Blue light damaged-retinal pigment epithelial cell derived-exosomes activate nod-like receptor protein inflammasome_Chinese Journal of Ocular Fundus Diseases

Authors：

Ma Yingxue ,  Chen Song , He Guanghui , Yang Jing , Chen Li , Jiang Jianhong , Song Jian

Tianjin Eye Hospital, Tianjin Key Laboratory of Ophthalmology and Visual Science, Tianjin Eye Institute, Clinical College of Ophthalmology Tianjin Medical University, Tianjin 300020, China;

Corresponding author：

Chen Song, Email: chensong20@hotmail.com

Keywords：

Retinal pigment epithelium/physiopathology; Exosomes; Inflammasomes; Photic stimulation/adverse effects

DOI：

10.3760/cma.j.issn.1005-1015.2017.05.014

Video：

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ObjectiveTo observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3).MethodsCultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR).ResultsBlue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04), IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P＜0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P＜0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P＜0.001).ConclusionThe expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.

Citation： Ma Yingxue, Chen Song, He Guanghui, Yang Jing, Chen Li, Jiang Jianhong, Song Jian. Blue light damaged-retinal pigment epithelial cell derived-exosomes activate nod-like receptor protein inflammasome. Chinese Journal of Ocular Fundus Diseases, 2017, 33(5): 498-502. doi: 10.3760/cma.j.issn.1005-1015.2017.05.014 Copy

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2.	Lykke-Andersen S, Brodersen DE, Jensen TH. Origins and activities of the eukaryotic exosome[J]. J Cell Sci, 2009, 122(Pt 10): 1487-1494. DOI: 10.1242/jcs.047399.
3.	Zhou H, Kajiyama H, Tsuji T, et al. Urinary exosomal Wilms’ tumor-1 as a potential biomarker for podocyte injury[J]. Am J Physiol Renal Physiol, 2013, 305(4): 553-559. DOI: 10.1152/ajprenal.00056.2013.
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5.	Tomany SC, Cruickshanks KJ, Klein R, et al. Sunlight and the 10-year incidence of age-related maculopathy: the Beaver Dam Eye Study[J]. Arch Ophthalmol, 2004, 122(5): 750-757.
6.	Fletcher AE, Bentham GC, Agnew M, et al. Sunlight exposure, antioxidants, and age-related macular degeneration[J]. Arch Ophthalmol, 2008, 126(10): 1396-1403. DOI: 10.1001/archopht.126.10.1396.
7.	Algvere PV, Marshall J, Seregard S. Age-related maculopathy and the impact of blue light hazard[J]. Acta Ophthalmol Scand, 2006, 84(1): 4-15.
8.	蔡善君, 严密, 毛咏秋, 等. 蓝光致人视网膜色素上皮细胞凋亡与线粒体膜电位和细胞色素C的关系[J]. 中华眼科杂志, 2006, 42(12): 1095-1102. DOI: 10.3760/j.issn.0412-4081.2006.12.009.Cai SJ, Yan M, Mao YQ, et al. Relationship between blue light-induced apoptosis and mitochondrial membrane potential and cytochrome C in cultured human retinal pigment epithelium cells[J]. Chin J Ophthalmol, 2006, 42(12): 1095-1102. DOI: 10.3760/j.issn.0412-4081.2006.12.009.
9.	Biasutto L, Chiechi A, Couch R, et al. Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress[J]. Exp Cell Res, 2013, 319(13): 2113-2123. DOI: 10.1016/j.yexcr.2013.05.005.
10.	Wang AL, Lukas TJ, Yuan M, et al. Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration [J/OL]. PLoS One, 2009, 4(1): 4160[2009-01-08]. https://doi.org/10.1371/journal.pone.0004160. DOI: 10.1371/journal.pone.0004160.
11.	Nozaki M, Raisler BJ, Sakurai E, et al. Drusen complement components C3a and C5a promote choroidal neovascularization[J]. Proc Natl Acad Sci USA, 2006, 103(7): 2328-2333.
12.	Hageman GS, Luthert PJ, Victor Chong NH, et al. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration[J]. Prog Retin Eye Res, 2001, 20(6): 705-732.
13.	Dentchev T, Milam AH, Lee VM, et al. Amyloid-beta is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas[J]. Mol Vis, 2003, 9: 184-190.
14.	Mevorach D. Clearance of dying cells and systemic lupus erythematosus: the role of C1q and the complement system[J]. Apoptosis, 2010, 15(9): 1114-1123. DOI: 10.1007/s10495-010-0530-8.
15.	Doyle SL, Campbell M, Ozaki E, et al. NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components[J]. Nat Med, 2012, 18(5): 791-798. DOI: 10.1038/nm.2717.
16.	Camelo S. Potential sources and roles of adaptive immunity in age-related macular degeneration: shall we rename AMD into autoimmune macular disease?[J/OL]. Autoimmune Dis, 2014, 2014: 532487[2014-04-30]. http://dx.doi.org/10.1155/2014/532487. DOI: 10.1155/2014/532487.
17.	Laudisi F, Spreafico R, Evrard M, et al. Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1β release[J]. J Immunol, 2013, 191(3): 1006-1110. DOI: 10.4049/jimmunol.1300489.
18.	Asgari E, Le Friec G, Yamamoto H, et al. C3a modulates IL-1β secretion in human monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome activation[J]. Blood, 2013, 122 (20): 3473-3481. DOI: 10.1182/blood-2013-05-502229.
19.	Anderson OA, Finkelstein A, Shima DT. A2E induces IL-1ß production in retinal pigment epithelial cells via the NLRP3 inflammasome[J/OL]. PLoS One, 2013, 8(6): 67263 [2013-06-28]. https://doi.org/10.1371/journal.pone.0067263. DOI: 10.1371/journal.pone.0067263.
20.	Schütt F, Bergmann M, Holz FG, et al. Isolation of intact lysosomes from human RPE cells and effects of A2-E on the integrity of the lysosomal and other cellular membranes[J]. Graefe’s Arch Clin Exp Ophthalmol, 2002, 240(12): 983-988.
21.	Kauppinen A, Niskanen H, Suuronen T, et al. Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells--implications for age-related macular degeneration (AMD)[J]. Immunol Lett, 2012, 147(1-2): 29-33. DOI: 10.1016/j.imlet.2012.05.005.
22.	Piippo N, Korkmaz A, Hytti M, et al. Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells[J]. Biochim Biophys Acta, 2014, 1843(12): 3038-3046. DOI: 10.1016/j.bbamcr.2014.09.015.

1. Tseng WA, Thein T, Kinnunen K, et al. NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: implications for age-related macular degeneration [J]. Invest Ophthalmol Vis Sci, 2013, 54(1): 110-120. DOI: 10.1167/iovs.12-10655.
2. Lykke-Andersen S, Brodersen DE, Jensen TH. Origins and activities of the eukaryotic exosome[J]. J Cell Sci, 2009, 122(Pt 10): 1487-1494. DOI: 10.1242/jcs.047399.
3. Zhou H, Kajiyama H, Tsuji T, et al. Urinary exosomal Wilms’ tumor-1 as a potential biomarker for podocyte injury[J]. Am J Physiol Renal Physiol, 2013, 305(4): 553-559. DOI: 10.1152/ajprenal.00056.2013.
4. Lopez-Verrilli MA, Court FA. Exosomes: mediators of communication in eukaryotes [J]. Biol Res, 2013, 46(1): 5-11. DOI: 10.4067/S0716-97602013000100001.
5. Tomany SC, Cruickshanks KJ, Klein R, et al. Sunlight and the 10-year incidence of age-related maculopathy: the Beaver Dam Eye Study[J]. Arch Ophthalmol, 2004, 122(5): 750-757.
6. Fletcher AE, Bentham GC, Agnew M, et al. Sunlight exposure, antioxidants, and age-related macular degeneration[J]. Arch Ophthalmol, 2008, 126(10): 1396-1403. DOI: 10.1001/archopht.126.10.1396.
7. Algvere PV, Marshall J, Seregard S. Age-related maculopathy and the impact of blue light hazard[J]. Acta Ophthalmol Scand, 2006, 84(1): 4-15.
8. 蔡善君, 严密, 毛咏秋, 等. 蓝光致人视网膜色素上皮细胞凋亡与线粒体膜电位和细胞色素C的关系[J]. 中华眼科杂志, 2006, 42(12): 1095-1102. DOI: 10.3760/j.issn.0412-4081.2006.12.009.Cai SJ, Yan M, Mao YQ, et al. Relationship between blue light-induced apoptosis and mitochondrial membrane potential and cytochrome C in cultured human retinal pigment epithelium cells[J]. Chin J Ophthalmol, 2006, 42(12): 1095-1102. DOI: 10.3760/j.issn.0412-4081.2006.12.009.
9. Biasutto L, Chiechi A, Couch R, et al. Retinal pigment epithelium (RPE) exosomes contain signaling phosphoproteins affected by oxidative stress[J]. Exp Cell Res, 2013, 319(13): 2113-2123. DOI: 10.1016/j.yexcr.2013.05.005.
10. Wang AL, Lukas TJ, Yuan M, et al. Autophagy and exosomes in the aged retinal pigment epithelium: possible relevance to drusen formation and age-related macular degeneration [J/OL]. PLoS One, 2009, 4(1): 4160[2009-01-08]. https://doi.org/10.1371/journal.pone.0004160. DOI: 10.1371/journal.pone.0004160.
11. Nozaki M, Raisler BJ, Sakurai E, et al. Drusen complement components C3a and C5a promote choroidal neovascularization[J]. Proc Natl Acad Sci USA, 2006, 103(7): 2328-2333.
12. Hageman GS, Luthert PJ, Victor Chong NH, et al. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE-Bruch’s membrane interface in aging and age-related macular degeneration[J]. Prog Retin Eye Res, 2001, 20(6): 705-732.
13. Dentchev T, Milam AH, Lee VM, et al. Amyloid-beta is found in drusen from some age-related macular degeneration retinas, but not in drusen from normal retinas[J]. Mol Vis, 2003, 9: 184-190.
14. Mevorach D. Clearance of dying cells and systemic lupus erythematosus: the role of C1q and the complement system[J]. Apoptosis, 2010, 15(9): 1114-1123. DOI: 10.1007/s10495-010-0530-8.
15. Doyle SL, Campbell M, Ozaki E, et al. NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components[J]. Nat Med, 2012, 18(5): 791-798. DOI: 10.1038/nm.2717.
16. Camelo S. Potential sources and roles of adaptive immunity in age-related macular degeneration: shall we rename AMD into autoimmune macular disease?[J/OL]. Autoimmune Dis, 2014, 2014: 532487[2014-04-30]. http://dx.doi.org/10.1155/2014/532487. DOI: 10.1155/2014/532487.
17. Laudisi F, Spreafico R, Evrard M, et al. Cutting edge: the NLRP3 inflammasome links complement-mediated inflammation and IL-1β release[J]. J Immunol, 2013, 191(3): 1006-1110. DOI: 10.4049/jimmunol.1300489.
18. Asgari E, Le Friec G, Yamamoto H, et al. C3a modulates IL-1β secretion in human monocytes by regulating ATP efflux and subsequent NLRP3 inflammasome activation[J]. Blood, 2013, 122 (20): 3473-3481. DOI: 10.1182/blood-2013-05-502229.
19. Anderson OA, Finkelstein A, Shima DT. A2E induces IL-1ß production in retinal pigment epithelial cells via the NLRP3 inflammasome[J/OL]. PLoS One, 2013, 8(6): 67263 [2013-06-28]. https://doi.org/10.1371/journal.pone.0067263. DOI: 10.1371/journal.pone.0067263.
20. Schütt F, Bergmann M, Holz FG, et al. Isolation of intact lysosomes from human RPE cells and effects of A2-E on the integrity of the lysosomal and other cellular membranes[J]. Graefe’s Arch Clin Exp Ophthalmol, 2002, 240(12): 983-988.
21. Kauppinen A, Niskanen H, Suuronen T, et al. Oxidative stress activates NLRP3 inflammasomes in ARPE-19 cells--implications for age-related macular degeneration (AMD)[J]. Immunol Lett, 2012, 147(1-2): 29-33. DOI: 10.1016/j.imlet.2012.05.005.
22. Piippo N, Korkmaz A, Hytti M, et al. Decline in cellular clearance systems induces inflammasome signaling in human ARPE-19 cells[J]. Biochim Biophys Acta, 2014, 1843(12): 3038-3046. DOI: 10.1016/j.bbamcr.2014.09.015.

Chinese Journal of Ocular Fundus Diseases

Blue light damaged-retinal pigment epithelial cell derived-exosomes activate nod-like receptor protein inflammasome

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