Therapeutic potential of histamine H4 receptor antagonist as a preventive treatment for diabetic retinopathy in mice

Abstract

Diabetic retinopathy (DR) is a prevalent complication of diabetes, often resulting in vision loss and blindness. Existing treatments primarily aim to control blood sugar levels and inhibit angiogenesis. However, current therapies for DR, such as anti-VEGF and laser photocoagulation, are frequently invasive, and can cause adverse side effects. Consequently, there is a critical need for new preventive therapeutics to address DR more effectively. This study aimed to examine the therapeutic potential of a histamine H₄ receptor (HRH4) antagonist as a preventive treatment for DR in mice. A mouse model of DR was established by intraperitoneally injecting 200 mg/kg of streptozotocin (STZ). Immune cell infiltration into the retina of mice with STZ-induced diabetes was measured using fluorescence-activated cell sorting (FACS) 12 weeks after STZ injection. The preventive effects of the HRH4 antagonist on inflammation and pathological retinal vessel leakage were determined in a mouse model of DR. Infiltration of HRH4-expressing macrophages increased in the retina of mice with STZ-induced DR. The HRH4 antagonist prevented macrophage infiltration and retinal vascular leakage to prevent STZ-induced DR in mice without causing any retinal toxicity. The infiltration of macrophages increased in the retina of mice with STZ-induced diabetes through HRH4, indicating that HRH4 is potentially a novel preventative therapeutic target in DR. These findings suggest that targeting HRH4 is a promising strategy for the prevention and treatment of DR.

Introduction

Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus (DM) that causes a significant proportion of blindness worldwide¹. The incidence of DR increases with the duration of diabetes; particularly, DR develops in 6.6% of patients whose duration of diabetes is ≤ 5 years and 75% in patients whose duration of diabetes is ≥ 15 years^2,3. All patients with diabetes are recommended to undergo an annual ophthalmic examination, even if no clinical signs of DR are present^4,5. Therefore, early diagnosis and preventive treatment through regular screening are the best strategies for preventing DR.

Patients with DM exhibit high blood glucose levels, leading to decreased vascular flexibility, increased oxidative stress, blood flow inhibition, and consequently, the destruction of microvasculature and subsequent neovascularization. The pathogenesis of DR parallels that of DM, as it involves disruption of the blood-retinal barrier and abnormal angiogenesis^6,7. Elevated levels of pleiotropic mediators such as histamine, vascular endothelial growth factor (VEGF), and interleukin-6 (IL-6) have been linked to blood-retina barrier disruption^8–10; this relationship highlights the importance of reducing retinal inflammation in preventing DR progression.

Current treatments for DR include anti-VEGF and laser photocoagulation therapy; however, these treatments are frequently invasive and have adverse side effects, including increased inflammation and reactive oxygen species formation^11,12. Therefore, new preventive therapeutics that focus on reducing retinal permeability and inflammation are necessary. Blocking the infiltration of inflammatory immune cells such as macrophages (early markers of DR) could be a potential treatment to prevent DR progression. For example, colony-stimulating factor 1 receptor (CSF-1R) inhibition reduces retinal inflammation by depleting retinal microglia and blocking macrophage infiltration¹³. However, antagonists of CSF-1 signaling have limited therapeutic applications because CSF-1 signaling promotes myeloid progenitor differentiation into macrophages and requires homeostasis¹⁴.

Histamine levels are elevated in the serum and vitreous fluid of patients with diabetes⁹. Histamine increases vascular permeability^15,16 and mediates chemotaxis through the histamine H₄ receptor (HRH4)¹⁷. Histamine binds to four different types of histamine receptors: HRH1, HRH2, HRH3, and HRH4. The major expression locations of HRH1 to HRH4 has been well known as follows: HRH1: nerve cells, smooth muscles, vascular endothelial cells, etc.; HRH2: gastric parietal cells, smooth muscles, endothelial cells, epithelial cells, etc.; HRH3: histaminergic neurons, eosinophils, dendritic cells, monocytes, etc.; HRH4: high expression on bone marrow and peripheral hematopoietic cells¹⁸. Among these four receptors, HRH4 is known for high expression in activated immune cells, including macrophages and dendritic cells¹⁹. Among the four receptors, HRH4 has been reported as important mediator of inflammatory reactions of macrophages including chemotaxis and migration to the site of inflammation, phagocytosis, and M1 differentiation^20,21. Based on this evidence, we hypothesized that histamine increases retinal inflammation and permeability by stimulating the retinal infiltration of activated macrophages expressing HRH4. These infiltrating macrophages secrete pro-inflammatory and pro-angiogenic cytokines, exacerbating the pathogenesis of DR.

In this study, we employed the high-dose streptozotocin (STZ) induction protocol to induce a DR mouse model following the guidelines by the Animal Models of Diabetic Complications Consortium. A single dose of STZ was administered to establish the DR model and subsequently, we conducted comprehensive analyses of the immune system and retinal tissue histology, focusing on HRH4 expression and the effects of HRH4 antagonist treatment.

Results

Increase in the infiltration of HRH4-expressing macrophages in the retina of mice with diabetic retinopathy

We established a DR mouse model by injecting STZ to confirm whether HDC, VEGF, and IL-6 levels increased in mice. To validate the diabetes model, blood glucose and weight were measured at 2 weeks intervals, which began 3 days after a single intraperitoneal injection of 200 mg/kg STZ. We confirmed that the mRNA levels of HDC, VEGF, and IL-6 increased in the retinas of STZ-induced DR mice (Fig. 1a–c). Immunohistochemistry staining of retinal sections (n = 5 in each group) revealed that the levels of macrophage marker F4/80 were increased in the retina of STZ-induced DR mice compared with those in normal mice (Fig. 1d,e). Additionally, we found that HRH4 and F4/80 co-localized in the retinas of mice with STZ-induced DR (Fig. 1f). The ratio of the F4/80⁺HRH4⁺ cells were higher in STZ group than Con group (Fig. 1g). Based on these results, we hypothesized that histamine promotes infiltration of macrophages expressing HRH4 into the retinas of mice with STZ-induced DR, leading to increased inflammation.

Fig. 1 [Images not available. See PDF.]

Inflammation and infiltration of HRH4-expressing macrophages are increased in the retina of mice with diabetic retinopathy. (a–c) Gene transcripts for HDC (a), VEGF (b), and IL-6 (c) in the retina from the controls (Con) and mice with STZ-induced diabetic retinopathy (STZ) were analyzed by real-time PCR. Relative expression levels were obtained and normalized to that of 18S rRNA (n = 6–10). (d) Immunohistochemistry of F4/80 (red arrows) in retinas from Con and STZ groups (n = 5). Scale bar = 50 μm. (e) The number of F4/80⁺ cells from the immunohistochemistry slides of Con and STZ groups. Cells were counted from randomly selected observed range of interest (n = 7) (f) Co-immunofluorescence (IF) staining of HRH4 (green) and F4/80 (red) in the retinas of Con and STZ groups. Scale bar = 50 μm. (g) Ratio of the cell numbers of F4/80⁺HRH4⁺ cells to F4/80⁺ cells of Con and STZ groups (n = 2, 3). Data are presented as the means ± standard errors of the mean and are representative of two independent experiments. *P < 0.05, **P < 0.01 by the t test.

The major infiltrated cell population in the retina of mice with diabetic retinopathy consists of macrophages expressing HRH4

Subsequently, we performed immune cell analysis of the retina from the control mice and mice with STZ-induced DR using flow cytometry (Fig. 2a). The number of CD45⁺ immune cells increased in the retina of mice with STZ-induced DR compared to that in the normal state; among the immune cells, the number of HRH4-expressing cells significantly increased (Fig. 2b). We hypothesized that macrophages are the major CD45⁺HRH4⁺ cell population in the retina of mice with DR; this was confirmed through various immune cell population analyses. Our results revealed that macrophages and T cells increased in the retina of mice with DR, with macrophages accounting for the majority of CD45⁺HRH4⁺ cells (Fig. 2c).

Fig. 2 [Images not available. See PDF.]

HRH4-expressing macrophages constitute the majority of infiltrated cell population in the retina of mice with diabetic retinopathy. (a,b) The population percentages of CD45⁺ and CD45⁺HRH4⁺ determined by flow cytometry. (a) Dot plots for flow cytometry gating of CD45⁺ and CD45⁺HRH4⁺ groups and (b) according population percentages in retinas from controls (Con) and diabetic mice induced with STZ (STZ) (n = 5). (c) Immune cell populations in retinas from Con and STZ groups analyzed by flow cytometry (n = 4–5). Data are presented as the means ± standard errors and are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by the one-way ANOVA test.

Histamine induces chemotaxis in bone marrow-derived macrophages exposed to high glucose via HRH4

Previous studies have demonstrated that histamine induces macrophage chemotaxis via HRH4¹⁷, and we confirmed that the number of HRH4-expressing macrophages increased in the retinas of mice with STZ-induced DR. To investigate whether macrophage infiltration in DR occurs via HRH4, we treated bone marrow derived macrophages (BMDMs) with high glucose for 48 h. We first evaluated the mRNA levels of HRH4 in BMDMs after exposure to LPS, low glucose (5 mM), or high glucose (25 mM) for 48 h. We found that exposure to high glucose upregulated the expression of HRH4 mRNA in BMDMs, even though LPS did not (Fig. 3a). We then treated BMDMs with the HRH4 antagonist JNJ7777120 (JNJ) after treating them with high glucose levels (25 mM) and evaluated histamine-induced chemotaxis (Fig. 3b,c). We observed that macrophages showed increased migration in response to histamine. However, macrophages treated with JNJ did not exhibit increased migration in response to histamine (Fig. 3b,c). These results suggest that histamine induces chemotaxis via HRH4 in macrophages exposed to high glucose.

Fig. 3 [Images not available. See PDF.]

Histamine induces chemotaxis in bone marrow-derived macrophages exposed to high glucose via HRH4. (a) Gene transcripts for HRH4 in bone marrow derived macrophages (BMDMs) exposed to LPS, low glucose (5 mM), and high glucose (25 mM) were analyzed by real-time PCR. Relative expression levels were normalized using 18S rRNA expression (n = 5). (b) Quantitative graph of the number of nuclei moved from the upper chamber to lower chamber by chemotaxis. Chemotaxis assay was performed with 25 mM glucose, with and without 10 μM JNJ7777120 (JNJ) treatment. After JNJ treatment, the BMDMs were added to the upper well. Chemotaxis medium containing 50 μM histamine (HIS) was added to the bottom wells. (c) Image of DAPI-stained nuclei of chemotaxis assay groups. Scale bar = 50 μm. Data are presented as the means ± standard errors and are representative of two independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001 by the one-way ANOVA test.

Treatment with a histamine H4 receptor antagonist suppressed inflammation and vessel leakage in the retina of mice with diabetic retinopathy

To determine the effect of JNJ, a selective HRH4 antagonist, on macrophage infiltration, mice were intraperitoneally administered JNJ every day from 8 to 12 weeks after STZ injection. The number of CD11b⁺F4/80⁺ macrophages decreased in the JNJ-treated group (Fig. 4a). Additionally, we confirmed that the HRH4⁺IL-6⁺ and HRH4⁺VEGF⁺ macrophages that infiltrated the retina were reduced in the JNJ-treated group (Fig. 4b,c). Moreover, the protein levels of IL-6 and VEGF were significantly decreased in the serum of the JNJ-treated group compared to those in the STZ-induced DR group (Fig. 4d,e). JNJ also decreased vessel leakage and blood vessel rupture in the retina of mice with STZ-induced DR (Fig. 4f–j). Retinal histology showed no evidence of retinal toxicity following JNJ treatment (Fig. 4j). These results suggested that HRH4-targeted treatment may be a promising therapeutic approach for DR.

Fig. 4 [Images not available. See PDF.]

Histamine H₄ receptor antagonist treatment suppressed inflammation and pathological vessel leakage in the retina of mice with diabetic retinopathy. (a) Flow cytometry analysis of population percentages of CD11b⁺F4/80⁺ cells of retinas from the controls (Con), mice with STZ-induced diabetic retinopathy (STZ), and JNJ7777120 (JNJ) treated STZ mice (n = 5–7). (b) Population percentages of HRH4⁺IL-6⁺ and HRH4^–IL-6⁺ in CD11b⁺F4/80⁺ cells analyzed by flow cytometry (n = 5–7). (c) Population percentages of HRH4⁺VEGF⁺ and HRH4^-VEGF⁺ in CD11b⁺F4/80⁺ cells analyzed by flow cytometry (n = 5–7). (d,e) ELISA of IL-6 (d) and VEGFA (e) in the serum of Con, STZ, JNJ treated STZ groups (n = 5–7). (f) Quantitative graph of the number of leakage points from fluorescein vessel images (h–j). (g) Quantitative graph of the fluorescence intensity of normal branches from Con group (h) and leakage points from STZ group (i). Fluorescence intensity was analyzed using ImageJ software. (h–j) Representative images of fluorescein vessel leakage assay from the Con (h), STZ (i), and JNJ-treated STZ (j) groups. Increased vascular permeability resulted in the vessel leakage in STZ group (white arrows). Scale bar = 100 μm. Data are presented as the means ± standard errors and are representative of two independent experiments. *P < 0.05, **P < 0.01, **P < 0.001, ****P < 0.0001 by the one-way ANOVA test.

Discussion

In this study, we demonstrated that infiltrated macrophages in the retinas of DR mice express HRH4 that contributes to inflammation and pathological vessel leakage. Treatment with an HRH4 antagonist reduced macrophage infiltration and endothelial permeability (Fig. 5); this result indicates the potential therapeutic efficacy of HRH4-targeted treatment for DR.

Fig. 5 [Images not available. See PDF.]

Schematic figure of the effects of HRH4 antagonist on the pathogenesis of diabetic retinopathy. The most pathologic key factors of DR are the blood-retinal barrier disruption and the abnormal angiogenesis. HRH4 is expressed in macrophages that infiltrate into the retina of mice with STZ-induced diabetes. Blocking HRH4 using JNJ7777120 (JNJ) suppressed the infiltration of macrophages and leakage of retinal vessels, indicating the potential of HRH4 as a novel preventative therapeutic target in diabetic retinopathy.

Histamine binds to four subtypes of G-protein-coupled receptors, including H1, H2, H3, and H4; furthermore, histamine acts as a potent mediator of immediate hypersensitivity reactions^22–24. HRH4 is a unique subtype, with little homology to classical histamine receptors 1 and 2²⁵. Based on these unique properties, HRH4 is mainly expressed in immune cells, including monocytes, macrophages, dendritic cells, and T cells, and its expression is regulated by the inflammatory response in peripheral tissues^26–29. HRH4 plays a major role in leukocyte chemotaxis at the sites of inflammation²⁹. Additionally, histamine induces chemotaxis and phagocytosis in murine macrophages via HRH4¹⁷.

Several studies have targeted HRH1 to regulate inflammation, but recent research has suggested that HRH4 could also be targeted and modulated in inflammatory diseases. Dunford et al. found that lung inflammation, lung eosinophil/lymphocyte infiltration, and T_H2 responses were reduced in HRH4^−/− and JNJ-treated mice³⁰. Additionally, in a laser-induced CNV mouse model, F4/80 and HRH4 were co-localized in the retina, and JNJ administration reduced VEGF volume³¹.

A previous study that showed elevated histamine levels in patients with DR⁹; considering this, we hypothesized that targeting HRH4 could regulate retinal inflammation and vessel permeability by inhibiting macrophage infiltration. In our study, we confirmed that macrophages expressing HRH4 secreted IL-6 and VEGF; additionally, administrating an HRH4 antagonist reduced macrophage infiltration, resulting in reduced inflammatory response and vascular permeability (Fig. 4). We also found that HRH4 was highly expressed in retinal macrophages but not in T cells (Fig. 2C), suggesting that HRH4 is a selective target for inflammatory macrophages. However, further studies are required to selectively target only macrophages.

In our study, we utilized JNJ, a highly selective antagonist of HRH4 with minimal cross-reactivity to other histamine receptors³². JNJ is characterized by a short half-life³³, which influenced our administration protocol. We administered the drug once daily for 4 weeks prior to the expected onset of DR. This approach was chosen to potentially minimize off-target side effects, capitalizing on the drug's short duration of action. The pharmacokinetic profile of JNJ, combined with its selective action on HRH4, suggests it could be developed into an oral medication for preventative treatment. Such a formulation could be particularly beneficial for diabetes patients who are at risk of developing DR but have not yet manifested retinal complications.

In conclusion, our study demonstrates the significant role of HRH4 in the pathogenesis of diabetic retinopathy, particularly through its expression on infiltrating macrophages. The selective targeting of HRH4 with JNJ effectively reduced inflammation and vascular permeability in our DR mouse model, highlighting its potential as a novel therapeutic approach.

Methods

Animals

Male C57BL/6J mice (7 weeks of age) were obtained from ORIENT BIO (Republic of Korea) and used for animal experiments at the Institute for Experimental Animals, College of Medicine. The mice were housed under specific pathogen-free conditions with a constant flow of air exchange in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institutional Animal Care and Use Committee of Seoul National University in Seoul (accession number SNU-190313-1-2). To establish a mouse model of type 1 diabetes, STZ (200 mg/kg) was dissolved in a 0.1 M sodium citrate buffer (pH 4.5) and injected into the peritoneal cavity of C57BL/6 mice. Control C57BL/6 mice were injected with a sham (0.1 M sodium citrate buffer). To measure blood glucose level, blood was collected from the tail vein of each mouse under anesthesia (ketamine-xylazine; 150 μL/mouse; intraperitoneal injection), starting from the third day of STZ injection and in every 2 weeks interval. Blood glucose levels were measured using Accu-Chek (Roche, Germany), and hyperglycemia was defined as blood glucose levels higher than 300 mg/dL. To evaluate the effect of HRH4 antagonist JNJ7777120 (JNJ) on DR, 1 mg/kg of JNJ in 100 μL volume or PBS in the same volume was intraperitoneally administered 8–12 weeks after STZ injection. All mice were euthanized 12 weeks after the STZ injection by CO₂ inhalation. All experiments were performed in accordance with the ARRIVE guidelines³⁴.

Cells

Primary macrophages were differentiated from bone marrow cells obtained from C57BL/6J male mice at 7–12 weeks of age. Bone marrow cells were differentiated into mature BMDMs for 7 days in RPMI 1640 containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mM L-glutamine (Gibco, MA, USA) supplemented with fresh recombinant murine macrophage colony-stimulating factor (M-CSF) (50 ng/mL; Miltenyi Biotec, Bergisch Gladbach, Germany). The medium was replaced on days three and five with fresh medium containing M-CSF.

Immunostaining of mouse retina

Twelve weeks after STZ injection, mouse eyes fixed in 4% paraformaldehyde and the anterior segments including cornea, lens, and vitreous humor were removed to get eyecup-shaped remaining tissue including retina and residual pigment epithelium. Paraffin-embedded tissues were sectioned to a thickness of 5 μm and the slides were deparaffinized, rehydrated, and antigen-retrieved in a citrate buffer (10 mM sodium citrate, 0.05% Tween 20, and pH 6.0) for further analysis. Primary antibodies were pre-diluted to 1:200 in a blocking buffer for F4/80 (Bio-Rad, MA, USA) and were applied to tissue sections overnight at 4 °C in a humidified chamber. Biotinylated secondary antibodies were applied, followed by signal development using liquid DAB substrate (Dako, CA, USA). The sections were counterstained with hematoxylin (Merck, Darmstadt, Germany). For immunofluorescence, slides were incubated with 1% bovine serum albumin (BSA) in phosphate-buffered saline with tween 20 (PBST) for 1 h to inhibit non-specific antibody binding. Primary antibodies were pre-diluted to 1:200 in a blocking buffer for F4/80 (Santa Cruz Biotechnology, CA, USA) and HRH4 (Biorbyt, NC, USA) and applied to tissue sections overnight at 4 °C in a humidified chamber. The next day, Alexa-488 or Alexa-594 secondary antibodies were applied, and the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen, MA, USA) before mounting. Fluorescence signals were detected using a TCS SP8 confocal microscope (Leica, Wetzlar, Germany). All images were obtained under the same imaging parameters including laser power, detector gain, and image resolution.

RNA isolation and RT-PCR

Mice were euthanized 12 weeks after the STZ injection. The cornea, lens, and vitreous humor were removed, and the retina was obtained through a cut together with the residual pigment epithelium. Retinal RNA was solubilized in the TRIzol reagent (Invitrogen, MA, USA) and extracted according to the manufacturer’s instructions. The concentration and purity of extracted total RNA were determined using NanoDrop (Thermo Fisher Scientific, MA, USA). cDNA was synthesized from 1 μg of total RNA using Reverse Transcription kits (Enzynomics, Daejeon, Korea) following the manufacturer’s instructions. The targeted genes’ mRNA quantity were determined using real-time PCR analysis (CFX96; Bio-Rad, CA, USA) with SYBR Green qPCR premix (Enzynomics, Daejeon, Republic of Korea). The primer sequences were as follows: mouse HDC forward, 5′-GATGCCGGATCCCTTTGATG-3′; mouse HDC reverse, 5′-AGATTCTGGCCCGGAAGGAG-3′; mouse VEGF forward, 5′-ACTGGACCCTGGCTTTACTG-3′; mouse VEGF reverse, 5′-TCTGCTCTCCTTCTGTCGTG-3′; mouse IL-6 forward, 5′-TGGAGTCACAGAAGGAGTGGCTAAG-3′; mouse IL-6 reverse, 5′-TCTGACCACAGTGAGGAATGTCCAC-3′; mouse histamine H₁ receptor forward, 5′-CCAGAGCTTCGGGAAGATAA-3′; mouse histamine H₁ receptor reverse, 5′-ACCACAGCATGAGCAAAGTG-3′; mouse histamine H₂ receptor forward, 5′-CGTCATGGGAGCATTCATCG-3′; mouse histamine H₂ receptor reverse, 5′-GGTACGCCATGCGGAAGTCT-3′; mouse histamine H₃ receptor forward, 5′- CTGTCGCGGGACAAGAAGGT-3′; mouse histamine H₃ receptor reverse, 5′-TGGGTAGAGGACGGGGTTGA-3′; mouse histamine H₄ receptor forward, 5′-GGGTGGCTTGCAGGACAAGT-3′; mouse histamine H₄ receptor reverse, 5′-CTTTCCGATCGCCAGAAGGA-3′; mouse 18S rRNA forward, 5′-GCAATTATTCC CATGAACG-3′; and mouse 18S rRNA reverse, 5′-GGCCTCACTAAACCATCCAA-3′.

Flow cytometry

The anterior portion of the mouse eye were removed and the remaining tissue were enzymatically digested with collagenase type I (Sigma-Aldrich, St Louis, MO, USA) for 1 h in 37 °C with 50 units/mL DNase I (Sigma-Aldrich, St Louis, MO, USA) added to obtain single cells. Dissociated cells were filtered with cell strainer with 40 µm pore size to get single cells for the following antibody staining and washed with PBS. The cells were then resuspended with staining buffer (1% BSA, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.1% NaN₃ in PBS) and anti-mouse CD16/32 antibody (clone number 93) was pre-added to inhibit non-specific binding of immunoglobulins to macrophage Fc receptors. After washing and resuspension with staining buffer, cells were stained with the following antibodies for surface staining (4 °C, 30 min): anti-mouse CD45 (30-F11), F4/80 (BM8), CD11b (M1/70), CD3 (145-2C11), CD19 (1D3), Ly6C (HK1.4), HRH4 (Biorbyt, NC, USA), and Ly6G (1A8-Ly6g). All antibodies were obtained from eBioscience (CA, USA), unless indicated. For intracellular staining, cells were fixed and permeabilized with BD Perm/Wash buffer (BD Pharmingen, San Diego, CA, USA) and labeled with anti-VEGFA (C-1) (Santa Cruz Biotechnology, CA, USA) or IL-6 (MP5-20F3); subsequently, the processed cells were stained with the appropriate secondary antibodies. Data were acquired using LSR Fortessa (BD Biosciences, CA, USA) and analyzed using FlowJo software (version 10.8; FlowJo LCC, OR, USA).

Chemotaxis assay

After educating BMDMs by incubating with high glucose (25 mM) containing media for 48 h, the cells were treated with 10 μM JNJ (HRH4 antagonist) (R&D Systems, MN, USA) for 2 h. After treatment with HRH4 antagonist, BMDMs were washed once with PBS by centrifugation at 1800 rpm for 5 min at 4 °C and then re-suspended at a cell density of 5 × 10⁶ cells/mL in a pre-warmed chemotaxis buffer (37 °C). Sterile-filtered chemotaxis medium consisted of RPMI 1640 supplemented with 0.1% essentially fatty acid-free BSA (Sigma-Aldrich, St Louis, MO, USA). Chemotaxis medium containing 50 μM histamine (Sigma-Aldrich, St Louis, MO, USA) was added to the lower chambers of a 24-well Transwell^® plate (8 μm pore size, Corning Costar), and 200 μL of the above-described cell suspensions including 1 × 10⁶ cells were added to the upper chambers. The plate was then incubated for 6 h at 37 °C and under 5% CO₂ to allow chemotaxis. The assay was terminated by detaching the upper chambers from the plate. Migrated cells in the lower chambers were then fixed with 1% formalin in PBS for 10 min, rinsed with PBS, and then stained with DAPI (1 μg/mL in distilled water) for 10 min in dark environment. DAPI-stained nuclei were detected using a DMi6000B CS TCS SP5 Inverted Confocal Microscope (Leica, Wetzlar, Germany) and three images were taken per a well. ImageJ software was used to count the number of nuclei in each image.

Perfusion with FITC dextran

Mice were anesthetized using ketamine-xylazine (10 mg/kg; intraperitoneal injection) and 70 kDa FITC-dextran (Sigma-Aldrich, St Louis, MO, USA) (25 mg/mL in sterile PBS) was injected into the left ventricle. The tracer was allowed to circulate for 15 min, and the mouse eyes were enucleated and immediately fixed in 4% PFA for 15 min. Retina was dissected and flat-mounted on microscopic slides. A few drops of the anti-fluorescent quencher were added, and the retinas were mounted with coverslips. The mounted slides were kept in a black wet box at 4 °C and analyzed using a DMi6000B CS TCS SP5 Inverted Confocal Microscope (Leica, Wetzlar, Germany) at 100×magnification.

Hematoxylin and eosin (H&E) staining of the retina

Mice eyes were fixed in 4% paraformaldehyde neutral buffer solution (pH 7.4) for 24 h. Tissue was proceeded for successive serial dehydration, embedding, and slicing (5 μm) for tissue slide production, and hematoxylin and eosin (H&E) staining was carried out according to standard protocols³⁵.

Enzyme-linked immunosorbent assay (ELISA)

Blood was collected from mouse and incubated in room temperature for 1 h for subsequent centrifuge step to separate serum (3000g, 15 min, 4 °C). Collected serum was subjected to ELISA for VEGFA and IL-6 levels analysis using DuoSet ELISA (DY406-05; R&D Systems, MN, USA) according to the manufacturer’s protocol.

Quantification and statistical analyses

All statistical analyses were conducted using GraphPad Prism software (version 8.0). Data were presented as the mean ± standard errors of the mean. A P-value less than 0.1 was considered to indicate a significant difference (*P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001).

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00356146), the Korea Research Institute of Bioscience and Biotechnology Research Initiative Program (KGM4572323), and the K-Bio Health R&D Project, Ministry of Health & Welfare, Republic of Korea (HO16C0001).

Author contributions

Study conception and design: J.W.K., K.L., and S.H.S. Acquisition of data: J.W.K., J.P., and S.W.K. Analysis and interpretation of data: J.W.K., K.L., J.P., J.C., S.W.K., and S.H.S. Manuscript drafting: J.W.K., J.P. and S.W. Critical revision: J.J.H., K.L., S.H.S., J.C. and S.W.K.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Approval for animal experiments

The use of experimental animals in this study was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (accession number: SNU-190313-1-2). All procedures were conducted in accordance with the institutional animal care guidelines.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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