Deficiency of C-X-C chemokine receptor type 5 (CXCR5) gene causes dysfunction of retinal pigment epithelium cells

Abstract

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.

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Fig. 1: CXCR5 expression in mouse RPE cells is reduced by oxidative stress environment.
Fig. 2: De-differentiation of the primary CXCR5-deficient RPE cells.
Fig. 3: The impaired barrier function of the CXCR5-deficient RPE cells.
Fig. 4: Increased EMT markers in CXCR5-deficient RPE cells.
Fig. 5: Increased EMT markers in the CXCR5 KO mice with age.
Fig. 6: Deregulated autophagy function of the CXCR5-deficient RPE cells.
Fig. 7: PI3K-AKT-FOXO1 signaling in CXCR5-deficient RPE cells.
Fig. 8: Extracellular deposition of β-amyloid and APOE of primary mouse CXCR5 KO RPE cells.

Data availability

All data generated and analyzed in the current study are included in this published article and its Supplementary information.

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Acknowledgements

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).

Funding

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.

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The study was conceived and designed by AL and HH; AL, AM, and HH performed primary RPE cell isolation and culture, in vitro experiments, RPE–Choroid–Sclera Complex Flat Mounts staining and analysis. The manuscript writing and figure design were performed by AL, AM, MSS, and HH. All authors reviewed and accepted the final version of the manuscript.

Corresponding author

Correspondence to Hu Huang.

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The authors declare that they have no conflict of interest.

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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.

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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

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