Homeostasis of the retinal pigment epithelium (RPE) is essential for the health and proper function of the retina. Regulation of RPE homeostasis is, however, largely unexplored, yet dysfunction of this process may lead to retinal degenerative diseases, including age-related macular degeneration (AMD). Here, we report that chemokine receptor CXCR5 regulates RPE homeostasis through PI3K/AKT signaling and by suppression of FOXO1 activation. We used primary RPE cells isolated from CXCR5-deficient mice and wild type controls, as well as ex vivo RPE–choroidal–scleral complexes (RCSC) to investigate the regulation of homeostasis. CXCR5 expression in mouse RPE cells was diminished by treatment with hydrogen peroxide. Lack of CXCR5 expression leads to an abnormal cellular shape, pigmentation, decreased expression of the RPE differentiation marker RPE65, an increase in the undifferentiated progenitor marker MITF, and compromised RPE barrier function, as well as compromised cell-to-cell interaction. An increase in epithelial-mesenchymal transition (EMT) markers (αSMA, N-cadherin, and vimentin) was noted in CXCR5-deficient RPE cells both in vitro and in age-progression specimens of CXCR5−/− mice (6, 12, 24-months old). Deregulated autophagy in CXCR5-deficient RPE cells was observed by decreased LC3B-II, increased p62, abnormal autophagosomes, and impaired lysosome enzymatic activity as shown by GFP-LC3-RFP reporter plasmid. Mechanistically, deficiency in CXCR5 resulted in the downregulation of PI3K and AKT signaling, but upregulation and nuclear localization of FOXO1. Additionally, inhibition of PI3K in RPE cells resulted in an increased expression of FOXO1. Inhibition of FOXO1, however, reverts the degradation of ZO-1 caused by CXCR5 deficiency. Collectively, these findings suggest that CXCR5 maintains PI3K/AKT signaling, which controls FOXO1 activation, thereby regulating the expression of genes involved in RPE EMT and autophagy deregulation.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
All data generated and analyzed in the current study are included in this published article and its Supplementary information.
Kaneko H, Dridi S, Tarallo V, Gelfand BD, Fowler BJ, Cho WG, et al. DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature. 2011;471:325–30.
Kerur N, Fukuda S, Banerjee D, Kim Y, Fu D, Apicella I, et al. cGAS drives noncanonical-inflammasome activation in age-related macular degeneration. Nat Med. 2018;24:50–61.
Ma W, Yan RT, Li X, Wang SZ. Reprogramming retinal pigment epithelium to differentiate toward retinal neurons with Sox2. Stem Cells. 2009;27:1376–87.
Zhao C, Yasumura D, Li X, Matthes M, Lloyd M, Nielsen G, et al. mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice. J Clin Invest. 2011;121:369–83.
Brown EE, DeWeerd AJ, Ildefonso CJ, Lewin AS, Ash JD. Mitochondrial oxidative stress in the retinal pigment epithelium (RPE) led to metabolic dysfunction in both the RPE and retinal photoreceptors. Redox Biol. 2019;24:101201.
Esteve-Rudd J, Hazim RA, Diemer T, Paniagua AE, Volland S, Umapathy A, et al. Defective phagosome motility and degradation in cell nonautonomous RPE pathogenesis of a dominant macular degeneration. Proc Natl Acad Sci USA. 2018;115:5468–73.
Golestaneh N, Chu Y, Xiao YY, Stoleru GL, Theos AC. Dysfunctional autophagy in RPE, a contributing factor in age-related macular degeneration. Cell Death Dis. 2017;8:e2537.
Dobner T, Wolf I, Emrich T, Lipp M. Differentiation-specific expression of a novel G protein-coupled receptor from Burkitt’s lymphoma. Eur J Immunol. 1992;22:2795–9.
Kazanietz MG, Durando M, Cooke M. CXCL13 and Its Receptor CXCR5 in Cancer: inflammation, immune response, and beyond. Front Endocrinol (Lausanne). 2019;10:471.
Chevalier N, Jarrossay D, Ho E, Avery DT, Ma CS, Yu D, et al. CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses. J Immunol. 2011;186:5556–68.
Xiao H, Luo G, Son H, Zhou Y, Zheng W. Upregulation of peripheral CD4+CXCR5+ T cells in osteosarcoma. Tumour Biol. 2014;35:5273–9.
Yan Q, Yuan Y, Yankui L, Jingjie F, Linfang J, Yong P, et al. The expression and significance of CXCR5 and MMP-13 in colorectal cancer. Cell Biochem Biophys. 2015;73:253–9.
Mitkin NA, Hook CD, Schwartz AM, Biswas S, Kochetkov DV, Muratova AM, et al. p53-dependent expression of CXCR5 chemokine receptor in MCF-7 breast cancer cells. Sci Rep. 2015;5:9330.
Zhang X, Huang W, Chen X, Lian Y, Wang J, Cai C, et al. CXCR5-overexpressing mesenchymal stromal cells exhibit enhanced homing and can decrease contact hypersensitivity. Mol Ther. 2017;25:1434–47.
Ma W, Cojocaru R, Gotoh N, Gieser L, Villasmil R, Cogliati T, et al. Gene expression changes in aging retinal microglia: relationship to microglial support functions and regulation of activation. Neurobiol Aging. 2013;34:2310–21.
Spindler J, Zandi S, Pfister IB, Gerhardt C, Garweg JG. Cytokine profiles in the aqueous humor and serum of patients with dry and treated wet age-related macular degeneration. PLoS ONE. 2018;13:e0203337.
Huang H, Liu Y, Wang L, Li W. Age-related macular degeneration phenotypes are associated with increased tumor necrosis-alpha and subretinal immune cells in aged Cxcr5 knockout mice. PLoS ONE. 2017;12:e0173716.
Lennikov A, Saddala MS, Mukwaya A, Tang S, Huang H. Autoimmune-mediated retinopathy in CXCR5-deficient mice as the result of age-related macular degeneration associated proteins accumulation. Front Immunol. 2019;10:1903.
Huang H, Lennikov A. CXCR5/NRF2 double knockout mice develop retinal degeneration phenotype at early adult age. Exp Eye Res. 2020;196:108061.
Saddala MS, Lennikov A, Mukwaya A, Huang H. Transcriptome-wide analysis of CXCR5 deficient retinal pigment epithelial (RPE) cells reveals molecular signatures of RPE homeostasis. Biomedicines. 2020;8:147–19.
Manicam C, Pitz S, Brochhausen C, Grus FH, Pfeiffer N, Gericke A. Effective melanin depigmentation of human and murine ocular tissues: an improved method for paraffin and frozen sections. PLoS ONE. 2014;9:e102512.
Johnson LV, Forest DL, Banna CD, Radeke CM, Maloney MA, Hu J, et al. Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration. Proc Natl Acad Sci USA. 2011;108:18277–82.
Barth S, Glick D, Macleod KF. Autophagy: assays and artifacts. J Pathol. 2010;221:117–24.
Kaizuka T, Morishita H, Hama Y, Tsukamoto S, Matsui T, Toyota Y, et al. An autophagic flux probe that releases an internal control. Mol Cell. 2016;64:835–49.
Yamamoto H, Itoh N, Kawano S, Yatsukawa Y-i, Momose T, Makio T, et al. Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria. Proc Natl Acad Sci USA. 2011;108:91–6.
Tzivion G, Dobson M, Ramakrishnan G. FoxO transcription factors; regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta. 2011;1813:1938–45.
Cao X, Li W, Liu Y, Huang H, Ye CH. The anti-inflammatory effects of CXCR5 in the mice retina following ischemia-reperfusion injury. Biomed Res Int. 2019;2019:3487607.
Busch C, Annamalai B, Abdusalamova K, Reichhart N, Huber C, Lin Y, et al. Anaphylatoxins activate Ca(2+), Akt/PI3-Kinase, and FOXO1/FoxP3 in the retinal pigment epithelium. Front Immunol. 2017;8:703.
Weng J, Mata NL, Azarian SM, Tzekov RT, Birch DG, Travis GH. Insights into the function of rim protein in photoreceptors and etiology of stargardt’s disease from the phenotype in abcr knockout mice. Cell. 1999;98:13–23.
Zhou M, Geathers JS, Grillo SL, Weber SR, Wang W, Zhao Y, et al. Role of epithelial-mesenchymal transition in retinal pigment epithelium dysfunction. Front Cell Dev Biol. 2020;8:501–13.
Fernandez-Godino R, Garland DL, Pierce EA. Isolation, culture and characterization of primary mouse RPE cells. Nat Protoc. 2016;11:1206–18.
Huang H, Shen J, Vinores SA. Blockade of VEGFR1 and 2 suppresses pathological angiogenesis and vascular leakage in the eye. PLoS ONE. 2011;6:e21411.
Huang H, Van de Veire S, Dalal M, Parlier R, Semba RD, Carmeliet P, et al. Reduced retinal neovascularization, vascular permeability, and apoptosis in ischemic retinopathy in the absence of prolyl hydroxylase-1 due to the prevention of hyperoxia-induced vascular obliteration. Invest Ophthalmol Vis Sci. 2011;52:7565–73.
Giaever I, Keese CR. Micromotion of mammalian cells measured electrically. Proc Natl Acad Sci USA. 1991;88:7896–900.
Matsuda T, Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA. 2004;101:16–22.
Huang H, Lennikov A, Saddala MS, Gozal D, Grab DJ, Khalyfa A, et al. Placental growth factor negatively regulates retinal endothelial cell barrier function through suppression of glucose-6-phosphate dehydrogenase and antioxidant defense systems. FASEB J. 2019;33:13695–709.
The authors would like to acknowledge the following contributors: Allen Raye (University of Missouri Department of Biomedical Sciences, Columbia, Missouri, USA) for assistance with animal resources; DeAna Grant Electron Microscopy core (University of Missouri, Columbia, Missouri, USA) for technical assistance with EM data acquisition. Ms. Lijuan Fan (Department of Ophthalmology, University of Missouri School of Medicine, Columbia, Missouri) for benchwork assistance. Ms. Catherine Brooks J. (Department of Ophthalmology, University of Missouri School of Medicine, Columbia, Missouri) for benchwork assistance and language corrections. Confocal images were acquired at the University of Missouri Molecular Cytology Core facility (University of Missouri, Columbia, Missouri, USA).
HH’s research was supported by NIH grant R01 EY027824 and Missouri University start-up funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest
The authors declare that they have no conflict of interest.
All experiments were approved by the Institutional Animal Care and Use Committee of the University of Missouri School of Medicine (protocol number: 9520) and were in accordance with the guidelines of the Association for Research in Vision and Ophthalmology Statement for the use of animals in ophthalmic and vision research.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Lennikov, A., Mukwaya, A., Saddala, M.S. et al. Deficiency of C-X-C chemokine receptor type 5 (CXCR5) gene causes dysfunction of retinal pigment epithelium cells. Lab Invest (2020). https://doi.org/10.1038/s41374-020-00491-4