Ni et al. Journal of Neuroinflammation(2024) 21:164
https://doi.org/10.1186/s12974-024-03155-y

Journal of Neuroinflammation

Open Access

RESEARCH

Therapeutic intervention
in neuroinflammation for neovascular oculardiseases through targeting the cGAS-STINGnecroptosis pathway
Biyan Ni1,2†, Ziqi Yang1,2†, Tian Zhou1,2†, Hong Zhou1,2, Yang Zhou1,2, Shiya Lin1,2, Huiyi Xu1,2, Xiaojing Lin1,2, Wei Yi1,2,
Chang He1,2* and Xialin Liu1,2*
Abstract
The microglia-mediated neuroinflammation have been shown to play a crucial role in the ocular pathologicalangiogenesis process, but specific immunotherapies for neovascular ocular diseases are still lacking. This study
proposed that targeting GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) might be a novel
immunotherapy for these angiogenesis diseases. We found a significant upregulation of CGAS and STING genes in
the RNA-seq data derived from retinal tissues of the patients with proliferative diabetic retinopathy. In experimental
models of ocular angiogenesis including laser-induced choroidal neovascularization (CNV) and oxygen-induced
retinopathy (OIR), the cGAS-STING pathway was activated as angiogenesis progressed. Either genetic deletion or
pharmacological inhibition of STING resulted in a remarkable suppression of neovascularization in both models.
Furthermore, cGAS-STING signaling was specifically activated in myeloid cells, triggering the subsequent RIP1RIP3-MLKL pathway activation and leading to necroptosis-mediated inflammation. Notably, targeted inhibitionof the cGAS-STING pathway with C-176 or SN-011 could significantly suppress pathological angiogenesis in CNV
and OIR. Additionally, the combination of C-176 or SN-011 with anti-VEGF therapy led to least angiogenesis,
markedly enhancing the anti-angiogenic effectiveness. Together, our findings provide compelling evidence forthe importance of the cGAS-STING-necroptosis axis in pathological angiogenesis, highlighting its potential as apromising immunotherapeutic target for treating neovascular ocular diseases.
Keywords Pathological angiogenesis, STING, Myeloid cells, Necroptosis, Neuroinflammation, Immunotherapy,
Neovascular ocular diseases†

Biyan Ni, Ziqi Yang and Tian Zhou contributed equally to this work.

*Correspondence:
Chang He
hech33@mail.sysu.edu.cn
Xialin Liu
liuxl28@mail.sysu.edu.cn
1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,
Sun Yat-sen University, Guangzhou 510060, China
2
Guangdong Provincial Key Laboratory of Ophthalmology and Visual
Science, Guangzhou 510060, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Ni et al. Journal of Neuroinflammation(2024) 21:164

Introduction
Pathological angiogenesis is a prevalent feature in various human neovascular ocular diseases, including diabetic retinopathy (DR), neovascular age-related maculardiseases (nAMD), etc [1]. During this pathological process, the myeloid cells including microglia and infiltrating
monocyte-derived macrophages are activated, inducing neuroinflammation in the retina and choroid. These
immune cells release pro-inflammatory factors and othermolecules that stimulate endothelial cell proliferationand migration, consequently leading to the formation ofabnormal neovessels [2]. Steroids, employed as non-specific anti-inflammatory therapies, have been widely used
to treat neovascular ocular diseases [3]. However, theirefficacy is limited with risks of potential side effects [4].
Therefore, mechanistic investigations into the immuneinflammatory pathways involved in angiogenesis and theidentification of potential specific immunotherapeutictargets are highly warranted.
The cGAS-STING pathway has recently emerged asa critical innate immune signaling mechanism in hostdefense. cGAS primarily recognizes cytosolic DNA,
particularly pathogenic DNA from invading viruses andbacteria, and triggers downstream IRF3/IRF7 or NF-κB
signaling, resulting in the production of type I interferons and other inflammatory cytokines [5, 6]. Unravelingthe potential implications of the cGAS-STING pathway
beyond infection could offer valuable insights into immunotherapy for various conditions, ranging from immune
disorders, metabolic ailments, and neural diseases tocancer. In the context of the retina, the activation of thecGAS-STING pathway has been linked to inflammationand degeneration [7]. The inhibition of cGAS-STING
pathway alleviates neuroinflammation-induced retinal
ganglion cell death after ischemia/reperfusion injury[8]. The cGAS-STING pathway contributes to the development of RPE senescence [9]. Ma et al. reported that
PPARα could regulate cGAS-STING pathway activationin the oxygen-induced retinopathy (OIR) model [10],highlighting its potential impact on inflammation-associated angiogenesis in the retina. However, the specificmechanisms regarding to the downstream of cGASSTING pathway and the intervention strategies within
the eye remain elusive.
In this study, our analysis of a published retinal RNAseq dataset revealed significant upregulation of cGAS and
STING expression in patients with proliferative diabetic
retinopathy (PDR). Further investigations in two wellestablished models of retinal/choroidal angiogenesis in
Sting deficiency mice, laser-induced choroidal neovascularization (CNV) and OIR, demonstrated the crucialrole of STING activity in retinal/choroidal angiogenesis.
Targeting cGAS-STING pathway may be a novel immunotherapy approach for neovascular ocular diseases, andPage 2 of 14

combination of STING inhibition and anti-VEGF drugs
holds better treatment prospects.

Methods
Patients’ dataset and data analysis

A published RNA-seq data (GSE160306) of retinal tissues
from healthy controls, diabetic patients, patients with
non-proliferative diabetic retinopathy (NPDR), and PDR
patients was downloaded and re-analyzed for screening
the regulatory factors involved [11]. The data of macular and peripheral retinal tissues were used for analysis.
DESeq2 (v1.30.1) was applied for identification of differentially expressed genes (DEGs) with the criteria of absolute fold change > 2 and adjusted P-value < 0.05. All the
DEGs were used to conduct Gene Ontology (GO) enrichment analysis by clusterProfiler (v4.0.2). GO terms withadjusted P-value < 0.05 were considered significantly.
Gene set enrichment analysis (GSEA) was conductedusing the GSEA software (http://www.gsea-msigdb.org/
gsea/index.jsp) with 1000 times gene-set permutations
and cutoff values of FDR < 0.25. A power analysis was
performed and statistical analysis was conducted using
one-way ANOVA with Turkey’s post hoc test for comparisons of these four groups. Pearson’s correlation test was
employed for correlation analysis between the expressionof STING and the average expression from GO terms.
CNV and OIR mouse models

All animal experiments were approved by Institutional
Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University (Ethic ID: 2020 − 102).
The Stinggt (#017537) mice were obtained from JacksonLaboratory and C57BL/6J mice were purchased from
GemPharmatech Company. CNV model was establishedas previously described [12]. Briefly, laser photocoagulation was performed using an argon laser (Lumenis,
Inc., Santa Clara, CA, USA) with a center wavelengthof 532 nm, an incident power of 200mW, a spot size of
100 μm, and a pulse duration of 100ms to induce ruptureof Bruch’s membrane. Intravitreal injections were performed using a 5-µL Hamilton syringe with a 33-gauge
needle. A concentration gradient of STING inhibitors
and activator was established to determine the optimal
dosage. The following concentrations were set for theexperiments: C-176 at 1 mM, 5 mM, and 25 mM; SN-011
at 0.8 mM, 4 mM, and 20 mM; and diABZI at 0.1 mM,
0.5 mM, and 2.5 mM. Fundus photography and fluorescein angiography by intraperitoneal injection of 2% fluorescein sodium solution (Alcon laboratories, TX, USA)
(5 µl/g) were captured using the Micron IV retinal imaging system (Phoenix Research Laboratories, Pleasanton,
CA, USA). OIR model was established as previously
described [13]. These mice received a single intravitreal

Ni et al. Journal of Neuroinflammation(2024) 21:164

injection of STING inhibitors and activator at P12 andthe extent of retinal angiogenesis was determined at P17.
Cell enrichment of myeloid cells

Myeloid cells from retinae or choroidal-scleral complexes
were isolated using the EasySep CD11b + Cell Isolation
Kit (STEMCELL, Canada). Tissues (n = 5–7) were initially
isolated on ice, mechanically fragmented, and digested
in a solution containing 25 µl papain (5 µl/ml, Worthington) and 35 µl Dnase (7 µl/ml, Sigma-Aldich) at 35 °C for
8 min. The resulting single-cell suspension underwent
EasySep Cell Isolation Kit protocol, including cell labeling, antibody incubation, magnetic bead binding, and cell
separation. A purified CD11b + myeloid cell population(> 85% purity) was obtained for subsequent experiments(Fig. S1).
Single-cell RNA sequencing data analysis

The scRNA-seq data for the retinae of control miceand OIR mice was retrieved from the GEO database(GSE152928) [13]. The filtering, clustering, and subclustering analysis were performed as described previously[13]. We further scored all subtypes of microglia using
the irGSEA package (v1.1.2) for pathways such as ROS,
IFN-α, Angiogenesis, and generated a graphical representation of the scores.
Cell culture and treatment

Bv2, N9, Raw264.7 or bEnd.3 cells were cultured in
DMEM/F12 containing 10% fetal bovine serum and 1%
Penicillin-Streptomycin (Invitrogen). Hypoxic condition
was produced in a Forma 3111 Series II Water Jacketed
Incubator (Thermo Fisher Scientific; Waltham, MA,
USA) with 5% CO2, 1.5% O2, and balance nitrogen gas.
The cells were pre-stimulated with either C-176 (10µM)
or vehicle before cultured in hypoxic condition for 24 h.
Quantitative PCR analysis and western blotting

The qPCR was performed as previously described[14]. The 20 µl reaction mix included 2 µl cDNA, 10 µl
2×SYBR Premix Ex Taq, 7 µl ddH2O, and 10 µmol/lof primer pairs. Roche LightCycler480 II was used for
qPCR, sequences of the primers used in this study werelisted in the supplementary Table 1. The western blotting
was performed as previously described [14], and the primary antibodies were listed in the supplementary Table
2.
Immunofluorescence and hematoxylin & eosin staining

The immunofluorescence on whole-mounts and cryosections were carried out as previously described [13].
The primary antibodies were listed in the supplementary Table 2. The TUNEL assay was performed following the manufacturer’s protocol (Roche Diagnostics,

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Indianapolis, IN, USA). Confocal microscope images
were captured using LSM980 (Carl Zeiss). H&E staining was conducted and the thickness of CNV lesions wasmeasured as previously described [12, 13].
Statistical analysis

The representative results were showed in the figures.
Data quantification was presented as mean ± standard
error of measurement (SEM). Statistical analysis was
conducted using one-way ANOVA with Turkey’s post
hoc test for comparisons of three or more groups, or a
2-tailed Student t-test for two-group comparisons. A
p-value < 0.05 was considered statistically significant.
(*p < 0.05; **p < 0.01; ***p < 0.001).

Results
cGAS-STING signaling involved in the development of PDR

To investigate the regulatory factors involved in thepathological process of retinal neovascularization, we
downloaded and re-analyzed the published RNA-seq
datasets of human retina [11]. Generally, the differentially expressed genes were enriched in the late DRstage of PDR but not other stages of diabetes and NPDR
(Fig. 1A), which was coincident with original study byBeker et al. [11]. Specifically, we focused on the differentially expressed genes in the PDR stage. The GO analysis revealed that the upregulated genes in PDR mainly
involved in the myeloid leukocyte activation, regulationof inflammatory response, innate immune response, type
I interferon (IFN) production, and so on (Fig. 1B). In addition, GSEA analysis also showed genes related to positive
regulation of type I IFN production and viral induced
cytoplasmic pattern recognition receptor signaling pathway were up-regulated in the PDR patients comparedwith healthy controls (Fig. 1C). Furthermore, the expressions of CGAS and STING, key molecules of cytoplasmic DNA sensing pathway that induces the productionof type I IFN and activates the innate immune system,
were significantly elevated in PDR patients compared toothers, whereas no significant difference was observed
between diabetic or NPDR patients and healthy controls(Fig. 1D). Then the relationships between STING and theangiogenesis or inflammatory responses were analyzed.
The expression of STING is positively correlated to genes
associated with angiogenesis or inflammatory responses(Fig. 1E), indicating the possible involvement of STING
signaling in the angiogenic process.
cGAS-STING pathway was activated in two models of
retinal/choroidal angiogenesis

Next, we examined the cGAS-STING pathway in twoclassical models of retinal/choroidal angiogenesis, theCNV and OIR models (Fig. 2A). The laser-induced
CNV is a self-limiting model of angiogenesis, with

Ni et al. Journal of Neuroinflammation(2024) 21:164

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Fig. 1 cGAS and STING was upregulated in the PDR patients. A The RNA-seq data was obtained from a published dataset (GSE160306) [11]. Comparedto the Healthy control, there were 68 downregulated and 47 upregulated DEGs in the Diabetic group, 12 downregulated and 5 upregulated DEGs in the
NPDR group, and 314 downregulated and 1369 upregulated DEGs in the PDR group. B Differentially expressed genes in retinae from PDR (n = 5 samples)and healthy control (n = 20 samples) were analyzed. The GO analysis of biological processes revealed that the differentially expressed genes in PDR
retinae were enriched in myeloid leukocyte activation, regulation of inflammatory response, and other processes. C The GSEA profile showed that genesets associated with the positive regulation of type I interferon production and the regulation of viral induced cytoplasmic pattern recognition receptor
signaling pathway were upregulated in PDR retinae. D The expressions of CGAS and STING were markedly increased in the retinae from PDR patients (n = 5
samples) compared to healthy controls (n = 20 samples), diabetic (n = 20 samples) and NPDR patients (n = 19 samples). Power analysis was conducted toevaluate the reliability of detecting expression differences among the four groups, yielding a 99% power for STING and 96% power for CGAS. These highlevels of statistical power provide confidence in the robustness of these comparisons using one-way ANVOA with Turkey’s post hoc test. E Linear regression analysis showed that the expression of STING was positively correlated with the up-regulated genes associated with angiogenesis and inflammatory
responses in PDR patients (n = 5 samples). Data are shown as mean ± SEM. **P < 0.01; ***P < 0.001

Ni et al. Journal of Neuroinflammation(2024) 21:164

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Fig. 2 cGAS-STING pathway was significantly activated in CNV and OIR mice. A The schematic diagram illustrates the modeling process of CNV and
OIR. B Western blot results display the expression changes of cGAS-STING pathway proteins, including cGAS, p-STING, TBK1 and p-TBK1 in CNV mousechoroids. The pathway proteins exhibit an initial increase, peak at D3, and gradually decrease thereafter. C The cGAS-STING pathway including p-γH2A.X,
STING, TBK1 and their phosphorylation forms were detected on D7 (n = 5 choroid-sclera complexes). The statistical results were depicted. D The expressions of cGAS, STING, TBK1 and their phosphorylation forms were also examined in the OIR-retinae at various time points. E The cGAS-STING pathway was
further detected on P17 (n = 5 retinae). The statistical results were depicted. D, day; P, postnatal day. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01;***P < 0.001

lesions peaking at D7 and then gradually regressing [15].
Another classical angiogenesis model, OIR, demonstrates
the development of retinal neovascular tufts from P12
to P17, reaching their peak at P17, followed by a gradualregression of retinal angiogenic tufts [16]. As illustrated
in Fig. 2B, the levels of cGAS and phosphorylated-STING(p-STING) in the choroid-scleral complex were notably up-regulated on D3 post laser administration andgradually declined thereafter, but still be elevated on D7.

The downstream molecules, TBK1 and IRF7 also exhibited heightened activation on D3 and D7 (Fig. 2B). The
expressions of p-γH2A.X, a marker of double-strand
break (DSB) damage response signaling, p-STING,
and p-TBK1 were significantly elevated on D3 and D7
post-CNV induction (Fig. 2C and S2A, B), indicating
the involvement of the cGAS-STING axis in the induction of CNV. Similarly, the expressions of p-STING and
p-TBK1 in OIR retinae were significantly increased as

Ni et al. Journal of Neuroinflammation(2024) 21:164

early as P13, with a prominent peak on P15, followed bya gradual decline (Fig. 2D). On P17, the peak time-pointof retinal angiogenesis development in OIR, the cGASSTING pathway was highly activated (Fig. 2E), indicating the contribution of the cGAS-STING pathway in the
progression of pathological retinal angiogenesis. Thesefindings collectively suggest that the activation of thecGAS-STING signaling contributes to the developmentof retinal/choroidal pathological angiogenesis in mice.
Sting deficiency significantly alleviated the pathologicalangiogenesis

To further evaluate the effect of cGAS-STING pathway
on the development of pathological angiogenesis, weestablished the CNV and OIR models utilizing Stinggt
mice, which harbor a mutant missense allele in the Stinggene resulting in the lack of STING protein [17]. As
shown in the representative fundus images of Fig. 3A,
laser burns were successfully induced in both WT and
Stinggt mice. The Bruch’s membrane was damaged by
focal laser photocoagulation and the new formed andleaky choroidal blood vessels invaded into the retina in
the CNV model [15]. When monitored by FFA, The
Stinggt-CNV mice present relatively less leakage area
(Fig. 3A). Choroid-sclera flat mounts and histologicalsections stained with HE also revealed that Stinggt-CNV
mice displayed a smaller area of neovascularization anda thinner lesion thickness in both the transverse andlongitudinal sections of the eyeball (Fig. 3B). Similarly,
areas of retinal neovascular tufts and avascular zone wereremarkably decreased in the Stinggt-OIR mice (Fig. 3C),
and neovascular cells anterior to the internal limiting
membrane (ILM), hallmarks of angiogenesis in OIR, weresignificantly reduced in the Stinggt-OIR mice (Fig. 3C).
The pharmacological activation and inhibition of STING
were also utilized to further evaluate the role of STING
on retinal angiogenesis. We conducted dose-response
experiments to determine the optimal dosage of C-176,
SN-011, and diABZI. As shown in Fig. S3A-C, concentrations of 5 mM for C-176, 4 mM for SN-011, and 0.5
mM for diABZI were selected as the lowest effective concentrations for further study. Intravitreal injection of the
STING inhibitor C-176 resulted in effects similar to the
Stinggt group, while administration of diABZI, a STING
agonist that binds to the cGAMP binding pocket of
STING, led to STING activation and larger area of neovascular tufts, indicating the essential role of STING activation for promoting pathological angiogenesis (Fig. 3D).
In conclusion, these results demonstrated that the cGASSTING signaling is required for the development of pathological angiogenesis.

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cGAS-STING signaling was required for myeloid cells
activation during retinal/choroidal angiogenesis

To uncover the potential mechanisms underlying cGASSTING activation during retinal/choroidal angiogenesis, we re-analyzed our previously published single-celldata in OIR on P17 [13]. Interestingly, the genes related
to cGAS-STING signaling were enriched in the retinal myeloid cells (Fig. S4A), consistent with previous
studies that STING is mainly expressed in myeloid cells
located in central nervous system (CNS) [18]. Immunofluorescence staining of cryosection showed that thecGAS was specifically expressed on the Iba-1 + microgliathat adjacent to neovascular tufts, while lack of cGAS
expression was observed in the Iba-1 + microglia cells
away from neovascular tufts, indicating the involvementof cGAS-STING signaling in the microglia activationand migration towards the neovascular tufts (Fig. 4A).
The choroid-sclera flat-mounts staining showed that
Iba-1 + myeloid cells around the vessels in WT-CNV
mice exhibited the amoeboid activation morphology
and expressed TNF-α and IFN-β, of which production
were triggered by cGAS-STING signaling. By contrast,
the Stinggt mice presented much less TNF-α and IFN-β
expressed on the Iba-1 + myeloid cells (Fig. 4B). Similarly, the Stinggt-OIR mice also exhibited less amoeboid
Iba-1 + microglia around the CD31 + tufts, and the TNF-α
and IFN-β expressed on the Iba-1 + microglia were significantly reduced (Fig. S4B) indicating the STING activation contributed to the microglia activation and theinflammatory responses during angiogenesis. In addition,
the qPCR analysis demonstrated lower secretion of proangiogenic factors and reduced activation of interferonstimulated genes (ISGs) in the Stinggt group (Fig. 4G).
Collectively, these results demonstrated STING activity
in myeloid cells directed inflammatory responses andangiogenesis in the CNV and OIR models.
Hypoxia-induced DNA damage and activation of cGASSTING signaling in myeloid cells

Given that cytoplasmic DNA serves as a trigger for
cGAS-STING signaling [19, 20], we aim to further investigate whether hypoxic stress induces cytoplasmic DNA
leakage and downstream STING signaling activation
in myeloid cells. Two microglia cell lines, Bv2 and N9,
as well as a macrophage cell line, Raw264.7, were cultured under the hypoxic condition for 24 h. As expected,
upregulation of p-γH2A.X was observed in all threecell lines in response to hypoxic insults (Fig. 4C, D). In
addition, the cGAS-STING signaling was activated with
up-regulation of cGAS, p-STING, and p-TBK1 in the
hypoxia-stimulated myeloid cells. However, no such
response was observed in the bEnd.3 endothelial cellline under hypoxic condition (Fig. 4C, D). Consistently,
immunofluorescence staining revealed that hypoxia led

Ni et al. Journal of Neuroinflammation(2024) 21:164

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Fig. 3 Sting deficiency suppressed the pathological angiogenesis in both the CNV and OIR mice. A Fundus photography (a) and fluorescein angiography(b) in the CNV models from WT and Stinggt mice. The significantly alleviated laser burn lesions and reduced vascular leakage were observed in Stinggt-CNV
mice. B CD31 staining on choroid-sclera complex flat-mounts showed that the CNV angiogenesis area (white circle) was much smaller in the Stinggt CNV
mice (a, n = 6 choroid-sclera complexes). HE staining on sections showing the CNV lesion areas delineated by the yellow dashed line (b, n = 5 eyes). It isquantified as Ta/Tb ratio and lesion area. Ta indicated the thickness of the lesion (black bar), while Tb indicated the thickness of adjacent normal choroid(white bar). C CD31 staining of retinal whole mounts (a, n = 5 retinae) and H&E staining (b, n = 7 eyes) in OIR mice and quantification results. Stinggt micesignificantly reduced the area of neovascular tufts compared with WT-OIR mice. D CD31 staining of retinal whole mounts (a, n = 5 retinae) and H&E
staining (b, n = 7 eyes) in OIR mice and quantification results. Mice subjected to intravitreal injection of C-176 exhibited a reduction in neovascularization
similar to Stinggt mice, while mice injected with diABZI showed a significantly increased area of neovascularization compared to the vehicle controls. R,
retina; Ch, choroid; S, sclera. ILM, internal limiting membrane; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars wereshown in the figures. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

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Fig. 4 (See legend on next page.)(2024) 21:164

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Fig. 4 The cGAS-STING pathway was specifically activated in the myeloid cells. A In the cryosection immunofluorescence staining of WT-OIR mice,Iba-1 + myeloid cells adjacent to neovessels showed more co-localization with cGAS (yellow arrows), whereas Iba-1 + myeloid cells away from neovessels were lack of cGAS (white arrows). In addition, the Stinggt-OIR mice also showed less cGAS expression on the Iba-1 + myeloid cells. Yellow rectangles
demonstrated the enlarged images. B Immunostaining of the choroid-sclera complex flat-mount revealed that WT-CNV mice exhibited more Iba-1 + myeloid cells infiltration, larger cell bodies, and a greater number of cells co-expressing TNF-α and IFN-β (yellow arrowheads). In contrast, Stinggt-CNV micedisplayed a branched morphology similar to that of the controls and exhibited less co-localization with TNF-α and IFN-β. C and D In response to hypoxia
stimulation, the expressions of p-γH2A.X, p-STING, and p-TBK1 were markedly elevated in Bv2 cells, N9 cells, and Raw264.7 cells, whereas these expression
elevations were not observed in the bEnd.3 cells (n = 3 cultures). E and F Under hypoxia condition, cytosolic micronucleus with both DAPI and dsDNA
stanning were co-localized with p-STING in N9 cells (E, yellow arrows). In addition, there were also some scattered cytosolic p-STING signaling co-localize
with scattered cytoplasmic dsDNA signaling, indicating a possible leakage of mtDNA under hypoxic conditions. (F, yellow rectangles). Scale bars wereshown in the figures. G qPCR results showed that compared to the WT-CNV and WT-OIR groups, Stinggt groups exhibit lower expression of Il-6, Isg15, andIfna4. (n = 3 eyes). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no significanceto the accumulation of cytoplasmic DNA, possibly as the
micronuclei from nuclear DNA leakage or mtDNA leakage from mitochondria (Fig. 4E, F and S5A-C). These
cytoplasmic DNA were co-stained with p-STING, indicating the STING activation upon the recognition ofcytosolic DNA in myeloid cells.

these findings suggested that activation of cGAS-STING
triggered necroptosis in myeloid cells, resulting in thedevelopment of pathological neovascularization.

cGAS-STING activation induced necroptosis in ocularmyeloid cells

Finally, we explored the therapeutic potential of targeting
cGAS-STING signaling in neovascular ocular diseases.
Two STING inhibitors, C-176 and SN-011 [21, 22], were
employed to block the cGAS-STING axis through intravitreal injections in OIR and CNV mice. Both C-176 andSN-011 showed remarkable suppression of pathologicalvascular lesions, along with reduced FFA leakage areasin the CNV model (Fig. 6A, B). Moreover, combination
therapy of C-176 or SN-011 with anti-VEGF neutralizing
antibody resulted in the least leakage area (Fig. 6A, B),
further highlighting the therapeutic potential of targeting the cGAS-STING axis in neovascular ocular diseases.
Similarly, the intravitreal injection of C-176 or SN-011
could significantly inhibit the neovascular tufts areas andtheir combination therapy with anti-VEGF neutralizing
antibody also present with least neovascular tufts areasin the OIR model (Fig. 6C, D). Collectively, our findings
demonstrated that targeting STING by C-176 or SN-011
could effectively suppress retinal/choroidal pathologicalangiogenesis, and combination therapy with anti-VEGF
could achieve enhanced efficacy.

Activation of cGAS-STING pathway leads to increased
expression of downstream inflammatory factors, including I-IFN and TNF-α, which serves as a classical inducerof cell necroptosis. Our previous studies have found thatmicroglia promoted the retinal angiogenesis throughRIP3-mediated necroptosis [13]. Here, we hypothesizethat the cGAS-STING signaling in ocular myeloid cells
would induce their necroptosis, resulting in the development of angiogenesis. To validate this hypothesis,
we firstly performed the GSEA analysis on the scRNAseq data of the CD11b + cells. Among the five different
CD11b clusters (Fig. 5A), sMG2 (small microglia cluster2), a previously identified subset associated with necroptosis [13], was highly responded to hypoxia and exhibited increased IFN-α responses (Fig. 5A), indicating the
involvement of cGAS-STING signaling in myeloid cells
necroptosis. Furthermore, the expression of key proteins involved in the necroptosis pathway was examinedin enriched CD11b + myeloid cells from both the CNV
and OIR models. As expected, the p-RIP3, p-MLKL, andFGF2 were highly expressed in the myeloid cells enrichedfrom WT-CNV and WT-OIR, which were abrogated by
Sting deficiency (Fig. 5B), indicating the requirement of
STING activity for necroptosis. Immunofluorescencestaining and 3D co-localization analysis also showedthat the Iba-1 + myeloid cells in Stinggt-CNV and StinggtOIR mice presented with less p-MLKL expression andreduced TUNEL positivity in comparison to WT groups(Fig. 5D, E). In vitro, hypoxia induced necroptosis withhigh expression of RIP1, p-RIP3, and p-MLKL in N9 and
Raw264.7, which was suppressed by STING inhibitor of
C-176, indicating the essential role of STING activation
on necroptosis in myeloid cells (Fig. 5C). Taken together,

Enhanced inhibition of retinal/choroidal angiogenesis bycombination treatment with anti-STING and anti-VEGF
agentsDiscussion
Increasing evidence suggests that immune responses andinflammation play a crucial role in exacerbating neovascular ocular diseases [23]. Intraocular injections ofsteroids like dexamethasone and triamcinolone acetonide have shown promising effects in alleviating macularedema associated with various neovascular ocular diseases [3]. However, their non-specific property brings
about side effects such as elevated intraocular pressure and cataract formation [3]. Understanding specificimmune signaling pathways that mediate angiogenesis
formation holds significant promise in exploring targeted immunotherapy for intervening in neovascularNi et al. Journal of Neuroinflammation

Fig. 5 (See legend on next page.)(2024) 21:164

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Fig. 5 The cGAS-STING activation induces necroptosis in myeloid cells. A The single-cell data was obtained from a published dataset (GSE152928) [13].
The tSNE plot showed five CD11b + clusters: MG0, homeostatic microglia; LcMG, a large cluster of microglia; MF, monocyte-derived and perivascular macrophages; and sMG1 and sMG2, two smaller clusters identified under hypoxic conditions. The irGSEA analysis revealed that the sMG2 subpopulation (redcircles), associated with necroptotic processes, exhibited higher scores in hypoxia, ROS pathway, type I interferon, and angiogenesis biological processes,
indicating the possible relation of necroptosis and cGAS-STING. B The expressions of necroptosis pathway molecules, including p-RIP3, p-MLKL, and FGF2
were detected in the enriched CD11b + myeloid cells from WT and Stinggt choroid-sclera complexes on CNV-D7, as well as in the enriched CD11b + myeloid cells from WT and Stinggt retinae on OIR-P17. C The expressions of necroptosis pathway molecules in Raw264.7 and N9 cell lines. D, E The TUNEL + pMLKL + Iba-1 + necroptotic myeloid cells (yellow arrowheads) were significantly reduced in the CNV (n = 5 choroid-sclera complexes) and OIR (n = 6 eyes)from Stinggt mice and the statistical results were depicted. nv, new vessels; GCL, ganglion cell layer; INL, inner nuclear layer. Scale bars were shown in the
figures. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01

ocular diseases. Our study reveals the pivotal role ofcGAS-STING signaling in promoting pathological angiogenesis through inducing necroptosis in myeloid cells.
STING inhibitors, SN-011 or C-176, effectively mitigatedretinal/choroid inflammation and neovascularization,
indicating targeting cGAS-STING signaling may be a
promising avenue for novel immunotherapeutic interventions in the management of neovascular ocular diseases (Fig. 7).
The emerging approach of immunotherapy has demonstrated considerable potential in the treatment of various
diseases, including tumors and autoimmune conditions[24, 25]. By either stimulating or suppressing the body’s
immune response, it educates the immune system to protect itself and prevent damage. In this study, we observed
STING activation in retinal Iba-1 + microglia and choroidal Iba-1 + macrophages contributed to the pathologicalangiogenesis. Targeting the abnormally activated STING
in myeloid cells could educate the immune system tostop the inflammation-related angiogenesis process,presenting as a novel immunotherapy approach. As weknown, within the CNS, myeloid cells collectively referto microglia, other CNS-associated macrophages, andmonocytes due to their closely related cell types [26].
Here microglia are considered to be the predominant
immune cells in the retinal neovascular environment,
while monocyte-derived macrophages are the main
immune cells in the choroidal neovascular condition.
Our results demonstrated that the selective activation of
STING in retinal microglia and choroidal macrophages
emerge as a distinctive therapeutic target.
Our findings revealed that STINGgt mice were resistant to hypoxia-induced necroptosis, highlighting theessential role of STING signaling in triggering microglial
necroptosis. In fact, the STING pathway has a multifaceted involvement in the regulation of diverse cell deathmechanisms, including apoptosis, necroptosis, pyroptosis, and PANoptosis [27, 28]. For instance, administrationof the STING agonist diABZI in the respiratory tract ofmice triggers PANcroptosis in respiratory epithelial cells,
resulting in the establishment of an ARDS model [29].
Here our results demonstrated that aberrant activationof STING in myeloid cells increased necroptosis eventsand subsequently resulted in pathological angiogenesis.

Elucidating the intricate interplay between STING andnecroptosis holds potential for uncovering innovative
therapeutic strategies for angiogenic diseases. Interestingly, the Sting deficiency induce a reduction in cGAS
expression in Fig. 4A, indicating the presence of a potential feedback loop between cGAS and STING. Previous
studies have shown that activation of cGAS-STING pathway induces type I IFN expression, which in turn triggers
the transcriptional activation of a series of downstream
IFN-stimulated genes [27]. The promoter region of thecGAS gene contains two IFN-sensitive response elementsthat can potentially induce cGAS synthesis in response totype I IFN [30, 31]. Therefore, upon activation of STING
by cGAS, downstream genes could subsequently activate
cGAS, forming a feedback loop. Further research into
regulation of cGAS-STING would elucidate the intricate
relationship between these two molecules.
Hypoxia-induced VEGF expression and release havebeen considered to be the key mechanism mediating
retinal/choroidal angiogenesis. Actually, anti-VEGF
drugs have been widely used in the clinical treatmentof neovascular ocular diseases such as PDR, wet AMD,etc. However, their efficacy is limited, and long-termsustainability still faces challenges. Other factors thatmediate neovascularization need to be further considered. In this study, we found STING-activated myeloid
cells orchestrated the pro-inflammatory microenvironment that favors an increased rate of angiogenesis. Ourprior investigation has unveiled necroptosis-mediated
FGF2 release from myeloid cells, suggesting a synergistic promotion of ocular pathological angiogenesis with
VEGF [13]. This indicates that STING-necroptosis may
function as an independent pathway, complementing the
VEGF-mediated angiogenesis. Based on this background,the combination of immunotherapy targeting STING signaling and anti-VEGF agents was attempted. The results
showed that this combination therapy could enhance theanti-angiogenic efficacy, indicating a promising clinicaltherapy for neovascular ocular diseases.
In this study, C-176 and SN-011, two small-moleculecompounds of STING inhibitors were examined fortreating pathological angiogenesis. As we known, STING
inhibitors have seen application in diverse animal disease models, including systemic lupus erythematosusNi et al. Journal of Neuroinflammation(2024) 21:164

Page 12 of 14

Fig. 6 Combination therapy of STING inhibitors and Anti-VEGF agents in CNV and OIR Models. A Single intravitreal injection of inhibitors for STING (C-176
or SN-011) resulted in a notable reduction in angiogenesis development in CNV. The choroid-sclera complex flat-mounts displayed minimal angiogenic
lesions following combined treatment with C-176 or SN-011 and anti-VEGF Nab. The statistical results are shown in B (n = 6 choroid-sclera complexes). C
Single intravitreal injection of C-176 or SN-011 effectively suppressed angiogenesis in OIR. Notably, OIR retinae exhibited reduced angiogenic tufts following combined treatment with C-176 or SN-011 and anti-VEGF Nab. The statistical results were depicted in D (n = 6 retinae). Scale bars were shown in the
figures. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001(SLE) [32], Parkinson’s disease [33], and ischemic stroke[34]. Notably, a recent clinical trial has been conducted
for using the first inhibitor targeting the cGAS-STING
pathway in SLE [35]. Here we found STING inhibitorspresented promising candidates for neovascular ocular diseases. STING inhibitors typically falling into twoclasses: those binding to cysteine residues C88 or C91
near STING’s transmembrane domain, exemplified by
C-176 and H-151 [36], and those occupying the CDNbinding site, acting as competitive antagonists of STING
activators, including SN-011 and Astin C [22, 37]. Here
we found both types of inhibitors achieved similar degreeof angiogenesis suppression, but C-176 required a higher
concentration (Fig. 6A, C). Studies have shown that
C-176 and its analogs are Cys-reactive drugs, known for

nonspecific binding to unpaired Cys in various proteins,suggesting more off-target activities than SN-011 [21].
Collectively, we proposed that SN-011 might offer betterselectivity and safety for suppressing angiogenesis. Moreover, unlike systemic administration in previous studies,
the intravitreal injection method in this study allows forlower drug doses, minimizing the potential systemic risk
[37].

Conclusion
In summary, our findings demonstrated the activationof the cGAS-STING pathway in retinal microglia andchoroidal macrophage facilitated the process of RIP3MLKL-mediated necroptosis, ultimately leading to neuroinflammation and pathological angiogenesis. TargetedNi et al. Journal of Neuroinflammation(2024) 21:164

Page 13 of 14

Acknowledgements
We thank the staff of Laboratory Animal Center and the staff of Core Facilities
at State Key Laboratory of Ophthalmology, Zhongshan ophthalmic Center fortechnical support.
Author contributions
C. H. and X. L. designed the research. B. N., Z. Y., T. Z., H. Z., Y. Z., S. L., H. X., X. L.,
and W. Y.conducted all the experiments, analyzed the data and performed
statistical analysis. B. N., Z.Y., T. Z., C. H. and X. L. prepared the figures anddrafted the manuscript. All authors read andapproved the final manuscript.
Funding
This work was supported by National Natural Science Foundation of China(No. 82322016, 82171068, 82271095) and Guangdong Provincial Key Area R&D
Program (No. 2023B1111050004).
Data availability
A published RNA-seq data (GSE160306) of retinal tissues was downloaded andre-analyzed.

Declarations
Ethics approval and consent to participate
All procedures were approved by the Animal Use and Care Committee ofZhongshan Ophthalmic Center at the Sun Yat-Sen University, Guangzhou,
China (Ethic ID: 2020 − 102).
Consent for publication
All the authors have approved the manuscript.

Fig. 7 Schematic diagram. The activation of the cGAS-STING pathway
in myeloid cells leads to necroptosis and subsequent pathological angiogenesis in neovascular ocular diseases. Under hypoxic conditions, thedamaged double-stranded DNA triggers the activation of cytosolic cGASSTING and release of I-IFN, TNF-α, and other inflammatory factors. In addition, the cGAS-STING activation upregulates the expression of necroptotic
proteins, including RIP1, RIP3, and MLKL, leading to the secretion of proangiogenic factors such as FGF2 and VEGF. These events collectively
promote the development of neovascular ocular diseases. Employing immunotherapy via SN-011 or C-176, which target distinct sites of the STING
molecule, combined with anti-VEGF treatment, could effectively modulate the immune microenvironment for angiogenesis and suppress pathological neovascularization. Some icons were sourced from BioRender.com

inhibition of the cGAS-STING pathway with C-176 or
SN-011 could significantly suppress the myeloid cell
necroptosis-mediated neuroinflammation and pathological angiogenesis. Additionally, combination of STING
inhibitors, SN-011 and C-176, with anti-VEGF agents
exhibited enhanced anti-angiogenic efficacy. These findings pivotally position the cGAS-STING-necroptosis axis
as a novel and promising target for immunotherapeuticintervention.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03155-y.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3

Competing interests
The authors declare no competing interests.
Received: 30 March 2024 / Accepted: 17 June 2024

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