CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis
Lingjun Zhang, … , Rachel R. Caspi, Feng Lin
Lingjun Zhang, … , Rachel R. Caspi, Feng Lin
Published August 11, 2022
Citation Information: JCI Insight. 2022;7(18):e157038. https://doi.org/10.1172/jci.insight.157038.
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Research Article

CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis

Abstract

Cub domain-containing protein 1 (CDCP1) is a protein that is highly expressed on the surface of many cancer cells. However, its distribution in normal tissues and its potential roles in nontumor cells are poorly understood. We found that CDCP1 is present on both human and mouse retinal pigment epithelial (RPE) cells. CDCP1-KO mice developed attenuated retinal inflammation in a passive model of autoimmune uveitis, with disrupted tight junctions and infiltrating T cells detected in RPE flat mounts from WT but not CDCP1-KO mice during EAU development. Mechanistically, we discovered that CDCP1 on RPE cells was upregulated by IFN-γ in vitro and after EAU induction in vivo. CD6 stimulation induced increased RPE barrier permeability of WT but not CDCP1-knockdown (CDCP1-KD) RPE cells, and activated T cells migrated through WT RPE monolayers more efficiently than the CDCP1-KD RPE monolayers. In addition, CD6 stimulation of WT but not the CDCP1-KD RPE cells induced massive stress fiber formation and focal adhesion disruption to reduce cell barrier tight junctions. These data suggest that CDCP1 on RPE cells interacts with CD6 on T cells to induce RPE cytoskeleton remodeling and focal adhesion disruption, which open up the tight junctions to facilitate T cell infiltration for the development of uveitis.

Authors

Lingjun Zhang, Nozha Borjini, Yu Lun, Sweta Parab, Gospel Asonye, Rupesh Singh, Brent A. Bell, Vera L. Bonilha, Andrei Ivanov, David A. Fox, Rachel R. Caspi, Feng Lin

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Figure 6

CDCP1 knockdown on RPE cells impairs T cell transmigration by reducing CD6-stimulated cytoskeletal remodeling and tight junction disruption.

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CDCP1 knockdown on RPE cells impairs T cell transmigration by reducing C...
(A) Flow cytometry analysis showed decreased CDCP1 levels on CDCP1-knockdown (CDCP1-KD) RPE cells. (B) WT and CDCP1-KD RPE cells were cultured in top chambers of Transwell inserts to form monolayers for T cell migration assays. CFSE-labeled mouse T cells were activated and overlaid on RPE monolayers. After 18 hours, numbers of T cells in the top and bottom chambers were quantified using flow cytometry, and the percentages of migration were calculated. Experiments were repeated 3 times. **P < 0.01, t test. (C) After CD6 stimulation, permeabilities of WT and CDCP1-KD RPE monolayers were measured using a FITC-dextran leakage assay. CDCP1-KO RPE monolayer had decreased barrier permeability. Experiments were repeated twice. *P < 0.05, 2-way ANOVA. (D) WT and CDCP1-KD RPE cells were analyzed by confocal microscopy after a 6-hour incubation with hCD6-Fc or human IgG protein. Representative confocal images of ZO-1 (green) and DAPI (magenta). White arrowheads marked ZO-1 loss. (E) CDCP1-KD RPE cells have increased ZO-1 expression compared with WT cells after CD6 treatment. (F) Representative images of actin filaments (green) and activated myosin light chain (pMLC, red) in WT and CDCP1-KD RPE cells after a 4-hour incubation with 1 μg/mL recombinant hCD6-Fc fusion protein or human IgG. (G and H) CD6 treatment induced robust actin filament bundle and stress fiber formation but had a milder effect on the CDCP1-KD cells, as shown by mean fluorescence intensity (MFI). Data were quantitated and analyzed using 2-way ANOVA and Tukey’s test after repeating the experiment 3 times (n = 6 images/condition, experiments were repeated twice) and are presented as the mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 1 μm.