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Cited by 142Retinal Development and DisordersRetinal Diseases and Treatmentsinterferon and immune responses

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Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration

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Zou, Ming, Ke, Qin, Nie, Qian, Qi, Ruili, Zhu, Xingfei, Liu, Wei, Hu, Xuebin, Sun, Qian, Fu, Jia-Ling, Tang, Xiangcheng, Liu, Yizhi, Li, David Wan-Cheng, Gong, Lili·2022·Cell Death & Differentiation
DOI 10.1038/s41418-022-00967-4PMC PMC9433402
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Abstract

Atrophic (“dry”) form of age-related macular degeneration (AMD) is a leading cause of vision loss characterized by macular retinal pigment epithelium (RPE) and the ensuing photoreceptor degeneration. cGAS-STING signaling is a key cytosolic DNA sensor system in innate immunity and have recently been shown promotes RPE degeneration. However, expression regulation and therapeutic potential of cGAS and STING are not explored in retina under dry AMD pathogenic conditions. Our analysis shows upregulated STING RNA and increased chromatin accessibility around cGAS and STING promoters in macular retinas from dry AMD patients. cGAS-STING activation was detected in oxidative stress-induced mouse retina degeneration, accompanied with cytosolic leakage of damaged DNA in photoreceptors. Pharmaceutical or genetic approaches indicates STING promotes retina inflammation and degeneration upon oxidative damage. Drug screening reveals that BRD4 inhibitor JQ1 reduces cGAS-STING activation, inflammation and photoreceptor degeneration in the injured retina. BRD4 inhibition epigenetically suppresses STING transcription, and promotes autophagy-dependent cytosolic DNA clearance. Together, our results show that activation of cGAS-STING in retina may present pivotal innate immunity response in GA pathogenesis, whereas inhibition of cGAS-STING signaling by JQ1 could serve as a potential therapeutic strategy.

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Figures

Fig. 1 STING is elevated in macular retina of dry AMD patients and cGAS-STING signaling is activated during mouse retina degeneration. A Gene set enrichment analysis (GSEA) profiles showing significant enrichment of gene sets associated with indicated pathway in dry AMD retinas (n = 41) compared to normal retinas (n = 55) (GSE29801) [24]. The false discovery rate (FDR) < 0.25 for pathways mentioned. B RNA-seq analysis of STING expression in extra-macular and macular retinas of normal (n = 26) and dry AMD patients (n = 20) [24]. Comparisons were made between retinas from normal and dry AMD patients (unpaired t-test), and the extra-macular and macular retinas of the same eye (paired t-test). C–J Mice were intraperitoneal (IP) injected with PBS or sodium iodate (SI) (35 mg/kg) and analysis was conducted 3-day post injection otherwise indicated. C Fundus photography (a, b) and fluorescein angiography (c, d) showing eye morphology. RPE flat mounts stained with phallodin-FITC to label the F-actin (e, f) and with IBA1 antibody to label the mononuclear phagocytes (MP) (g, h). Scale bar: 20 μM. (n = 3). (i, j) Immunohistochemistry (IHC) showing IBA1-positive MP in retina sections. Scale bar: 50 μM (n = 2). D Upper panels: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showing cell death. Arrow head: TUNEL-positive RPE cells. Lower panels: Hematoxylin and eosin (HE) staining. (n = 3) H: hour, D: day. Scale bar: 50 μM. Western blot (WB) analysis showing retina proteins at the indicated time (E) or 3-day (F, G) post injection. Right panels: quantification results of WB. M month. H qRT-PCR analysis of retina RNA 3-day post injection. I WB showing retina proteins. Right panels: quantification results of WB. J IHC analysis using rhodopsin antibody shows retina structure 1-day post SI injection. Arrows indicated destruction of photoreceptors. (n = 3). Scale bar:100 μM.
Fig. 1 STING is elevated in macular retina of dry AMD patients and cGAS-STING signaling is activated during mouse retina degeneration. A Gene set enrichment analysis (GSEA) profiles showing significant enrichment of gene sets associated with indicated pathway in dry AMD retinas (n = 41) compared to normal retinas (n = 55) (GSE29801) [24]. The false discovery rate (FDR) < 0.25 for pathways mentioned. B RNA-seq analysis of STING expression in extra-macular and macular retinas of normal (n = 26) and dry AMD patients (n = 20) [24]. Comparisons were made between retinas from normal and dry AMD patients (unpaired t-test), and the extra-macular and macular retinas of the same eye (paired t-test). C–J Mice were intraperitoneal (IP) injected with PBS or sodium iodate (SI) (35 mg/kg) and analysis was conducted 3-day post injection otherwise indicated. C Fundus photography (a, b) and fluorescein angiography (c, d) showing eye morphology. RPE flat mounts stained with phallodin-FITC to label the F-actin (e, f) and with IBA1 antibody to label the mononuclear phagocytes (MP) (g, h). Scale bar: 20 μM. (n = 3). (i, j) Immunohistochemistry (IHC) showing IBA1-positive MP in retina sections. Scale bar: 50 μM (n = 2). D Upper panels: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showing cell death. Arrow head: TUNEL-positive RPE cells. Lower panels: Hematoxylin and eosin (HE) staining. (n = 3) H: hour, D: day. Scale bar: 50 μM. Western blot (WB) analysis showing retina proteins at the indicated time (E) or 3-day (F, G) post injection. Right panels: quantification results of WB. M month. H qRT-PCR analysis of retina RNA 3-day post injection. I WB showing retina proteins. Right panels: quantification results of WB. J IHC analysis using rhodopsin antibody shows retina structure 1-day post SI injection. Arrows indicated destruction of photoreceptors. (n = 3). Scale bar:100 μM.CC BY · PubMed Central
Fig. 2 STING promotes retina inflammation upon oxidative injury. A HE staining of mouse retina. Subretinal bleeding was observed in retinas from 4 out of 6 diABZI-injected mice. Scale bar: 100 μM. B IF analysis showing IBA1-positive MPs. Note that diABZI alone increased MP infiltration around the optic nerve. Right panel: quantification of IBA1-positive cells. Scale bar: 200 μM. WB (C) or qRT-PCR (D) showing indicated retinal proteins or gene expression. WB (E) or qRT-PCR (F) showing indicated retinal proteins or gene expression. G HE staining of mouse retina. Arrows indicate retina disorganization and arrow heads indicate swelling and bundling of RPE cells. Scale bar: 200 μM. H IF analysis showing IBA1-positive MPs in retina. Arrows: destructive retina, arrow heads: subretinal localization of MPs. n = 4. Scale bar: 200 μM. I IF analysis shows IBA1-positive MPs in RPE flat mounts. n = 4. Scale bar: 500 μM. Right panel: IBA1-positive cell number in four randomly selected regions. J WB showing indicated proteins in ARPE-19 cells. Cells were exposed to 600 μM H2O2 for 2 h and recovered for 2 h before analysis.
Fig. 2 STING promotes retina inflammation upon oxidative injury. A HE staining of mouse retina. Subretinal bleeding was observed in retinas from 4 out of 6 diABZI-injected mice. Scale bar: 100 μM. B IF analysis showing IBA1-positive MPs. Note that diABZI alone increased MP infiltration around the optic nerve. Right panel: quantification of IBA1-positive cells. Scale bar: 200 μM. WB (C) or qRT-PCR (D) showing indicated retinal proteins or gene expression. WB (E) or qRT-PCR (F) showing indicated retinal proteins or gene expression. G HE staining of mouse retina. Arrows indicate retina disorganization and arrow heads indicate swelling and bundling of RPE cells. Scale bar: 200 μM. H IF analysis showing IBA1-positive MPs in retina. Arrows: destructive retina, arrow heads: subretinal localization of MPs. n = 4. Scale bar: 200 μM. I IF analysis shows IBA1-positive MPs in RPE flat mounts. n = 4. Scale bar: 500 μM. Right panel: IBA1-positive cell number in four randomly selected regions. J WB showing indicated proteins in ARPE-19 cells. Cells were exposed to 600 μM H2O2 for 2 h and recovered for 2 h before analysis.CC BY · PubMed Central
Fig. 3 OS-induced DNA damage and cytosolic leakage provoke retinal cell inflammation. Immunofluorescence (IF) staining for dsDNA A and dsDNA/rhodopsin B in retina. The DNA was counterstained with DAPI. Scale bar: 200 μM. C Schematic diagram for preparation of nuclei- and mitochondria-free retinal cytosol. D WB showing separation of retina nuclear and cytosolic fractions. E q-PCR analysis of cytosolic nuclear and mitochondrial (Mito) DNA. F, G IF staining in 661W cells using the indicated antibodies. Arrows: cytosolic DNA with positive γH2Ax labeling. Scale bar: 5 μM. WB showing retina proteins at the indicated time points (H) or 3-day (I) post SI injection. J WB showing indicated proteins in 661W cells. Cells were treated with or without Ara-C (10 μM) and collected at the indicated time points. K, L qRT-PCR analysis showing gene expression in ARPE cells. Cells were treated with DMSO or NAC (1 mM) or MitoQ (1 μM) for 24 h before collection. M, N WB analysis showing protein expression. M 661W cells were untreated (Ctrl) or, transfected with lipofectamine or linearized GFP-C3 plasmid (1 ug/ml) and harvested at the indicated time points. N ARPE cells were transfected with lipofectamine (Ctrl), or genomic DNA isolated from normal (nor-gDNA) or H2O2-exposred (H2O2-gDNA) ARPE or 661W cells (2 ug/ml) and collected 16 h post transfection. O qRT-PCR analysis showing indicated genes in ARPE cells transfected with or without ARPE genomic DNA as described as Fig. 6N.
Fig. 3 OS-induced DNA damage and cytosolic leakage provoke retinal cell inflammation. Immunofluorescence (IF) staining for dsDNA A and dsDNA/rhodopsin B in retina. The DNA was counterstained with DAPI. Scale bar: 200 μM. C Schematic diagram for preparation of nuclei- and mitochondria-free retinal cytosol. D WB showing separation of retina nuclear and cytosolic fractions. E q-PCR analysis of cytosolic nuclear and mitochondrial (Mito) DNA. F, G IF staining in 661W cells using the indicated antibodies. Arrows: cytosolic DNA with positive γH2Ax labeling. Scale bar: 5 μM. WB showing retina proteins at the indicated time points (H) or 3-day (I) post SI injection. J WB showing indicated proteins in 661W cells. Cells were treated with or without Ara-C (10 μM) and collected at the indicated time points. K, L qRT-PCR analysis showing gene expression in ARPE cells. Cells were treated with DMSO or NAC (1 mM) or MitoQ (1 μM) for 24 h before collection. M, N WB analysis showing protein expression. M 661W cells were untreated (Ctrl) or, transfected with lipofectamine or linearized GFP-C3 plasmid (1 ug/ml) and harvested at the indicated time points. N ARPE cells were transfected with lipofectamine (Ctrl), or genomic DNA isolated from normal (nor-gDNA) or H2O2-exposred (H2O2-gDNA) ARPE or 661W cells (2 ug/ml) and collected 16 h post transfection. O qRT-PCR analysis showing indicated genes in ARPE cells transfected with or without ARPE genomic DNA as described as Fig. 6N.CC BY · PubMed Central
Fig. 4 BRD4 inhibitors repress STING expression and STING-mediated NFκB activation. A Schematic diagram shows drug screening process. The red triangles represent identified BRD4 inhibitors. B Representative images from high-content analysis. Arrow heads: γH2Ax-positive cytosolic DNA. Right panel: relative cytosolic DNA number after normalized to nuclei numbers, 30–50 cells counted. WB or qRT-PCR analysis showing protein C or gene D expression in ARPE-19 cells. Selection of other small molecules in WB was based on results from high-content analysis. Treatment conducted as described in A. WB E or qRT-PCR F analysis conducted in 661W treated with or without 10 μM of JQ1 for 24 h.
Fig. 4 BRD4 inhibitors repress STING expression and STING-mediated NFκB activation. A Schematic diagram shows drug screening process. The red triangles represent identified BRD4 inhibitors. B Representative images from high-content analysis. Arrow heads: γH2Ax-positive cytosolic DNA. Right panel: relative cytosolic DNA number after normalized to nuclei numbers, 30–50 cells counted. WB or qRT-PCR analysis showing protein C or gene D expression in ARPE-19 cells. Selection of other small molecules in WB was based on results from high-content analysis. Treatment conducted as described in A. WB E or qRT-PCR F analysis conducted in 661W treated with or without 10 μM of JQ1 for 24 h.CC BY · PubMed Central
Fig. 5 BRD4 inhibitors epigenetically represses STING transcription. A, B qRT-PCR analysis for gene expression. The detailed treatment was described in “Methods” section. C ATAC-Seq shows chromatin accessibility in the indicated genes. (macular: n = 5 for normal, n = 2 for early dry AMD, n = 5 for GA; peripheral macular: n = 6 for normal, n = 4 for early dry AMD, n = 4 for GA) [37]. D Micrococcal nuclease digestion assay shows chromatin compaction in 661W cells. Treatment was the same in B. TSS transcription start site. E Epigenetic profiles of STING in mouse macrophage treated with 5 μM of I-BET762 (+) or DMSO (−). Original data from published ChIP-Seq [39]. The y-axes represent the average number of tags per gene per 25 base pairs per 1,000,000 mapped reads. Scale values are indicated in parentheses. F WB analysis of retina proteins in the indicated treatment. G ChIP assay shows association of the indicated proteins with the STING.
Fig. 5 BRD4 inhibitors epigenetically represses STING transcription. A, B qRT-PCR analysis for gene expression. The detailed treatment was described in “Methods” section. C ATAC-Seq shows chromatin accessibility in the indicated genes. (macular: n = 5 for normal, n = 2 for early dry AMD, n = 5 for GA; peripheral macular: n = 6 for normal, n = 4 for early dry AMD, n = 4 for GA) [37]. D Micrococcal nuclease digestion assay shows chromatin compaction in 661W cells. Treatment was the same in B. TSS transcription start site. E Epigenetic profiles of STING in mouse macrophage treated with 5 μM of I-BET762 (+) or DMSO (−). Original data from published ChIP-Seq [39]. The y-axes represent the average number of tags per gene per 25 base pairs per 1,000,000 mapped reads. Scale values are indicated in parentheses. F WB analysis of retina proteins in the indicated treatment. G ChIP assay shows association of the indicated proteins with the STING.CC BY · PubMed Central
Fig. 6 OS-induced BRD4 promotes STING expression and inflammation. A Heat map depicting upregulated genes involved in inflammatory response in OS-exposed ARPE cells (p < 0.05, with 1.5-fold change) [40]. B–D WB analysis showing induction of BRD4 upon OS. B 661W cells were treated with or without 600 μM H2O2 for 2 h and recover for 1 day before analysis. Mouse retinas were collected at the indicated time points (C) or 3 days (D) after SI injection for WB. qRT-PCR (E) or WB (F) analysis in ARPE cells. G, H Live/dead cell viability assay shows dead (red) and live (green) ARPE cells. For H2O2 treatment, 1.8 mM used in G and 0.6 mM used in H. I Relative ROS levels in ARPE cells. ROS levels were normalized by reads from cell counting kit-8 assay. J–L qRT-PCR showing gene expression in ARPE cells. After transfection, cells were treated with or without H2O2 (0.6 mM, 2 h) (K), or NAC (1 mM) or MitoQ (1 μM) for 6 h before analysis. M Schematic diagram summarizes OS-induced BRD4 activates STING and inflammation.
Fig. 6 OS-induced BRD4 promotes STING expression and inflammation. A Heat map depicting upregulated genes involved in inflammatory response in OS-exposed ARPE cells (p < 0.05, with 1.5-fold change) [40]. B–D WB analysis showing induction of BRD4 upon OS. B 661W cells were treated with or without 600 μM H2O2 for 2 h and recover for 1 day before analysis. Mouse retinas were collected at the indicated time points (C) or 3 days (D) after SI injection for WB. qRT-PCR (E) or WB (F) analysis in ARPE cells. G, H Live/dead cell viability assay shows dead (red) and live (green) ARPE cells. For H2O2 treatment, 1.8 mM used in G and 0.6 mM used in H. I Relative ROS levels in ARPE cells. ROS levels were normalized by reads from cell counting kit-8 assay. J–L qRT-PCR showing gene expression in ARPE cells. After transfection, cells were treated with or without H2O2 (0.6 mM, 2 h) (K), or NAC (1 mM) or MitoQ (1 μM) for 6 h before analysis. M Schematic diagram summarizes OS-induced BRD4 activates STING and inflammation.CC BY · PubMed Central
Fig. 7 JQ1 inhibits cGAS-STING activation and retina degeneration after SI injury. WB (A, B, D) and qRT-PCR (C) analysis of indicated protein or gene expression. IF showing IBA1-postive cells in retina cryosections (E) and retina flat mounts (F). Right panels: quantification results of IBA1-positive cells in 10 (E) or 15 (F) randomly selected regions. Scale bar: 200 μM, n = 3. G RPE flat mounts were stained with IBA1 and FITC Phalloidin to label F-actin. Scale bar: 100 μM. Right panels: quantification results of IBA1-positive cells in five randomly selected regions. H IHC analysis of the mouse retina. n = 4. Scale bar: 100 μM. I Fundus photography (upper panels) and fluorescein angiography (lower panels) analysis. n = 4.
Fig. 7 JQ1 inhibits cGAS-STING activation and retina degeneration after SI injury. WB (A, B, D) and qRT-PCR (C) analysis of indicated protein or gene expression. IF showing IBA1-postive cells in retina cryosections (E) and retina flat mounts (F). Right panels: quantification results of IBA1-positive cells in 10 (E) or 15 (F) randomly selected regions. Scale bar: 200 μM, n = 3. G RPE flat mounts were stained with IBA1 and FITC Phalloidin to label F-actin. Scale bar: 100 μM. Right panels: quantification results of IBA1-positive cells in five randomly selected regions. H IHC analysis of the mouse retina. n = 4. Scale bar: 100 μM. I Fundus photography (upper panels) and fluorescein angiography (lower panels) analysis. n = 4.CC BY · PubMed Central
Fig. 8 BRD4 inhibition promotes autophagy-dependent clearance of cytosolic DNA. A IF analysis showing cytosolic dsDNA in retina photoreceptors. Scale bar: 200 μM. WB analysis showing indicated proteins in 661W cells (B) or ARPE cells (C, D). E Live cell imaging showing JQ1 promoted exogenous cytosolic dsDNA clearance. Cy3- DNA was transfected into ARPE LC3-GFP cells and live cell imaging was performed 6 h after transfection with or without the addition of JQ1 (1 or 10 μM). The still frames were indicated at the indicated time points. Lower panel: mean fluorescence intensity of Cy3-dsDNA calculated from live cell images using ImageJ from 20 different fields. F IF for dsDNA in 661W cells. Scale bar: 50 μM. Lower panels: the relative cytosolic dsDNA foci number and area in the intact nuclei were calculated from six randomly selected fields. G IF analysis showing dsDNA and DNA damage. Scale bar: 20 μM. Lower panel: relative cytosolic DNA number after normalized to nuclei numbers. For each group, 30–50 cells were counted. H, I qRT-PCR analysis showing indicated gene expression in ARPE cells. JQ1 (10 μM), and or CQ (10 μM) were added 6 h after transfection.
Fig. 8 BRD4 inhibition promotes autophagy-dependent clearance of cytosolic DNA. A IF analysis showing cytosolic dsDNA in retina photoreceptors. Scale bar: 200 μM. WB analysis showing indicated proteins in 661W cells (B) or ARPE cells (C, D). E Live cell imaging showing JQ1 promoted exogenous cytosolic dsDNA clearance. Cy3- DNA was transfected into ARPE LC3-GFP cells and live cell imaging was performed 6 h after transfection with or without the addition of JQ1 (1 or 10 μM). The still frames were indicated at the indicated time points. Lower panel: mean fluorescence intensity of Cy3-dsDNA calculated from live cell images using ImageJ from 20 different fields. F IF for dsDNA in 661W cells. Scale bar: 50 μM. Lower panels: the relative cytosolic dsDNA foci number and area in the intact nuclei were calculated from six randomly selected fields. G IF analysis showing dsDNA and DNA damage. Scale bar: 20 μM. Lower panel: relative cytosolic DNA number after normalized to nuclei numbers. For each group, 30–50 cells were counted. H, I qRT-PCR analysis showing indicated gene expression in ARPE cells. JQ1 (10 μM), and or CQ (10 μM) were added 6 h after transfection.CC BY · PubMed Central

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Authors
Zou, M., Ke, Q., Nie, Q., Qi, R., Zhu, X., Liu, W., Hu, X., Sun, Q., Fu, J.-L., Tang, X., Liu, Y., Li, D. W.-C., & Gong, L.
Affiliations
Sun Yat-sen University (CN)
Corresponding author
  • Li, David Wan-Cheng
  • Gong, Lili
ORCID
Venue
Cell Death & Differentiation
Publisher
Springer Science and Business Media LLC
Published
2022-03-28
Volume
29
Issue
9
Pages
1816-1833
ISSN
  • 1350-9047 (ISSN-L)
  • 1476-5403
DOI
10.1038/s41418-022-00967-4
PMC
PMC9433402
OpenAlex
W4220995455
Cited by
138
References
50
Citations by year
2026: 28 · 2025: 37 · 2024: 45 · 2023: 20 · 2022: 8
FWCI
9.30
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Differentiation (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (81770910, 81970787, 82070969, 82000876, 81970784, 81970910)
  • Natural Science Foundation of Guangdong Province (2019B1515120014)
  • Sun Yat-sen University (3030901009090)
Topics
  • Health Sciences › Medicine › Ophthalmology › Retinal Development and Disorders
  • Health Sciences › Medicine › Ophthalmology › Retinal Diseases and Treatments
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
Keywords
cGAS-STING pathway, JQ1, BRD4 inhibition, dry age-related macular degeneration, retinal pigment epithelium degeneration, photoreceptor degeneration, oxidative stress, cytosolic DNA sensing, chromatin accessibility, autophagy, innate immunity
MeSH terms
STING Protein, Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase, Animals, Inflammation, Membrane Proteins, Nuclear Proteins, Nucleotidyltransferases, Retina, Transcription Factors, Oxidative Stress, Mice, Retinal Pigment Epithelium

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