cell Biologics, Chicago, IL) and human retinas (ACBRI 181, Cell Systems, Kirkland, WA).

Primary retinal microvascular endothelial cells of C57BL/6 mice were isolated from the retinal tissue of pathogen-free laboratory mice. The cells were negative for bacteria, yeast, fungi, and mycoplasma. Primary human retinal microvascular endothelial cells were also examined for absence of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) contaminations by serologic or PCR test (by CLIA Licensed Clinical Lab) and Mycoplasma spp. contaminations (ATCC method by CLIA Licensed Clinical Lab). They were propagated to confluence on gelatin-coated 96-well plates in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, endothelial cell growth supplement (Sigma, St. Louis, MO), heparin, and non-essential amino acids (Gibco).

To create endothelial progenitor cells (EPCs), isolated bone marrow cells of mice were plated on fibronectin-coated plates. The nonadherent cells were removed, and the attached cells were cultured for 2 weeks in DME/F-12 supplement with 15% FBS and recombinant GMCSF (10 ng/ml, Peprotec, Rocky Hill, NJ) (Wang et al., 1998). Colony forming units were formed and attached at 1 week. To confirm an endothelial cell lineage, the cells were stained with endothelial cell markers including CD31, VCAM-1, and von Willebrand factor. Briefly, cells plated on temperature-responsive dishes (CellSeed, Tokyo, Japan) were non-enzymatically dispersed and stained for FACS analysis.

Tube formation assay of endothelial cells and microarray analysis

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To examine the roles played by cytokines in angiogenesis, vascular endothelial cells were assayed for in vitro tube formation as described in detail (DeCicco-Skinner et al., 2014). Briefly, bone marrow-derived EPCs or retinal vascular endothelial cells were plated on Matrigel-coated plates with or without recombinant mouse IL-4 (R and D Systems) or recombinant human IL-4 (Peprotec, for human endothelial cells), and the presence of tube networks was quantified by digitization of the photographs by Photoshop (Adobe, San Jose, CA) after 24 hr.

The gene and the pathway associated with the tube formation were determined by microarray analysis of tube forming EPCs. EPCs derived from wild type or Il4ra-/- mice were plated on Matrigel plates with or without IL-4 (10 ng/ml) to examine for tube formation. The total RNA was extracted using RNeasy mini kit (Qiagen) and analyzed using SurePrint G3 Mouse GE 8 × 60K Microarray (Agilent Technologies, Santa Clara, CA) (Miyazaki et al., 2017). The microarray data were analyzed using GeneSpring Software with setting of single color array and a fold change cut off of 3 and a p<0.05. A set of the IL-4-induced genes in the bone marrow-derived EPCs was analyzed to identify the canonical pathways and upstream regulators using Ingenuity Pathway Analysis software (IPA, Qiagen, accessed on 2020/4/5). The transcriptional networks of IL-4-stimulated EPCs were also constructed using IPA and evaluated by the P value as likelihood that assembly of the genes in a network could be explained by random chance alone.

Enzyme-linked immunosorbent assay (ELISA)

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The supernatants of EPC were assayed with a commercial ELISA kit (ThermoFisher Scientific, Waltham MA). The levels of IL-4 and IL-13 in the aqueous humor samples were measured using commercial ELISA kit as described in detail (Chono et al., 2018; Sasaki et al., 2012).

Statistical analyses

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Data are presented as the means ± standard error of the means (SEMs). The significance of the differences was determined by two-tailed t tests, linear mixed-effects regression analysis, or ANOVA with post hoc tests. Logistic regression analysis was used to compute the odds ratios based on quintiles of each cytokine levels. A p<0.05 was taken to be significant.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
    Expression of CCL2 and its receptor in activation and migration of microglia and monocytes induced by photoreceptor apoptosis
    1. C Feng
    2. X Wang
    3. T Liu
    4. M Zhang
    5. G Xu
    6. Y Ni
    (2017)
    Molecular Vision 23:765–777.
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40

Decision letter

  1. Satyajit Rath
    Senior Editor; Indian Institute of Science Education and Research (IISER), India
  2. Lois Smith
    Reviewing Editor; Boston Children's Hospital, Harvard Medical School, United States
  3. Ye Sun
    Reviewer; Boston Children's Hospital, Harvard Medical School, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The authors studied the role of IL-4 in choroidal neovascularization (CNV) in a mouse model. CNV is vision threatening in age related macular degeneration, commonly seen in the elderly population. They found that the IL-4/IL-4Rα axis contributed to CNV through communications with CCR2+ bone marrow cells which were recruited to CNV lesions. CCL2 enhanced recruitment of CCR2+ bone marrow cells. For CNV development, IL-4 in bone marrow cells is required, and IL-4 promotes CNV formation.

Decision letter after peer review:

Thank you for submitting your article "Role of IL-4 in bone marrow driven dysregulated angiogenesis and age-related macular degeneration" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board Reviewing Editors, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Ye Sun (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript by Bab et al. identifies high levels of interleukin-4 in the aqueous of patients presenting with the neovascular form of AMD. From this observation, the authors go on to use a series of experimental techniques, both in vitro and in vivo, to suggest that IL-4 is rapidly expressed in experimental laser induced CNV lesions in a mouse model. This occurs in tandem with increases in CCL2 and CCR2 and the authors suggest that IL-4 is critical for the formation of CNV. They go on to show that systemic injection of IL-4 can exacerbate CNV lesions while inhibition of IL-4 can prevent CNV development. Finally, they show, using a series of bone marrow chimeric mice, that IL-4 is critical for CNV development and in its absence (systemically), CNV lesions cannot adequately form. While this study is of interest and the data presented are intriguing, there are numerous issues that need to be addressed that will clarify the datasets and make the study more robust.

Essential revisions:

1) In all figure legends n=8-9 eyes (not mice) per group were mentioned for each mouse strain/treatment. However, the description of the laser-induced CNV procedure was unclear, e.g. the number of laser shots per eye, as well as the number of mice per group was not stated. The laser-CNV model can have very large variations (PMID: 24136346; 26161975). Therefore, at least 8-10 mice per group, with 3-4 laser shots per eye have been recommended to obtain reliable results.

2) Please use scatter plots in all figures.

3) The authors claim that bone marrow-derived cells were recruited and differentiated into endothelial cells in CNV lesions (Figure 2E). However, isolectin is not a specific endothelial cell marker, as it also stains microglia (PMID: 23413818). As the authors have demonstrated, CCL2 is localized with microglia (Iba1 staining) in CNV lesions (Figure 1C). Therefore, the authors need to use more specific markers for endothelial cells, and for macrophage/microglia to further validate the type of cells that bone marrow-derived cells differentiate into. If it is macrophage/microglia cells, the authors need to re-validate the data in these cells instead of endothelial progenitor cells in Figure 3-4.

4) In Figure 2E and Figure 5B, the authors showed the co-staining of bone marrow marker and endothelial cell marker and concluded that bone marrow derived cells incorporated into CNV lesion. But those cells may just simply stick on top of the CNV lesion through cell fusion instead of incorporation into CNV endothelium. If the authors can provide Z-stack confocal images to show those cells in same location are also in the same layer, not on top of endothelial cells.

5) The authors examined how IL-4 affected the differentiation of vascular endothelial progenitor cells from bone marrow cells in Figure 3 by measuring the induction of angiogenesis related factors including CCL2, VEGF, VEGF receptors, angiopoietin-1, endothelial receptor, thrombin receptors, P-selectin, and vascular endothelial cadherin, and found that CCL2 and VEGFR1 were significantly induced upon IL-4 exposure. This experiment was done in cell culture and EPC markers were not used to confirm the cell lineage of EPC. Do IL-4 exposure changes the amounts/levels or timing of differentiation of vascular endothelial progenitor cells from bone marrow cells?

6) In Figure 1C, the authors showed the colocalization of IL-4 and Iba-1 and concluded that IL-4 was associated mainly with Iba-1-positive retinal microglial cells. Can the authors rule out the possibility of endothelial cells?

Images in Figure 1B really need a counterstain to show the demarcation of the lesions. For example, an isolectin stain or CD31 stain could be used to highlight the lesion independent of Il-4 or CD11b staining.

7) Why has a time course not been completed for Iba-1 and CCL2 staining? What does this staining look like at the times of peak IL4 expression? The authors state in the legend hat the Iba1 and CCL2 staining are in the retinal tissue. Was the retina left intact during these flat mounts or was it removed? Are these microglia adhered to the neovascular membrane or are they within the retina?

8) Figure 2: It would be beneficial to include similar time-points as those outlined in Figure 1A for each of the markers stained for in Figure 2E. As it stands, this only represents what appears to be occurring 2 weeks post laser injury. As this is a temporally developing lesion, it would be important to stain for these components at all other time points listed previously.

9) Figure 3: In the endothelial progenitors cells stimulated with IL-4 (Figure 3A), CCL2 is not increasing in a dose dependent manner as suggested by the authors, but rather is lower at 100 ng/ml than at 10 ng/ml. How is this explained?

While the authors show effects of IL-4 on endothelial CCL2 and VEGF mRNA expression, do they have data pertaining to protein expression from these similar experiments?

10) In Figure 3, it's strange that although IL-13 can induce CCL2 and VEGFR1 expression, inhibition of IL-13 has absolutely no effect on CNV formation as outlined in Figure 2. This should be elaborated upon in detail.

11) Figure 5: Images should be shown for each bone marrow chimera generated, not just for IL-4-/- mice.

12) Discussion: The authors need to address the apparent contradictory results between their study and the one they have cited in Wu et al., 2015. This study shows that systemic administration of IL-4 prevents CNV development whereas their own data suggests it augments it. This needs to be clarified with further discussion.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for re-submitting your article "Role of IL-4 in bone marrow driven dysregulated angiogenesis and age-related macular degeneration" for consideration by eLife. Your revised article has been evaluated by a Reviewing Editor and Satyajit Rath as the Senior Editor. The Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) is a major cause of vision loss. CNV is associated with high local levels of IL-4. This study examined the role of IL-4 in CNV formation in a murine model. IL-4/IL-4 receptors (IL4Rs) controlled proangiogenic responses of bone marrow cells. CCR2+ bone marrow cells were recruited to very early CNV lesions. IL-4 rapidly induces CCL2, which enhances recruitment of CCR2+ bone marrow cells. For CNV development, IL-4 in bone marrow cells is required, and IL-4 promotes CNV formation. The IL-4/IL-4Rα axis contributes to pathological angiogenesis through communications with bone marrow cells leading to retinal degeneration.

Essential revisions:

1) The interactome in Figure 4—figure supplement 1 needs to be simplified as it is almost illegible in its complexity.

2) The authors still need to do some editing to tone down the conclusion that bone marrow-derived cells differentiated into endothelial cells as the data does not completely support this conclusion. Although the staining with endothelial cell CD31 lends some support, CD31 is expressed not only in vascular endothelial cells but also in T cells, B cells, dendritic cells (DCs), neutrophils, monocytes, and macrophage (PMID: 31732534). Please note this in the text.

3) In the text, the authors need to change their conclusion after adding CD31 staining in Figure 2: "In the CNV lesion, bone marrow-derived cells (green) were localized to isolectin-positive vascular endothelial cells indicating that the bone marrow-derived cells had differentiated into endothelial cells." The conclusion needs to be changed to "…CD31-positive…indicating that the bone marrow-derived cells may be able to differentiate into endothelial cells." and discuss the specificity of CD31 in endothelial cells.

https://doi.org/10.7554/eLife.54257.sa1

Author response

Essential revisions:

1) In all figure legends n=8-9 eyes (not mice) per group were mentioned for each mouse strain/treatment. However, the description of the laser-induced CNV procedure was unclear, e.g. the number of laser shots per eye, as well as the number of mice per group was not stated. The laser-CNV model can have very large variations (PMID: 24136346; 26161975). Therefore, at least 8-10 mice per group, with 3-4 laser shots per eye have been recommended to obtain reliable results.

Because there were three laser irradiation sites/eye, the nested ANOVA test was used to compare the size of the CNV area between groups. We showed each CNV area/spot with dots in all figures.

Our experiments were conducted using at least 8-10 eyes (8-12 mice), and 3 laser spots were made in each eye. The descriptions were changed to spots and eyes. Because the spots were nested in the eyes, nested ANOVA analysis was used.

Subsection “Induction of choroidal neovascularization”: “Three laser spots were created in each eye.”

Figure 2 legend: “Nested ANOVA with post hoc test.”

Figure 5 legend: “Nested ANOVA with post hoc test.”

Some of the IL-4 chimeric mice did not survive through the experimental period. This suggested that IL-4 plays a critical role in the survival of bone marrow cells. The number of dead mice is shown in the figure legends.

Figure 5 legend: “Six out of 10 Il4-/- bone marrow chimeric mice in each group did not survive through procedures, and/or euthanized.”

2) Please use scatter plots in all figures.

Plots were changed to scatter graphs.

3) The authors claim that bone marrow-derived cells were recruited and differentiated into endothelial cells in CNV lesions (Figure 2E). However, isolectin is not a specific endothelial cell marker, as it also stains microglia (PMID: 23413818). As the authors have demonstrated, CCL2 is localized with microglia (Iba1 staining) in CNV lesions (Figure 1C). Therefore, the authors need to use more specific markers for endothelial cells, and for macrophage/microglia to further validate the type of cells that bone marrow-derived cells differentiate into. If it is macrophage/microglia cells, the authors need to re-validate the data in these cells instead of endothelial progenitor cells in Figure 3-4.

The purpose of our study was to determine how IL-4 contributed to bone marrow-mediated CNV formation. To understand the kinetics and role of bone marrow cells, we conducted another time series analysis after bone marrow transfer. For this series of analyses, CD31 staining was used to identify endothelial cells (Figure 2—figure supplement 1).

We showed that the initial recruitment of bone marrow cells peaked at 3 days after the irradiation. Bone marrow cells in this priming phase are CD11b+, and they secrete CCL2. This population are not positive for iba1 and are morphological distinct from the accumulated microglial cells. This suggested that these cells will enhance the recruitment using CCL2 signals.

After the priming phase subsides, CD31-positive bone marrow cells are observed in the center of the CNVs. Because these cells secrete IL-4, endothelial cells, either bone marrow derived or not, are stimulated. Our in vitro tube formation experiments using endothelial cells as well as bone marrow-derived cells supports this stimulatory effect of IL-4.

“Recruitment of GFP+ bone marrow derived cells (green) peaked at 3 days after irradiation. Thus, bone marrow-derived cell recruitment also contributed to the inductive phase process. Bone marrow-derived cells in this phase were CD11b+ lineage, and they were positive for CCL2. These bone marrow cells did not express iba1 and were morphologically distinct from microglial cells. This suggested that these cells will amplify the recruitment of CCR2+ lineage cells (Figure 2—figure supplement 1).”

4) In Figure 2E and Figure 5B, the authors showed the co-staining of bone marrow marker and endothelial cell marker and concluded that bone marrow derived cells incorporated into CNV lesion. But those cells may just simply stick on top of the CNV lesion through cell fusion instead of incorporation into CNV endothelium. If the authors can provide Z-stack confocal images to show those cells in same location are also in the same layer, not on top of endothelial cells.

We conducted 3D analysis on bone marrow chimeric mice (Figure 2—video 1). In the priming phase, the bone marrow cells were seen attached to the budding CNV. This phase appeared as the amplification phase. CD31 positive bone marrow cells with endothelial morphology were seen after 7 days.

“Seven days after irradiation, bone marrow-derived cells were incorporated into CD31+ endothelial cells, and they had endothelial cell morphology (Figure 2—video 1).”

5) The authors examined how IL-4 affected the differentiation of vascular endothelial progenitor cells from bone marrow cells in Figure 3 by measuring the induction of angiogenesis related factors including CCL2, VEGF, VEGF receptors, angiopoietin-1, endothelial receptor, thrombin receptors, P-selectin, and vascular endothelial cadherin, and found that CCL2 and VEGFR1 were significantly induced upon IL-4 exposure. This experiment was done in cell culture and EPC markers were not used to confirm the cell lineage of EPC. Do IL-4 exposure changes the amounts/levels or timing of differentiation of vascular endothelial progenitor cells from bone marrow cells?

The differentiation protocol was based on previous studies (Wang et al., 1998), and was also reviewed by Calzi et al. Microvascular Res 2010 (doi:10.1016/j.mvr.2010.02.011).

Currently, the described protocols do not require the addition of IL-4. We used IL-4 after differentiation. We assumed that the intrinsic IL-4 was sufficient for full differentiation or IL-4 will activate endothelial cells.

6) In Figure 1C, the authors showed the colocalization of IL-4 and Iba-1 and concluded that IL-4 was associated mainly with Iba-1-positive retinal microglial cells. Can the authors rule out the possibility of endothelial cells?

Images in Figure 1B really need a counterstain to show the demarcation of the lesions. For example, an isolectin stain or CD31 stain could be used to highlight the lesion independent of Il-4 or CD11b staining.

Microglial cells were co-stained with Iba-1 and CD11b (not IL-4) and were located on the surface of the CNVs beneath the retinal vessels 1 to 3 days after laser treatment. (Figure 1—video 1).

“The microglial cells migrated to surface of the CNV (Figure 1—video 1).”

“Figure 1—video 1

Localization of bone marrow-derived cells and microglial cells in the retinal tissue of GFP bone marrow chimeric mouse in 3D rendering at 3 days after laser irradiation.

Bone marrow-derived cells (green) are located in the subretinal layer around the laser irradiation lesion 3 days after laser irradiation. Bone marrow-derived cells (green) expressing CD11b (cyan) was spatially and morphologically distinct from accumulated microglial cells (arrows). CD11b (cyan) positive microglial cells migrated on CD31 (red) positive retinal and choroidal vessels, and are observed on the surface of choroidal neovascularization. Scale 50 μm.”

7) Why has a time course not been completed for Iba-1 and CCL2 staining? What does this staining look like at the times of peak IL4 expression? The authors state in the legend hat the Iba1 and CCL2 staining are in the retinal tissue. Was the retina left intact during these flat mounts or was it removed? Are these microglia adhered to the neovascular membrane or are they within the retina?

We showed the time course for Iba-1 and CCL2 staining in retinal flat mounts (Figure 1—figure supplement 1). Lesional IL-4 peaks at 3 days after the laser irradiation. IL-4 expressing bone marrow cells were mainly CD11b+, and they had monocytic morphology.

“The kinetics of Iba1, CCL2, and CD11b positive cells after laser exposure was consistent with that of the mRNA induction (Figure 1—figure supplement 1).”

8) Figure 2: It would be beneficial to include similar time-points as those outlined in Figure 1A for each of the markers stained for in Figure 2E. As it stands, this only represents what appears to be occurring 2 weeks post laser injury. As this is a temporally developing lesion, it would be important to stain for these components at all other time points listed previously.

We prepared kinetics panel staining as shown in Figure 2—figure supplement 1. IL-4 secretion is generally a very small in amount. Using immunohistochemistry, IL-4 was not obviously expressed during the one to three days and was not shown.

Recruitment of GFP+ bone marrow derived cells (green) peaked at 3 days after irradiation. Thus, bone marrow-derived cell recruitment also contributed to the inductive phase process. Bone marrow-derived cells in this phase were CD11b+ lineage, and they were positive for CCL2. These bone marrow cells did not express iba1 and were morphologically distinct from microglial cells. This suggested that these cells will amplify the recruitment of CCR2+ lineage cells (Figure 2—figure supplement 1).

9) Figure 3: In the endothelial progenitors cells stimulated with IL-4 (Figure 3A), CCL2 is not increasing in a dose dependent manner as suggested by the authors, but rather is lower at 100 ng/ml than at 10 ng/ml. How is this explained?

IL-4 of 100 ng/ml is a high in concentration for in vivo conditions. The optimal concentration of IL-4 is within 10 ng/ml at most. The IL-4 concentration of 100 ng/ml was used to confirm the blocking effect of anti-IL-4Rα antibody.

While the authors show effects of IL-4 on endothelial CCL2 and VEGF mRNA expression, do they have data pertaining to protein expression from these similar experiments?

We confirmed that the protein expression of endothelial CCL2 and VEGFR-1 stimulated 1 ng/mL or 10 ng/mL by ELISA (Figure 3—figure supplement 2).

“An upregulation of the translation of CCL2 and VEGFR-1 in EPCs was confirmed by ELISA. IL-4 exposed EPCs had a significant increase in the secretion of CCL2 (P = 0.000) and VEGFR-1 (P = 0.000) after 24 h exposure to IL-4 (Figure 3—figure supplement 2).”

10) In Figure 3, it's strange that although IL-13 can induce CCL2 and VEGFR1 expression, inhibition of IL-13 has absolutely no effect on CNV formation as outlined in Figure 2. This should be elaborated upon in detail.

CCL2 and VEGFR1 are representatives of important genes for the angiogenesis process. However, angiogenesis requires numerous factors, which operate in coordination kinetically and spatially. We assume that the IL-13/IL-13Rα axis may not induce some other essential factors.

11) Figure 5: Images should be shown for each bone marrow chimera generated, not just for IL-4-/- mice.

We prepared panels of bone marrow cells and CNV as Figure 5—figure supplement 1.

Figure 5—figure supplement 1

Localization of bone marrow-derived cells (green) and endothelial cells (red) in the retinal tissue of wild type and Ilra-/- bone marrow chimeric mice on Il4ra-/- background and wild type, Il4-/- and Il4ra-/- bone marrow chimeric mice on wild type background. Endothelial cells in the CNV were labeled with IB4 (red). Reduced sized CNV contained wild type bone-derived cells. Scale 10 μm.

12) Discussion: The authors need to address the apparent contradictory results between their study and the one they have cited in Wu et al., 2015. This study shows that systemic administration of IL-4 prevents CNV development whereas their own data suggests it augments it. This needs to be clarified with further discussion.

The direct suppressive effect of IL-4 on CNV development is shown using vitreous injection of IL-4 by Wu et al. However, the concentration used by Wu et al. was extremely high (600 ng/ml). This extremely high concentration is not necessary under in vivo conditions, and IL-4 is not expressed in such high concentration in the eye. This level is highly toxic and can damage endothelial cells or recruited cells. Other experiments showing the suppressive effect of IL-4 was conducted using in vitro differentiated macrophages presumably reflecting the stimulating differentiation into regulatory phenotype by IL-4.

“Wu et al. also showed a contradictory role of IL-4 for CNV formation (Wu et al., 2015). This was shown using vitreous injection of IL-4 at very high concentration (600 ng/ml). This may cause toxic damage to endothelial cells or recruited cells which may not reflect physiological role of IL-4.”

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1) The interactome in Figure 4—figure supplement 1 needs to be simplified as it is almost illegible in its complexity.

Figure 4—figure supplement 1B

The primary networks were confined to the top 3 major networks. Fewer genes and molecules are shown in the merged network. To improve the understanding, the layout of the genes was changed to a hierarchical view.

Subsection “Tube formation assay of endothelial cells and microarray analysis”: “... (IPA, Qiagen, accessed on 2020/4/5).”

Legend to Figure 4—figure supplement 1:

“A: IL-4Rα-mediated transcriptional networks associated with angiogenesis in bone marrow-derived EPCs after IL-4 treatment. Z score = 2.781, P = 1.8×10-4

B: IL-4Rα-mediated transcriptional networks of bone marrow-derived EPCs. The top 3 highest significant networks (P <1×10-27) were merged and are shown in a hierarchical layout. Amyloid-β A4 protein (App) and cellular tumor antigen p53 (Tp53) are shown in the top and bottom positions. Red indicates an upregulation and green indicates a downregulation. The IPA was accessed on 2020/4/5.”

2) The authors still need to do some editing to tone down the conclusion that bone marrow-derived cells differentiated into endothelial cells as the data does not completely support this conclusion. Although the staining with endothelial cell CD31 lends some support, CD31 is expressed not only in vascular endothelial cells but also in T cells, B cells, dendritic cells (DCs), neutrophils, monocytes, and macrophage (PMID: 31732534). Please note this in the text.

Definitive proof of differentiation of bone marrow-derived cells requires further meticulous experimentations. Our major focus was to determine the contribution of bone marrow cells and IL-4 to CNV formation. Although our data are consistent with the bone marrow cells differentiation into endothelial cells, the context was changed to suggest other possibility that bone marrow cells may behave as non-endothelial lineage.

“To identify an endothelial lineage, we used CD31 or isolectin staining, because CD31 is highly expressed on endothelial cells and is commonly used as an endothelial cell marker. However, CD31 can also be expressed in other lineage cells including T cells, B cells, dendritic cells (DCs) (Clement et al., 2014), neutrophils, monocytes, and macrophage (Merchand-Reyes et al., 2019).”

“Our data support the idea that bone marrow-derived cells may be able to differentiate into endothelial cells. However in the CNV lesion, whether endothelial differentiation is complete or bone marrow cells serve as immature or a different lineage is uncertain. Importantly, bone marrow-derived cells do play pivotal roles in the CNV formation.”

3) In the text, the authors need to change their conclusion after adding CD31 staining in Figure 2. "In the CNV lesion, bone marrow-derived cells (green) were localized to isolectin-positive vascular endothelial cells indicating that the bone marrow-derived cells had differentiated into endothelial cells." The conclusion need to be changed to "…CD31-positive…indicating that the bone marrow-derived cells may be able to differentiate into endothelial cells." and discuss the specificity of CD31 in endothelial cells.

Immunohistochemistry showed a colocalization of CD31 and bone marrow cells. However, a co-expression of CD31 does not completely exclude the possibility that bone marrow cells may have differentiated into another lineage or remain relatively immature. The description in the Results section was revised to that shown below to suggest this other possibility. We also added text that non-endothelial cells may express CD31 in the Discussion section.

“... bone marrow-derived cells were incorporated into the structures formed by CD31+ endothelial cells” (Figure 2—video 1).

“In the CNV lesion, bone marrow-derived cells (green) were localized to the structures formed by isolectin-positive vascular endothelial cells, although this may not indicate complete differentiation into endothelial lineage. However, the co-localization of the marrow-derived cells to structures formed by CD31+ endothelial cells indicates that the bone marrow-derived cells might be able to differentiate into endothelial cells.”

“Alternatively, bone marrow cells will also differentiate into a non-endothelial cell lineage serving as provider of CNV forming signals.”

“... the IL-4R+ bone marrow-derived cells were incorporated into the CNV presumably as late EPCs together with the resident cell-derived endothelial cells.”

https://doi.org/10.7554/eLife.54257.sa2

Article and author information

Author details

  1. Takashi Baba

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    For correspondence
    babatakashi8@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7318-9420
  2. Dai Miyazaki

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Supervision, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    miyazaki-ttr@umin.ac.jp
    Competing interests
    No competing interests declared
  3. Kodai Inata

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  4. Ryu Uotani

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  5. Hitomi Miyake

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  6. Shin-ichi Sasaki

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  7. Yumiko Shimizu

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  8. Yoshitsugu Inoue

    Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago, Japan
    Contribution
    Supervision, Funding acquisition, Project administration, Writing - review and editing
    Competing interests
    medical advisor of Senju Pharmaceutical Company, Japan.
  9. Kazuomi Nakamura

    Division of Pathological Biochemistry, Department of Biomedical Sciences, Faculty of Medicine, Tottori University, Tottori, Japan
    Contribution
    Resources, Formal analysis, Supervision, Methodology
    Competing interests
    No competing interests declared

Funding

Japan Society for the Promotion of Science (JP16K15733)

  • Takashi Baba

Japan Society for the Promotion of Science (JP25670733)

  • Takashi Baba

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to Dr. Marc E Rothenberg who supplied the animal for the experiments using IL-13 receptor deficient mice. Dr. Tetsuya Ohbayashi kindly supported animal experiments conducted in Research Center for Bioscience and technology and animal care. Tottori Bio Frontier managed by Tottori prefecture kindly allowed access to confocal microscope, LSM730.

Ethics

Human subjects: The study protocol was approved by the Ethics Committee of the Tottori University (protocol #: 2699), and the procedures used conformed to the tenets of the Declaration of Helsinki. An informed consent was obtained from all of the participants.

Animal experimentation: All mice were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and protocols approved by the Institutional Animal Care and Use Committee of Tottori University (protocol #: 12-Y-7, 13-Y-25, 16-Y-22, and 19-Y-50).

Senior Editor

  1. Satyajit Rath, Indian Institute of Science Education and Research (IISER), India

Reviewing Editor

  1. Lois Smith, Boston Children's Hospital, Harvard Medical School, United States

Reviewer

  1. Ye Sun, Boston Children's Hospital, Harvard Medical School, United States

Publication history

  1. Received: December 7, 2019
  2. Accepted: April 19, 2020
  3. Version of Record published: May 5, 2020 (version 1)

Copyright

© 2020, Baba et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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