Preliminary study on prevention and treatment of experimental autoimmune uveitis by blocking CD73 detachment from the surface of retinal pigment epithelial cells with matrix metalloprotein-9 inhibitor_Chinese Journal of Ocular Fundus Diseases

Authors：

Zhou Shumin ¹ , Kong Fanqiang ² ,  Chen Song ³

1. The Second Hospital of Tianjin Medical University, Tianjin 300211, China;
2. Laboratory Medicine, General Hospital of Tianjin Medical University, Tianjin 300052, China;
3. Department of Ophthalmology, General Hospital of Tianjin Medical University, Tianjin 300052, China;

Corresponding author：

Chen Song, Email: chszyy248@163.com

Keywords：

Matrix metalloproteinase 9/antagonists & inhibitors; 5'-nucleotidase; Retinal pigment epithelium; Cells, cultured; Uveitis/physiopathology; Animal experimentation

DOI：

10.3760/cma.j.cn511434-20180502-00141

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Objective To preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).Methods RPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73^-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4⁺ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73^-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.Results When the stimulation mode was AMP, the proliferation of CD4⁺ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4⁺ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4⁺ T cells between the CD73^-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).Conclusion MMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.

Citation： Zhou Shumin, Kong Fanqiang, Chen Song. Preliminary study on prevention and treatment of experimental autoimmune uveitis by blocking CD73 detachment from the surface of retinal pigment epithelial cells with matrix metalloprotein-9 inhibitor. Chinese Journal of Ocular Fundus Diseases, 2020, 36(4): 289-294. doi: 10.3760/cma.j.cn511434-20180502-00141 Copy

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16.	Percopo CM, Hooks JJ, Shinohara T, et al. Cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation[J]. J Immunol, 1990, 145(12): 4101-4107.
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18.	Saze Z, Schuler PJ, Hong CS, et al. Adenosine production by human B cells and B cell-mediated suppression of activated T cells[J]. Blood, 2013, 122(1): 9-18. DOI: 10.1182/blood-2013-02-482406.
19.	Benson HL, Mobashery S, Chang M, et al. Endogenous matrix metalloproteinases 2 and 9 regulate activation of CD4⁺ and CD8⁺ T cells[J]. Am J Respir Cell Mol Biol, 2011, 44(5): 700-708. DOI: 10.1165/rcmb.2010-0125OC.
20.	Park SL, Hwang B, Lee SY, et al. p21WAF1 is required for interleukin-16-induced migration and invasion of vascular smooth muscle cells via the p38MAPK/Sp-1/MMP-9 pathway[J/OL]. PLoS One, 2015, 10(11): 0142153[2015-11-06]. https://doi.org/10.1371/journal.pone.0142153. DOI: 10.1371/journal.pone.0142153.

1. Elliott MR, Chekeni FB, Trampont PC, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance[J]. Nature, 2009, 461(7261): 282-286. DOI: 10.1038/nature08296.
2. Stagg J, Smyth MJ. Extracellular adenosine triphosphate and adenosine in cancer[J]. Oncogene, 2010, 29(39): 5346-5358. DOI: 10.1038/onc.2010.292.
3. Velasquez S, Malik S, Lutz SE, et al. Pannexin1 Channels are required for chemokine-mediated migration of CD4⁺ T lymphocytes: role in inflammation and experimental autoimmune encephalomyelitis[J]. J Immunol, 2016, 196(10): 4338-4347. DOI: 10.4049/jimmunol.1502440.
4. Pandolfi JB, Ferraro AA, Sananez I, et al. ATP-induced inflammation drives tissue-resident Th17 cells in metabolically unhealthy obesity[J]. J Immunol, 2016, 196(8): 3287-3296. DOI: 10.4049/jimmunol.1502506.
5. Cauwels A, Rogge E, Vandendriessche B, et al. Extracellular ATP drives systemic inflammation, tissue damage and mortality[J]. Cell Death Dis, 2014, 5(3): 1102. DOI: 10.1038/cddis.2014.70.
6. Liu Y, Zou H, Zhao P, et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels[J]. Neuroscience, 2016, 330: 150-161. DOI: 10.1016/j.neuroscience.2016.05.028.
7. Li N, Mu L, Wang J, et al. Activation of the adenosine A2A receptor attenuates experimental autoimmune myasthenia gravis severity[J]. Eur J Immunol, 2012, 42(5): 1140-1151. DOI: 10.1002/eji.201142088.
8. Yao SQ, Li ZZ, Huang QY, et al. Genetic inactivation of the adenosine A(2A) receptor exacerbates brain damage in mice with experimental autoimmune encephalomyelitis[J]. J Neurochem, 2012, 23(1): 100-112. DOI: 10.1111/j.1471-4159.2012.07807.x.
9. Chen S, Zhou S, Zang K, et al. CD73 expression in RPE cells is associated with the suppression of conventional CD4 cell proliferation[J]. Exp Eye Res, 2014, 127: 26-36. DOI: 10.1016/j.exer.2014.05.008.
10. 孔繁强, 周树民, 陈松. 基质金属蛋白酶-9调控CD73自视网膜色素上皮细胞膜表面脱落机制的初步研究[J]. 中华眼底病杂志, 2017, 33(5): 518-522. DOI: 10.3760/cma.j.issn.1005-1015.2017.05.018.Kong FQ, Zhou SM, Chen S. Matrix metalloproteinase-9 regulates the shedding of CD73 from retinal pigment epithelium[J]. Chin J Ocul Fundus Dis, 2017, 33(5): 518-522. DOI: 10.3760/cma.j.issn.1005-1015.2017.05.018.
11. Crane IJ, Kuppner MC, McKillop-Smith S, et al. Cytokine regulation of RANTES production by human retinal pigment epithelial cells[J]. Cell Immunol, 1998, 184(1): 37-44. DOI: 10.1006/cimm.1997.1235.
12. Chang L, Mei C, Heping X. Complement gene expression and regulation in mouse retina and retinal pigment epithelium/choroid[J]. Mol Vis, 2011, 17: 1588-1597.
13. Thurau SR, Chan CC, Nussenblatt RB, et al. Oral tolerance in a murine model of relapsing experimental autoimmune uveoretinitis (EAU): induction of protective tolerance in primed animals[J]. Clin Exp Immunol, 1997, 109(2): 370-376.
14. Horie S, Sugita S, Futagami Y, et al. Human retinal pigment epithelium-induced CD4⁺ CD25⁺ regulatory T cells suppress activation of intraocular effector T cells[J]. Clin Immunol, 2010, 136(1): 83-95. DOI: 10.1016/j.clim.2010.03.001.
15. Sugita S, Horie S, Yamada Y, et al. Suppression of interleukin-17-producing T-helper 17 cells by retinal pigment epithelial cells[J]. Jpn J Ophthalmol, 2011, 55(5): 565-575. DOI: 10.1007/s10384-011-0064-9.
16. Percopo CM, Hooks JJ, Shinohara T, et al. Cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation[J]. J Immunol, 1990, 145(12): 4101-4107.
17. Palma-Nicolás JP, López E, López-Colomé AM. Thrombin stimulates RPE cell motility by PKC-zeta-and NF-kappaB-dependent gene expression of MCP-1 and CINC-1/GRO chemokines[J]. J Cell Biochem, 2010, 110(4): 948-967. DOI: 10.1002/jcb.22608.
18. Saze Z, Schuler PJ, Hong CS, et al. Adenosine production by human B cells and B cell-mediated suppression of activated T cells[J]. Blood, 2013, 122(1): 9-18. DOI: 10.1182/blood-2013-02-482406.
19. Benson HL, Mobashery S, Chang M, et al. Endogenous matrix metalloproteinases 2 and 9 regulate activation of CD4⁺ and CD8⁺ T cells[J]. Am J Respir Cell Mol Biol, 2011, 44(5): 700-708. DOI: 10.1165/rcmb.2010-0125OC.
20. Park SL, Hwang B, Lee SY, et al. p21WAF1 is required for interleukin-16-induced migration and invasion of vascular smooth muscle cells via the p38MAPK/Sp-1/MMP-9 pathway[J/OL]. PLoS One, 2015, 10(11): 0142153[2015-11-06]. 10.1371/journal.pone.0142153">https://doi.org/10.1371/journal.pone.0142153. DOI: 10.1371/journal.pone.0142153.

Chinese Journal of Ocular Fundus Diseases

Preliminary study on prevention and treatment of experimental autoimmune uveitis by blocking CD73 detachment from the surface of retinal pigment epithelial cells with matrix metalloprotein-9 inhibitor

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