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. 2025 Sep 19;66(12):44. doi: 10.1167/iovs.66.12.44

Targeting RPE Senescence Via Suppressing IL-6/IL-6R Signaling for Treating Retinal Degenerative Diseases

Tian Zhou 1,2, Ziqi Yang 1,2, Biyan Ni 1,2, Hong Zhou 1,2, Yang Zhou 1,2, Huiyi Xu 1,2, Xiaojing Lin 1,2, Shiya Lin 1,2, Wei Yi 1,2, Chang He 1,2,✉, Xialin Liu 1,2,✉
PMCID: PMC12453063  PMID: 40970669

Abstract

Purpose

Progressive dysfunction of retinal pigment epithelium (RPE) cells is a crucial factor for retinal degeneration, leading to irreversible blindness with limited therapeutic options. Cellular senescence of RPE cells and inflammation are important hallmarks for retinal degeneration, but the underlying molecular mechanisms and potential interventions remain largely unexplored. This study aims to explore whether the IL-6/ IL-6R axis establishes a senescence-inducing circuit in RPE cells, and to evaluate the therapeutic efficacy of its inhibition in rescuing senescent RPE cells and degenerative retina.

Methods

Sodium iodate (NaIO₃)-induced retinal degeneration mouse models were established and subjected to intravitreal injections of IL-6 neutralizing antibody, or an IL-6R inhibitor tocilizumab, respectively. Conditional deletion of Stat3 in RPE cells was achieved via subretinal delivery of AAV vectors. RPE cells were isolated for single-cell RNA sequencing (scRNA-seq), qPCR, Western blotting, and immunofluorescence staining. Retinal structure and function were assessed using optical coherence tomography (OCT), hematoxylin and eosin (H&E) staining, and electroretinography (ERG).

Results

RPE underwent cellular senescence in NaIO3-induced degeneration, which was dependent on activation of the IL-6/IL-6R axis. IL-6 promoted the senescence of RPE and exacerbated retinal degeneration. In contrast, inhibition of IL-6 suppressed RPE senescence and facilitated recovery of retinal structure and function. Mechanistically, STAT3 activation was essential for IL-6-mediated cellular senescence. Notably, tocilizumab effectively blocked the IL-6/IL-6R/STAT3 signaling cascade, attenuated RPE senescence, and protected against retinal degeneration, expanding the indications of tocilizumab.

Conclusions

IL-6 and IL-6R/STAT3 signaling played an essential role in RPE senescence, and tocilizumab presents a translational opportunity in treating retinal degenerative diseases.

Keywords: IL-6, retinal pigment epithelium (RPE), senescence, tocilizumab, retinal degenerative diseases

Retinal degenerative diseases encompass a variety of disorders that lead to vision impairment, with age-related macular degeneration (AMD) being particularly significant due to the global trend of an aging population.1,2 As the prevalence of AMD is projected to rise exponentially, understanding the interplay between aging and AMD pathophysiology has become an urgent priority.3 The retinal pigment epithelium (RPE), a crucial monolayer of cells that supports the photoreceptors, is particularly vulnerable in retinal degenerative diseases. They would undergo functional impairment and degeneration under chronic stress conditions such as aging, oxidative stress, and so on. Mounting evidence suggests that RPE dysfunction serves as an early and central event in the pathogenesis of AMD, positioning it as a key cellular target for mechanistic investigation and therapeutic intervention.

RPE cells experience an accelerated cellular senescence process in response to oxidative stress, DNA damage, and mitochondrial reactive oxygen species during retinal degeneration.4 Senescent RPE cells are characterized by irreversible growth arrest and distinct biochemical and structural changes, including accumulation of lipofuscin granules, reduction in phagocytic capacity, and alterations in cytokine secretion.5 The senescence-associated secretory phenotype (SASP) of RPE cells including pro-inflammatory cytokines, chemokines, proteases, and other molecules creates a pro-inflammatory microenvironment within the retina, exacerbating oxidative stress, and promoting further degenerative changes in neighboring cells.6 Consequently, targeting RPE senescence may represent a promising therapeutic strategy for preventing retinal degenerative diseases. However, the specific mechanisms by which RPE cells undergo senescence remain poorly understood, hindering the development of targeted treatments.

The importance of chronic inflammation for cellular senescence is increasingly recognized and some inflammatory cytokines themselves constitute the SASP, a central hallmark of senescence. Although studies have identified the SASP components in various cells, the precise composition of SASP remains elusive and is the subject of ongoing research. SASP factors, including inflammatory cytokines, chemokines, and specific proteases, can reinforce and spread senescence through both autocrine and paracrine mechanisms,7,8 orchestrating a vicious cycle of inflammation and senescence. Among the SASP, IL-6, a classic pro-inflammatory cytokine, is notably elevated in the serum of patients with dry AMD.9,10 Here, we propose that IL-6 could form a senescence-inducing circuit to initiate and reinforce the cellular senescence of RPE cells.

In this study, we utilized the sodium iodate (NaIO3)-induced retinal degeneration mouse model, of which the degeneration progression is similar to that observed in patients with dry AMD.11 We found RPE experienced cellular senescence, with IL-6 emerging as a key component of SASP. Particularly, IL-6 reinforced the senescent state of RPE cells and could potentially orchestrate a pro-inflammatory circumstance within the retina, thereby accelerating the retinal degenerative process. Additionally, we utilized tocilizumab, the first US Food and Drug Administration (FDA)-approved monoclonal antibody that inhibits the IL-6 receptor, to target the IL-6/IL-6R/STAT3 pathway, which is crucial for RPE senescence. The significant protective effects of tocilizumab highlight its potential as a therapeutic approach for treating retinal degenerative diseases such as AMD.

METHODS

Animals

All the experiments were carried out in accordance with the ARVO Statement and followed the guidelines of Animal Care and Use Committee of Zhongshan Ophthalmic Center (Ethics ID: Z2023056). Eight- to 12-week-old mice with a male-to-female ratio of 1:1, including C57BL/6J (Gempharmatech Co., Ltd) and Stat3fl/fl mice (Jackson Laboratory, Strain: #016923) were used in this study. These mice were verified without rd8 mutation by PCR and DNA sequencing as reported12 (Supplementary Fig. S1). Mice received tail vein injections of NaIO₃ (20 mg/kg) or PBS as control,13,14 followed by immediate intravitreal injection of IL-6 Nab (Selleck, A2118) or tocilizumab (Selleck, A2012), each compared with their respective isotype controls (Selleck A2119 or A2051). A concentration of 1 µg/µL was used, determined as the lowest effective dose from preliminary testing. For conditional knockout, Stat3fl/fl mice received subretinal injections of 1.5 µL AAV2/8 vectors (EGFP or EGFP-Cre driven by the hVMD2 promoter; OBiO Technology) immediately after NaIO3 injection. Transduction efficiency was confirmed by immunofluorescence (Supplementary Fig. S2A). Eyes were collected 7 days later for analysis.

Single Cell RNA-Sequencing Preparation and Data Analysis

RPE single cells were prepared from eyes of six mice per group (PBS or NaIO3 treated C57BL/6J mice). After digestion in 2% dispase II for 30 minutes in 37°C, anterior structures with neural retina were removed. RPE cells were collected by gently tapping the eyecup, and the hexagonal pigmented cells were selected under microscopy. Single-cell RNA sequencing (scRNA-seq) libraries were generated using the Chromium Single Cell 30 Reagent Kits version 3 (10x Genomics) and sequenced on an Illumina HiSeg X Ten at 50K to 100K reads/cell. Reads were de-multiplexed and mapped to the mouse genome (mm10) using Cell Ranger (version 3.0.2). Downstream analysis was performed using R software (version 4.1.2) and Seurat (version 3.0). Cells with <200 genes or >15% mitochondrial UMI were excluded. Data were log-normalized, integrated, and scaled using Seurat. Clustering was visualized by tSNE and annotated by marker gene expression. Enrichment analysis (UCell, Singscore) were conducted according to the SenMayo gene set, which was used to reflect the degree of cellular senescence, or the Cellular Senescence gene set from Reactome (https://www.reactome.org/content/detail/R-MMU-2559583), and the mouse gene set of “HALLMARK_IL6_JAK_STAT3_SIGNALING” according to Gene Set Enrichment Analysis (GSEA). Pictures were further drawn by “irGSEA”. Transcriptomic differences of senescence-related cytokines/chemokines were statistically compared and analyzed using visualization tools of DotPlot. Our scRNA-seq data are accessible at Gene Expression Omnibus (GEO) under the accession number GSE270658 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE270658).

Electroretinogram Recordings and Optical Coherence Tomography

Electroretinogram (ERG) was performed as described in previous reports.15 The amplitudes of the a- and b-waves were measured using the Celeris-Diagnosys system. Optical coherence tomography (OCT) images were obtained using the SPECTRALIS-OCT (Heidelberg, Germany) with a mouse objective lens. The scanning and quantitative analysis was performed following the previous procedures16 to measure the thickness of outer nuclear layer (ONL).

Visual Acuity

Visual acuity in mice was assessed as previously described.14 The contrast was set at 100% with a grating of low spatial frequency (0.042 cycles per degree), and the tracking behavior of the mice was observed and recorded. The stimuli were then rotated anticlockwise to test the other eye. The acuity threshold was defined as the highest spatial frequency to which the mouse responded.

Retinal Fundus Imaging and Grading

Retinal fundus images were captured using the Micron IV fundus camera (Phoenix Research Labs, Pleasanton, CA, USA) with a mouse objective lens and a 50-degree field of view (1.8-mm diameter) centered on the optic cup. Retinal infiltrates and folds were counted as previously reported.14

Hematoxylin and Eosin Staining and Thickness Analysis

Hematoxylin and Eosin (H&E) staining was carried out and retinal morphology was evaluated as previously described.17 The number of melanin-rich aggregations in RPE layer was manually counted in a masked fashion in six fields/eye from at least three eyes.

ARPE-19 Cell Line Culture

The ARPE-19 cells (ScienCell, China) were treated with IL-6 (100 ng/mL, 216-16; PeproTech), combined with either IL-6 Nab (50 ng/mL; Selleck, A2118) or IgG1 isotype (50 ng/mL; Selleck, A2119) for 24 hours. Hydrogen peroxide (H2O2, 300 uM) stimulated ARPE-19 cells received either tocilizumab (3 µg/mL; Selleck, A2012) or isotype (3 µg/mL; Selleck, A2051) treatment for 24 hours. The cells were harvested for further analysis.

Quantitative PCR Analysis and Western Blotting

The qPCR of RPE-sclera-extracted RNA was performed as previously described.18 The sequences of the used primers are listed in Supplementary Table S1. The Western blotting was conducted following previous reports,17 and the primary antibodies are listed in Supplementary Table S2.

Immunofluorescence

The immunofluorescence on the RPE-scleral whole-mounts, cryosections, and ARPE-19 cells were carried out as previously described.14 The primary antibodies are listed in Supplementary Table S2. The SA-β-Gal staining was performed following the manufacturer's protocol (Cell Signaling Technology; #9860) after depigmentation steps, as previously reported,19 and the percentage of SA-β-Gal positive cells were analyzed using ImageJ software (National Institutes of Health [NIH], Bethesda, MD, USA). Confocal microscope images were captured using LSM980 (Carl Zeiss).

Statistics

Data were presented as mean ± standard error of mean (SEM) with sample sizes indicated in the figure legends. Normality was assessed using the Shapiro-Wilk test. For normally distributed data, unpaired Student's t-test or 1-way ANOVA with Tukey's post hoc test were used. For non-normal data, the Mann-Whitney U test or Kruskal–Wallis test with Dunn's post hoc test were applied. P values < 0.05 were considered statistically significant.

Results

RPE Undergoes Senescence in the NaIO3-Induced Retinal Degeneration Model

NaIO3-induced retinal degeneration mouse is a widely used model for investigating the pathological processes of retinal degeneration and evaluating potential therapeutic interventions.20,21 Here, single cell-RNA sequencing was performed by collecting RPE cells from the RPE-choroid complex of the NaIO3-treated and PBS-treated control mice. The majority of the cells were RPE cells, accompanied by a few microglia/macrophages, fibroblasts, photoreceptors, pericytes, and endothelium (Fig. 1A). Notably, a significant reduction of RPE cells was observed in the NaIO3 mouse group, whereas the amounts of microglia/macrophages and fibroblasts increased (see Fig. 1A). The RPE cells were further unbiasedly subdivided into four subgroups, with a markable increase in the R1 cluster in the NaIO₃ group (Fig. 1B). Interestingly, cellular senescence scores assessed using the SenMayo gene set22 (Fig. 1C) and the Cellular Senescence pathway from Reactome23 (Fig. 1D) revealed the highest senescence scores in the R1 cluster, indicating senescence in this subcluster. Expressions of senescence-associated-β-galactosidase (SA-β-Gal) and other senescence markers were analyzed. The SA-β-gal signaling was enriched along the RPE layer, and p16, p21, as well as p-p38 were elevated in the RPE-choroidal complex from NaIO3 mice (Figs. 1E, 1F), confirming cellular senescence in RPE cells during retinal degeneration. Then, the senescence-related cytokines/chemokines were analyzed from scRNA-seq. IL-6 and its receptor IL-6R were enriched in the R1 senescent cluster (Fig. 1G). In order to verify the elevation of IL-6/IL-6R in retinal degeneration, we determined their expressions by Western blotting and immunofluorescent staining. As expected, the IL-6 and IL-6R were significantly increased in the RPE-scleral complex from NaIO3-induced retinal degeneration mouse (Fig. 1H). IL-6R was co-stained with RPE cell markers (ZO-1 and Rpe65) in the retinal flat-mount and cryosection (Figs. 1I, 1J). Together, these results suggested that RPE experienced cellular senescence during NaIO3-induced retinal degeneration.

Figure 1.

Figure 1.

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A specific subtype of RPE cells experiences senescence in the NaIO3-induced retinal degeneration model. (A) Cell types and amounts of the collected RPE-choroid cells from NaIO3- and PBS-treated control mice in scRNA-seq. (B) The t-SNE plot depicting the separation of RPE cells into four sub-clusters, and bar graphs indicating the fold change of cell amount in each subpopulation. (C, D) Evaluation of senescent scores according to the SenMayo gene set (C) and the Cellular Senescence pathway from Reactome (D) by irGSEA analysis. (E). Representative images of SA-β-gal staining (blue) by retinal cryosection from PBS and NaIO3 treated mice. (F) Expression of senescent marker genes in the RPE-choroidal complex by Western blotting (n = 3). (G) Dot plot comparing the expression of typical senescence-related cytokines/chemokines in RPE subclusters from scRNA-seq. (H) Increased IL-6 and IL-6R expression in the NaIO3 treated RPE-scleral complex by Western blotting (n = 3). (I, J) Representative images of RPE flat-mount sheet (I) and frozen retinal section (J) showing IL-6R expression (red), and its co-staining with RPE cells indicated by ZO-1/ Rpe65 (green) staining. Scale bar = 20 µm. Data are shown as means ± SEM, *P < 0.05, **P < 0.01, unpaired Student's t-test.

IL-6 Promotes the Senescence of RPE and Accelerates Retinal Degeneration

IL-6 is not only an important part of SASP, but also may be a candidate for promoting cell senescence.24,25 In order to evaluate the role of IL-6 on cellular senescence of RPE, we cultured RPE cells in vitro and stimulated them with IL-6 cytokines. The in vitro system showed that IL-6 could significantly increase the activity of SA-β-Gal, and the expressions of typical senescent markers, including the p16, p21, p-p38, the serine/threonine kinase mTOR, and PAI-1 (Figs. 2A, 2B). Importantly, IL-6 neutralizing antibody (IL-6 Nab) treatment could nearly abrogate these high expressions (see Figs. 2A, 2B), indicating the requirement of IL-6 signaling for induction of RPE senescence.

Figure 2.

Figure 2.

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IL-6 stimulation accelerated the senescence of RPE cells and exacerbated retinal degeneration. (A) Representative figures of SA-β-gal staining in ARPE-19 cells treated by PBS, IL-6 + isotype, and IL-6 + IL-6 Nab for 24 hours. Scale bar = 100 µm. The percentage of SA-β-gal positive cells was calculated and compared (n = 14). (B) Western blotting analysis compared the expression of senescent markers following IL-6 incubation in vitro with isotype or IL-6 Nab treatment. (C) RPE flat-mount sheet revealing the expression of IL-6R (red) on RPE cells (ZO-1, green) under IL-6 stimulation and IL-6 Nab treatment. Scale bar = 20 µm. (D) SA-β-gal staining of PBS or NaIO3 treated mice receiving IL-6 Nab or isotype injection on retinal cryosection with the percentage of SA-β-gal positive cells (blue) calculated. Scale bar = 100 µm (n = 6). (E, F) expression of senescent markers from the isotype- or IL-6 Nab- treated NaIO3 RPE-scleral complexes by Western blotting (E) and qPCR analysis (F) (n = 4 in E and n = 5 in F). (G) RPE flat mount showing expression state of p21 (red) and ZO-1+ (green) under NaIO3 stimulation treated by isotype or IL-6 Nab. Scale bar = 50 µm. The amount of hexagonal ZO-1+ cells and percentage of p21 + ZO-1+ cells in comparison to total ZO-1+ cells were calculated (n = 7). (H) Discontinued RPE layer with highly reflective granules (red arrows) presented in the isotype treated NaIO3 model by OCT examination, with improvement in the IL-6 Nab treated group. The ONL thickness was calculated and compared (n = 6). (I) Representative figures of H&E staining showing ONL folds (yellow stars) and melanin rich aggregation (yellow arrows) in the RPE layer of the isotype-NaIO3-retina, particularly in the center and mid-peripheral locations. The IL-6 Nab treated group presented preserved RPE layer with less melanin rich aggregation and increased nucleus amount in the ONL layer (n = 6). Scale bar = 50 µm. (J) ERG showed the amplitudes of a- and b-waves after isotype or IL-6 Nab treated NaIO3 mice (n = 6). (K) Visual acuity response improved after IL-6 Nab treatment in comparison to the isotype treated mice from the optomotor experiment (n = 6). Data are shown as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, unpaired Student's t-test and Mann-Whitney U test in F, 1-way ANOVA test followed by Tukey's multiple comparisons test and Kruskal–Wallis test with Dunn's post hoc test in A, D, E, and G to K.

We also investigated the effect of IL-6 signaling in vivo. IL-6 Nab was injected intravitreally to the NaIO3-induced mice, and the isotype injected as the corresponding controls. The IL-6 Nab treatment could suppress the IL-6R expression in RPE cells and protect the barrier-damaged RPE cells with hexagonal-packaging ZO-1 recovery (Fig. 2C). Particularly, the IL-6 Nab injection inhibited the RPE cellular senescence with less SA-β-Gal activity along the RPE layer in NaIO3 mouse model (Fig. 2D) and a notable reduction of senescent markers, such as p16, p21, mTOR, Trp53, Angptl4, and so on (Figs. 2E, 2F, Supplementary Fig. S3). On flat mount RPE sheet, IL-6 inhibition decreased the amount of p21+ZO-1+ senescent RPE cells and remarkably rescued the barrier function of considerable hexagonal-packaging ZO-1 recovery in RPE cells (Fig. 2G).

From structural and functional analysis, we found RPE lesions displayed as aberrant hyper-reflective discrete foci on RPE band in the isotype-treated NaIO3-eyes as observed by OCT scan (Fig. 2H). In contrast, after IL-6 inhibition by the Nab, the RPE layer appeared smooth, continuous, and uniformly reflective, resembling that of the normal controls. In addition, the reduced thickness of ONL in NaIO3-isotype-induced retina was recovered after IL-6 Nab treatment (see Fig. 2H). Consistently, many crumby-structure patches (yellow arrows), possibly the pathologic aggregation of the melanin granules in the RPE cells, were observed in the NaIO3-retina in H&E staining. Besides, the discontinued degenerated RPE layer was usually associated with retinal folds (yellow stars) in the NaIO3-retina, an indication of photoreceptor damage.26 Notably, IL-6 Nab treatment largely preserved the well-organized RPE layer with increased amounts of nucleus in the ONL layers and reduced the aggregation of melanin granules and ONL folds, indicating a protective effect on overall retinal structure (Fig. 2I). Functionally, the amplitudes in both a and b waves increased significantly, and visual acuity improved after IL-6 Nab treatment (Figs. 2J, 2K). Taken together, IL-6 potentially promoted the senescence of RPE cells and accelerated the retinal degeneration process.

STAT3 Activation Is Required for IL-6 Induced RPE Senescence

STAT3 is a vital transcription factor activated downstream of IL-6 signaling.27 Here, we also found the highest score of IL6-JAK-STAT3 signaling gene set in the R1 subcluster of RPE cells determined by scRNA-seq data, indicating the potential attribution of STAT3 in the R1 senescence (Fig. 3A). Western blotting results demonstrated the substantial STAT3 activation in the isotype group and its abolishment after IL-6 Nab treatment (Fig. 3B). To explore whether STAT3 activation mediated the IL-6-induced RPE senescence during the NaIO3-induced retinal degeneration, RPE-cre AAV was injected subretinally in the Stat3fl/fl mice to generate conditional knockout mice that lack STAT3 specifically in the RPE. Western blotting analysis confirmed the decreased p-STAT3 and STAT3 expression in RPE cells after RPE-cre AAV injection (see Supplementary Fig. S2B). Notably, the mice with Stat3-CKO from RPE exhibited much less expressions of senescent genes in RPE cells, including p16, p21, mTOR, PAI-1, p-p38, and Histone H2A (Figs. 3C, 3D), indicating STAT3 activation was required for RPE senescence during the NaIO3-induced retinal degeneration. Particularly, these mice presented with partially rescued RPE phenotypes with more regular hexagonal-packaging morphology (see Fig. 3D).

Figure 3.

Figure 3.

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IL-6 induced RPE senescence via STAT3 activation. (A) Half-violin plot presenting the expression of IL6-JAK-STAT3 signaling scores among different RPE subclusters by irGSEA analysis. (B) The increase of p-STAT3 expression induced by NaIO3 stimulation were diminished after IL-6 Nab treatment from Western blotting analysis (n = 4). (C) In Stat3 fl/fl mice, the conditional knockout of Stat3 in RPE cells via cre-AAV decreased the abnormal elevation of senescent markers induced by NaIO3, as observed through Western blotting (n = 3). (D) Expression patterns of senescent markers (red) and RPE integrity (green) on immunofluorescent RPE flat-mounts from vector AAV or RPE-cre AAV treated Stat3fl/fl mice under NaIO3 stimulation. Scale bar = 20 µm. The amount of hexagonal ZO-1+ cells and percentage of p21 + ZO-1+, p-p38 + ZO-1+, mTOR + ZO-1+, and Histone H2A + ZO-1+ cells in comparison to total ZO-1+ cells were calculated (n = 7). Data are shown as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, 1-way ANOVA test followed by Tukey's multiple comparisons test and Kruskal-Wallis test followed by Dunn's multiple comparisons test were used in B to D.

Consistently, pale-colored retinas with dot-like infiltrates and retinal folds were observed by fundoscopy in Vector AAV-treated NaIO3 retinas, whereas RPE-cre AAV treatment significantly alleviated these pathological changes (Fig. 4A). The hyper-reflective foci along the RPE layer in OCT scans were reduced after RPE-cre AAV treatment, which group displayed a smooth, continuous, and uniformly reflective RPE band (Fig. 4B). The thickness of the ONL was also increased, suggesting the recovery of photoreceptors (see Fig. 4B). From histological analysis, crumby structure patches and melanin-rich aggregation dots in NaIO3 mice were significantly decreased after RPE-cre AAV treatment (Fig. 4C). Functionally, the mice with Stat3-CKO from RPE had active waves with elevated amplitudes of both the a-wave and b-wave in ERG examination and improved responses in visual acuity measurement in comparison to the vector AAV controls (Figs. 4D, 4E). These results indicated that STAT3 mediated the IL-6-induced RPE senescence in NaIO3-induced retinal degeneration mouse model.

Figure 4.

Figure 4.

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STAT3 activation in RPE cells promoted retinal degeneration. (A) The fundoscopy of vector AAV treated Stat3fl/fl mice after NaIO3 induction was characterized by obvious structural damage with pale appearance and some dot-like or linear lesions, which were alleviated after RPE-cre AAV treatment (n = 6). (B) Representative OCT images from vector AAV or RPE-cre AAV treated NaIO3-Stat3fl/fl mice. In the vector group, the RPE layer showed discontinuities with high-reflective deposits (red arrows). In contrast, the RPE-cre AAV treated group exhibited a smooth, continuous, and uniform reflectiveness RPE layer. The ONL thickness (the distance between the yellow lines) was measured and compared (n = 6). (C) H&E staining displayed retinal folds (yellow stars), crumby structure patches and melanin rich aggregation (yellow arrows) of the vector AAV treated NaIO3-retina, whereas RPE-cre AAV treatment relieved these structural damages (n = 6). Scale bar = 50 µm. (D) As ERG examinations shown, a- and b-waves were almost extinct in vector AAV treated NaIO3- Stat3fl/fl retina, whereas both amplitudes increased after RPE-cre AAV treatment (n = 6). (E) The optomotor experiment demonstrated improved visual acuity response after RPE-cre AAV treatment in comparison to the vector AAV treated NaIO3- Stat3fl/fl mice (n = 10). Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, unpaired Student's t-test.

Tocilizumab Could Suppress the IL-6R/STAT3 Activation and Prevent RPE Cells From Senescence

Next, we intended to evaluate the effects of tocilizumab, the first FDA-approved IL-6R neutralizing antibody, in the NaIO3-induced retinal degeneration mouse model. As expected, tocilizumab treatment suppressed the activation of IL-6R and STAT3 pathway in the RPE-choroid complex from NaIO3 mice (Fig. 5A). On the RPE-sclera flat-mount, tocilizumab also decreased the expression of IL-6R co-staining with ZO-1, as well as the level of p-STAT3 in NaIO3-RPE (Fig. 5B). In vitro, H2O2 was utilized to stimulate the RPE cells. Tocilizumab treatment could also suppress the elevated expressions of p-STAT3 and IL-6R under H2O2 insult (Figs. 5C, 5D). These data suggested that tocilizumab could target on IL-6R receptor and suppress STAT3 activation in RPE cells.

Figure 5.

Figure 5.

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Tocilizumab suppressed the IL-6R/STAT3 activation in RPE cells in vivo and in vitro. (A) Tocilizumab (TCZ) treatment induced decrease of p-STAT3 and IL-6R expression in NaIO3 model by Western blotting (n = 3). (B) Representative images of RPE flat-mount sheet revealing IL-6R (blue) and p-STAT3 (red) decreased close to normal control after tocilizumab treatment, with recovered hexagonal tight junction structures (green). Scale bar = 20 µm. (C, D) Expression of p-STAT3 and IL-6R in ARPE-19 cells after H2O2 induction and tocilizumab treatment by Western blotting (n = 3), (C) and immunofluorescence (D). Scale bar = 50 µm. Data are shown as means ± SEM, *P < 0.05, **P < 0.01, 1-way ANOVA test followed by Tukey's multiple comparisons test.

To further explore whether tocilizumab could prevent the senescent processes in RPE cells, the expressions of cellular senescence markers were examined. In vitro, ARPE-19 cells stimulated by H2O2 or IL-6 displayed increased activity of SA-β-Gal than cells in PBS control, which was abolished by tocilizumab treatment (Fig. 6A, Supplementary Fig. S4). Other senescent markers including p21, mTOR, p-p38, and PAI-1 were elevated by H2O2 stress, but were abrogated after tocilizumab treatment (Figs. 6B, 6C). As shown in Figure 6D, the NaIO3-induced mice with tocilizumab treatment were characterized by significant reductions of p21, p-p38, Histone H2A, and mTOR expressions, as well as barrier recovery with more hexagonal-packaging ZO-1+ RPE cells. The Western blotting data showed that tocilizumab treatment suppressed the activation of p21, p16, and p-p38 signaling (Fig. 6E). In tocilizumab-treated mice, a noticeable reduction in SA-β-gal–positive cells were also observed in the subretinal space of compared with controls (Fig. 6F). These in vivo data confirmed that tocilizumab treatment could protect the RPE cells from cellular senescence.

Figure 6.

Figure 6.

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Tocilizumab alleviated the senescence of RPE cells. (A) Representative SA-β-gal staining figures of ARPE-19 cells under H2O2 stimulation. (B, C) Immunofluorescence (B) and Western blotting analysis (C) measuring the expression state of senescent markers of cultured ARPE-19 cells under oxidative stress insults in the presence or absence of tocilizumab treatment. (D, E) Detection of senescent markers in NaIO3 treated RPE flat mount under Isotype or tocilizumab treatment by immunofluorescence on RPE flat-mount (D) and Western blotting (E) of the RPE-choroidal complex (n = 3). (F) SA-β-gal staining of PBS or NaIO3 treated mice receiving intravitreal tocilizumab or isotype injection on retinal cryosection. The percentage of SA-β-gal positive cells were measured by ImageJ software (n = 6). Scale bar = 50 µm. Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, 1-way ANOVA test followed by Tukey's multiple comparisons test.

Intravitreal Injection of Tocilizumab Protects Against the NaIO3-Induced Retinal Degeneration

Finally, the effects of tocilizumab on NaIO3-induced retinal degeneration were investigated. Notably, the intravitreal injection of tocilizumab suppressed the presence of hyper-reflective foci in the RPE layer and increased the thickness of the ONL by OCT examination (Figs. 7A, 7B), indicating the protective role of tocilizumab on RPE and photoreceptors. The H&E images revealed discontinuous RPE structures and retinal folds and reduced nucleus amounts in the ONL in isotype-treated NaIO3 mice, which were inhibited by tocilizumab treatment (Fig. 7C). From ERG examination, we found that the tocilizumab treated group showed significantly elevated amplitudes of a-wave and b-wave compared with the isotype-treated NaIO3 mice, whereas the retinal function could not be restored to a normal level as the PBS mice (Fig. 7D). The measurement of visual acuity also demonstrated improved responses after tocilizumab treatment (Fig. 7E). In addition, the amount of infiltrated of Iba1+ cells also decreased after IL-6R blockade, with the retinal IL-6 level decreased, suggesting this treatment would concomitantly dampen the inflammatory response (Supplementary Fig. S5). Moreover, intravitreal injection of tocilizumab showed little effect on the weight of the kidneys, liver, and spleen (Supplementary Figs. S6A–C), and with no obvious infiltrated lymphocytes in liver (Supplementary Fig. S6D). These results suggested that intravitreal injection of tocilizumab could be a safe and effective approach for suppressing retinal degenerative diseases.

Figure 7.

Figure 7.

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Tocilizumab protected neuroretina structure and function in the NaIO3-induced retinal degenerative mice. (A, B) In OCT examination, isotype treated NaIO3 mice presented discontinuity of RPE layer with highly reflective granules (red arrows). Importantly, the calculated ONL thickness (the distance between the yellow lines) was reduced in the isotype treated NaIO3 model. Tocilizumab treatment could partially restore the ONL thickness in NaIO3 model (n = 6). (C) H&E staining revealed melanin rich aggregation (yellow arrows) and ONL folds (yellow stars) accumulated in the NaIO3-isotype treated RPE. Tocilizumab treatment rescued RPE injuries with much less melanin rich aggregation and ONL folds, whereas the amount of nucleus increased in the ONL layer. Scale bar = 20 µm (n = 3). (D) ERG showed increased amplitudes of a- and b-waves after tocilizumab treatment. The isotype treated NaIO3 mice presented almost extinct waves (n = 6). (E) The optomotor experiment demonstrated improved visual acuity response after tocilizumab treatment in comparison to the isotype treated NaIO3 mice (n = 6). Data are shown as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, 1-way ANOVA test followed by Tukey's multiple comparisons test.

Discussion

Chronic inflammation is an important hallmark of retinal degenerative diseases that are intimately linked with RPE senescence. In this study, we identified a population of senescent RPE cells characterized by a strong IL-6/IL-6R signature by scRNA-seq. Furthermore, we demonstrated that IL-6, the classic pro-inflammatory factor of the SASP, could itself induce RPE cell senescence by activating the IL-6R and subsequently the STAT3 pathway. This created a vicious cycle of inflammation and senescence, leading to irreversible RPE degeneration and atrophy. Furthermore, tocilizumab, the first approved IL-6 receptor neutralizing antibody,28 not only protected RPE cells from senescence, but also preserved neuroretinal function in the NaIO3-induced retinal degeneration mouse model. This underscores tocilizumab’s therapeutic potential and additional functions beyond its anti-inflammatory effects.

Single-cell RNA sequencing identified a senescent RPE subpopulation with high IL-6 and IL-6R expression, directly implicating the IL-6/IL-6R axis as a key component of the SASP response in RPE. In parallel, NaIO₃-induced injury induced a relative increase in microglia/macrophages and fibroblast-like cells, indicating local retinal microenvironment remodeling. The accumulation of immune and stromal cells may amplify inflammation and fibrosis, exacerbating neuroretinal damage. These findings support a model in which IL-6-driven RPE senescence promotes both intrinsic degeneration and paracrine-mediated disruption of retinal homeostasis.

Targeting cellular senescence has been attempted to develop drugs to treat age-related diseases. The senolytics, which selectively eliminate senescent cells, have shown some benefits in preclinical studies as well as small clinical trials such as Alzheimer’s Disease and cartilage degeneration.29,30 The senomorphics, which suppress the SASP and other markers of senescence, are being developed to reduce age-associated pathologies and promote the lifespan.31 In this study, we found that targeting IL-6, a classic pro-inflammatory cytokine and a critical key of SASP, could prevent the RPE cell from senescence, leading to inhibition of SASP propagation and inflammatory vicious cycle. Therefore, IL-6 turns to play a crucial role in multiple aspects of RPE cell senescence, and locally targeting IL-6 signaling could suppress RPE cell senescence unlike conventional senolytics or senomorphics.

IL-6 activates classical signaling by binding to membrane-bound IL-6R (mIL-6R), which predominantly expressed on the surface of certain cell types, including hepatocytes, monocytes, macrophages, neutrophils, and some subsets of T cells. The soluble form of IL-6R (sIL-6R), generated by proteolytic cleavage of mIL-6R or alternative splicing, can bind IL-6 in the circulation and interact with gp130, which is ubiquitously expressed on various cell types, to initiate trans-signaling. Recent studies suggested that some types of epithelial cells also expressed mIL-6R.32 In our study, IL-6R expression was significantly upregulated in RPE cells, suggesting that IL-6 may activate classical signaling via membrane-bound IL-6R in this context.33 It is of interest to further investigate the downstream pathways of IL-6/mIL-6R on RPE senescence.

Tocilizumab, the first FDA-approved monoclonal antibody targeting IL-6R, blocks all three IL-6 signaling modes: classic, trans-signaling, and trans-presentation. Although primarily approved for systemic inflammatory diseases, such as rheumatoid arthritis (RA), giant cell arteritis, and cytokine release syndrome,28 Tocilizumab has also shown promise in off-label treatment of refractory ocular inflammation, including RA-associated scleritis and juvenile idiopathic arthritis (JIA)-associated uveitis. Here, we demonstrate for the first time that tocilizumab attenuates RPE cell senescence in a retinal degeneration model, suggesting its potential as a targeted senotherapeutic beyond its anti-inflammatory role. This expands the clinical relevance of tocilizumab to age-related retinal diseases.

Systemic tocilizumab administration carries risks such as hypertension, liver enzyme elevation, cytopenias, and serious infections. Attempts to reduce toxicity via subcutaneous dosing have shown limited success.34 In contrast, our study shows that intravitreal injection achieves local efficacy with minimal complications in vivo, supporting a safer and more efficient delivery route. Interestingly, tocilizumab reduced IL-6R protein levels in RPE but not in the retina, possibly due to receptor internalization, degradation, or negative feedback, although further investigation is needed. In addition, other FDA-approved agents targeting IL-6/IL-6R/STAT3 pathway, such as Sarilumab or Bazedoxifene, might offer similar benefits, although their efficacy and long-term effects require further clinical evaluation.

In conclusion, our study provided evidence that IL-6 is not only a classic effector of cellular senescence but also a potent inducer of RPE senescence in retinal degeneration mouse models. IL-6 activates mIL-6Ra on RPE cells, triggering STAT3 phosphorylation and initiating classic signaling. By targeting this signaling, tocilizumab protects senescent RPE cells and preserves neuroretinal function during retinal degeneration. These findings underscore the therapeutic potential of tocilizumab in treating AMD, offering a promising new approach for managing this condition.

Supplementary Material

Supplement 1
iovs-66-12-44_s001.docx (8.6MB, docx)

Acknowledgments

The authors thank the staff of the Animal Center and Core Facilities at State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, for their technical support.

Supported by the National Natural Science Foundation of China (Nos. 82322016, 82171068, and 82271095), the Guangdong Provincial Key Area R&D Program (No. 2023B1111050004), the Fundamental Research Funds of SYSU (No. 24pnpy242), and the Natural Science Foundation of Guangdong Province, China (No. 2023A1515012656). Guangzhou Science and Technology Plan Project (2025A03J3981, 2024B01J1121, and 2025A03J3988), and GBRCE for Major Blinding Eye Diseases Prevention and Treatment.

Disclosure: T. Zhou, None; Z. Yang, None; B. Ni, None; H. Zhou, None; Y. Zhou, None; H. Xu, None; X. Lin, None; S. Lin, None; W. Yi, None; C. He, None; X. Liu, None

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Supplementary Materials

Supplement 1
iovs-66-12-44_s001.docx (8.6MB, docx)

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