The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye

doi:10.1038/s41467-020-16400-8

. 2020 Jun 1;11(1):2709.

doi: 10.1038/s41467-020-16400-8.

The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye

Anne Wolf^#¹, Marc Herb^#², Michael Schramm², Thomas Langmann^{3

4}

Affiliations

¹ Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, D-50931, Cologne, Germany.
² Institute for Medical Microbiology, Immunology and Hygiene, D-50931, Cologne, Germany.
³ Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, D-50931, Cologne, Germany. thomas.langmann@uk-koeln.de.
⁴ Center for Molecular Medicine Cologne (CMMC), University of Cologne, D-50931, Cologne, Germany. thomas.langmann@uk-koeln.de.

^# Contributed equally.

PMID: 32483169
PMCID: PMC7264151
DOI: 10.1038/s41467-020-16400-8

The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye

Anne Wolf et al. Nat Commun. 2020.

. 2020 Jun 1;11(1):2709.

doi: 10.1038/s41467-020-16400-8.

Authors

Anne Wolf^#¹, Marc Herb^#², Michael Schramm², Thomas Langmann^{3

4}

Affiliations

¹ Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, D-50931, Cologne, Germany.
² Institute for Medical Microbiology, Immunology and Hygiene, D-50931, Cologne, Germany.
³ Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Faculty of Medicine and University Hospital Cologne, D-50931, Cologne, Germany. thomas.langmann@uk-koeln.de.
⁴ Center for Molecular Medicine Cologne (CMMC), University of Cologne, D-50931, Cologne, Germany. thomas.langmann@uk-koeln.de.

^# Contributed equally.

PMID: 32483169
PMCID: PMC7264151
DOI: 10.1038/s41467-020-16400-8

Abstract

Aberrant immune responses including reactive phagocytes are implicated in the etiology of age-related macular degeneration (AMD), a major cause of blindness in the elderly. The translocator protein (18 kDa) (TSPO) is described as a biomarker for reactive gliosis, but its biological functions in retinal diseases remain elusive. Here, we report that tamoxifen-induced conditional deletion of TSPO in resident microglia using Cx3cr1^CreERT2:TSPO^fl/fl mice or targeting the protein with the synthetic ligand XBD173 prevents reactivity of phagocytes in the laser-induced mouse model of neovascular AMD. Concomitantly, the subsequent neoangiogenesis and vascular leakage are prevented by TSPO knockout or XBD173 treatment. Using different NADPH oxidase-deficient mice, we show that TSPO is a key regulator of NOX1-dependent neurotoxic ROS production in the retina. These data define a distinct role for TSPO in retinal phagocyte reactivity and highlight the protein as a drug target for immunomodulatory and antioxidant therapies for AMD.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. XBD173 dampens phagocyte reactivity in laser-CNV.**
a Representative images of Iba1⁺ phagocytes within retinal laser lesions. Scale bar: 50 μm. b Quantification of Iba1⁺ cell morphology within lesions. DMSO/XBD173 n = 17/13 spots. c Quantification of Iba1⁺ area in lesions. n = 13 spots. d TSPO protein in retinas of naïve and lasered mice at indicated time points. Each lane represents an individual retina. Dotted line indicates individual blots processed in parallel. e Densitometry of western blots. LMW TSPO signals were normalized to β-Actin and HMW:LMW TSPO ratio determined. n = 6 retinas from two independent experiments. f Cytokines in retinas of naïve and lasered mice at indicated time points. CCL2 (n = 8 retinas from individual mice); IL-6 (naïve n = 32; 6 h n = 25; 3 d/7 d/14 d n = 17 retinas from individual mice), IL-1β (DMSO/XBD173 naïve n = 33/25; 6 h n = 25; 3 d, 7 d, and 14 d n = 17 retinas from individual mice) and TNF (DMSO/XBD173 naïve n = 33/24; 6 h, 3d, 7d, and 14d n = 17 retinas from individual mice). g Representative images of Iba1⁺ cells in RPE/choroidal laser lesions. Scale bar: 50 μm. h Quantification of Iba1⁺ cell morphology within lesions. i Quantification of Iba1⁺ area in laser lesions. n = 13 spots. j TSPO protein in RPE/choroids of naïve and lasered mice at the indicated time points. k Densitometry of Western blots. n = 6 RPE/choroids from two independent experiments. LMW lower molecular weight, HMW, higher molecular weight, n.t. non-treated. l Cytokine levels in RPE/choroids of naïve and lasered mice. CCL2 (n = 8 RPE/choroids from individual mice); IL-6, IL-1β, and TNF (DMSO/XBD173 naïve n = 30/20; 6 h n = 23; 3 d, 7 d, 14 d n = 17 RPE/choroids from individual mice. m Quantification of extracellular ROS production by primary microglia from wild type mice. Kinetics of ROS production and the area under the curve (AUC) are shown. n = 11 independent experiments. Data show mean ± SEM. ROS data were analyzed using two-tailed unpaired Student’s t test. A linear mixed model was used for laser-CNV data; **P < 0.01 and ***P ≤ 0.001. Source data are provided as a Source Data file.

**Fig. 2. XBD173 inhibits laser-induced vascular leakage and pathological choroidal neovascularization (CNV) in mice.**
a Representative late phase fundus fluorescein angiography (FFA) images at indicated time points post laser injury. Scale bar: 200 µm. b Quantification of vascular leakage intensity after laser-induced CNV. 3 d n = 56; 7 d, 14 d n = 25 eyes from individual mice. c Quantification of vascular leakage area after laser-induced CNV. 3 d n = 56; 7 d, 14 d n = 25 eyes from individual mice. d Representative laser-induced CNV stained with isolectin B4 in RPE/choroidal flat mounts. Scale bar: 100 µm; FA fluorescein angiography. e Quantification of laser-induced CNV area in RPE/choroidal flat mounts. DMSO/XBD173 3 d n = 18/34; 7 d n = 38/17; 14 d n = 17/23 RPE/choroids from individual mice. f Pro-angiogenic growth factor levels in retinas of naïve and lasered mice at indicated time points. n = 8 eyes from individual mice. g Pro-angiogenic growth factor levels in RPE/choroids of naïve and lasered mice at indicated time points. n = 8 eyes from individual mice. h Representative infrared (IR) fundus images at indicated time points post laser injury. Lower panel shows OCT scan from one laser spot marked by a red line. Scale bar: 200 µm. I Quantification of laser spot size. DMSO/XBD173 0 d n = 106/71; 3 d n = 72/42; 7 d, 14 d n = 26 eyes from individual mice. Data show mean ± SEM and a linear mixed model was used for statistical analyses; *P < 0.05; **P < 0.01; and ***P ≤ 0.001. n.s., not significant. Source data are provided as a Source Data file.

**Fig. 3. TSPO deficiency dampens phagocyte reactivity in laser-CNV.**
a Representative images of Iba1⁺ cells within retinal laser lesions. Scale bar: 50 μm. b Quantification of Iba1⁺ cell morphology within lesions. n = 18 spots; TSPO^ΔMG 7 d n = 21 spots. c Quantification of Iba1⁺ area in lesions. n = 18 spots; TSPO^ΔMG 7 d n = 21 spots. d TSPO protein in retinas of naïve and lasered TSPO^fl/fl and TSPO^ΔMG mice at indicated time points. Each lane represents an individual retina. Dotted line indicates individual blots processed in parallel. e Densitometry of western blots. LMW TSPO signals were normalized to β-Actin and HMW:LMW TSPO ratio determined. n = 6 retinas from two independent experiments. f Cytokines in retinas of naïve and lasered TSPO^fl/fl and TSPO^ΔMG mice at indicated time points. CCL2 (n = 8 retinas from individual mice); IL-6, IL-1β, and TNF (naïve n = 13 retinas from individual mice); IL-6, TNF (3 d–14 d n = 8 retinas from individual mice) and IL-1β (6 h–14 d n = 10 retinas from individual mice). g Representative images of Iba1⁺ cells in RPE/choroidal lesions. Scale bar: 50 μm. h Quantification of Iba1⁺ cell morphology within lesions. n = 18 spots. i Quantification of Iba1⁺ area of the laser lesions. n = 18 spots. j TSPO protein in RPE/choroids of naïve and lasered TSPO^fl/fl and TSPO^ΔMG mice at indicated time points. k Densitometry of Western blots. n = 6 RPE/choroids from two independent experiments. LMW lower molecular weight; HMW higher molecular weight. l Cytokines in RPE/choroids of naïve and lasered TSPO^fl/fl and TSPO^ΔMG mice. CCL2 (n = 8 RPE/choroids from individual mice); IL-6 TSPO^fl/fl/TSPO^ΔMG 6 h n = 11/12, 3–14 d n = 8; IL-1β TSPO^fl/fl/TSPO^ΔMG 6 h n = 10/12, 3–14 d n = 10; TNF TSPO^fl/fl/TSPO^ΔMG 6 h n = 9/12, 3 d n = 12 and 7–14 d n = 8 RPE/choroids from individual mice. m Extracellular ROS production by microglia from TSPO^fl/fl and TSPO^ΔMG mice. Kinetics and area under the curve (AUC) are shown. n = 10 independent experiments (+XBD173, n = 3 independent experiments). Data show mean ± SEM. ROS data were analyzed using two-tailed unpaired Student’s t test. A linear mixed model was used for laser-CNV data; **P < 0.01 and ***P ≤ 0.001. Source data are provided as a Source Data file.

**Fig. 4. TSPO deficiency inhibits laser-induced vascular leakage and pathological CNV in mice.**
a Representative late phase fundus fluorescein angiography (FFA) images at indicated time points post laser injury. Scale bar: 200 µm. b Quantification of vascular leakage intensity after laser-induced CNV. 3 d n = 85; 7 d n = 36; and 14 d n = 22 retinas from individual mice. FA fluorescein angiography. c Quantification of vascular leakage area after laser-induced CNV. DMSO/XBD173 3 d n = 91/79; 7 d n = 42/33; 14 d n = 30 retinas from individual mice. d Representative laser-induced CNV stained with isolectin B4 in RPE/choroidal flat mounts. Scale bar: 100 µm; e Quantification of laser-induced CNV area in RPE/choroidal flat mounts. n = 22 RPE/choroids from individual mice. f Pro-angiogenic growth factor levels in retinas of naïve and lasered TSPO^fl/fl and TSPO^ΔMG mice at indicated time points. n = 8 retinas/RPEs from individual mice. g Pro-angiogenic growth factor levels in RPE/choroids of naïve and lasered TSPO^fl/fl and TSPO^ΔMG mice at indicated time points. n = 8 retinas/RPEs from individual mice. h Representative infrared (IR) fundus images at indicated time points post laser injury. Lower panel shows OCT scan from one laser spot marked by a red line. Scale bar: 200 µm. i Quantification of laser spot size. 0 d n = 45, 3 d n = 39, 7 d n = 33, and 14 d n = 25 eyes from individual mice. Data show mean ± SEM and a linear mixed model was used for statistical analyses; *P < 0.05; **P < 0.01; and ***P ≤ 0.001. n.s., not significant. Source data are provided as a Source Data file.

**Fig. 5. ROS production by primary microglia involves TSPO-dependent NOX1 activation.**
a Laser-induced gene expression of *Nox1* in retina and RPE/choroid of DMSO-treated or XBD173-treated mice. Transcript levels for each enzyme were normalized to β-Actin. n = 6 retinas/RPEs from individual mice. b and c Quantification of extracellular ROS production by primary microglia from WT and p22^phox-KO mice b, Nox1-KO mice c. Kinetics of ROS production and the area under the curve (AUC) are shown. Where indicated, primary microglia were stimulated with photoreceptor cell debris. n = 3 independent experiments. d Laser-induced gene expression of *Nox*1 in retina and RPE/choroid of TSPO^fl/fl and TSPO^ΔMG mice. Transcript levels for each enzyme was normalized to β-Actin. n = 6 retinas/RPEs from individual mice. Data shown as mean ± SEM. ROS data were analyzed using two-tailed unpaired Student’s t test. A linear mixed model was used for laser-CNV data; *P < 0.05; **P < 0.01; and ***P ≤ 0.001. Source data are provided as a Source Data file.

**Fig. 6. TSPO associated increase in cytosolic calcium is essential for Nox1-derived extracellular ROS production.**
a and b Quantification of extracellular ROS production by primary microglia from WT a, TSPO^fl/fl, and TSPO^ΔMG mice b. Kinetics of ROS production and the area under the curve (AUC) are shown. Where indicated, primary microglia were stimulated with photoreceptor cell debris and cytosolic Ca²⁺ was increased with the Ca²⁺ ionophore ionomycin as a positive control. n = 4 a, 3 b independent experiments. c and d Quantification of cytosolic calcium levels in primary microglia from WT c, TSPO^fl/fl, and TSPO^ΔMG mice d. Where indicated, primary microglia were stimulated with photoreceptor cell debris. n = 3 independent experiments. Data show mean ± SEM; two-tailed unpaired Student’s t test, *P < 0.05, **P < 0.01. n.t. non-treated. Source data are provided as a Source Data file.

**Fig. 7. NOX1 deficiency reduces mononuclear phagocyte reactivity in laser-induced CNV in mice.**
a Representative images show accumulation of Iba1⁺ cells within the laser lesion in retinal flat mounts. Scale bar: 50 μm. b Quantification of Iba1⁺ cell morphology within laser lesions. 3 d n = 18; 7 d n = 22 spots. c Quantification of Iba1⁺ area of the laser lesions. 3 d n = 18; 7 d n = 22 spots. d TSPO protein levels in retinal cell extracts of naïve and lasered WT and Nox1-KO mice at indicated time points. Each lane represents an individual retina. Dotted line indicates individual blots, which were processed in parallel. e Densitometric analysis of western blots. LMW TSPO signals were normalized to β-Actin and HMW:LMW TSPO ratio determined. n = 6 retinas from two independent experiments. f Cytokine levels in retinas of naïve and lasered WT and Nox1-KO mice at indicated time points. n = 8 retinas from individual mice. g Representative images of Iba1⁺ cells within the laser lesion in RPE/choroidal flat mounts. Scale bar: 50 μm. h Quantification of Iba1⁺ cell morphology within laser lesions. 3 d n = 18, 7 d n = 22 spots. i Quantification of Iba1⁺ area of the laser lesions. 3 d n = 18, 7 d n = 22 spots. j Western blots showing TSPO expression in RPE/choroidal cell extracts of naïve and lasered WT and Nox1-KO mice at indicated time points. k Densitometric analysis of western blots. n = 6 RPE/choroids from two independent experiments. LMW lower molecular weight; HMW higher molecular weight. l Pro-inflammatory cytokine levels in RPE/choroids of naïve and lasered WT and Nox1-KO mice. n = 8 RPE/choroids from individual mice. Data show mean ± SEM. A linear mixed model was used for statistical analyses, *P < 0.05; **P < 0.01; and ***P ≤ 0.001. Source data are provided as a Source Data file.

**Fig. 8. NOX1 deficiency limits laser-induced vascular leakage and pathological CNV in mice.**
a Representative late phase fundus fluorescein angiography (FFA) images at indicated time points post laser injury. Scale bar: 200 µm. b Quantification of vascular leakage intensity after laser-induced CNV. 3 d/7 d n = 32 and 14 d n = 22 eyes from individual mice. c Quantification of vascular leakage area after laser-induced CNV. 3 d/7 d n = 32 and 14 d n = 22 eyes from individual mice. d Representative laser-induced CNV stained with isolectin B4 in RPE/choroidal flat mounts. Scale bar: 100 µm; FA fluorescein angiography. e Quantification of laser-induced CNV area in RPE/choroidal flat mounts. WT/Nox1-KO n = 12/18 RPE/choroids from individual mice. f Pro-angiogenic growth factor levels in retinas of naïve and lasered WT and Nox1-KO mice at indicated time points. n = 8 eyes from individual mice. g Pro-angiogenic growth factor levels in RPE/choroids of naïve and lasered WT and Nox1-KO mice at indicated time points. n = 8 eyes from individual mice. h Representative infrared (IR) fundus images at indicated time points post laser injury. Lower panel shows OCT scan from one laser spot marked by a red line. Scale bar: 200 µm. I Quantification of laser spot size. WT/Nox1-KO 0 d n = 28/68, 3 d n = 22/51, 7 d n = 22/45,14 d n = 22/25, n = 25 eyes from individual mice. Data show mean ± SEM. A linear mixed model was used for statistical analyses, *P < 0.05; **P < 0.01; and ***P ≤ 0.001. n.s., not significant. Source data are provided as a Source Data file.

**Fig. 9. Model of TSPO-mediated ROS production in retinal phagocytes.**
a In the healthy retina, resident microglia populate the plexiform layers. With their long protrusions, they constantly scan their environment and phagocytose cell debris. Different insults in the RPE and photoreceptor layer rapidly alert microglia. Resident microglia transform into ameboid phagocytes, migrate to the lesion sites and recruit macrophages from the periphery. b In response to these pathological signals, microglia increase pro-inflammatory and pro-angiogenic cytokine expression to resolve neuroinflammation and promote tissue recovery. Reactive microglia also upregulate mitochondrial TSPO leading to increased cytosolic calcium levels, which is essential for NOX1-mediated ROS production. Chronic activation of microglia may be detrimental and promote retinal degeneration. Binding of the synthetic ligand XBD173 to TSPO limits inflammatory responses and inhibits the increase of cytosolic calcium levels thus preventing from ROS damage. XBD173 supports the conversion of reactive microglia towards a neuroprotective phenotype, limiting pathological CNV. BM Bruch’s membrane; OS outer segment; IS inner segment; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; GCL ganglion cell layer; NFL nerve fiber layer.

See this image and copyright information in PMC

Cited by

Targeting TSPO Reduces Inflammation and Apoptosis in an In Vitro Photoreceptor-Like Model of Retinal Degeneration.
Corsi F, Baglini E, Barresi E, Salerno S, Cerri C, Martini C, Da Settimo Passetti F, Taliani S, Gargini C, Piano I. Corsi F, et al. ACS Chem Neurosci. 2022 Nov 16;13(22):3188-3197. doi: 10.1021/acschemneuro.2c00582. Epub 2022 Oct 27. ACS Chem Neurosci. 2022. PMID: 36300862 Free PMC article.
Role of the TSPO-NOX4 axis in angiogenesis in glioblastoma.
Jiang H, Li F, Cai L, Chen Q. Jiang H, et al. Front Pharmacol. 2022 Oct 7;13:1001588. doi: 10.3389/fphar.2022.1001588. eCollection 2022. Front Pharmacol. 2022. PMID: 36278207 Free PMC article.
The efficient generation of knockout microglia cells using a dual-sgRNA strategy by CRISPR/Cas9.
Zhang M, Yi F, Wu J, Tang Y. Zhang M, et al. Front Mol Neurosci. 2022 Oct 13;15:1008827. doi: 10.3389/fnmol.2022.1008827. eCollection 2022. Front Mol Neurosci. 2022. PMID: 36311032 Free PMC article.
Single cell RNA sequencing confirms retinal microglia activation associated with early onset retinal degeneration.
Kumari A, Ayala-Ramirez R, Zenteno JC, Huffman K, Sasik R, Ayyagari R, Borooah S. Kumari A, et al. Sci Rep. 2022 Sep 10;12(1):15273. doi: 10.1038/s41598-022-19351-w. Sci Rep. 2022. PMID: 36088481 Free PMC article.
Microglia depletion/repopulation does not affect light-induced retinal degeneration in mice.
Laudenberg N, Kinuthia UM, Langmann T. Laudenberg N, et al. Front Immunol. 2024 Jan 15;14:1345382. doi: 10.3389/fimmu.2023.1345382. eCollection 2023. Front Immunol. 2024. PMID: 38288111 Free PMC article.

See all "Cited by" articles

References

1. Wong WL, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob. Health. 2014;2:e106–e116. - PubMed
1. Witmer A. Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 2003;22:1–29. - PubMed
1. Ba J, et al. Intravitreal anti-VEGF injections for treating wet age-related macular degeneration: a systematic review and meta-analysis. Drug Des. Dev. Ther. 2015;9:5397–5405. - PMC - PubMed
1. Yang S, Zhao J, Sun X. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review. Drug Des. Dev. Ther. 2016;10:1857–1867. - PMC - PubMed
1. Fritsche LG, et al. Age-related macular degeneration: genetics and biology coming together. Annu. Rev. Genom. Hum. Genet. 2014;15:151–171. - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions
Actions

LinkOut - more resources

Full Text Sources
Molecular Biology Databases
- Mouse Genome Informatics (MGI)

[1] Wong WL, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob. Health. 2014;2:e106–e116. - PubMed

[2] Wong WL, et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. Lancet Glob. Health. 2014;2:e106–e116. - PubMed

[3] Witmer A. Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 2003;22:1–29. - PubMed

[4] Witmer A. Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 2003;22:1–29. - PubMed

[5] Ba J, et al. Intravitreal anti-VEGF injections for treating wet age-related macular degeneration: a systematic review and meta-analysis. Drug Des. Dev. Ther. 2015;9:5397–5405. - PMC - PubMed

[6] Ba J, et al. Intravitreal anti-VEGF injections for treating wet age-related macular degeneration: a systematic review and meta-analysis. Drug Des. Dev. Ther. 2015;9:5397–5405. - PMC - PubMed

[7] Yang S, Zhao J, Sun X. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review. Drug Des. Dev. Ther. 2016;10:1857–1867. - PMC - PubMed

[8] Yang S, Zhao J, Sun X. Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review. Drug Des. Dev. Ther. 2016;10:1857–1867. - PMC - PubMed

[9] Fritsche LG, et al. Age-related macular degeneration: genetics and biology coming together. Annu. Rev. Genom. Hum. Genet. 2014;15:151–171. - PMC - PubMed

[10] Fritsche LG, et al. Age-related macular degeneration: genetics and biology coming together. Annu. Rev. Genom. Hum. Genet. 2014;15:151–171. - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye

Affiliations

The TSPO-NOX1 axis controls phagocyte-triggered pathological angiogenesis in the eye

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources

Molecular Biology Databases

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

LinkOut - more resources

Full Text Sources

Molecular Biology Databases